1. Br J Cancer. 2018 Mar 20;118(6):839-846. doi: 10.1038/bjc.2017.459. Epub 2018 Feb
13.

IKKα is required in the intestinal epithelial cells for tumour stemness.

Colomer C(1), Margalef P(1)(2), Gonzalez J(1), Vert A(1), Bigas A(1), Espinosa
L(1).

Author information: 
(1)Cancer Research Program, Institut Mar d'Investigacions Mèdiques, CIBERONC,
Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain.
(2)DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT,
UK.

BACKGROUND: Colorectal cancer is a common cause of death in developed countries. 
Progression from adenoma to invasive carcinoma requires accumulation of mutations
starting with the Adenomatous Polyposis Coli (Apc) gene. NF-κB signalling is a
key element in cancer, mainly related to the activity of IKKβ. IKKα kinase also
participates in this process by mechanisms that are primarily unknown.
METHODS: We generated a compound mouse model with mutation in Apc and lacking
intestinal epithelial IKKα, produced intestinal organoids and tumour spheroids
with different genetic backgrounds, and performed immunohistochemistry and
RNA-seq analysis.
RESULTS: Deficiency of IKKα prevents adenoma formation, with adenomas lacking
IKKα showing reduced proliferation. In contrast, IKKα status did not affect
normal intestinal function. The same divergent phenotype was found in the
organoid-spheroid model. We also found that epithelial IKKα controls stemness,
proliferation and apoptosis-related expression.
CONCLUSIONS: IKKα is a potential therapeutic target for Apc mutant colorectal
cancer patients.

DOI: 10.1038/bjc.2017.459 
PMID: 29438366 


2. Gene. 2018 Mar 20;647:21-30. doi: 10.1016/j.gene.2018.01.001. Epub 2018 Jan 3.

Gene expression profiling provides insights into the immune mechanism of Plutella
xylostella midgut to microbial infection.

Lin J(1), Xia X(2), Yu XQ(3), Shen J(2), Li Y(2), Lin H(2), Tang S(2), Vasseur
L(4), You M(5).

Author information: 
(1)State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou
350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; Fujian Vocational College of Bioengineering,
Fuzhou, 350002, China; Fujian-Taiwan Joint Innovation Centre for Ecological
Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou 350002,
China; Joint International Research Laboratory of Ecological Pest Control,
Ministry of Education, Fuzhou 350002, China.
(2)State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou
350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; Fujian-Taiwan Joint Innovation Centre for
Ecological Control of Crop Pests, Fujian Agriculture and Forestry University,
Fuzhou 350002, China; Joint International Research Laboratory of Ecological Pest 
Control, Ministry of Education, Fuzhou 350002, China.
(3)State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou
350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; Joint International Research Laboratory of
Ecological Pest Control, Ministry of Education, Fuzhou 350002, China; School of
Biological Sciences, University of Missouri-Kansas city, Kansas City, MO
64110-2499, USA.
(4)State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou
350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; Department of Biological Sciences, Brock
University, 1812 Sir Isaac Brock Way, St. Catharines, ON L2S 3A1, Canada.
(5)State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou
350002, China; Institute of Applied Ecology, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; Fujian-Taiwan Joint Innovation Centre for
Ecological Control of Crop Pests, Fujian Agriculture and Forestry University,
Fuzhou 350002, China; Joint International Research Laboratory of Ecological Pest 
Control, Ministry of Education, Fuzhou 350002, China. Electronic address:
msyou@iae.fjau.edu.cn.

Insect gut immunity plays a key role in defense against microorganism infection. 
The knowledge of insect gut immunity has been obtained mostly from Drosophila
melanogaster. Little is known about gut immunity in the diamondback moth,
Plutella xylostella (L.), a pest destroying cruciferous crops worldwide. In this 
study, expressions of the immune-related genes in the midgut of P. xylostella
orally infected with Staphylococcus aureus, Escherichia coli and Pichia pastoris 
were profiled by RNA-seq and qRT-PCR approaches. The results revealed that the
Toll, IMD, JNK and JAK-STAT pathways and possibly the prophenoloxidase activation
system in P. xylostella could be activated by oral infections, and moricins,
gloverins and lysozyme2 might act as important effectors against microorganisms. 
Subsequent knock-down of IMD showed that this gene was involved in regulating the
expression of down-stream genes in the IMD pathway. Our work indicates that the
Toll, IMD, JNK and JAK-STAT pathways may synergistically modulate immune
responses in the P. xylostella midgut, implying a complex and diverse immune
system in the midgut of insects.

Copyright © 2018 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.gene.2018.01.001 
PMID: 29305978  [Indexed for MEDLINE]


3. Gene. 2018 Mar 10;646:136-142. doi: 10.1016/j.gene.2018.01.002. Epub 2018 Jan 3.

LEA proteins from Gastrodia elata enhance tolerance to low temperature stress in 
Escherichia coli.

Zeng X(1), Ling H(1), Yang J(1), Li Y(1), Guo S(2).

Author information: 
(1)Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences,
Beijing 100193, PR China.
(2)Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences,
Beijing 100193, PR China. Electronic address: sxguo2015@163.com.

BACKGROUND: Late embryogenesis abundant (LEA) proteins were initially discovered 
about 35years ago as accumulating late in embryogenesis of cotton seeds. Although
abundant in seeds and pollens, these proteins have been found to protect cells
against desiccation, cold, high temperature, and high salinity.
OBJECTIVE: Here, we present the first comprehensive survey of LEA proteins and
their encoding genes in Gastrodia elata, a well-known medicinal orchid in China. 
Moreover, we researched on LEA family evolutionary relationships and functional
characteristics.
METHODS: The LEA gene family in G. elata (GeLEAs) was cloned based on RNA-Seq
data. In addition, all of GeLEA genes were introduced into Escherichia coli to
assess the function of GeLEAs under low temperature stress.
RESULTS: Based on the phylogenetic analysis with Arabidopsis and Oryza LEA
proteins, we identified 8 LEA protein encoding genes in G. elata that could be
classified into 6 distinct subgroups. The motif composition of these proteins was
an important feature specific to LEA groups. Compared with control, the
expressions of 5 GeLEAs in E. coli exhibited enhanced cold resistance and
viability, indicating that GeLEAs protein could play a protective role in cells
under low temperature stress.
CONCLUSION: Our results suggest that LEAs from G. elata play an important role in
responses to abiotic stress.

Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

DOI: 10.1016/j.gene.2018.01.002 
PMID: 29305972  [Indexed for MEDLINE]


4. J Nanosci Nanotechnol. 2018 Mar 1;18(3):1599-1605. doi: 10.1166/jnn.2018.14212.

Surface Plasmon Resonance Aptamer Biosensor for Discriminating Pathogenic
Bacteria Vibrio parahaemolyticus.

Ahn JY(1), Lee KA(1), Lee MJ(1), Sekhon SS(1), Rhee SK(1), Cho SJ(1), Ko JH(2),
Lee L(2), Han J(2), Kim SY(3), Min J(4), Kim YH(1).

Author information: 
(1)School of Biological Sciences, Chungbuk National University 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, South Korea.
(2)College of Veterinary Medicine, Western University of Health Sciences, Pomona 
CA 91766, USA.
(3)Department of Food Science and Biotechnology, Shin Ansan University, 135,
Sinansandaehak-ro, Danwon-Gu, Ansan 425-792, South Korea.
(4)Department of Bioprocess Engineering, Chonbuk National University, 567
Baekje-daero, Deokjin-Gu Jeonju, Jeonbuk 54896, South Korea.

In this paper, whole-bacteria SELEX (WB-SELEX) strategy was adopted to isolate
specific aptamers against Vibrio parahaemolyticus. Round selection for V.
parahaemolyticus was conducted 11 rounds, including two negative selection
rounds. It was determined through real-time PCR amplification and post-SELEX
experiment. The selected aptmers had high binding property and specificity to V. 
parahaemolyticus. Of 28 aptamers tested, VPCA-apta#1 had the highest binding
affinity compared to other aptamer candidates obtained. To detect V.
parahaemolyticus, aptamer based SPR biosensor platform was constructed and
pathogenic bacteria sensing was conducted in two steps. The first step was to
construct 5'-biotinylated VPCA-apta#1 binding probe. The second step was to
incubate V. parahaemolyticus and test microbes in functionalized SA sensor chip
in parallel. Our platform showed significant activity for detecting and
discriminating V. parahaemolyticus from other enteric species such as Escherichia
coli, Listeria monocytogenes, Sigella sonnei, and Vibrio fischeri. This is the
first report on the use of whole-SELEX to isolate DNA aptamers specific for V.
parahaemolyticus. We demonstrated the feasibility of using aptamer platform for
the detection of V. parahaemolyticus in various food supplies. It might be used
in multiple points of care for diagnosing Vibriosis.

DOI: 10.1166/jnn.2018.14212 
PMID: 29448635 


5. ACS Synth Biol. 2018 Feb 16;7(2):553-564. doi: 10.1021/acssynbio.7b00328. Epub
2018 Jan 5.

Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli.

Liu Q(1)(2)(3), Schumacher J(4), Wan X(1)(2), Lou C(5), Wang B(1)(2).

Author information: 
(1)School of Biological Sciences, University of Edinburgh , Edinburgh, EH9 3FF,
U.K.
(2)Centre for Synthetic and Systems Biology, University of Edinburgh , Edinburgh,
EH9 3JR, U.K.
(3)Department of Chemistry and Biology, National University of Defense Technology
, Changsha, 410073, China.
(4)Department of Life Sciences, Imperial College London , London, SW7 2AZ, U.K.
(5)CAS Key Laboratory of Microbial Physiological and Metabolic Engineering,
Institute of Microbiology, Chinese Academy of Sciences , Beijing, 100101, China.

Synthetic biology approaches commonly introduce heterologous gene networks into a
host to predictably program cells, with the expectation of the synthetic network 
being orthogonal to the host background. However, introduced circuits may
interfere with the host's physiology, either indirectly by posing a metabolic
burden and/or through unintended direct interactions between parts of the circuit
with those of the host, affecting functionality. Here we used RNA-Seq
transcriptome analysis to quantify the interactions between a representative
heterologous AND gate circuit and the host Escherichia coli under various
conditions including circuit designs and plasmid copy numbers. We show that the
circuit plasmid copy number outweighs circuit composition for their effect on
host gene expression with medium-copy number plasmid showing more prominent
interference than its low-copy number counterpart. In contrast, the circuits have
a stronger influence on the host growth with a metabolic load increasing with the
copy number of the circuits. Notably, we show that variation of copy number, an
increase from low to medium copy, caused different types of change observed in
the behavior of components in the AND gate circuit leading to the unbalance of
the two gate-inputs and thus counterintuitive output attenuation. The study
demonstrates the circuit plasmid copy number is a key factor that can
dramatically affect the orthogonality, burden and functionality of the
heterologous circuits in the host chassis. The results provide important guidance
for future efforts to design orthogonal and robust gene circuits with minimal
unwanted interaction and burden to their host.

DOI: 10.1021/acssynbio.7b00328 
PMCID: PMC5820654
PMID: 29240998 


6. Front Microbiol. 2018 Feb 6;9:136. doi: 10.3389/fmicb.2018.00136. eCollection
2018.

Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and
Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism.

Zhang S(1), Liu S(1), Wu N(1), Yuan Y(1), Zhang W(1), Zhang Y(1)(2).

Author information: 
(1)Key Lab of Molecular Virology, Department of Infectious Diseases, Huashan
Hospital, Fudan University, Shanghai, China.
(2)Department of Molecular Microbiology and Immunology, Bloomberg School of
Public Health, Johns Hopkins University, Baltimore, MD, United States.

As dormant phenotypic variants of bacteria, persisters account for many chronic
infections affecting human health. Despite numerous studies, the role of small
non-coding RNA (sRNA) in bacterial persistence has not been reported. To
investigate the role of Hfq-interacting sRNA in persistence, we constructed the
deletion mutants of 20 Hfq-interacting sRNAs (RyhB, GcvB, MgrR, RybB, MicF, SgrS,
RprA, DicF, SsrS, FnrS, GadY, DsrA, OmrB, ArcZ, RyeB, RydC, OmrA, MicA, MicC, and
ChiX) to assess their persistence capacity in uropathogenic Escherichia coli
strain UTI89 and identified a new sRNA RyhB being involved in persister
formation. The ryhB-knockout mutant had significant defect in persistence to a
diverse range of antibiotics (levofloxacin, cefotaxime, gentamicin) and stresses 
(hyperosmosis, acid, and heat) in both exponential phase and stationary phase. In
addition, the effect of RyhB on persistence was synergistic with ppGpp and Fur
protein. RNA-Seq analysis indicated that the ryhB-knockout mutant had a
hyperactive metabolic state compared with the parent strain. Interestingly,
increased adenosine triphosphate (ATP) levels and altered NAD+/NADH ratios were
observed in the ryhB-knockout mutant. Our findings represent a new level of
persistence regulation via sRNA and may provide novel therapeutic targets for
interventions.

DOI: 10.3389/fmicb.2018.00136 
PMCID: PMC5808207
PMID: 29467745 


7. J Mol Med (Berl). 2018 Feb;96(2):147-157. doi: 10.1007/s00109-017-1609-2. Epub
2017 Nov 13.

Identification of generic and pathogen-specific cord blood monocyte
transcriptomes reveals a largely conserved response in preterm and term newborn
infants.

de Jong E(1), Hancock DG(2), Hibbert J(3), Wells C(4)(5), Richmond P(3), Simmer
K(3), Burgner D(6)(7)(8), Strunk T(3)(9), Currie AJ(10)(3).

Author information: 
(1)Medical & Molecular Sciences, School of Veterinary & Life Sciences, Murdoch
University, South Street, Murdoch, Western Australia, 6150, Australia.
emma.dejong@outlook.com.
(2)School of Medicine, Flinders University, Adelaide, Australia.
(3)Centre for Neonatal Research & Education and Division of Paediatrics,
University of Western Australia, Perth, Australia.
(4)Centre for Stem Cell Systems, Department of Anatomy and Neuroscience, MDHS,
University of Melbourne, Melbourne, Australia.
(5)The Walter and Eliza Hall Institute, Melbourne, Australia.
(6)Royal Children's Hospital, Murdoch Children's Research Institute, Melbourne,
Australia.
(7)Department of Paediatrics, University of Melbourne, Melbourne, Australia.
(8)Department of Paediatrics, Monash University, Melbourne, Australia.
(9)Neonatal Directorate, King Edward Memorial and Princess Margaret Hospitals,
Subiaco, Australia.
(10)Medical & Molecular Sciences, School of Veterinary & Life Sciences, Murdoch
University, South Street, Murdoch, Western Australia, 6150, Australia.

Escherichia coli and Staphylococcus epidermidis are predominant causes of
neonatal sepsis, particularly affecting preterm infants. Susceptibility to
infection has been attributed to "immature" innate monocyte defences, but no
studies have assessed global transcriptional responses of neonatal monocytes to
these pathogens. Here, we aimed to identify and characterise the neonatal
monocyte transcriptional responses to E. coli and S. epidermidis and the role of 
common modifiers such as gestational age (GA) and exposure to chorioamnionitis (a
common complication of preterm birth) to better understand early life innate
immune responses. RNA-sequencing was performed on purified cord blood monocytes
from very preterm (< 32 weeks GA) and term infants (37-40 weeks GA) following
standardised challenge with live S. epidermidis or E. coli. The major
transcriptional changes induced by either pathogen were highly conserved between 
infant groups and stimuli, highlighting a common extant neonatal monocyte
response to infection, largely mediated by TLR/NF-κB/TREM-1 signalling. In
addition, we observed an activated interferon-centred immune response specific to
stimulation with E. coli in both preterm and term infants. These data provide
novel insights into the functionality of neonatal monocytes at birth and
highlight potential pathways that could be targeted to reduce the harmful effects
of bacterial-induced inflammation in sepsis. E. coli and S. epidermidis elicit
common transcriptional changes in cord monocytes. The common transcriptional
response is mediated by TLR/NF-κB/TREM-1 signalling. IFN genes are differentially
regulated by E. coli and S. epidermidis in monocytes. These responses are largely
unaffected by GA or exposure to chorioamnionitis.KEY MESSAGES: E. coli and S.
epidermidis elicit common transcriptional changes in cord monocytes. The common
transcriptional response is mediated by TLR/NF-κB/TREM-1 signalling. IFN-genes
are differentially regulated by E. coli and S. epidermidis in monocytes. These
responses are largely unaffected by GA or exposure to chorioamnionitis.

DOI: 10.1007/s00109-017-1609-2 
PMID: 29134255 


8. J Biotechnol. 2018 Jan 20;266:39-49. doi: 10.1016/j.jbiotec.2017.12.011. Epub
2017 Dec 12.

Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using
a QCM sensor.

Yu X(1), Chen F(2), Wang R(3), Li Y(4).

Author information: 
(1)Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 
72701, USA.
(2)Rice Research Center, Shenyang Agricultural University, Shenyang, China.
(3)Department of Biological & Agricultural Engineering, University of Arkansas,
Fayetteville, AR, 72701, USA.
(4)Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 
72701, USA; Department of Biological & Agricultural Engineering, University of
Arkansas, Fayetteville, AR, 72701, USA; Center of Excellence for Poultry Science,
University of Arkansas, Fayetteville, AR, 72701, USA. Electronic address:
yanbinli@uark.edu.

The rapid detection of foodborne pathogens is critical to ensure food safety. The
objective of this study is to select aptamers specifically bound to Escherichia
coli O157:H7 using the whole-bacterium SELEX (Systematic Evolution of Ligands by 
Exponential Enrichment) and apply the selected aptamer to a QCM (quartz crystal
microbalance) sensor for rapid and sensitive detection of target bacteria. A
total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of
counter selection against a mixture of Staphylococcus aureus, Listeria
monocytogenes, and Salmonella Typhimurium, were performed. The aptamer pool from 
the last round was cloned and sequenced. One sequence S1 that appeared 16 times
was characterized and a dissociation constant (Kd) of 10.30nM was obtained.
Subsequently, a QCM aptasensor was developed for the rapid detection of E. coli
O157:H7. The limit of detection (LOD) and the detection time of the aptasensor
was determined to be 1.46×103 CFU/ml and 50min, respectively. This study
demonstrated that the ssDNA aptamer selected by the whole-bacterium SELEX
possessed higher sensitivity than previous work and the potential use of the
constructed QCM aptasensor in rapid screening of foodborne pathogens.

Copyright © 2017 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jbiotec.2017.12.011 
PMID: 29242148 


9. BMC Genomics. 2018 Jan 16;19(1):52. doi: 10.1186/s12864-017-4413-z.

Transcriptional responses of Escherichia coli during recovery from inorganic or
organic mercury exposure.

LaVoie SP(1), Summers AO(2).

Author information: 
(1)Department of Microbiology, University of Georgia, Athens, GA, 30602, USA.
slavoie5@uga.edu.
(2)Department of Microbiology, University of Georgia, Athens, GA, 30602, USA.
summers@uga.edu.

BACKGROUND: The protean chemical properties of mercury have long made it
attractive for diverse applications, but its toxicity requires great care in its 
use, disposal, and recycling. Mercury occurs in multiple chemical forms, and the 
molecular basis for the distinct toxicity of its various forms is only partly
understood. Global transcriptomics applied over time can reveal how a cell
recognizes a toxicant and what cellular subsystems it marshals to repair and
recover from the damage. The longitudinal effects on the transcriptome of
exponential phase E. coli were compared during sub-acute exposure to mercuric
chloride (HgCl2) or to phenylmercuric acetate (PMA) using RNA-Seq.
RESULTS: Differential gene expression revealed common and distinct responses to
the mercurials throughout recovery. Cultures exhibited growth stasis immediately 
after each mercurial exposure but returned to normal growth more quickly after
PMA exposure than after HgCl2 exposure. Correspondingly, PMA rapidly elicited
up-regulation of a large number of genes which continued for 30 min, whereas
fewer genes were up-regulated early after HgCl2 exposure only some of which
overlapped with PMA up-regulated genes. By 60 min gene expression in PMA-exposed 
cells was almost indistinguishable from unexposed cells, but HgCl2 exposed cells 
still had many differentially expressed genes. Relative expression of energy
production and most metabolite uptake pathways declined with both compounds, but 
nearly all stress response systems were up-regulated by one or the other
mercurial during recovery.
CONCLUSIONS: Sub-acute exposure influenced expression of ~45% of all genes with
many distinct responses for each compound, reflecting differential biochemical
damage by each mercurial and the corresponding resources available for repair.
This study is the first global, high-resolution view of the transcriptional
responses to any common toxicant in a prokaryotic model system from exposure to
recovery of active growth. The responses provoked by these two mercurials in this
model bacterium also provide insights about how higher organisms may respond to
these ubiquitous metal toxicants.

DOI: 10.1186/s12864-017-4413-z 
PMCID: PMC5769350
PMID: 29338696 


10. J Gen Appl Microbiol. 2018 Jan 15;63(6):311-324. doi: 10.2323/jgam.2017.01.002.
Epub 2017 Sep 12.

Building a complete image of genome regulation in the model organism Escherichia 
coli.

Ishihama A(1).

Author information: 
(1)Research Institute of Micro-Nano Technology, Hosei University.

The model organism, Escherichia coli, contains a total of more than 4,500 genes, 
but the total number of RNA polymerase (RNAP) core enzyme or the transcriptase is
only about 2,000 molecules per genome. The regulatory targets of RNAP are,
however, modulated by changing its promoter selectivity through two-steps of
protein-protein interplay with 7 species of the sigma factor in the first step,
and then 300 species of the transcription factor (TF) in the second step.
Scientists working in the field of prokaryotic transcription in Japan have made
considerable contributions to the elucidation of genetic frameworks and
regulatory modes of the genome transcription in E. coli K-12. This review
summarizes the findings by this group, first focusing on three sigma factors, the
stationary-phase sigma RpoS, the heat-shock sigma RpoH, and the
flagellar-chemotaxis sigma RpoF, as examples. It also presents an overview of the
current state of the systematic research being carried out to identify the
regulatory functions of all TFs from a single and the same bacterium E. coli
K-12, using the genomic SELEX and PS-TF screening systems. All these studies have
been undertaken with the aim of understanding the genome regulation in E. coli
K-12 as a whole.

DOI: 10.2323/jgam.2017.01.002 
PMID: 28904250 


11. J Cell Mol Med. 2018 Jan;22(1):152-162. doi: 10.1111/jcmm.13303. Epub 2017 Aug 7.

A novel mutation of adenomatous polyposis coli (APC) gene results in the
formation of supernumerary teeth.

Yu F(1), Cai W(2)(3), Jiang B(4), Xu L(3), Liu S(3), Zhao S(3)(4).

Author information: 
(1)Department of Pediatric Dentistry, School & Hospital of Stomatology, Tongji
University, Shanghai Engineering Research Center of Tooth Restoration and
Regeneration, Shanghai, China.
(2)Center for Translational Neurodegeneration and Regenerative Therapy, Shanghai 
Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
(3)Department of Stomatology, Huashan Hospital, Fudan University, Shanghai,
China.
(4)Department of Endodontics, School & Hospital of Stomatology, Shanghai
Engineering Research Center of Tooth Restoration and Regeneration, Tongji
University, Shanghai, China.

Supernumerary teeth are teeth that are present in addition to normal teeth.
Although several hypotheses and some molecular signalling pathways explain the
formation of supernumerary teeth, but their exact disease pathogenesis is
unknown. To study the molecular mechanisms of supernumerary tooth-related
syndrome (Gardner syndrome), a deeper understanding of the aetiology of
supernumerary teeth and the associated syndrome is needed, with the goal of
inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese
family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary
teeth and colonic polyp lesion biopsies revealed that these patients exhibited
significant pathological characteristics. APC gene mutations were detected by PCR
and direct sequencing. We revealed the pathological pathway involved in human
supernumerary tooth development and the mouse tooth germ development expression
profile by RNA sequencing (RNA-seq). Sequencing analysis revealed that an APC
gene mutation in exon 15, namely 4292-4293-Del GA, caused Gardner syndrome in
this family. This mutation not only initiated the various manifestations typical 
of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this
case. Furthermore, RNA-seq analysis of human supernumerary teeth suggests that
the APC gene is the key gene involved in the development of supernumerary teeth
in humans. The mouse tooth germ development expression profile shows that the APC
gene plays an important role in tooth germ development. We identified a new
mutation in the APC gene that results in supernumerary teeth in association with 
Gardner syndrome. This information may shed light on the molecular pathogenesis
of supernumerary teeth. Gene-based diagnosis and gene therapy for supernumerary
teeth may become available in the future, and our study provides a
high-resolution reference for treating other syndromes associated with
supernumerary teeth.

© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John 
Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

DOI: 10.1111/jcmm.13303 
PMCID: PMC5742724
PMID: 28782241 


12. BMC Microbiol. 2017 Dec 16;17(1):233. doi: 10.1186/s12866-017-1141-0.

Isolation and characterization of HepP: a virulence-related Pseudomonas
aeruginosa heparinase.

Dzvova N(1), Colmer-Hamood JA(1)(2), Griswold JA(3), Hamood AN(4)(5).

Author information: 
(1)Department of Immunology and Molecular Microbiology, Texas Tech University
Health Sciences Center, 3601 4th St. Mail Stop 6591, Lubbock, TX, 79430, USA.
(2)Department of Medical Education, Texas Tech University Health Sciences Center,
Lubbock, TX, USA.
(3)Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, 
TX, USA.
(4)Department of Immunology and Molecular Microbiology, Texas Tech University
Health Sciences Center, 3601 4th St. Mail Stop 6591, Lubbock, TX, 79430, USA.
abdul.hamood@ttuhsc.edu.
(5)Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, 
TX, USA. abdul.hamood@ttuhsc.edu.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes
serious infections in immunocompromised hosts including severely burned patients.
In burn patients, P. aeruginosa infection often leads to septic shock and death. 
Despite numerous studies, the influence of severe thermal injuries on the
pathogenesis of P. aeruginosa during systemic infection is not known. Through
RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain
UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients
significantly altered the expression of the PA14 transcriptome when compared with
its growth in blood from healthy volunteers. The expression of PA14_23430 and the
adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these
conditions.
RESULTS: Quantitative real-time PCR analysis confirmed the enhancement of
expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from
severely burned patients. Computer analysis revealed that PA14_23430 (hepP)
encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative
zinc-binding dehydrogenase. This analysis further suggested that the two genes
form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR
experiments. We characterized hepP and its protein product HepP. hepP was cloned 
from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP)
was purified using nickel column chromatography. Heparinase assays using
commercially available heparinase as a positive control, revealed that rHepP
exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle
formation at the air-liquid interface by PA14 under static growth conditions.
Biofilm formation by PA14ΔhepP was also significantly reduced. In the
Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a
significantly lower rate of killing than that of the parent strain PA14.
CONCLUSIONS: Changes within the blood of severely burned patients significantly
induced expression of hepP in PA14. The heparinase encoded by hepP is a potential
virulence factor for PA14 as HepP influences pellicle formation as well as
biofilm development by PA14 and the protein is required for full virulence in the
C. elegans model of slow killing.

DOI: 10.1186/s12866-017-1141-0 
PMCID: PMC5732420
PMID: 29246112 


13. Biosens Bioelectron. 2017 Dec 15;98:486-493. doi: 10.1016/j.bios.2017.07.004.
Epub 2017 Jul 5.

Bridged Rebar Graphene functionalized aptasensor for pathogenic E. coli
O78:K80:H11 detection.

Kaur H(1), Shorie M(1), Sharma M(1), Ganguli AK(2), Sabherwal P(3).

Author information: 
(1)Institute of Nano Science & Technology, Mohali 160062, India.
(2)Institute of Nano Science & Technology, Mohali 160062, India. Electronic
address: ashokganguliiitd@gmail.com.
(3)Institute of Nano Science & Technology, Mohali 160062, India. Electronic
address: psnanobiotech@gmail.com.

We report a novel fabrication method of functionalised Bridged Rebar Graphene
(BRG) onto newly designed nanostructured aptasensor for label free impedimetric
sensing of pathogenic bacteria E. coli O78:K80:H11. The chemical facilitated
unscrolling of MWCNT and subsequent bridging with terephthalaldehyde (TPA) to
form 3D-hierarchical BRG nanoconstruct exhibited synergistic effect by combining 
enhanced electrical properties and facile chemical functionality for stable
bio-interface. The bacteria-DNA interactions were captured on BRG nanostructured 
electrode by using specific anti-E.coli DNA aptamer (Kd~ 14nM), screened by new
in-situ developed SELEX method using phenylboronic acid on microtitre plate. The 
developed nanostructured aptasensor demonstrated a low detection limit and
sensitivity of ~ 101cfu/mL towards E. coli O78:K80:H11 with a dynamic response
range from 101 to 106cfu/mL in water, juice and milk samples.

Copyright © 2017 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.bios.2017.07.004 
PMID: 28728009 


14. Sci Rep. 2017 Dec 15;7(1):17639. doi: 10.1038/s41598-017-17918-6.

Elucidating the 16S rRNA 3' boundaries and defining optimal SD/aSD pairing in
Escherichia coli and Bacillus subtilis using RNA-Seq data.

Wei Y(1), Silke JR(1), Xia X(2)(3).

Author information: 
(1)Department of Biology, University of Ottawa, 30 Marie Curie, P.O. Box 450,
Station A, Ottawa, Ontario, Canada.
(2)Department of Biology, University of Ottawa, 30 Marie Curie, P.O. Box 450,
Station A, Ottawa, Ontario, Canada. xxia@uottawa.ca.
(3)Ottawa Institute of Systems Biology, Ottawa, Ontario, K1H 8M5, Canada.
xxia@uottawa.ca.

Bacterial translation initiation is influenced by base pairing between the
Shine-Dalgarno (SD) sequence in the 5' UTR of mRNA and the anti-SD (aSD) sequence
at the free 3' end of the 16S rRNA (3' TAIL) due to: 1) the SD/aSD sequence
binding location and 2) SD/aSD binding affinity. In order to understand what
makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3' TAIL
and 2) extent of the core aSD sequence within the 3' TAIL. Our approach to
characterize these components in Escherichia coli and Bacillus subtilis involves 
1) mapping the 3' boundary of the mature 16S rRNA using high-throughput RNA
sequencing (RNA-Seq), and 2) identifying the segment within the 3' TAIL that is
strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous
discrepancies in the reported 3' TAIL in B. subtilis and recovered the
established 3' TAIL in E. coli. Furthermore, we extend previous studies to
suggest that both highly and lowly expressed genes favor SD sequences with
intermediate binding affinity, but this trend is exclusive to SD sequences that
complement the core aSD sequences defined herein.

DOI: 10.1038/s41598-017-17918-6 
PMCID: PMC5732282
PMID: 29247194 


15. Elife. 2017 Dec 4;6. pii: e30433. doi: 10.7554/eLife.30433.

LAST, a c-Myc-inducible long noncoding RNA, cooperates with CNBP to promote CCND1
mRNA stability in human cells.

Cao L(#)(1), Zhang P(#)(1), Li J(2), Wu M(1)(2).

Author information: 
(1)CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for
Excellence in Molecular Cell Science, Innovation Center for Cell Signaling
Network, School of Life Sciences, University of Science & Technology of China,
Hefei, China.
(2)Translational Research Institute, Henan Provincial People's Hospital, School
of Medicine, Henan University, Zhengzhou, China.
(#)Contributed equally

Cyclin D1 is a critical regulator of cell cycle progression and works at the G1
to S-phase transition. Here, we report the isolation and characterization of the 
novel c-Myc-regulated lncRNA LAST (LncRNA-Assisted Stabilization of Transcripts),
which acts as a CCND1 mRNA stabilizer. Mechanistically, LAST was shown to
cooperate with CNBP to bind to the 5'UTR of CCND1 mRNA to protect against
possible nuclease targeting. In addition, data from CNBP RIP-seq and LAST RNA-seq
showed that CCND1 mRNA might not be the only target of LAST and CNBP; three
additional mRNAs were shown to be post-transcriptional targets of LAST and CNBP. 
In a xenograft model, depletion of LAST diminished and ectopic expression of LAST
induced tumor formation, which are suggestive of its oncogenic function. We thus 
report a previously unknown lncRNA involved in the fine-tuned regulation of CCND1
mRNA stability, without which CCND1 exhibits, at most, partial expression.

DOI: 10.7554/eLife.30433 
PMCID: PMC5739540
PMID: 29199958 


16. FEMS Microbiol Lett. 2017 Dec 1;364(22). doi: 10.1093/femsle/fnx220.

Regulatory role of XynR (YagI) in catabolism of xylonate in Escherichia coli
K-12.

Shimada T(1)(2), Momiyama E(3), Yamanaka Y(1)(3), Watanabe H(3), Yamamoto
K(1)(3), Ishihama A(1)(3).

Author information: 
(1)Research Center for Micro-Nano Technology, Hosei University, Kajino-cho 3-7-2,
Koganei, Tokyo 184-0003, Japan.
(2)School of Agriculture, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571,
Japan.
(3)Department of Frontier Bioscience, Hosei University, Kajino-cho 3-7-2,
Koganei, Tokyo 184-0003, Japan.

The genome of Escherichia coli K-12 contains ten cryptic phages, altogether
constituting about 3.6% of the genome in sequence. Among more than 200 predicted 
genes in these cryptic phages, 14 putative transcription factor (TF) genes exist,
but their regulatory functions remain unidentified. As an initial attempt to make
a breakthrough for understanding the regulatory roles of cryptic phage-encoded
TFs, we tried to identify the regulatory function of CP4-6 cryptic
prophage-encoded YagI with unknown function. After SELEX screening, YagI was
found to bind mainly at a single site within the spacer of bidirectional
transcription units, yagA (encoding another uncharacterized TF) and yagEF
(encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively)
within this prophage region. YagEF enzymes are involved in the catabolism of
xylose downstream from xylonate. We then designated YagI as XynR (regulator of
xylonate catabolism), one of the rare single-target TFs. In agreement with this
predicted regulatory function, the activity of XynR was suggested to be
controlled by xylonate. Even though low-affinity binding sites of XynR were
identified in the E. coli K-12 genome, they all were inside open reading frames, 
implying that the regulation network of XynR is still fixed within the CR4-6
prophage without significant influence over the host E. coli K-12.

© FEMS 2017. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.

DOI: 10.1093/femsle/fnx220 
PMID: 29087459  [Indexed for MEDLINE]


17. Int J Mol Med. 2017 Dec;40(6):1792-1802. doi: 10.3892/ijmm.2017.3175. Epub 2017
Oct 5.

Internal driving factors leading to extrahepatic manifestation of the hepatitis C
virus infection.

Wu ZY(1), Li JR(1), Huang MH(1), Cheng JJ(1), Li H(1), Chen JH(1), Lv XQ(1), Peng
ZG(1), Jiang JD(1).

Author information: 
(1)Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and 
Peking Union Medical College, Beijing 100050, P.R. China.

The hepatitis C virus (HCV) infection is associated with various extrahepatic
manifestations, which are correlated with poor outcomes, and thus increase the
morbidity and mortality of chronic hepatitis C (CHC). Therefore, understanding
the internal linkages between systemic manifestations and HCV infection is
helpful for treatment of CHC. Yet, the mechanism by which the virus evokes the
systemic diseases remains to be elucidated. In the present study, using gene set 
enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA), a
comprehensive analysis of microarray data of mRNAs was conducted in HCV-infected 
and -uninfected Huh7.5 cells, and signaling pathways (which are significantly
activated or inhibited) and certain molecules (which are commonly important in
those signaling pathways) were selected. Forty signaling pathways were selected
using GSEA, and eight signaling pathways were selected with SPIA. These pathways 
are associated with cancer, metabolism, environmental information processing and 
organismal systems, which provide important information for further clarifying
the intrinsic associations between syndromes of HCV infection, of which seven
pathways were not previously reported, including basal transcription factors,
pathogenic Escherichia coli infection, shigellosis, gastric acid secretion,
dorso-ventral axis formation, amoebiasis and cholinergic synapse. Ten genes,
SOS1, RAF1, IFNA2, IFNG, MTHFR, IGF1, CALM3, UBE2B, TP53 and BMP7 whose
expression may be the key internal driving molecules, were selected using the
online tool Anni 2.1. Furthermore, the present study demonstrated the internal
linkages between systemic manifestations and HCV infection, and presented the
potential molecules that are key to those linkages.

DOI: 10.3892/ijmm.2017.3175 
PMCID: PMC5716440
PMID: 29039494  [Indexed for MEDLINE]


18. Nucleic Acids Res. 2017 Dec 1;45(21):12140-12151. doi: 10.1093/nar/gkx999.

sRNAnalyzer-a flexible and customizable small RNA sequencing data analysis
pipeline.

Wu X(1), Kim TK(1), Baxter D(1), Scherler K(1), Gordon A(1), Fong O(2), Etheridge
A(2), Galas DJ(2), Wang K(1).

Author information: 
(1)Institute for Systems Biology, Seattle, WA 98109, USA.
(2)Pacific Northwest Research Institute, Seattle, WA 98122, USA.

Although many tools have been developed to analyze small RNA sequencing
(sRNA-Seq) data, it remains challenging to accurately analyze the small RNA
population, mainly due to multiple sequence ID assignment caused by short read
length. Additional issues in small RNA analysis include low consistency of
microRNA (miRNA) measurement results across different platforms, miRNA mapping
associated with miRNA sequence variation (isomiR) and RNA editing, and the origin
of those unmapped reads after screening against all endogenous reference sequence
databases. To address these issues, we built a comprehensive and customizable
sRNA-Seq data analysis pipeline-sRNAnalyzer, which enables: (i) comprehensive
miRNA profiling strategies to better handle isomiRs and summarization based on
each nucleotide position to detect potential SNPs in miRNAs, (ii) different
sequence mapping result assignment approaches to simulate results from
microarray/qRT-PCR platforms and a local probabilistic model to assign mapping
results to the most-likely IDs, (iii) comprehensive ribosomal RNA filtering for
accurate mapping of exogenous RNAs and summarization based on taxonomy
annotation. We evaluated our pipeline on both artificial samples (including
synthetic miRNA and Escherichia coli cultures) and biological samples (human
tissue and plasma). sRNAnalyzer is implemented in Perl and available at:
http://srnanalyzer.systemsbiology.net/.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkx999 
PMCID: PMC5716150
PMID: 29069500  [Indexed for MEDLINE]


19. Anal Biochem. 2017 Nov 1;536:36-44. doi: 10.1016/j.ab.2017.08.005. Epub 2017 Aug 
14.

DNA aptamer identification and characterization for E. coli O157 detection using 
cell based SELEX method.

Amraee M(1), Oloomi M(2), Yavari A(1), Bouzari S(1).

Author information: 
(1)Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave.,
Tehran 13164, Iran.
(2)Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave.,
Tehran 13164, Iran. Electronic address: manaoloomi@yahoo.com.

Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms
in humans. Its rapid identification should be considered to avoid toxic effects
of the pathogen. In this study, systematic evolution of ligands by exponential
enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli
strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex
procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, 
DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The
specific interaction between selected DNA aptamers and targeted cell was
assessed. After applying six rounds of SELEX for selection of DNA aptamers, the
candidate sequences were obtained. Finally, specific aptamer was selected as an
ideal aptamer for detection and capturing of E. coli O157. Dissociation constant 
of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the
secondary structure prediction and cross reactivity assays were performed. The
isolated aptamer efficiency was confirmed and it was shown that the new DNA
aptamer sequence has the ability to use for detection. This specific O157:H7
aptamer have the potential for application as a diagnostic ligand and could be
used for detection of the related food borne diseases.

Copyright © 2017 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ab.2017.08.005 
PMID: 28818557  [Indexed for MEDLINE]


20. J Antibiot (Tokyo). 2017 Nov 1. doi: 10.1038/ja.2017.130. [Epub ahead of print]

Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls
negatively expression of genes of PHO regulon in Streptomyces coelicolor.

Martín-Martín S(1), Rodríguez-García A(1), Santos-Beneit F(2), Franco-Domínguez
E(1), Sola-Landa A(1), Martín JF(3).

Author information: 
(1)Instituto de Biotecnología de León (INBIOTEC), León, Spain.
(2)Centre for Bacterial Cell Biology, Institute for Cell and Molecular
Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, UK.
(3)Área de Microbiología, Departamento de Biología Molecular, Facultad de
Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n,
León, Spain.

Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is
mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster, 
and expressed in the opposite orientation, is a phoU-like encoding gene with low 
identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene 
is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and
used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its
transcription start site and regulation. RNA-seq studies identified two
transcription start sites, one upstream of phoU and the second upstream of the
mptA gene. Whereas transcription of PhoU is entirely dependent on PhoP,
expression of the downstream mtpA gene is only partially dependent on PhoP
activation. The phoU mutant grows more slowly than the parental strain,
sporulates poorly and the spores lack pigmentation. Production of actinorhodin
and undecylprodigiosin decreased in the phoU mutant, indicating that PhoU has a
positive modulating effect on production of these antibiotics. Indeed,
transcriptional studies of expression of the actII-ORF4 and redD genes indicated 
that the PhoU protein activates expression of these antibiotic regulators. Using 
the glpQ1 promoter as in vivo reporter of the activity of the PHO regulon genes, 
we observed that expression of glpQ1 is negatively modulated by PhoU. These
results were confirmed by reverse transcription-PCR studies of three genes of the
PHO regulon; that is, glpQ1, pstS and phoR. In conclusion, PhoU acts as a
negative modulator of expression of the PHO regulon genes and as phoU expression 
is strictly dependent on PhoP activation, this mechanism appears to work as a
feed-back control mechanism (self-regulation).The Journal of Antibiotics advance 
online publication, 1 November 2017; doi:10.1038/ja.2017.130.

DOI: 10.1038/ja.2017.130 
PMID: 29089595 


21. BMC Bioinformatics. 2017 Oct 12;18(1):450. doi: 10.1186/s12859-017-1860-0.

Partitioning of functional gene expression data using principal points.

Kim J(1), Kim H(2).

Author information: 
(1)Department of Statistics, Duksung Women's University, Seoul, South Korea.
jaehee@duksung.ac.kr.
(2)Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon,
South Korea.

BACKGROUND: DNA microarrays offer motivation and hope for the simultaneous study 
of variations in multiple genes. Gene expression is a temporal process that
allows variations in expression levels with a characterized gene function over a 
period of time. Temporal gene expression curves can be treated as functional data
since they are considered as independent realizations of a stochastic process.
This process requires appropriate models to identify patterns of gene functions. 
The partitioning of the functional data can find homogeneous subgroups of
entities for the massive genes within the inherent biological networks. Therefor 
it can be a useful technique for the analysis of time-course gene expression
data. We propose a new self-consistent partitioning method of functional
coefficients for individual expression profiles based on the orthonormal basis
system.
RESULTS: A principal points based functional partitioning method is proposed for 
time-course gene expression data. The method explores the relationship between
genes using Legendre coefficients as principal points to extract the features of 
gene functions. Our proposed method provides high connectivity in connectedness
after clustering for simulated data and finds a significant subsets of genes with
the increased connectivity. Our approach has comparative advantages that fewer
coefficients are used from the functional data and self-consistency of principal 
points for partitioning. As real data applications, we are able to find
partitioned genes through the gene expressions found in budding yeast data and
Escherichia coli data.
CONCLUSIONS: The proposed method benefitted from the use of principal points,
dimension reduction, and choice of orthogonal basis system as well as provides
appropriately connected genes in the resulting subsets. We illustrate our method 
by applying with each set of cell-cycle-regulated time-course yeast genes and E. 
coli genes. The proposed method is able to identify highly connected genes and to
explore the complex dynamics of biological systems in functional genomics.

DOI: 10.1186/s12859-017-1860-0 
PMCID: PMC5639779
PMID: 29025390  [Indexed for MEDLINE]


22. Nat Commun. 2017 Oct 12;8(1):896. doi: 10.1038/s41467-017-00884-y.

Extracellular matrix stiffness and cell contractility control RNA localization to
promote cell migration.

Wang T(1), Hamilla S(1)(2), Cam M(3), Aranda-Espinoza H(2), Mili S(4).

Author information: 
(1)Laboratory of Cellular and Molecular Biology, Center for Cancer Research,
National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
(2)Fischell Department of Bioengineering, University of Maryland, College Park,
MD, 20742, USA.
(3)CCR Collaborative Bioinformatics Resource, National Cancer Institute, NIH,
Bethesda, MD, 20892, USA.
(4)Laboratory of Cellular and Molecular Biology, Center for Cancer Research,
National Cancer Institute, NIH, Bethesda, MD, 20892, USA. voula.mili@nih.gov.

Numerous RNAs are enriched within cellular protrusions, but the underlying
mechanisms are largely unknown. We had shown that the APC (adenomatous polyposis 
coli) protein controls localization of some RNAs at protrusions. Here, using
protrusion-isolation schemes and RNA-Seq, we find that RNAs localized in
protrusions of migrating fibroblasts can be distinguished in two groups, which
are differentially enriched in distinct types of protrusions, and are
additionally differentially dependent on APC. APC-dependent RNAs become enriched 
in high-contractility protrusions and, accordingly, their localization is
promoted by increasing stiffness of the extracellular matrix. Dissecting the
underlying mechanism, we show that actomyosin contractility activates a
RhoA-mDia1 signaling pathway that leads to formation of a
detyrosinated-microtubule network, which in turn is required for localization of 
APC-dependent RNAs. Importantly, a competition-based approach to specifically
mislocalize APC-dependent RNAs suggests that localization of the APC-dependent
RNA subgroup is functionally important for cell migration.Adenomatous polyposis
coli (APC) regulates the localization of some mRNAs at cellular protrusions but
the underlying mechanisms and functional roles are not known. Here the authors
show that APC-dependent RNAs are enriched in contractile protrusions, via
detyrosinated microtubules, and enhance cell migration.

DOI: 10.1038/s41467-017-00884-y 
PMCID: PMC5638855
PMID: 29026081  [Indexed for MEDLINE]


23. Biotechnol Bioeng. 2017 Oct;114(10):2235-2244. doi: 10.1002/bit.26340. Epub 2017 
Jun 5.

Naringenin-responsive riboswitch-based fluorescent biosensor module for
Escherichia coli co-cultures.

Xiu Y(1)(2)(3), Jang S(4), Jones JA(3)(5), Zill NA(3), Linhardt RJ(3), Yuan Q(1),
Jung GY(4)(6), Koffas MAG(3).

Author information: 
(1)State Key Laboratory of Chemical Resource Engineering, Beijing University of
Chemical Technology, Beijing, China.
(2)Beijing Key Laboratory of Bioactive Substances and Functional Food, Beijing
Union University, Beijing, China.
(3)Department of Chemical and Biological Engineering, Center for Biotechnology
and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
(4)Department of Chemical Engineering, Pohang University of Science and
Technology, Pohang, Gyeongbuk, Korea.
(5)Department of Chemistry, Hamilton College, Clinton, New York.
(6)School of Interdisciplinary Bioscience and Bioengineering, Pohang University
of Science and Technology, Pohang, Gyeongbuk, Korea.

The ability to design and construct combinatorial synthetic metabolic pathways
has far exceeded our capacity for efficient screening and selection of the
resulting microbial strains. The need for high-throughput rapid screening
techniques is of upmost importance for the future of synthetic biology and
metabolic engineering. Here we describe the development of an RNA
riboswitch-based biosensor module with dual fluorescent reporters, and
demonstrate a high-throughput flow cytometry-based screening method for
identification of naringenin over producing Escherichia coli strains in
co-culture. Our efforts helped identify a number of key operating parameters that
affect biosensor performance, including the selection of promoter and linker
elements within the sensor-actuator domain, and the effect of host strain,
fermentation time, and growth medium on sensor dynamic range. The resulting
biosensor demonstrates a high correlation between specific fluorescence of the
biosensor strain and naringenin titer produced by the second member of the
synthetic co-culture system. This technique represents a novel application for
synthetic microbial co-cultures and can be expanded from naringenin to any
metabolite if a suitable riboswitch is identified. The co-culture technique
presented here can be applied to a variety of target metabolites in combination
with the SELEX approach for aptamer design. Due to the compartmentalization of
the two genetic constructs responsible for production and detection into separate
cells and application as independent modules of a synthetic microbial co-culture 
we have subsequently reduced the need for re-optimization of the producer module 
when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114:
2235-2244. © 2017 Wiley Periodicals, Inc.

© 2017 Wiley Periodicals, Inc.

DOI: 10.1002/bit.26340 
PMID: 28543037  [Indexed for MEDLINE]


24. Genomics Proteomics Bioinformatics. 2017 Oct;15(5):324-330. doi:
10.1016/j.gpb.2017.04.005. Epub 2017 Oct 12.

Ribosome Profiling Reveals Genome-wide Cellular Translational Regulation upon
Heat Stress in Escherichia coli.

Zhang Y(1), Xiao Z(2), Zou Q(2), Fang J(2), Wang Q(1), Yang X(3), Gao N(4).

Author information: 
(1)MOE Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for
Structural Biology, Tsinghua-Peking Center for Life Sciences, School of Life
Sciences, Tsinghua University, Beijing 100084, China.
(2)MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life
Sciences, Center for Synthetic and Systems Biology, School of Life Sciences,
Tsinghua University, Beijing 100084, China.
(3)MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life
Sciences, Center for Synthetic and Systems Biology, School of Life Sciences,
Tsinghua University, Beijing 100084, China. Electronic address:
yangxuerui@tsinghua.edu.cn.
(4)MOE Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for
Structural Biology, Tsinghua-Peking Center for Life Sciences, School of Life
Sciences, Tsinghua University, Beijing 100084, China; State Key Laboratory of
Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life
Sciences, Peking University, Beijing 100871, China. Electronic address:
gaon@pku.edu.cn.

Heat shock response is a classical stress-induced regulatory system in bacteria, 
characterized by extensive transcriptional reprogramming. To compare the impact
of heat stress on the transcriptome and translatome in Escherichia coli, we
conducted ribosome profiling in parallel with RNA-Seq to investigate the
alterations in transcription and translation efficiency when E. coli cells were
exposed to a mild heat stress (from 30 °C to 45 °C). While general changes in
ribosome footprints correlate with the changes of mRNA transcripts upon heat
stress, a number of genes show differential changes at the transcription and
translation levels. Translation efficiency of a few genes that are related to
environment stimulus response is up-regulated, and in contrast, some genes
functioning in mRNA translation and amino acid biosynthesis are down-regulated at
the translation level in response to heat stress. Moreover, our ribosome
occupancy data suggest that in general ribosomes accumulate remarkably in the
starting regions of ORFs upon heat stress. This study provides additional
insights into bacterial gene expression in response to heat stress, and suggests 
the presence of stress-induced but yet-to-be characterized cellular regulatory
mechanisms of gene expression at translation level.

Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights 
reserved.

DOI: 10.1016/j.gpb.2017.04.005 
PMCID: PMC5673677
PMID: 29031842  [Indexed for MEDLINE]


25. Res Microbiol. 2017 Oct;168(8):700-709. doi: 10.1016/j.resmic.2017.08.003. Epub
2017 Sep 9.

Global effect of CsrA on gene expression in enterohemorrhagic Escherichia coli
O157:H7.

Wang S(1), Yang F(2), Yang B(3).

Author information: 
(1)TEDA Institute of Biological Sciences and Biotechnology, Nankai University,
TEDA, Tianjin 300457, PR China. Electronic address:
wangshaomeng@mail.nankai.edu.cn.
(2)Department of Neurosurgery, Tianjin First Central Hospital, Tianjin 300192, PR
China. Electronic address: yangfanfirst123@163.com.
(3)TEDA Institute of Biological Sciences and Biotechnology, Nankai University,
TEDA, Tianjin 300457, PR China. Electronic address: yangbin@nankai.edu.cn.

The post-transcriptional regulator CsrA regulates multiple unrelated processes
such as central carbon metabolism, motility, biofilm formation and bacterial
virulence in different bacteria. However, regulation by CsrA in enterohemorrhagic
Escherichia coli (EHEC) O157:H7 is still largely unknown. In this study, we
performed a detailed analysis of gene expression differences between the EHEC
O157:H7 wild-type strain and a corresponding csrA::kan mutant using RNA-seq
technology. Genes whose expression was affected by CsrA were identified and
grouped into different clusters of orthologous group categories. Genes located in
the locus of enterocyte effacement (LEE) pathogenicity island were significantly 
upregulated, whereas expression of flagella-related genes was significantly
reduced in the csrA::kan mutant. Subsequent bacterial adherence and motility
assays showed that inactivation of CsrA in EHEC O157:H7 resulted in a significant
increase in bacterial adherence to host epithelial cells, with a concomitant loss
of swimming motility on semi-solid agar plates. Furthermore, we also found that
CsrA regulates genes not previously identified in other bacterial species,
including genes encoding cytochrome oxidases and those required for nitrogen
metabolism. Our results provide essential insight into the regulatory function of
CsrA.

Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights
reserved.

DOI: 10.1016/j.resmic.2017.08.003 
PMID: 28870757 


26. mSphere. 2017 Sep 27;2(5). pii: e00375-17. doi: 10.1128/mSphereDirect.00375-17.
eCollection 2017 Sep-Oct.

The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a
TolC-Deficient Strain of Escherichia coli.

Donner J(1), Reck M(1), Bunk B(2)(3), Jarek M(4), App CB(1), Meier-Kolthoff
JP(2)(3), Overmann J(2)(3), Müller R(5), Kirschning A(6), Wagner-Döbler I(1).

Author information: 
(1)Department of Medical Microbiology, Group Microbial Communication,
Helmholtz-Centre for Infection Research, Braunschweig, Germany.
(2)Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, 
Braunschweig, Germany.
(3)German Centre for Infection Research (DZIF), Partner Site
Hannover-Braunschweig, Braunschweig, Germany.
(4)Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig,
Germany.
(5)Department of Microbial Natural Products, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research 
and Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany.
(6)Institute of Organic Chemistry and Center of Biomolecular Drug Research
(BMWZ), Leibniz Universität Hannover, Hannover, Germany.

The myxobacterial secondary metabolite carolacton inhibits growth of
Streptococcus pneumoniae and kills biofilm cells of the caries- and
endocarditis-associated pathogen Streptococcus mutans at nanomolar
concentrations. Here, we studied the response to carolacton of an Escherichia
coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing 
of the laboratory E. coli strain TolC revealed the integration of an insertion
element, IS5, at the tolC locus and a close phylogenetic relationship to the
ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and 
determination of MIC values that carolacton penetrates the phospholipid bilayer
of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar
concentrations as for streptococci. This inhibition is completely lost for a C-9 
(R) epimer of carolacton, a derivative with an inverted stereocenter at carbon
atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also
inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of
native carolacton with a conserved cellular target present in bacterial phyla as 
distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor
(EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits 
AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that
carolacton has potential for use in antimicrobial chemotherapy against
Gram-negative bacteria, as a single drug or in combination with EPIs. Strain
E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq
data and MIC values, it can be used as a reference during future screenings for
novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant
against most or all of the antibiotics currently used in human therapy is a
global threat, and therefore the search for antimicrobials with novel targets and
modes of action is of utmost importance. The myxobacterial secondary metabolite
carolacton had previously been shown to inhibit biofilm formation and growth of
streptococci. Here, we investigated if carolacton could act against Gram-negative
bacteria, which are difficult targets because of their double-layered cytoplasmic
envelope. We found that the model organism Escherichia coli is susceptible to
carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its
multidrug efflux system AcrAB-TolC is either inactivated genetically, by
disruption of the tolC gene, or physiologically by coadministering an efflux pump
inhibitor. A carolacton epimer that has a different steric configuration at
carbon atom 9 is completely inactive, suggesting that carolacton may interact
with the same molecular target in both Gram-positive and Gram-negative bacteria.

DOI: 10.1128/mSphereDirect.00375-17 
PMCID: PMC5615136
PMID: 28959742 


27. J Bacteriol. 2017 Sep 18. pii: JB.00484-17. doi: 10.1128/JB.00484-17. [Epub ahead
of print]

Circuitry linking the global Csr and σE-dependent cell envelope stress response
systems.

Yakhnin H(1)(2), Aichele R(1)(2), Ades SE(1), Romeo T(3), Babitzke P(4)(2).

Author information: 
(1)Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, Pennsylvania, 16802.
(2)Center for RNA Molecular Biology, The Pennsylvania State University,
University Park, Pennsylvania, 16802.
(3)Department of Microbiology and Cell Science, Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, Florida 32611.
(4)Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, Pennsylvania, 16802 pxb28@psu.edu.

CsrA of Escherichia coli is an RNA-binding protein that globally regulates a wide
variety of cellular processes and behaviors including carbon metabolism,
motility, biofilm formation, and the stringent response. CsrB and CsrC are sRNAs 
that sequester CsrA, thereby preventing CsrA-mRNA interaction. RpoE (σE) is the
extracytoplasmic stress response sigma factor of E. coli Previous RNA-seq studies
identified rpoE mRNA as a CsrA target. Here we explored the regulation of rpoE by
CsrA and found that CsrA represses rpoE translation. Gel mobility shift,
footprint and toeprint studies identified three CsrA binding sites in the rpoE
leader transcript, one of which overlaps the rpoE Shine-Dalgarno (SD) sequence,
while another overlaps the rpoE translation initiation codon. Coupled in vitro
transcription-translation experiments showed that CsrA represses rpoE translation
by binding to these sites. We further demonstrate that σE indirectly activates
transcription of csrB and csrC, leading to increased sequestration of CsrA such
that repression of rpoE by CsrA is reduced. We propose that the Csr system
fine-tunes the σE-dependent cell envelope stress response. We also identified a
51 amino acid coding sequence whose stop codon overlaps the rpoE start codon, and
demonstrate that rpoE is translationally coupled with this upstream open reading 
frame (ORF51). Loss of coupling reduces rpoE translation by more than 50%.
Identification of a translationally coupled ORF upstream of rpoE suggests that
this previously unannotated protein may participate in the cell envelope stress
response. In keeping with existing nomenclature, we name ORF51 as rseD, resulting
in an operon arrangement of rseD-rpoE-rseA-rseB-rseCIMPORTANCE CsrA
posttranscriptionally represses genes required for bacterial stress responses,
including the stringent response, catabolite repression, and the RpoS
(σS)-mediated general stress response. We show that CsrA represses translation of
rpoE, encoding the extracytoplasmic stress response sigma factor and that σE
indirectly activates transcription of csrB and csrC, resulting in reciprocal
regulation of these two global regulatory systems. These findings suggest that
extracytoplasmic stress leads to derepression of rpoE translation by CsrA, and
CsrA-mediated repression helps to reset RpoE abundance to pre-stress levels once 
envelope damage is repaired. The discovery of an ORF, RseD, translationally
coupled with rpoE adds further complexity to translational control of rpoE.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/JB.00484-17 
PMCID: PMC5686586 [Available on 2018-04-30]
PMID: 28924029 


28. BioData Min. 2017 Sep 5;10:30. doi: 10.1186/s13040-017-0150-8. eCollection 2017.

RNA-sequence data normalization through in silico prediction of reference genes: 
the bacterial response to DNA damage as case study.

Berghoff BA(1), Karlsson T(2), Källman T(3)(4), Wagner EGH(5), Grabherr MG(3)(4).

Author information: 
(1)Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität,
Giessen, Germany.
(2)Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala,
Sweden.
(3)Department of Medical Biochemistry and Microbiology, Uppsala University,
Uppsala, Sweden.
(4)Bioinformatics Infrastructure for Life Sciences (BILS), Science for Life
Laboratories, Uppsala University, Uppsala, Sweden.
(5)Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

BACKGROUND: Measuring how gene expression changes in the course of an experiment 
assesses how an organism responds on a molecular level. Sequencing of RNA
molecules, and their subsequent quantification, aims to assess global gene
expression changes on the RNA level (transcriptome). While advances in
high-throughput RNA-sequencing (RNA-seq) technologies allow for inexpensive data 
generation, accurate post-processing and normalization across samples is required
to eliminate any systematic noise introduced by the biochemical and/or technical 
processes. Existing methods thus either normalize on selected known reference
genes that are invariant in expression across the experiment, assume that the
majority of genes are invariant, or that the effects of up- and down-regulated
genes cancel each other out during the normalization.
RESULTS: Here, we present a novel method, moose2 , which predicts invariant genes
in silico through a dynamic programming (DP) scheme and applies a quadratic
normalization based on this subset. The method allows for specifying a set of
known or experimentally validated invariant genes, which guides the DP. We
experimentally verified the predictions of this method in the bacterium
Escherichia coli, and show how moose2 is able to (i) estimate the expression
value distances between RNA-seq samples, (ii) reduce the variation of expression 
values across all samples, and (iii) to subsequently reveal new functional groups
of genes during the late stages of DNA damage. We further applied the method to
three eukaryotic data sets, on which its performance compares favourably to other
methods. The software is implemented in C++ and is publicly available from
http://grabherr.github.io/moose2/.
CONCLUSIONS: The proposed RNA-seq normalization method, moose2 , is a valuable
alternative to existing methods, with two major advantages: (i) in silico
prediction of invariant genes provides a list of potential reference genes for
downstream analyses, and (ii) non-linear artefacts in RNA-seq data are handled
adequately to minimize variations between replicates.

DOI: 10.1186/s13040-017-0150-8 
PMCID: PMC5584328
PMID: 28878825 


29. Front Microbiol. 2017 Sep 1;8:1671. doi: 10.3389/fmicb.2017.01671. eCollection
2017.

DNA Adenine Methyltransferase (Dam) Overexpression Impairs Photorhabdus
luminescens Motility and Virulence.

Payelleville A(1), Lanois A(1), Gislard M(2), Dubois E(2), Roche D(3), Cruveiller
S(3), Givaudan A(1), Brillard J(1).

Author information: 
(1)Diversité, Génomes Interactions Microorganismes Insectes (DGIMI), Institut
National De La Recherche Agronomique, Université de MontpellierMontpellier,
France.
(2)MGX-Montpellier GenomiX, Institut de Génomique FonctionnelleMontpellier,
France.
(3)Le Commissariat à l'énergie atomique et aux énergies alternatives (CEA),
Genoscope, Université d'Evry, Centre National De La Recherche
Scientifique-UMR8030, Université Paris-SaclayEvry, France.

Dam, the most described bacterial DNA-methyltransferase, is widespread in
gamma-proteobacteria. Dam DNA methylation can play a role in various genes
expression and is involved in pathogenicity of several bacterial species. The
purpose of this study was to determine the role played by the dam ortholog
identified in the entomopathogenic bacterium Photorhabdus luminescens.
Complementation assays of an Escherichia coli dam mutant showed the restoration
of the DNA methylation state of the parental strain. Overexpression of dam in P. 
luminescens did not impair growth ability in vitro. In contrast, compared to a
control strain harboring an empty plasmid, a significant decrease in motility was
observed in the dam-overexpressing strain. A transcriptome analysis revealed the 
differential expression of 208 genes between the two strains. In particular, the 
downregulation of flagellar genes was observed in the dam-overexpressing strain. 
In the closely related bacterium Xenorhabdus nematophila, dam overexpression also
impaired motility. In addition, the dam-overexpressing P. luminescens strain
showed a delayed virulence compared to that of the control strain after injection
in larvae of the lepidopteran Spodoptera littoralis. These results reveal that
Dam plays a major role during P. luminescens insect infection.

DOI: 10.3389/fmicb.2017.01671 
PMCID: PMC5585154
PMID: 28919886 


30. J Vet Diagn Invest. 2017 Sep;29(5):721-724. doi: 10.1177/1040638717700689. Epub
2017 Apr 5.

Evaluation of applicability of DNA microarray-based characterization of bovine
Shiga toxin-producing Escherichia coli isolates using whole genome sequence
analysis.

Barth SA(1)(2)(3)(4), Menge C(1)(2)(3)(4), Eichhorn I(1)(2)(3)(4), Semmler
T(1)(2)(3)(4), Pickard D(1)(2)(3)(4), Geue L(1)(2)(3)(4).

Author information: 
(1)Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health,
Institute of Molecular Pathogenesis, Jena, Germany (Barth, Menge, Geue).
(2)Free University Berlin, Institute of Microbiology and Epizootics, Berlin,
Germany (Eichhorn, Semmler).
(3)Robert Koch Institute, Berlin, Germany (Semmler).
(4)The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus,
Hinxton, Cambridge, UK (Pickard).

We assessed the ability of a commercial DNA microarray to characterize bovine
Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results 
using in silico hybridization of the microarray probes within whole genome
sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178
isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico 
probe hybridization. Results were more congruent when detecting the genoserotype 
(209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance
genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes
(VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the
microarray, only 37.2% of the isolates could be genoserotyped. However, the
microarray proved suitable to rapidly screen bovine STEC strains for the
occurrence of high numbers of VAGs and AMRGs and is suitable for molecular
surveillance workflows.

DOI: 10.1177/1040638717700689 
PMID: 28381114  [Indexed for MEDLINE]


31. PLoS One. 2017 Aug 25;12(8):e0183862. doi: 10.1371/journal.pone.0183862.
eCollection 2017.

Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane
proteins in Escherichia coli.

Benhalevy D(1), Biran I(1), Bochkareva ES(1), Sorek R(2), Bibi E(1).

Author information: 
(1)Department of Biological Chemistry, Weizmann Institute of Science, Rehovot,
Israel.
(2)Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel.

Translation-independent mRNA localization represents an emerging concept in cell 
biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs)
are targeted to the membrane where they are translated by membrane associated
ribosomes and the produced proteins are inserted into the membrane
co-translationally. In order to better understand aspects of the biogenesis and
localization of MPRs, we investigated their subcellular distribution using cell
fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented
in the membrane fraction, as expected, and depletion of the signal recognition
particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane.
Surprisingly, however, MPRs were also found relatively abundant in the soluble
ribosome-free fraction and their amount in this fraction is increased upon
overexpression of CspE, which was recently shown to interact with MPRs. CspE also
conferred a positive effect on the membrane-expression of integral membrane
proteins. We discuss the possibility that the effects of CspE overexpression may 
link the intriguing subcellular localization of MPRs to the cytosolic
ribosome-free fraction with their translation into membrane proteins and that the
ribosome-free pool of MPRs may represent a stage during their targeting to the
membrane, which precedes translation.

DOI: 10.1371/journal.pone.0183862 
PMCID: PMC5571963
PMID: 28841711  [Indexed for MEDLINE]


32. Infect Immun. 2017 Aug 18;85(9). pii: e00307-17. doi: 10.1128/IAI.00307-17. Print
2017 Sep.

Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection
in a Murine Model of Lower Genital Tract Infection.

Gangaiah D(1), Raterman EL(2), Wu H(2), Fortney KR(1), Gao H(3), Liu Y(3), Jerse 
AE(2), Spinola SM(4)(5)(6).

Author information: 
(1)Department of Microbiology and Immunology, Indiana University School of
Medicine, Indianapolis, Indiana, USA.
(2)Department of Microbiology and Immunology, F. Edward Herbert School of
Medicine, Uniformed Services University, Bethesda, Maryland, USA.
(3)Department of Medical and Molecular Genetics, Indiana University School of
Medicine, Indianapolis, Indiana, USA.
(4)Department of Microbiology and Immunology, Indiana University School of
Medicine, Indianapolis, Indiana, USA sspinola@iu.edu.
(5)Department of Pathology and Laboratory Medicine, Indiana University School of 
Medicine, Indianapolis, Indiana, USA.
(6)Department of Medicine, Indiana University School of Medicine, Indianapolis,
Indiana, USA.

During infection, Neisseria gonorrhoeae senses and responds to stress; such
responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system
that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to
envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response
regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is
phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in
constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J.
L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M.
Shafer, Antimicrob Agents Chemother 60:4690-4700, 2016,
https://doi.org/10.1128/AAC.00823-16) showed that MisR (CpxR) is required for the
maintenance of membrane integrity and resistance to antimicrobial peptides,
suggesting a role in gonococcal survival in vivo Here, we evaluated the
contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model
of cervicovaginal colonization and identified MisR-regulated genes using RNA
sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity 
of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and 
reduced microbial fitness after exposure to heat shock. Compared to the wild type
(WT), the inactivation of misR identified 157 differentially regulated genes,
most of which encoded putative envelope proteins. The inactivation of misS
identified 17 differentially regulated genes compared to the WT and 139
differentially regulated genes compared to the misR mutant, 111 of which
overlapped those differentially expressed in the comparison of the WT versus the 
misR mutant. These data indicate that an intact MisRS system is required for
gonococcal infection of mice. Provided the MisR is constitutively phosphorylated 
in the misS mutant, the data suggest that controlled but not constitutive
activation is required for gonococcal infection in mice.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/IAI.00307-17 
PMCID: PMC5563589
PMID: 28652307  [Indexed for MEDLINE]


33. PLoS One. 2017 Aug 11;12(8):e0182800. doi: 10.1371/journal.pone.0182800.
eCollection 2017.

The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random
manner.

Antipov SS(1)(2)(3)(4), Tutukina MN(1)(5)(6)(7), Preobrazhenskaya EV(1),
Kondrashov FA(5)(6)(8), Patrushev MV(4), Toshchakov SV(4), Dominova I(4),
Shvyreva US(1), Vrublevskaya VV(9), Morenkov OS(9), Sukharicheva NA(1), Panyukov 
VV(7)(10), Ozoline ON(1)(2)(7).

Author information: 
(1)Department of Functional Genomics and Cellular Stress, Institute of Cell
Biophysics of Russian Academy of Sciences, Pushchino, Moscow Region, Russian
Federation.
(2)Department of Cell Biology, Pushchino State Institute of Natural Sciences,
Pushchino, Moscow Region, Russian Federation.
(3)Department of Biophysics and Biotechnology, Voronezh State University,
Voronezh, Russian Federation.
(4)Department of Genomics of Microorganisms, Immanuel Kant Baltic Federal
University, Kaliningrad, Russian Federation.
(5)Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG)
Barcelona, Spain.
(6)Department of Evolutionary Genomics, Universitat Pompeu Fabra (UPF),
Barcelona, Spain.
(7)Department of Structural and Functional Genomics,-Pushchino Research Center of
the Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.
(8)Institució Catalana de Recerca i Estudis Avançats (ICREA), 23 Pg. Lluís
Companys, Barcelona, Spain.
(9)Department of Cell Culture and Cell Engeneering, Institute of Cell Biophysics 
of Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.
(10)Department of Bioinformatics, Institute of Mathematical Problems of
Biology-the Branch of Keldysh Institute of Applied Mathematics of Russian Academy
of Sciences, Pushchino, Moscow Region, Russian Federation.

Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions
accumulating them in the form of Fe2O3 within its protein cavity, interacts with 
DNA tightly condensing bacterial nucleoid upon starvation and performs some other
functions. During the last two decades from discovery of this protein, its
ferroxidase activity became rather well studied, but the mechanism of Dps
interaction with DNA still remains enigmatic. The crucial role of lysine residues
in the unstructured N-terminal tails led to the conventional point of view that
Dps binds DNA without sequence or structural specificity. However, deletion of
dps changed the profile of proteins in starved cells, SELEX screen revealed
genomic regions preferentially bound in vitro and certain affinity of Dps for
artificial branched molecules was detected by atomic force microscopy. Here we
report a non-random distribution of Dps binding sites across the bacterial
chromosome in exponentially growing cells and show their enrichment with inverted
repeats prone to form secondary structures. We found that the Dps-bound regions
overlap with sites occupied by other nucleoid proteins, and contain
overrepresented motifs typical for their consensus sequences. Of the two types of
genomic domains with extensive protein occupancy, which can be highly expressed
or transcriptionally silent only those that are enriched with RNA polymerase
molecules were preferentially occupied by Dps. In the dps-null mutant we,
therefore, observed a differentially altered expression of several targeted genes
and found suppressed transcription from the dps promoter. In most cases this can 
be explained by the relieved interference with Dps for nucleoid proteins
exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial
cells from different stresses during exponential growth, Dps can modulate
transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis 
from some genes via competition with RNA polymerase or, vice versa, competing
with inhibitors to activate transcription.

DOI: 10.1371/journal.pone.0182800 
PMCID: PMC5553809
PMID: 28800583  [Indexed for MEDLINE]


34. Sci Rep. 2017 Aug 9;7(1):7630. doi: 10.1038/s41598-017-06737-4.

Host mechanisms involved in cattle Escherichia coli O157 shedding: a fundamental 
understanding for reducing foodborne pathogen in food animal production.

Wang O(1), McAllister TA(2), Plastow G(1), Stanford K(3), Selinger B(4), Guan
LL(5).

Author information: 
(1)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, T6G 2P5, Canada.
(2)Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, 
T1J 4B1, Canada.
(3)Alberta Agriculture and Forestry, Lethbridge, AB, T1J 4V6, Canada.
(4)Department of Biological Sciences, University of Lethbridge, Lethbridge, AB,
T1K 3M4, Canada.
(5)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, T6G 2P5, Canada. lguan@ualberta.ca.

The host mechanisms involved in Escherichia coli O157 super-shedding in cattle is
largely unknown. In this study, the comparison of transcriptomes of intestinal
tissues between super-shedders (SS) and cattle negative for E. coli O157 (NS) was
performed, aiming to identify genes that are potentially associated with
super-shedding. In total, 16,846 ± 639 (cecum) to 18,137 ± 696 (distal jejunum)
were expressed throughout the intestine, with the expressed genes associated with
immune functions more pronounced in the small intestine. In total, 351
differentially expressed (DE) genes were identified throughout the intestine
between SS and NS, with 101 being up-regulated and 250 down-regulated in SS.
Functional analysis revealed DE genes were involved in increased T-cell responses
and cholesterol absorption in the distal jejunum and descending colon, and
decreased B-cell maturation in the distal jejunum of SS. RNA-Seq based SNP
discovery revealed that the mutations in seven DE genes involved in leukocyte
activation and cholesterol transportation were associated with E. coli O157
shedding. Our findings suggest that T-cell responses and cholesterol metabolism
in the intestinal tract may be associated with super-shedding phenomenon, and the
SNPs in the DE genes are possibly associated with the observed gene expression
difference between SS and NS.

DOI: 10.1038/s41598-017-06737-4 
PMCID: PMC5550497
PMID: 28794460 


35. BMC Genomics. 2017 Aug 3;18(1):574. doi: 10.1186/s12864-017-3957-2.

Strand-specific transcriptomes of Enterohemorrhagic Escherichia coli in response 
to interactions with ground beef microbiota: interactions between microorganisms 
in raw meat.

Galia W(1)(2)(3)(4), Leriche F(5)(6), Cruveiller S(7), Garnier C(8), Navratil
V(9), Dubost A(10), Blanquet-Diot S(11), Thevenot-Sergentet D(8)(12).

Author information: 
(1)UMR 5557 Ecologie Microbienne, Research Group on Bacterial Opportunistic
Pathogens and Environment, CNRS, VetAgro Sup and Université de Lyon, Lyon,
France. wessam.galia@vetagro-sup.fr.
(2)Université Clermont Auvergne, INRA, UMRF, F-15000, Aurillac, France.
wessam.galia@vetagro-sup.fr.
(3)UMR UCA INRA 454 MEDIS Microbiota Digestive environment and Health, Université
Clermont Auvergne, 63000, Clermont-Ferrand, France. wessam.galia@vetagro-sup.fr.
(4)VetAgro Sup, Campus Agronomique de Lempdes, Lempdes, France.
wessam.galia@vetagro-sup.fr.
(5)Université Clermont Auvergne, INRA, UMRF, F-15000, Aurillac, France.
(6)VetAgro Sup, Campus Agronomique de Lempdes, Lempdes, France.
(7)Alternative Energies and Atomic Energy Commission (CEA), Genomic Institute
Genoscope & CNRS-UMR8030 & Evry University, Laboratory of Bioinformatics Analysis
in Genomics and Metabolism, Evry, France.
(8)UMR 5557 Ecologie Microbienne, Research Group on Bacterial Opportunistic
Pathogens and Environment, CNRS, VetAgro Sup and Université de Lyon, Lyon,
France.
(9)PRABI, Rhône Alpes Bioinformatics Center, UCBL, Lyon1, Université de Lyon,
Lyon, France.
(10)UMR 5557 Ecologie Microbienne, CNRS, Université de Lyon, Lyon, France.
(11)UMR UCA INRA 454 MEDIS Microbiota Digestive environment and Health,
Université Clermont Auvergne, 63000, Clermont-Ferrand, France.
(12)Reference Laboratory for Escherichia coli including Shiga Toxin-Producing E. 
coli, VetAgro Sup, Campus Vétérinaire de Lyon, Université de Lyon, Marcy
l'Etoile, Lyon, France.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are zoonotic agents
associated with outbreaks worldwide. Growth of EHEC strains in ground beef could 
be inhibited by background microbiota that is present initially at levels greater
than that of the pathogen E. coli. However, how the microbiota outcompetes the
pathogenic bacteria is unknown. Our objective was to identify metabolic pathways 
of EHEC that were altered by natural microbiota in order to improve our
understanding of the mechanisms controlling the growth and survival of EHECs in
ground beef.
RESULTS: Based on 16S metagenomics analysis, we identified the microbial
community structure in our beef samples which was an essential preliminary for
subtractively analyzing the gene expression of the EHEC strains. Then, we applied
strand-specific RNA-seq to investigate the effects of this microbiota on the
global gene expression of EHEC O2621765 and O157EDL933 strains by comparison with
their behavior in beef meat without microbiota. In strain O2621765, the
expression of genes connected with nitrate metabolism and nitrite detoxification,
DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous
genes involved in amino acid metabolism were down-regulated. Further, the
observed repression of ftsL and murF, involved respectively in building the
cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell 
wall peptidoglycan, might help to explain the microbiota's inhibitory effect on
EHECs. For strain O157EDL933, the induced expression of the genes implicated in
detoxification and the general stress response and the repressed expression of
the peR gene, a gene negatively associated with the virulence phenotype, might be
linked to the survival and virulence of O157:H7 in ground beef with microbiota.
CONCLUSION: In the present study, we show how RNA-Seq coupled with a 16S
metagenomics analysis can be used to identify the effects of a complex microbial 
community on relevant functions of an individual microbe within it. These
findings add to our understanding of the behavior of EHECs in ground beef. By
measuring transcriptional responses of EHEC, we could identify putative targets
which may be useful to develop new strategies to limit their shedding in ground
meat thus reducing the risk of human illnesses.

DOI: 10.1186/s12864-017-3957-2 
PMCID: PMC5543532
PMID: 28774270  [Indexed for MEDLINE]


36. Clin Transl Oncol. 2017 Aug;19(8):976-988. doi: 10.1007/s12094-017-1629-y. Epub
2017 Feb 21.

Selection of DNA aptamers for extra cellular domain of human epidermal growth
factor receptor 2 to detect HER2 positive carcinomas.

Sett A(1), Borthakur BB(2), Bora U(3)(4).

Author information: 
(1)Bioengineering Research Laboratory, Department of Biosciences and
Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039,
India.
(2)Department of Surgical Oncology, Dr. B. Borooah Cancer Institute, Guwahati,
Assam, 781016, India.
(3)Bioengineering Research Laboratory, Department of Biosciences and
Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039,
India. ubora@iitg.ernet.in.
(4)Mugagen Laboratories Pvt. Ltd., Technology Incubation Centre, Indian Institute
of Technology Guwahati, Guwahati, Assam, 781039, India. ubora@iitg.ernet.in.

BACKGROUND: Human epidermal growth factor receptor 2 (Her2, an orphan receptor of
ErbB family) is considered as an important biomarker as it plays a key role in
the development and progression of aggressive types of breast, ovarian, stomach
and gastric cancer. In the present study, we developed novel DNA aptamers against
the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive
carcinomas.
METHODS: We cloned and expressed Her2-ECD protein in E. coli system. After
purification, the protein was used as a bait for screening of specific DNA
aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro
Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The
aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The
specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was
estimated by FACS and immunofluorescence assay. The specificity of the candidate 
was also verified with the tissue samples of breast cancer patients by
immunohistochemistry process.
RESULTS: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24)
showed the highest binding affinity (K d = 6.33 ± 0.86 nM) to Her2-ECD protein.
The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 
490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection
limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic
staining in Her2-positive breast cancer cell lines (SKBR3) compared to
Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue
sections, it showed specific and selective localization in the cytoplasmic niche 
of malignant duct cancer cells without any cross-reactivity to fibroblasts,
inflammatory cells and adipocytes.
CONCLUSIONS: Binding assays, cytochemical and histochemical studies support
ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 
could be an effective low-cost alternative to conventional anti-Her2 antibody in 
solid phase immunoassays for cancer diagnosis and related applications.

DOI: 10.1007/s12094-017-1629-y 
PMID: 28224267 


37. Environ Microbiol Rep. 2017 Aug;9(4):441-448. doi: 10.1111/1758-2229.12553. Epub 
2017 Jun 27.

Interspecies cross-talk between co-cultured Pseudomonas putida and Escherichia
coli.

Molina-Santiago C(1), Udaondo Z(1), Cordero BF(1), Ramos JL(1).

Author information: 
(1)Department of Environmental Protection, Consejo Superior de Investigaciones
Científicas, C/Profesor Albareda 1, Granada, E-18008, Spain.

Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be
isolated from soil rhizosphere, the surface of vegetables, fresh waters and
wastewaters - environments in which they likely co-exist. Despite this, the
potential interactions between these microbes have not been studied in detail. To
analyse these interactions, we carried out RNA-seq transcriptomic analysis of
these microbes as monocultures and as co-cultures. Our results show that
co-culture of these microbes significantly alters transcriptional profiles. The
most dramatic transcriptional changes in both microorganisms were involved in
central carbon metabolism, as well as adhesion to surfaces and the activation of 
drug efflux pumps. We also found that acetate production was one of the
mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida
DOT-T1E.

© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

DOI: 10.1111/1758-2229.12553 
PMID: 28585781  [Indexed for MEDLINE]


38. mSystems. 2017 Jul 25;2(4). pii: e00024-17. doi: 10.1128/mSystems.00024-17.
eCollection 2017 Jul-Aug.

Transcriptional Variation of Diverse Enteropathogenic Escherichia coli Isolates
under Virulence-Inducing Conditions.

Hazen TH(1)(2), Daugherty SC(1), Shetty AC(1), Nataro JP(3), Rasko DA(1)(2).

Author information: 
(1)Institute for Genome Sciences, University of Maryland School of Medicine,
Baltimore, Maryland, USA.
(2)Department of Microbiology and Immunology, University of Maryland School of
Medicine, Baltimore, Maryland, USA.
(3)Department of Pediatrics, University of Virginia School of Medicine,
Charlottesville, Virginia, USA.

Comment in
    mSystems. 2017 Jul 25;2(4):.

Enteropathogenic Escherichia coli (EPEC) bacteria are a diverse group of
pathogens that cause moderate to severe diarrhea in young children in developing 
countries. EPEC isolates can be further subclassified as typical EPEC (tEPEC)
isolates that contain the bundle-forming pilus (BFP) or as atypical EPEC (aEPEC) 
isolates that do not contain BFP. Comparative genomics studies have recently
highlighted the considerable genomic diversity among EPEC isolates. In the
current study, we used RNA sequencing (RNA-Seq) to characterize the global
transcriptomes of eight tEPEC isolates representing the identified genomic
diversity, as well as one aEPEC isolate. The global transcriptomes were
determined for the EPEC isolates under conditions of laboratory growth that are
known to induce expression of virulence-associated genes. The findings
demonstrate that unique genes of EPEC isolates from diverse phylogenomic lineages
contribute to variation in their global transcriptomes. There were also
phylogroup-specific differences in the global transcriptomes, including genes
involved in iron acquisition, which had significant differential expression in
the EPEC isolates belonging to phylogroup B2. Also, three EPEC isolates from the 
same phylogenomic lineage (EPEC8) had greater levels of similarity in their
genomic content and exhibited greater similarities in their global transcriptomes
than EPEC from other lineages; however, even among closely related isolates there
were isolate-specific differences among their transcriptomes. These findings
highlight the transcriptional variability that correlates with the previously
unappreciated genomic diversity of EPEC. IMPORTANCE Recent studies have
demonstrated that there is considerable genomic diversity among EPEC isolates;
however, it is unknown if this genomic diversity leads to differences in their
global transcription. This study used RNA-Seq to compare the global
transcriptomes of EPEC isolates from diverse phylogenomic lineages. We
demonstrate that there are lineage- and isolate-specific differences in the
transcriptomes of genomically diverse EPEC isolates during growth under in vitro 
virulence-inducing conditions. This study addressed biological variation among
isolates of a single pathovar in an effort to demonstrate that while each of
these isolates is considered an EPEC isolate, there is significant
transcriptional diversity among members of this pathovar. Future studies should
consider whether this previously undescribed transcriptional variation may play a
significant role in isolate-specific variability of EPEC clinical presentations.

DOI: 10.1128/mSystems.00024-17 
PMCID: PMC5527300
PMID: 28766584 


39. Int J Food Microbiol. 2017 Jul 17;253:66-74. doi:
10.1016/j.ijfoodmicro.2017.05.005. Epub 2017 May 9.

Development of a gold nanoparticle-based universal oligonucleotide microarray for
multiplex and low-cost detection of foodborne pathogens.

Wang X(1), Ying S(2), Wei X(2), Yuan J(2).

Author information: 
(1)Xi'an Center for Disease Control and Prevention, 599 Xiying Road, Xi'an
710054, China. Electronic address: wang_xqiang@126.com.
(2)Xi'an Center for Disease Control and Prevention, 599 Xiying Road, Xi'an
710054, China.

Bacterial foodborne diseases remain major threats to food safety and public
health, especially in developing countries. In this study a novel assay,
combining gold nanoparticle (GNP)-based multiplex oligonucleotide ligation-PCR
and universal oligonucleotide microarray technology, was developed for
inexpensive, specific, sensitive, and multiplex detection of eight common
foodborne pathogens, including Shigella spp., Campylobacter jejuni, Bacillus
cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica,
Staphylococcus aureus, and Vibrio parahaemolyticus. The target fragments of the
eight pathogens were enriched by multiplex PCR and subjected to multiplex ligase 
detection reaction. Ligation products were enriched and labeled with GNPs by
universal asymmetric PCR, using excess GNP-conjugated primers. The labeled
single-stranded amplicons containing complementary tag sequences were captured by
the corresponding tag sequences immobilized on microarrays, followed by silver
staining for signal enhancement. Black images of microarray spots were visualized
by naked eyes or scanned on a simple flatbed scanner, and quantified. The results
indicated that this assay could unambiguously discriminate all eight pathogens in
single and multiple infections, with detection sensitivity of 3.3-85CFU/mL for
pure cultures. Microarray results of ninety-five artificially contaminated and
retail food samples were consistent with traditional culture, biochemical and
real-time PCR findings. Therefore, the novel assay has the potential to be used
for routine detection due to rapidity, low cost, and high specificity and
sensitivity.

Copyright © 2017 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.ijfoodmicro.2017.05.005 
PMID: 28505584  [Indexed for MEDLINE]


40. Nucleic Acids Res. 2017 Jul 7;45(12):7441-7454. doi: 10.1093/nar/gkx405.

Protein-only RNase P function in Escherichia coli: viability, processing defects 
and differences between PRORP isoenzymes.

Gößringer M(1), Lechner M(1), Brillante N(2), Weber C(2), Rossmanith W(2),
Hartmann RK(1).

Author information: 
(1)Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher 
Weg 6, 35037 Marburg, Germany.
(2)Center for Anatomy & Cell Biology, Medical University of Vienna, Währinger
Straße 13, 1090 Vienna, Austria.

The RNase P family comprises structurally diverse endoribonucleases ranging from 
complex ribonucleoproteins to single polypeptides. We show that the organellar
(AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes
from Arabidopsis thaliana confer viability to Escherichia coli cells with a
lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that
AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor 
tRNAs (pre-tRNAs) aberrantly in E. coli. Aberrant cleavage by AtPRORP1 was mainly
observed for pre-tRNAs that can form short acceptor-stem extensions involving G:C
base pairs, including tRNAAsp(GUC), tRNASer(CGA) and tRNAHis. However, both
AtPRORP1 and 3 were defective in processing of E. coli pre-tRNASec carrying an
acceptor stem expanded by three G:C base pairs. Instead, pre-tRNASec was
degraded, suggesting that tRNASec is dispensable for E. coli under laboratory
conditions. AtPRORP1, 2 and 3 are also essentially unable to process the primary 
transcript of 4.5S RNA, a hairpin-like non-tRNA substrate processed by E. coli
RNase P, indicating that PRORP enzymes have a narrower, more tRNA-centric
substrate spectrum than bacterial RNA-based RNase P enzymes. The cells' viability
also suggests that the essential function of the signal recognition particle can 
be maintained with a 5΄-extended 4.5S RNA.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkx405 
PMCID: PMC5499578
PMID: 28499021  [Indexed for MEDLINE]


41. Mol Cell. 2017 Jul 6;67(1):30-43.e6. doi: 10.1016/j.molcel.2017.05.025. Epub 2017
Jun 22.

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli.

Sedlyarova N(1), Rescheneder P(2), Magán A(1), Popitsch N(3), Rziha N(1), Bilusic
I(1), Epshtein V(4), Zimmermann B(5), Lybecker M(6), Sedlyarov V(7), Schroeder
R(8), Nudler E(9).

Author information: 
(1)Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories,
University of Vienna, Dr. Bohrgasse 9/5, 1030 Vienna, Austria.
(2)Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories,
University of Vienna & Medical University of Vienna, 1030 Vienna, Austria.
(3)Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, 1090
Vienna, Austria.
(4)Department of Biochemistry and Molecular Pharmacology, New York University
School of Medicine, New York, NY 10016, USA.
(5)Department of Molecular Evolution and Development, University of Vienna,
Althanstrasse 14, 1090 Vienna, Austria.
(6)University of Colorado, 1420 Austin Bluffs Parkway, Colorado Springs, CO
80918, USA.
(7)CeMM Research Center for Molecular Medicine of the Austrian Academy of
Sciences, 1090 Vienna, Austria.
(8)Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories,
University of Vienna, Dr. Bohrgasse 9/5, 1030 Vienna, Austria. Electronic
address: renee.schroeder@univie.ac.at.
(9)Department of Biochemistry and Molecular Pharmacology, New York University
School of Medicine, New York, NY 10016, USA; Howard Hughes Medical Institute, New
York University School of Medicine, New York, NY 10016, USA. Electronic address: 
evgeny.nudler@nyumc.org.

In search for RNA signals that modulate transcription via direct interaction with
RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for 
RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped
to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining
in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs
(iRAPs) that promote Rho-dependent transcription termination. A representative
iRAP within the coding region of the essential gene, nadD, greatly reduces its
transcriptional output in stationary phase and under oxidative stress,
demonstrating that iRAPs control gene expression in response to changing
environment. The mechanism of iRAPs involves active uncoupling of transcription
and translation, making nascent RNA accessible to Rho. iRAPs encoded in the
antisense strand also promote gene expression by reducing transcriptional
interference. In essence, our work uncovers a broad class of cis-acting RNA
signals that globally control bacterial transcription.

Copyright © 2017 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.molcel.2017.05.025 
PMCID: PMC5535762 [Available on 2018-07-06]
PMID: 28648779  [Indexed for MEDLINE]


42. Toxins (Basel). 2017 Jul 4;9(7). pii: E211. doi: 10.3390/toxins9070211.

Mechanisms for Differential Protein Production in Toxin-Antitoxin Systems.

Deter HS(1)(2), Jensen RV(3), Mather WH(4), Butzin NC(5).

Author information: 
(1)Department of Physics, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061-0435, USA. hdeter@vt.edu.
(2)Center for Soft Matter and Biological Physics, Virginia Polytechnic Institute 
and State University, Blacksburg, VA 24061-0435, USA. hdeter@vt.edu.
(3)Department of Biology, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061-0435, USA. rvjensen@vt.edu.
(4)Quantitative Biosciences, Inc., Solana Beach, CA 92075, USA.
will.mather@qbisci.com.
(5)Department of Biology and Microbiology, South Dakota State University,
Brookings, SD 57006, USA. nicholas.butzin@gmail.com.

Toxin-antitoxin (TA) systems are key regulators of bacterial persistence, a
multidrug-tolerant state found in bacterial species that is a major contributing 
factor to the growing human health crisis of antibiotic resistance. Type II TA
systems consist of two proteins, a toxin and an antitoxin; the toxin is
neutralized when they form a complex. The ratio of antitoxin to toxin is
significantly greater than 1.0 in the susceptible population (non-persister
state), but this ratio is expected to become smaller during persistence. Analysis
of multiple datasets (RNA-seq, ribosome profiling) and results from translation
initiation rate calculators reveal multiple mechanisms that ensure a high
antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms
include both translational and transcriptional regulation. We classified E. coli 
type II TA systems into four distinct classes based on the mechanism of
differential protein production between toxin and antitoxin. We find that the
most common regulation mechanism is translational regulation. This classification
scheme further refines our understanding of one of the fundamental mechanisms
underlying bacterial persistence, especially regarding maintenance of the
antitoxin-to-toxin ratio.

DOI: 10.3390/toxins9070211 
PMCID: PMC5535158
PMID: 28677629  [Indexed for MEDLINE]

Conflict of interest statement: The authors declare no conflict of interest.


43. FEMS Microbiol Ecol. 2017 Jul 1;93(7). doi: 10.1093/femsec/fix090.

The CTX-M-14 plasmid pHK01 encodes novel small RNAs and influences host growth
and motility.

Jiang X(1), Liu X(1), Law COK(1), Wang Y(2), Lo WU(2), Weng X(1), Chan TF(3), Ho 
PL(2), Lau TCK(1).

Author information: 
(1)Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong
Kong Special Administrative Region.
(2)Department of Microbiology, University of Hong Kong, Hong Kong, Hong Kong
Special Administrative Region.
(3)School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong
Kong Special Administrative Region.

The dissemination of extended-spectrum β-lactamases (ESBLs) genes among bacteria 
is commonly achieved by plasmid conjugation. In the last decade, the CTX-M type
enzyme was the most widespread and prevalent ESBLs in the world. In Hong Kong and
mainland China, among the commonly found CTX-M-carrying plasmids were pHK01 and
pHK01-like plasmids, which belong to incompatibility group FII (IncFII). In this 
work, we studied the physiological effect caused by the pHK01 plasmid in
bacterial host Escherichia coli J53. The plasmid did not affect cell growth of
the host but reduced their motility. The reduction of host motility was
attributed to downregulation of genes that encode the flagellar system. We also
identified several plasmid-encoded sRNAs, and showed that the overexpression of
one of them, AS-traI, in the presence of pHK01 plasmid shortened the lag phase of
host growth. In addition to the study of pHK01 in bacteria, we also developed a
fast and incompatibility group-specific curing method using countertranscribed
RNA, which could be of general usage for studying plasmid-host interaction in
clinical aspects.

© FEMS 2017. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.

DOI: 10.1093/femsec/fix090 
PMID: 28854680  [Indexed for MEDLINE]


44. PLoS One. 2017 Jun 30;12(6):e0179181. doi: 10.1371/journal.pone.0179181.
eCollection 2017.

The whole set of the constitutive promoters recognized by four minor sigma
subunits of Escherichia coli RNA polymerase.

Shimada T(1)(2), Tanaka K(2), Ishihama A(1).

Author information: 
(1)Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo,
Japan.
(2)Laboratory for Chemistry and Life Science, Institute of Innovative Research,
Tokyo Institute of Technology, Nagatsuda, Yokohama, Japan.

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined 
by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven
species of the sigma subunit, each recognizing a specific set of promoters. For
identification of the "constitutive promoters" that are recognized by each RNAP
holoenzyme alone in the absence of other supporting factors, we have performed
the genomic SELEX screening in vitro for their binding sites along the E. coli
K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a
collection of genome DNA segments of E. coli K-12. The whole set of constitutive 
promoters for each RNAP holoenzyme was then estimated based on the location of
RNAP-binding sites. The first successful screening of the constitutive promoters 
was achieved for RpoD (σ70), the principal sigma for transcription of
growth-related genes. As an extension, we performed in this study the screening
of constitutive promoters for four minor sigma subunits, stationary-phase
specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis
specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total
number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 
for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared
with that of known promoters identified in vivo under various conditions and
using varieties of E. coli strains, altogether allowing the estimation of
"inducible promoters" in the presence of additional supporting factors.

DOI: 10.1371/journal.pone.0179181 
PMCID: PMC5493296
PMID: 28666008  [Indexed for MEDLINE]


45. Genet Mol Res. 2017 Jun 29;16(2). doi: 10.4238/gmr16029574.

Promoter identification and analysis of key glycosphingolipid biosynthesis-globo 
series pathway genes in piglets.

Qin WY(1), Gan LN(1), Xia RW(1), Dong WH(1), Sun SY(1), Zhu GQ(2), Wu SL(1), Bao 
WB(3).

Author information: 
(1)Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular
Design of Jiangsu Province, College of Animal Science and Technology, , , China.
(2)College of Veterinary Medicine, , , China.
(3)Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular
Design of Jiangsu Province, College of Animal Science and Technology, , , China
wbbao@yzu.edu.cn.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1,
HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense 
against Escherichia coli F18 in piglets. In this study, we identified the
transcription initiation site and promoter of this gene cluster by mined previous
RNA-seq results using bioinformatics tools. The FUT1 transcription initiation
region included five alternative splicing sites and two promoter regions, whereas
each of the six other genes had one promoter. Dual luciferase reporter results
revealed significantly higher transcriptional activity by FUT1 promoter 2,
indicating that it played a more important role in transcription. The promoters
of glycosphingolipid biosynthesis genes identified contained a CpG island within 
the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG
sites. These results provide a deeper insight into methylation and the regulatory
mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in
piglets.

DOI: 10.4238/gmr16029574 
PMID: 28671252  [Indexed for MEDLINE]


46. ACS Synth Biol. 2017 Jun 16;6(6):1065-1075. doi: 10.1021/acssynbio.7b00006. Epub 
2017 Mar 9.

Obtaining a Panel of Cascade Promoter-5'-UTR Complexes in Escherichia coli.

Zhou S(1), Ding R(1), Chen J(1), Du G(1), Li H(1), Zhou J(1).

Author information: 
(1)Key Laboratory of Industrial Biotechnology, Ministry of Education, School of
Biotechnology, and ‡National Engineering Laboratory for Cereal Fermentation
Technology, Jiangnan University , 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

A promoter is one of the most important and basic tools used to achieve diverse
synthetic biology goals. Escherichia coli is one of the most commonly used model 
organisms in synthetic biology to produce useful target products and establish
complicated regulation networks. During the fine-tuning of metabolic or
regulation networks, the limited number of well-characterized inducible promoters
has made implementing complicated strategies difficult. In this study, 104 native
promoter-5'-UTR complexes (PUTR) from E. coli were screened and characterized
based on a series of RNA-seq data. The strength of the 104 PUTRs varied from
0.007% to 4630% of that of the PBAD promoter in the transcriptional level and
from 0.1% to 137% in the translational level. To further upregulate gene
expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by
integrating strong transcriptional promoters with strong translational 5'-UTRs.
Finally, two combinatorial PUTRs (PssrA-UTRrpsT and PdnaKJ-UTRrpsT) and two
cascade PUTRs (PUTRssrA-PUTRinfC-rplT and PUTRalsRBACE-PUTRinfC-rplT) were
identified as having the highest activity, with expression outputs of 170%, 137%,
409%, and 203% of that of the PBAD promoter, respectively. These engineered PUTRs
are stable for the expression of different genes, such as the red fluorescence
protein gene and the β-galactosidase gene. These results show that the PUTRs
characterized and constructed in this study may be useful as a plug-and-play
synthetic biology toolbox to achieve complicated metabolic engineering goals in
fine-tuning metabolic networks to produce target products.

DOI: 10.1021/acssynbio.7b00006 
PMID: 28252945  [Indexed for MEDLINE]


47. Cell. 2017 Jun 15;169(7):1249-1262.e13. doi: 10.1016/j.cell.2017.05.036.

Microbial Genetic Composition Tunes Host Longevity.

Han B(1), Sivaramakrishnan P(2), Lin CJ(1), Neve IAA(1), He J(3), Tay LWR(3),
Sowa JN(1), Sizovs A(4), Du G(3), Wang J(4), Herman C(5), Wang MC(6).

Author information: 
(1)Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030,
USA; Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
(2)Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
(3)Department of Integrative Biology and Pharmacology, University of Texas Health
Science Center at Houston, Houston, TX 77030, USA.
(4)Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030,
USA.
(5)Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA; Dan L. Duncan Comprehensive Cancer Center, Baylor College
of Medicine, Houston, TX 77030, USA.
(6)Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030,
USA; Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA; Dan L. Duncan Comprehensive Cancer Center, Baylor College
of Medicine, Houston, TX 77030, USA. Electronic address: wmeng@bcm.edu.

Comment in
    Cell. 2017 Jun 15;169(7):1168-1169.

Homeostasis of the gut microbiota critically influences host health and aging.
Developing genetically engineered probiotics holds great promise as a new
therapeutic paradigm to promote healthy aging. Here, through screening 3,983
Escherichia coli mutants, we discovered that 29 bacterial genes, when deleted,
increase longevity in the host Caenorhabditis elegans. A dozen of these bacterial
mutants also protect the host from age-related progression of tumor growth and
amyloid-beta accumulation. Mechanistically, we discovered that five bacterial
mutants promote longevity through increased secretion of the polysaccharide
colanic acid (CA), which regulates mitochondrial dynamics and unfolded protein
response (UPRmt) in the host. Purified CA polymers are sufficient to promote
longevity via ATFS-1, the host UPRmt-responsive transcription factor.
Furthermore, the mitochondrial changes and longevity effects induced by CA are
conserved across different species. Together, our results identified molecular
targets for developing pro-longevity microbes and a bacterial metabolite acting
on host mitochondria to promote longevity.

Copyright © 2017 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.cell.2017.05.036 
PMCID: PMC5635830 [Available on 2018-06-15]
PMID: 28622510  [Indexed for MEDLINE]


48. Cell Discov. 2017 Jun 6;3:17018. doi: 10.1038/celldisc.2017.18. eCollection 2017.

Multiplex gene regulation by CRISPR-ddCpf1.

Zhang X(1)(2)(3), Wang J(4)(5), Cheng Q(6), Zheng X(1), Zhao G(1), Wang J(1).

Author information: 
(1)Key Laboratory of Synthetic Biology, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai, China.
(2)School of Life Science and Technology, Shanghai Tech University, Shanghai,
China.
(3)University of Chinese Academy of Sciences, Beijing, China.
(4)State Engineering Laboratory of Medical Key Technologies Application of
Synthetic Biology, Shenzhen Second People's Hospital, The First Affiliated
Hospital of Shenzhen University, Shenzhen, China.
(5)Sun Yat-sen University Cancer Center, Guangzhou, China.
(6)Shanghai Tolo Biotechnology Company Limited, Shanghai, China.

The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9
system has been widely applied in both transcriptional regulation and epigenetic 
studies. However, for multiple targets, independent expression of multiple single
guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, 
we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We
demonstrated that ddCpf1 alone could be employed for gene repression in
Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs)
specifically targeting to the template strand of its target genes, which was
different from that of dCas9. When targeting the promoter region, both strands
showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome 
RNA-seq technique was further employed to demonstrate the high specificity of
ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity 
in ddCpf1 was capable of processing a precursor CRISPR array to simply generate
multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the 
employment of this multiplex gene regulation strategy, we also showed how to
quickly screen a library of candidate targets, that is, the two-component systems
in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may
be further developed and widely applied in both biological research and clinical 
studies.

DOI: 10.1038/celldisc.2017.18 
PMCID: PMC5460296
PMID: 28607761 

Conflict of interest statement: The authors declare no conflict of interest.


49. Appl Environ Microbiol. 2017 May 31;83(12). pii: e00442-17. doi:
10.1128/AEM.00442-17. Print 2017 Jun 15.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and 
Reregulates Catabolism.

He A(1), Penix SR(1), Basting PJ(1), Griffith JM(1), Creamer KE(1), Camperchioli 
D(1), Clark MW(1), Gonzales AS(1), Chávez Erazo JS(1), George NS(2), Bhagwat
AA(2), Slonczewski JL(3).

Author information: 
(1)Department of Biology, Kenyon College, Gambier, Ohio, USA.
(2)Environmental Microbiology and Food Safety Laboratory, Beltsville Agricultural
Research Center, U.S. Department of Agriculture, Beltsville, Maryland, USA.
(3)Department of Biology, Kenyon College, Gambier, Ohio, USA
slonczewski@kenyon.edu.

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial
culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W.
Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L.
Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015,
https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains 
revealed insertion sequence (IS)-driven insertions and deletions that knocked out
regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of
glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain
showed loss of one or more amino acid decarboxylase systems, which normally help 
neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH
2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated
deletion in cadC, while population B11 had a point mutation affecting the
arginine activator adiY The cadC and adiY mutants failed to neutralize acid in
the presence of exogenous lysine or arginine. In strain B11-1, reversion of an
rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization.
All eight strains showed deletion or downregulation of the Gad acid fitness
island. Strains with the Gad deletion lost the ability to produce GABA
(gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome
sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of
diverse substrates but downregulated acid stress genes (the biofilm regulator
ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption
mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 
had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC,
frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in
buffered acid showed loss or downregulation of systems that neutralize unbuffered
acid and showed altered regulation of catabolism.IMPORTANCE Experimental
evolution of an enteric bacterium under a narrow buffered range of acid pH leads 
to loss of genes that enhance fitness above or below the buffered pH range,
including loss of enzymes that may raise external pH in the absence of buffer.
Prominent modes of evolutionary change involve IS-mediated insertions and
deletions that knock out key regulators. Over generations of acid stress,
catabolism undergoes reregulation in ways that differ for each evolving strain.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/AEM.00442-17 
PMCID: PMC5452808
PMID: 28389540  [Indexed for MEDLINE]


50. PLoS One. 2017 May 25;12(5):e0178483. doi: 10.1371/journal.pone.0178483.
eCollection 2017.

ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched
RNA-seq data.

Promworn Y(1), Kaewprommal P(1), Shaw PJ(1), Intarapanich A(2), Tongsima S(1),
Piriyapongsa J(1).

Author information: 
(1)National Center for Genetic Engineering and Biotechnology (BIOTEC), National
Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.
(2)National Electronics and Computer Technology Center (NECTEC), National Science
and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

BACKGROUND: Biochemical methods are available for enriching 5' ends of RNAs in
prokaryotes, which are employed in the differential RNA-seq (dRNA-seq) and the
more recent Cappable-seq protocols. Computational methods are needed to locate
RNA 5' ends from these data by statistical analysis of the enrichment. Although
statistical-based analysis methods have been developed for dRNA-seq, they may not
be suitable for Cappable-seq data. The more efficient enrichment method employed 
in Cappable-seq compared with dRNA-seq could affect data distribution and thus
algorithm performance.
RESULTS: We present Transformation of Nucleotide Enrichment Ratios (ToNER), a
tool for statistical modeling of enrichment from RNA-seq data obtained from
enriched and unenriched libraries. The tool calculates nucleotide enrichment
scores and determines the global transformation for fitting to the normal
distribution using the Box-Cox procedure. From the transformed distribution,
sites of significant enrichment are identified. To increase power of detection,
meta-analysis across experimental replicates is offered. We tested the tool on
Cappable-seq and dRNA-seq data for identifying Escherichia coli transcript 5'
ends and compared the results with those from the TSSAR tool, which is designed
for analyzing dRNA-seq data. When combining results across Cappable-seq
replicates, ToNER detects more known transcript 5' ends than TSSAR. In general,
the transcript 5' ends detected by ToNER but not TSSAR occur in regions which
cannot be locally modeled by TSSAR.
CONCLUSION: ToNER uses a simple yet robust statistical modeling approach, which
can be used for detecting RNA 5'ends from Cappable-seq data, in particular when
combining information from experimental replicates. The ToNER tool could
potentially be applied for analyzing other RNA-seq datasets in which enrichment
for other structural features of RNA is employed. The program is freely available
for download at ToNER webpage (http://www4a.biotec.or.th/GI/tools/toner) and
GitHub repository (https://github.com/PavitaKae/ToNER).

DOI: 10.1371/journal.pone.0178483 
PMCID: PMC5444824
PMID: 28542466  [Indexed for MEDLINE]


51. Sci Rep. 2017 May 23;7(1):2277. doi: 10.1038/s41598-017-02628-w.

Generation and Characterization of Acid Tolerant Fibrobacter succinogenes S85.

Wu CW(1)(2), Spike T(3), Klingeman DM(1)(4), Rodriguez M(1)(4), Bremer VR(5),
Brown SD(6)(7).

Author information: 
(1)Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
(2)Department of Pathobiological Sciences, University of Wisconsin-Madison,
Madison, WI, USA.
(3)Biota Biosciences, Inc., Cambridge City, IN, USA.
(4)BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
(5)Elanco Animal Health, Greenfield, IN, USA.
(6)Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
brownsd@ornl.gov.
(7)BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
brownsd@ornl.gov.

Microorganisms are key components for plant biomass breakdown within rumen
environments. Fibrobacter succinogenes have been identified as being active and
dominant cellulolytic members of the rumen. In this study, F. succinogenes type
strain S85 was adapted for steady state growth in continuous culture at pH 5.75
and confirmed to grow in the range of pH 5.60-5.65, which is lower than has been 
reported previously. Wild type and acid tolerant strains digested corn stover
with equal efficiency in batch culture at low pH. RNA-seq analysis revealed 268
and 829 genes were differentially expressed at pH 6.10 and 5.65 compared to pH
6.70, respectively. Resequencing analysis identified seven single nucleotide
polymorphisms (SNPs) in the sufD, yidE, xylE, rlmM, mscL and dosC genes of acid
tolerant strains. Due to the absence of a F. succinogenes genetic system,
homologues in Escherichia coli were mutated and complemented and the resulting
strains were assayed for acid survival. Complementation with wild-type or acid
tolerant F. succinogenes sufD restored E. coli wild-type levels of acid
tolerance, suggesting a possible role in acid homeostasis. Recent genetic
engineering developments need to be adapted and applied in F. succinogenes to
further our understanding of this bacterium.

DOI: 10.1038/s41598-017-02628-w 
PMCID: PMC5442110
PMID: 28536480 


52. Molecules. 2017 May 17;22(5). pii: E825. doi: 10.3390/molecules22050825.

Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent
Biosensor Platform.

Song MS(1), Sekhon SS(2), Shin WR(3), Kim HC(4), Min J(5), Ahn JY(6), Kim YH(7).

Author information: 
(1)School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, Korea. smst04@nate.com.
(2)School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, Korea. simranjeet261@gmail.com.
(3)School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, Korea. aomaya91@nate.com.
(4)Technology Transfer Center, Korea Research Institute of Bioscience &
Biotechnology, 125 Gwahak-Ro, Yuseong-Gu, Daejeon 34141, Korea. hcgimm@gmail.com.
(5)Department of Bioprocess Engineering, Chonbuk National University, 567
Baekje-daero, Deokjin-Gu Jeonju, Jeonbuk 54896, Korea. jihomin@chonbuk.ac.kr.
(6)School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, Korea. jyahn@chungbuk.ac.kr.
(7)School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 28644, Korea. kyh@chungbuk.ac.kr.

In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate 
specific aptamers against Shigella sonnei. Real-time PCR amplification and
post-SELEX experiment revealed that the selected aptmers possessed a high binding
affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t)
values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively)
had the lowest value compared to other aptamer candidates. The SS-3 and SS-4
aptamers also displayed a binding affinity (KD) of 39.32 ± 5.02 nM and 15.89 ±
1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed 
to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 
and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor
platform consisted of three elements: (1) 5'amine-SS-4 modification in a 96-well 
type microtiter plate surface (N-oxysuccinimide, NOS) as capture probes; (2) the 
incubation with S. sonnei and test microbes in functionalized 96 assay wells in
parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3
aptamer as the detector. Our platform showed a significant ability to detect and 
discriminate S. sonnei from other enteric species such as E. coli, Salmonella
typhimurium and other Shigella species (S. flexneri, S. boydii). In this study,
we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei
in a variety of foods and pave the way for its use in diagnosing shigellosis
through multiple, portable designs.

DOI: 10.3390/molecules22050825 
PMID: 28513559  [Indexed for MEDLINE]

Conflict of interest statement: The authors declare no conflict of interest.


53. PLoS One. 2017 May 17;12(5):e0177939. doi: 10.1371/journal.pone.0177939.
eCollection 2017.

SPOt: A novel and streamlined microarray platform for observing cellular tRNA
levels.

Grelet S(1), McShane A(2), Hok E(2), Tomberlin J(2), Howe PH(1), Geslain R(2).

Author information: 
(1)Department of Biochemistry and Molecular Biology, MUSC, Charleston, SC, United
States of America.
(2)Laboratory of tRNA Biology, Department of Biology, College of Charleston,
Charleston, SC, United States of America.

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the
heart of fundamental cellular processes such as embryonic development and tumor
progression. Such discoveries were contingent on the concomitant development of
methods able to deliver high-quality tRNA profiles. The present study describes
the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA 
analysis platform named SPOt (Streamlined Platform for Observing tRNA). This
approach comprises three steps. First, E. coli cultures are spiked with
radioactive orthophosphate; second, labeled total RNAs are trizol-extracted;
third, RNA samples are hybridized on in-house printed microarrays and spot
signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features
such as reproducibility and specificity were assessed using several tRNA
subpopulations. Dynamic range and sensitivity were evaluated by overexpressing
specific tRNA species. SPOt does not require any amplification or post-extraction
labeling and can be adapted to any organism. It is modular and easily streamlined
with popular techniques such as polysome fractionation to profile tRNAs
interacting with ribosomes and actively engaged in translation. The biological
relevance of these data is discussed in regards to codon usage, tRNA gene copy
number, and position on the genome.

DOI: 10.1371/journal.pone.0177939 
PMCID: PMC5435355
PMID: 28545122  [Indexed for MEDLINE]


54. Dev Comp Immunol. 2017 May;70:80-87. doi: 10.1016/j.dci.2017.01.008. Epub 2017
Jan 7.

Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during
Escherichia coli infection in Drosophila.

Li S(1), Shen L(1), Sun L(1), Xu J(1), Jin P(1), Chen L(2), Ma F(3).

Author information: 
(1)Laboratory for Comparative Genomics and Bioinformatics, Jiangsu Key Laboratory
for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal
University, Nanjing 210046, China.
(2)The Key Laboratory of Developmental Genes and Human Disease, College of Life
Science, Nanjing Normal University, Nanjing 210046, China.
(3)Laboratory for Comparative Genomics and Bioinformatics, Jiangsu Key Laboratory
for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal
University, Nanjing 210046, China. Electronic address: mafei01@tsinghua.org.cn.

Drosophila have served as a model for research on innate immunity for decades.
However, knowledge of the post-transcriptional regulation of immune gene
expression by microRNAs (miRNAs) remains rudimentary. In the present study, using
small RNA-seq and bioinformatics analysis, we identified 67 differentially
expressed miRNAs in Drosophila infected with Escherichia coli compared to injured
flies at three time-points. Furthermore, we found that 21 of these miRNAs were
potentially involved in the regulation of Imd pathway-related genes. Strikingly, 
based on UAS-miRNAs line screening and Dual-luciferase assay, we identified that 
miR-9a and miR-981 could both negatively regulate Drosophila antibacterial
defenses and decrease the level of the antibacterial peptide, Diptericin. Taken
together, these data support the involvement of miRNAs in the regulation of the
Drosophila Imd pathway.

Copyright © 2017 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.dci.2017.01.008 
PMID: 28069431  [Indexed for MEDLINE]


55. PLoS One. 2017 Apr 24;12(4):e0174704. doi: 10.1371/journal.pone.0174704.
eCollection 2017.

Relationship of the CreBC two-component regulatory system and inner membrane
protein CreD with swimming motility in Stenotrophomonas maltophilia.

Huang HH(1), Chen WC(1)(2), Lin CW(3), Lin YT(4)(5), Ning HC(6)(7), Chang YC(3), 
Yang TC(1).

Author information: 
(1)Department of Biotechnology and Laboratory Science in Medicine, National
Yang-Ming University, Taipei, Taiwan.
(2)Super Laboratory Co. Ltd., New Taipei City, Taiwan.
(3)Department of Medical Laboratory Science and Biotechnology, China Medical
University, Taichung, Taiwan.
(4)Division of Infectious Diseases, Department of Medicine, Taipei Veterans
General Hospital, Taipei, Taiwan.
(5)School of Medicine, National Yang-Ming University, Taipei, Taiwan.
(6)Department of Laboratory Medicine, Chang Gung Memorial Hospital Linkou Branch,
Taoyuan, Taiwan.
(7)Department of Medical Biotechnology and Laboratory Science, Chang Gung
University, Taoyuan, Taiwan.

The CreBC two-component system (TCS) is a conserved regulatory system found in
Escherichia coli, Aeromonas spp., Pseudomonas aeruginosa, and Stenotrophomonas
maltophilia. In this study, we determined how CreBC TCS regulates secreted
protease activities and swimming motility using creB, creC, and creBC in-frame
deletion mutants (KJΔCreB, KJΔCreC, and KJΔBC) of S. maltophilia KJ. Compared to 
wild-type KJ, KJΔCreB had a comparable secreted protease activity; however, the
secreted protease activities were obviously reduced in KJΔCreC and KJΔBC,
suggesting that CreC works together with another unidentified response regulator 
(not CreB) to regulate secreted protease activity. Single gene inactivation of
creB or creC resulted in mutants with an enhanced swimming motility, and this
phenotype was exacerbated in a double mutant KJΔBC. To elucidate the underlying
mechanism responsible for the ΔcreBC-mediated swimming enhancement, flagella
morphology observation, RNA-seq based transcriptome assay, qRT-PCR, and membrane 
integrity and potential assessment were performed. Flagella morphological
observation ruled out the possibility that swimming enhancement was due to
altered flagella morphology. CreBC inactivation upregulated the expression of
creD and flagella-associated genes encoding the basal body- and motor-associated 
proteins. Furthermore, KJΔBC had an increased membrane susceptibility to Triton
X-100 and CreD upregulation in KJΔBC partially alleviated the compromise of
membrane integrity. The impact of creBC TCS on bacterial membrane potential was
assessed by carbonyl cyanide m-chlorophenyl hydrazine (CCCP50) concentration at
which 50% of bacterial swimming is inhibited. CCCP50 of wild-type KJ increased
when creBC was deleted, indicating an association between the higher membrane
potential of KJΔBC cells and enhanced motility. Upregulation of the basal body-
and motor-associated genes of flagella in KJΔBC cells may explain the increased
membrane potential. Collectively, inactivation of creBC increased swimming
motility through membrane potential increase and creD upregulation in S.
maltophilia. The increased membrane potential may supply more energy for flagella
propelling and CreD upregulation supports membrane stability, providing a strong 
membrane for flagellum function.

DOI: 10.1371/journal.pone.0174704 
PMCID: PMC5402928
PMID: 28437463  [Indexed for MEDLINE]


56. Microbiology. 2017 Apr;163(4):611-621. doi: 10.1099/mic.0.000448. Epub 2017 Apr
13.

Genome amplification and promoter mutation expand the range of csgD-dependent
biofilm responses in an STEC population.

Uhlich GA(1), Chen CY(1), Cottrell BJ(1), Andreozzi E(1), Irwin PL(1), Nguyen
LH(1).

Author information: 
(1)Molecular Characterization of Foodborne Pathogens Research Unit, Eastern
Regional Research Center, Agricultural Research Service, U.S. Department of
Agriculture, 600 East Mermaid Lane, Wyndmoor, PA, USA.

Expression of the major biofilm components of E. coli, curli fimbriae and
cellulose, requires the CsgD transcription factor. A complex regulatory network
allows environmental control of csgD transcription and biofilm formation.
However, most clinical serotype O157 : H7 strains contain prophage insertions in 
the csgD regulator, mlrA, or mutations in other regulators that restrict csgD
expression. These barriers can be circumvented by certain compensating mutations 
that restore higher csgD expression. One mechanism is via csgD promoter mutations
that switch sigma factor utilization. Biofilm-forming variants utilizing RpoD
rather than RpoS have been identified in glycerol freezer stocks of the
non-biofilm-forming food-borne outbreak strain, ATCC 43894. In this study we used
whole genome sequencing and RNA-seq to study genotypic and transcriptomic
differences between those strains. In addition to defining the consequences of
the csgD promoter switch and identifying new csgD-controlled genes, we discovered
a region of genome amplification in our laboratory stock of 43894 (designated
43894OW) that contributed to the regulation of csgD-dependent properties.

DOI: 10.1099/mic.0.000448 
PMID: 28406080  [Indexed for MEDLINE]


57. Plant Physiol Biochem. 2017 Apr;113:78-88. doi: 10.1016/j.plaphy.2017.02.002.
Epub 2017 Feb 5.

A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene
ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana.

Lu X(1), Yang L(2), Yu M(3), Lai J(4), Wang C(5), McNeil D(6), Zhou M(7), Yang
C(8).

Author information: 
(1)Tasmanian Institute of Agriculture, University of Tasmania, PO Box 46, Kings
Meadows, TAS 7249, Australia; Guangdong Provincial Key Laboratory of
Biotechnology for Plant Development, School of Life Sciences, South China Normal 
University, Guangzhou 510631, China; College of Pratacultural Science, Gansu
Agriculture University, Lanzhou 730070, China. Electronic address:
Xiang.lu@utas.edu.au.
(2)College of Life Science, Anhui Normal University, Wuhu 241000, China.
Electronic address: cricleno@hotmail.com.
(3)Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
School of Life Sciences, South China Normal University, Guangzhou 510631, China. 
Electronic address: chelseaYMY@126.com.
(4)Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
School of Life Sciences, South China Normal University, Guangzhou 510631, China. 
Electronic address: laijianbin@hotmail.com.
(5)Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
School of Life Sciences, South China Normal University, Guangzhou 510631, China. 
Electronic address: wangchao850304@163.com.
(6)Tasmanian Institute of Agriculture, University of Tasmania, PO Box 46, Kings
Meadows, TAS 7249, Australia. Electronic address: david.mcneil@utas.edu.au.
(7)Tasmanian Institute of Agriculture, University of Tasmania, PO Box 46, Kings
Meadows, TAS 7249, Australia. Electronic address: meixue.zhou@utas.edu.au.
(8)Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
School of Life Sciences, South China Normal University, Guangzhou 510631, China; 
Dongli Planting and Farming Industrial Co., LTD, Lianzhou 513400, China.
Electronic address: yangchw@scnu.edu.cn.

The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close 
wild relative of maize and thus can be effectively used in maize improvement. In 
this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of
RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The
deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix
(bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein
localizes to the nucleus and shows sumoylation when expressed in an Escherichia
coli reconstitution system. In addition, yeast one hybrid assays indicated that
ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1
in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1
overexpressed plants showed lower electrolyte leakage (EL), reduced contents of
malondialdehyde (MDA). The expression of downstream cold related genes of
Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive
genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to
desiccation gene (AtRD29A) was significantly induced when compared with wild type
under low temperature treatment. Taken together, these results indicated that
ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2) 
and plays an important role in the regulation of freezing stress response.

Copyright © 2017 Elsevier Masson SAS. All rights reserved.

DOI: 10.1016/j.plaphy.2017.02.002 
PMID: 28189052  [Indexed for MEDLINE]


58. Front Plant Sci. 2017 Mar 22;8:389. doi: 10.3389/fpls.2017.00389. eCollection
2017.

Genome-Wide Identification, Expression Patterns, and Functional Analysis of UDP
Glycosyltransferase Family in Peach (Prunus persica L. Batsch).

Wu B(1), Gao L(1), Gao J(1), Xu Y(1), Liu H(1), Cao X(1), Zhang B(1), Chen K(1).

Author information: 
(1)Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative
Biology/Laboratory of Fruit Quality Biology, Zhejiang University Hangzhou, China.

Peach (Prunus persica L. Batsch) is a commercial grown fruit trees, important
because of its essential nutrients and flavor promoting secondary metabolites.
The glycosylation processes mediated by UDP-glycosyltransferases (UGTs) play an
important role in regulating secondary metabolites availability. Identification
and characterization of peach UGTs is therefore a research priority. A total of
168 peach UGT genes that distributed unevenly across chromosomes were identified 
based on their conserved PSPG motifs. Phylogenetic analysis of these genes with
plant UGTs clustered them into 16 groups (A-P). Comparison of the patterns of
intron-extron and their positions within genes revealed one highly conserved
intron insertion event in peach UGTs. Tissue specificity, temporal expression
patterns in peach fruit during development and ripening, and in response to
abiotic stress UV-B irradiation was investigated using RNA-seq strategy. The
relationship between UGTs transcript levels and concentrations of glycosylated
volatiles was examined to select candidates for functional analysis. Heterologous
expressing these candidate genes in Escherichia coli identified UGTs that were
involved in the in vitro volatile glycosylation. Our results provide an important
source for the identification of functional UGT genes to potential manipulate
secondary biosynthesis in peach.

DOI: 10.3389/fpls.2017.00389 
PMCID: PMC5360731
PMID: 28382047 


59. Nucleic Acids Res. 2017 Mar 17;45(5):2746-2756. doi: 10.1093/nar/gkw894.

Landscape of RNA polyadenylation in E. coli.

Maes A(1), Gracia C(1), Innocenti N(2)(3), Zhang K(4), Aurell E(2)(5), Hajnsdorf 
E(1).

Author information: 
(1)CNRS UMR8261 (previously FRE3630) associated with University Paris Diderot,
Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, 13 rue P. et M.
Curie, 75005 Paris, France.
(2)Department of Computational Biology, KTH Royal Institute of Technology,
AlbaNova University Center, Roslagstullsbacken 17, SE-10691 Stockholm, Sweden.
(3)Combient AB, Nettovägen 6, SE-175 41 Järfälla, Sweden.
(4)Systems Biology Laboratory, Research Programs Unit,Genome-Scale Biology,
Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finlandepts of 
Computer Science and Applied Physics, Aalto University, Konemiehentie 2, FI-02150
Espoo, Finland.
(5)Departments of Computer Science and Applied Physics, AaltoUniversity,
Konemiehentie 2, FI-02150 Espoo, Finlandombient AB, Nettovägen 6, SE-175 41
Järfälla, Sweden.

Polyadenylation is thought to be involved in the degradation and quality control 
of bacterial RNAs but relatively few examples have been investigated. We used a
combination of 5΄-tagRACE and RNA-seq to analyze the total RNA content from a
wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178
transcripts were either up- or down-regulated in the mutant when compared to the 
wild-type strain. Poly(A)polymerase up-regulates the expression of all genes
related to the FliA regulon and several previously unknown transcripts, including
numerous transporters. Notable down-regulation of genes in the expression of
antigen 43 and components of the type 1 fimbriae was detected. The major
consequence of the absence of poly(A)polymerase was the accumulation of numerous 
sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the 
processing of entire transcripts. A new algorithm to analyze the position and
composition of post-transcriptional modifications based on the sequence of
unencoded 3΄-ends, was developed to identify polyadenylated molecules. Overall
our results shed new light on the broad spectrum of action of polyadenylation on 
gene expression and demonstrate the importance of poly(A) dependent degradation
to remove structured RNA fragments.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkw894 
PMCID: PMC5389530
PMID: 28426097  [Indexed for MEDLINE]


60. PLoS Genet. 2017 Mar 16;13(3):e1006676. doi: 10.1371/journal.pgen.1006676.
eCollection 2017 Mar.

Global analysis of translation termination in E. coli.

Baggett NE(1), Zhang Y(1), Gross CA(1)(2)(3).

Author information: 
(1)Department of Microbiology and Immunology, University of California, San
Francisco, San Francisco, California, United States of America.
(2)Department of Cell and Tissue Biology, University of California, San
Francisco, San Francisco, California, United States of America.
(3)California Institute of Quantitative Biology, University of California, San
Francisco, San Francisco, California, United States of America.

Terminating protein translation accurately and efficiently is critical for both
protein fidelity and ribosome recycling for continued translation. The three
bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons
and terminate translation; and RF3 promotes disassociation of bound release
factors. Probing release factors mutations with reporter constructs containing
programmed frameshifting sequences or premature stop codons had revealed a
propensity for readthrough or frameshifting at these specific sites, but their
effects on translation genome-wide have not been examined. We performed ribosome 
profiling on a set of isogenic strains with well-characterized release factor
mutations to determine how they alter translation globally. Consistent with their
known defects, strains with increasingly severe release factor defects exhibit
increasingly severe accumulation of ribosomes over stop codons, indicative of an 
increased duration of the termination/release phase of translation. Release
factor mutant strains also exhibit increased occupancy in the region following
the stop codon at a significant number of genes. Our global analysis revealed
that, as expected, translation termination is generally efficient and accurate,
but that at a significant number of genes (≥ 50) the ribosome signature after the
stop codon is suggestive of translation past the stop codon. Even native E. coli 
K-12 exhibits the ribosome signature suggestive of protein extension, especially 
at UGA codons, which rely exclusively on the reduced function RF2 variant of the 
K-12 strain for termination. Deletion of RF3 increases the severity of the
defect. We unambiguously demonstrate readthrough and frameshifting protein
extensions and their further accumulation in mutant strains for a few select
cases. In addition to enhancing recoding, ribosome accumulation over stop codons 
disrupts attenuation control of biosynthetic operons, and may alter expression of
some overlapping genes. Together, these functional alterations may either augment
the protein repertoire or produce deleterious proteins.

DOI: 10.1371/journal.pgen.1006676 
PMCID: PMC5373646
PMID: 28301469  [Indexed for MEDLINE]


61. J Bacteriol. 2017 Mar 14;199(7). pii: e00755-16. doi: 10.1128/JB.00755-16. Print 
2017 Apr 1.

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli
K-12.

Wong GT(1), Bonocora RP(2), Schep AN(1), Beeler SM(1), Lee Fong AJ(1), Shull
LM(1), Batachari LE(1), Dillon M(1), Evans C(3), Becker CJ(1), Bush EC(1), Hardin
J(3), Wade JT(4)(5), Stoebel DM(6).

Author information: 
(1)Department of Biology, Harvey Mudd College, Claremont, California, USA.
(2)Wadsworth Center, New York State Department of Health, Albany, New York, USA.
(3)Department of Mathematics, Pomona College, Claremont, California, USA.
(4)Wadsworth Center, New York State Department of Health, Albany, New York, USA
joseph.wade@health.ny.gov stoebel@g.hmc.edu.
(5)Department of Biomedical Sciences, School of Public Health, University at
Albany, SUNY, Albany, New York, USA.
(6)Department of Biology, Harvey Mudd College, Claremont, California, USA
joseph.wade@health.ny.gov stoebel@g.hmc.edu.

The alternative sigma factor RpoS is a central regulator of many stress responses
in Escherichia coli The level of functional RpoS differs depending on the stress.
The effect of these differing concentrations of RpoS on global transcriptional
responses remains unclear. We investigated the effect of RpoS concentration on
the transcriptome during stationary phase in rich media. We found that 23% of
genes in the E. coli genome are regulated by RpoS, and we identified many
RpoS-transcribed genes and promoters. We observed three distinct classes of
response to RpoS by genes in the regulon: genes whose expression changes linearly
with increasing RpoS level, genes whose expression changes dramatically with the 
production of only a little RpoS ("sensitive" genes), and genes whose expression 
changes very little with the production of a little RpoS ("insensitive"). We show
that sequences outside the core promoter region determine whether an
RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive
and insensitive genes are enriched for specific functional classes and that the
sensitivity of a gene to RpoS corresponds to the timing of induction as cells
enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to
coordinate specific cellular processes with growth phase and may also contribute 
to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor
RpoS is a global regulator that controls the response to many stresses in
Escherichia coli Different stresses result in different levels of RpoS
production, but the consequences of this variation are unknown. We describe how
changing the level of RpoS does not influence all RpoS-regulated genes equally.
The cause of this variation is likely the action of transcription factors that
bind the promoters of the genes. We show that the sensitivity of a gene to RpoS
levels explains the timing of expression as cells enter stationary phase and that
genes with different RpoS sensitivities are enriched for specific functional
groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates
specific cellular processes in response to stresses.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/JB.00755-16 
PMCID: PMC5350281
PMID: 28115545  [Indexed for MEDLINE]


62. Sci Rep. 2017 Mar 10;7:44050. doi: 10.1038/srep44050.

Improved drought tolerance in wheat plants overexpressing a synthetic bacterial
cold shock protein gene SeCspA.

Yu TF(1), Xu ZS(1), Guo JK(2), Wang YX(2), Abernathy B(3), Fu JD(1), Chen X(1),
Zhou YB(1), Chen M(1), Ye XG(1), Ma YZ(1).

Author information: 
(1)Institute of Crop Science, Chinese Academy of Agricultural Sciences
(CAAS)/National Key Facility for Crop Gene Resources and Genetic Improvement, Key
Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of
Agriculture, Beijing 100081, China.
(2)Shijiazhuang Academy of Agricultural and Forestry Sciences, Research Center of
Wheat Engineering Technology of Hebei, Shijiazhuang, Hebei 050041, China.
(3)Center for Applied Genetic Technologies, Department of Plant Sciences,
University of Georgia, 30602, Athens, GA, United States.

Cold shock proteins (CSPs) enhance acclimatization of bacteria to adverse
environmental circumstances. The Escherichia coli CSP genes CspA and CspB were
modified to plant-preferred codon sequences and named as SeCspA and SeCspB.
Overexpression of exogenous SeCspA and SeCspB in transgenic Arabidopsis lines
increased germination rates, survival rates, and increased primary root length
compared to control plants under drought and salt stress. Investigation of
several stress-related parameters in SeCspA and SeCspB transgenic wheat lines
indicated that these lines possessed stress tolerance characteristics, including 
lower malondialdehyde (MDA) content, lower water loss rates, lower relative Na+
content, and higher chlorophyll content and proline content than the control
wheat plants under drought and salt stresses. RNA-seq and qRT-PCR expression
analysis showed that overexpression of SeCsp could enhance the expression of
stress-responsive genes. The field experiments showed that the SeCspA transgenic 
wheat lines had great increases in the 1000-grain weight and grain yield compared
to the control genotype under drought stress conditions. Significant differences 
in the stress indices revealed that the SeCspA transgenic wheat lines possessed
significant and stable improvements in drought tolerance over the control plants.
No such improvement was observed for the SeCspB transgenic lines under field
conditions. Our results indicated that SeCspA conferred drought tolerance and
improved physiological traits in wheat plants.

DOI: 10.1038/srep44050 
PMCID: PMC5345034
PMID: 28281578 


63. BMC Microbiol. 2017 Mar 8;17(1):56. doi: 10.1186/s12866-017-0966-x.

Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli
O157:H7 results in differential gene expression in relation to biofilm formation,
stress responses and metabolism.

Sharma VK(1), Bayles DO(2), Alt DP(2), Looft T(3), Brunelle BW(3), Stasko JA(4).

Author information: 
(1)Food Safety and Enteric Pathogens Research Unit, National Animal Disease
Center, ARS-USDA, P. O. Box 70, 1920 Dayton Avenue, Ames, IA, 50010, USA.
vijay.sharma@ars.usda.gov.
(2)Infectious Bacterial Diseases Research Unit, National Animal Disease Center,
ARS-USDA, Ames, IA, 50010, USA.
(3)Food Safety and Enteric Pathogens Research Unit, National Animal Disease
Center, ARS-USDA, P. O. Box 70, 1920 Dayton Avenue, Ames, IA, 50010, USA.
(4)Microscopy Services Unit, National Animal Disease Center, ARS-USDA, Ames, IA, 
50010, USA.

BACKGROUND: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986
disease outbreak, displays curli- and biofilm-negative phenotypes that are
correlated with the lack of Congo red (CR) binding and formation of white
colonies (CR-) on a CR-containing medium. However, on a CR medium this strain
produces red isolates (CR+) capable of producing curli fimbriae and biofilms.
RESULTS: To identify genes controlling differential expression of curli fimbriae 
and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the
CR- parental isolate. Of the 242 genes expressed differentially in the CR+
isolate, 201 genes encoded proteins of known functions while the remaining 41
encoded hypothetical proteins. Among the genes with known functions, 149 were
down- and 52 were up-regulated. Some of the upregulated genes were linked to
biofilm formation through biosynthesis of curli fimbriae and flagella. The genes 
encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD,
BssR and FlhDC, which modulate biofilm formation, were significantly altered in
their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, 
htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR
and pst) response pathways, and amino acid metabolism were downregulated in the
CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, 
which influence biofilm formation directly or indirectly, were also
down-regulated. Comparative genomics of CR+ and CR- isolates revealed the
presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The 
alignment of the amino acid sequences of RcsB of the two isolates showed
truncation of RcsB in the CR+ isolate at the insertion site of the duplicated
sequence. Complementation of CR+ isolate with rcsB of the CR- parent restored
parental phenotypes to the CR+ isolate.
CONCLUSIONS: The results of this study indicate that RcsB is a global regulator
affecting bacterial survival in growth-restrictive environments through
upregulation of genes promoting biofilm formation while downregulating certain
metabolic functions. Understanding whether rcsB inactivation enhances persistence
and survival of O157 in carrier animals and the environment would be important in
developing strategies for controlling this bacterial pathogen in these niches.

DOI: 10.1186/s12866-017-0966-x 
PMCID: PMC5343319
PMID: 28274217  [Indexed for MEDLINE]


64. Sci Rep. 2017 Mar 8;7:43641. doi: 10.1038/srep43641.

Broadly reactive aptamers targeting bacteria belonging to different genera using 
a sequential toggle cell-SELEX.

Song MY(1), Nguyen D(2), Hong SW(2)(3), Kim BC(1)(2).

Author information: 
(1)Center for Environment, Health and Welfare Research, Korea Institute of
Science and Technology (KIST), Seoul 02792, Republic of Korea.
(2)Department of Energy and Environmental Engineering, University of Science and 
Technology (UST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of
Korea.
(3)Center for Water Resources Cycle Research, Korea Institute of Science and
Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of
Korea.

Conventional cell-SELEX aims to isolate aptamers to a single unique target
bacteria species. We propose a method to isolate single-stranded DNA aptamers
that have broad reactivity to multiple bacterial targets belonging to different
genera. The key of the proposed method is that targets of interest are changed
sequentially at each SELEX round. The general scheme was examined using six
bacteria from different genera, Escherichia coli, Enterobacter aerogenes,
Klebsiella pneumoniae, Citrobacter freundii, Bacillus subtilis, and
Staphylococcus epidermidis (four gram-negative and two gram-positive bacteria).
In the first round of SELEX, the DNA library was incubated with E. coli and
amplicons bound to E. coli were separated. The amplicons were sequentially
separated by incubation with E. aerogenes, K. pneumoniae, C. freundii, B.
subtilis, and S. epidermidis at each SELEX. The amplicons obtained using the last
bacterial species were incubated again with the first bacterial species and this 
loop was repeated two more times. We refer to this method as sequential toggle
cell-SELEX (STC-SELEX). The isolated aptamers had dissociation constants of
9.22-38.5 nM and had no affinity to other bacteria that were not included in
STC-SELEX. These results demonstrate the potential to isolate aptamers with broad
affinity to bacterial taxa in different genera.

DOI: 10.1038/srep43641 
PMCID: PMC5341558
PMID: 28272554 


65. Sci Rep. 2017 Mar 7;7:43925. doi: 10.1038/srep43925.

SeqTU: A Web Server for Identification of Bacterial Transcription Units.

Chen X(1)(2)(3)(4), Chou WC(5), Ma Q(6)(7), Xu Y(1)(2)(3).

Author information: 
(1)College of Computer Science and Technology, Jilin University, Changchun,
Jilin, China.
(2)Computational Systems Biology Lab, Department of Biochemistry and Molecular
Biology, and Institute of Bioinformatics, University of Georgia, GA, USA.
(3)BioEnergy Science Center, USA.
(4)Center for Applied Mathematics, Tianjin University, Tianjin, China.
(5)Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
(6)Department of Agronomy, Horticulture and Plant Science, South Dakota State
University, Brookings, SD, 57007, USA.
(7)BioSNTR, Brookings, SD, USA.

A transcription unit (TU) consists of K ≥ 1consecutive genes on the same strand
of a bacterial genome that are transcribed into a single mRNA molecule under
certain conditions. Their identification is an essential step in elucidation of
transcriptional regulatory networks. We have recently developed a
machine-learning method to accurately identify TUs from RNA-seq data, based on
two features of the assembled RNA reads: the continuity and stability of RNA-seq 
coverage across a genomic region. While good performance was achieved by the
method on Escherichia coli and Clostridium thermocellum, substantial work is
needed to make the program generally applicable to all bacteria, knowing that the
program requires organism specific information. A web server, named SeqTU, was
developed to automatically identify TUs with given RNA-seq data of any bacterium 
using a machine-learning approach. The server consists of a number of utility
tools, in addition to TU identification, such as data preparation, data quality
check and RNA-read mapping. SeqTU provides a user-friendly interface and
automated prediction of TUs from given RNA-seq data. The predicted TUs are
displayed intuitively using HTML format along with a graphic visualization of the
prediction.

DOI: 10.1038/srep43925 
PMCID: PMC5339711
PMID: 28266571 


66. Arch Microbiol. 2017 Mar;199(2):203-213. doi: 10.1007/s00203-016-1291-8. Epub
2016 Sep 21.

Probiotic Enterococcus faecalis Symbioflor® down regulates virulence genes of
EHEC in vitro and decrease pathogenicity in a Caenorhabditis elegans model.

Neuhaus K(1), Lamparter MC(2)(3), Zölch B(1), Landstorfer R(1), Simon S(4),
Spanier B(5), Ehrmann MA(2), Vogel RF(6).

Author information: 
(1)Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Weihenstphaner Berg 3, 85350, Freising, Germany.
(2)Lehrstuhl für Technische Mikrobiologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Gregor-Mendel-Str. 4, 85354, Freising, Germany.
(3)Bundesintitut für Risikobewertung, Max-Dohrn-Str. 8-10, 10589, Berlin,
Germany.
(4)Datenanalyse und Visualisierung, Fachbereich Informatik und
Informationswissenschaft, Universität Konstanz, Universitätsstr. 10, 78457,
Constance, Germany.
(5)Lehrstuhl für Ernährungsphysiologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Gregor-Mendel-Str. 2, 85354, Freising, Germany.
(6)Lehrstuhl für Technische Mikrobiologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Gregor-Mendel-Str. 4, 85354, Freising, Germany.
rudi.vogel@wzw.tum.de.

Enterohemorrhagic E. coli O157:H7 (EHEC) shorten the lifespan of Caenorhabditis
elegans compared to avirulent bacteria. Co-feeding EHEC with Enterococcus
faecalis Symbioflor® significantly increased the worms' lifespan. The
transcriptome of EHEC grown in vitro with or without Symbioflor® was analyzed
using RNA-seq. The analysis revealed downregulation of several
virulence-associated genes in the presence of Symbioflor®, including virulence
key genes (e.g., LEE, flagellum, quorum-sensing). The downregulation of the LEE
genes was corroborated by lux-transposon mutants. Upregulated genes included acid
response genes, due to a decrease in pH exerted by Symbioflor®. Further genes
indicate cellular stress in EHEC (e.g. prophage/mobile elements involved in
excision, cell lysis, and cell division inhibition). Thus, the observed
protection of C. elegans during an EHEC infection by the probiotic Symbioflor® is
suggested to be caused by triggering concomitant transcriptomic changes. To
verify the biological relevance of this modulation, exemplary genes found to be
influenced by Symbioflor® were knocked out (fliD, espB, Z3136, Z3917, and L7052).
The lifespan of nematodes changed when using knock-outs as food source and the
effect could be complemented in trans. In summary, Symbioflor® appears to be a
protective probiotic in the nematode model.

DOI: 10.1007/s00203-016-1291-8 
PMID: 27655246  [Indexed for MEDLINE]


67. Org Biomol Chem. 2017 Mar 1;15(9):1980-1989. doi: 10.1039/c6ob02451c.

Whole cell-SELEX of aptamers with a tyrosine-like side chain against live
bacteria.

Renders M(1), Miller E(1), Lam CH(1), Perrin DM(1).

Author information: 
(1)Chemistry Department, University of British Columbia, 2036 Main Mall,
Vancouver, BC V6T 1Z1, Canada. dperrin@chem.ubc.ca.

In an effort to expand the binding and recognition capabilities of aptamers, a
nucleoside triphosphate modified with a phenol that mimics the side chain of
tyrosine was used in the selection of DNA aptamers against live bacteria. Of
multiple modified aptamers that were isolated against Escherichia coli DH5α
cells, one aptamer displays high selectivity and affinity for the target cells
and is greatly enriched for phenol-modified dU nucleotides (dUy, 47.5%). When the
same sequences are synthesized with TTP, no binding is observed. Taken together, 
these findings highlight the value of using modified nucleotide triphosphates in 
aptamer selections and portends success in SELEX against an array of whole cells 
as targets.

DOI: 10.1039/c6ob02451c 
PMID: 28009914  [Indexed for MEDLINE]


68. Zhongguo Zhong Yao Za Zhi. 2017 Mar;42(5):890-895. doi:
10.19540/j.cnki.cjcmm.20170217.005.

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene
from Andrographis paniculata].

[Article in Chinese]

Wang J(1)(2), Qi MD(3), Guo J(2), Shen Y(2), Lin HX(2), Huang LQ(2).

Author information: 
(1)College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038,
China.
(2)State Key Laboratory of Dao-di Herbs Breeding Base, National Resource Center
for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China.
(3)School of Traditional Chinese Medicine, Beijing University of Chinese
Medicine, Beijing 100029, China.

Andrographis paniculata is widely used as medicinal herb in China for a long time
and andrographolide is its main medicinal constituent. To investigate the
underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata
leaves with MeJA treatment was performed. In A. paniculata transcriptomic data,
the expression pattern of one member of NAC transcription factor family (ApNAC1) 
matched with andrographolide accumulation. The coding sequence of ApNAC1 was
cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of
bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a 
predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To
confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. 
paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized
protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene
predominantly expresses in leaves. Compared with control sample, its expression
abundance sharply increased with methyl jasmonate treatment. Based on its
expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis.
ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein
was purified by Ni-NTA agarose. Further study will help us to understand the
function of ApNAC1 in andrographolide biosynthesis.

Copyright© by the Chinese Pharmaceutical Association.


PMID: 28994531 

Conflict of interest statement: The authors of this article and the planning
committee members and staff have no relevant financial relationships with
commercial interests to disclose.


69. ACS Cent Sci. 2017 Feb 22;3(2):117-123. doi: 10.1021/acscentsci.6b00330. Epub
2017 Jan 25.

Two-Way Chemical Communication between Artificial and Natural Cells.

Lentini R(1), Martín NY(1), Forlin M(1), Belmonte L(1), Fontana J(1), Cornella
M(1), Martini L(1), Tamburini S(1), Bentley WE(2), Jousson O(1), Mansy SS(1).

Author information: 
(1)CIBIO, University of Trento , via Sommarive 9, 38123 Povo, Italy.
(2)Institute for Bioscience and Biotechnology Research, University of Maryland , 
College Park, Maryland 20742, United States.

Artificial cells capable of both sensing and sending chemical messages to
bacteria have yet to be built. Here we show that artificial cells that are able
to sense and synthesize quorum signaling molecules can chemically communicate
with V. fischeri, V. harveyi, E. coli, and P. aeruginosa. Activity was assessed
by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications
for this technology were demonstrated. First, the extent to which artificial
cells could imitate natural cells was quantified by a type of cellular Turing
test. Artificial cells capable of sensing and in response synthesizing and
releasing N-3-(oxohexanoyl)homoserine lactone showed a high degree of likeness to
natural V. fischeri under specific test conditions. Second, artificial cells that
sensed V. fischeri and in response degraded a quorum signaling molecule of P.
aeruginosa (N-(3-oxododecanoyl)homoserine lactone) were constructed, laying the
foundation for future technologies that control complex networks of natural
cells.

DOI: 10.1021/acscentsci.6b00330 
PMCID: PMC5324081
PMID: 28280778 


70. BMC Biotechnol. 2017 Feb 13;17(1):10. doi: 10.1186/s12896-017-0337-6.

A novel point mutation in RpoB improves osmotolerance and succinic acid
production in Escherichia coli.

Xiao M(1)(2)(3), Zhu X(1)(2), Xu H(1)(2), Tang J(1)(2), Liu R(1)(2), Bi C(1)(2), 
Fan F(4)(5), Zhang X(6)(7).

Author information: 
(1)Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin, China.
(2)Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of
Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308, China.
(3)University of Chinese Academy of Sciences, Beijing, China.
(4)Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin, China. fan_fy@tib.cas.cn.
(5)Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of
Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308, China.
fan_fy@tib.cas.cn.
(6)Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin, China. zhang_xl@tib.cas.cn.
(7)Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of
Sciences, 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308, China.
zhang_xl@tib.cas.cn.

BACKGROUND: Escherichia coli suffer from osmotic stress during succinic acid (SA)
production, which reduces the performance of this microbial factory.
RESULTS: Here, we report that a point mutation leading to a single amino acid
change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB)
significantly improved the osmotolerance of E. coli. Importation of the D654Y
mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA
production by more than 40% compared to that of the control under high glucose
osmolality. The transcriptome profile, determined by RNA-sequencing, showed two
distinct stress responses elicited by the mutated RpoB that counterbalanced the
osmotic stress. Under non-stressed conditions, genes involved in the synthesis
and transport of compatible solutes such as glycine-betaine, glutamate or proline
were upregulated even without osmotic stimulation, suggesting a "pre-defense"
mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions,
genes encoding diverse sugar transporters, which should be down-regulated in the 
presence of high osmotic pressure, were derepressed in the rpoB mutant.
Additional genetic experiments showed that enhancing the expression of the mal
regulon, especially for genes that encode the glycoporin LamB and maltose
transporter, contributed to the osmotolerance phenotype.
CONCLUSIONS: The D654Y single amino acid substitution in RpoB rendered E. coli
cells resistant to osmotic stress, probably due to improved cell growth and
viability via enhanced sugar uptake under stressed conditions, and activated a
potential "pre-defense" mechanism under non-stressed conditions. The findings of 
this work will be useful for bacterial host improvement to enhance its resistance
to osmotic stress and facilitate bio-based organic acids production.

DOI: 10.1186/s12896-017-0337-6 
PMCID: PMC5307762
PMID: 28193207  [Indexed for MEDLINE]


71. Mol Genet Genomics. 2017 Feb;292(1):231-242. doi: 10.1007/s00438-016-1269-x. Epub
2016 Nov 8.

GWAS analysis of QTL for enteric septicemia of catfish and their involved genes
suggest evolutionary conservation of a molecular mechanism of disease resistance.

Zhou T(1), Liu S(1), Geng X(1), Jin Y(1), Jiang C(1), Bao L(1), Yao J(1), Zhang
Y(1), Zhang J(1), Sun L(1), Wang X(1), Li N(1), Tan S(1), Liu Z(2).

Author information: 
(1)Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit,
School of Fisheries, Aquaculture and Aquatic Sciences and Program of Cell and
Molecular Biosciences, Auburn University, 202 Samford Hall, Auburn, AL,
36849-5112, USA.
(2)Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit,
School of Fisheries, Aquaculture and Aquatic Sciences and Program of Cell and
Molecular Biosciences, Auburn University, 202 Samford Hall, Auburn, AL,
36849-5112, USA. liuzhan@auburn.edu.

Disease problems cause major economic losses for the aquaculture industries. In
catfish, enteric septicemia of catfish (ESC), caused by the bacterial pathogen
Edwardsiella ictaluri, is the leading disease problem, causing tens of millions
of dollars of annual economic losses. In this study, we conducted a genome-wide
association study to determine quantitative trait loci (QTL) for resistance
against ESC using an interspecific hybrid system. Five hundred fish were used in 
the analysis and 192 phenotypic extremes were used for genotyping with the
catfish 250K SNP arrays. A genomic region on linkage group (LG) 1 was found
significantly associated with ESC disease resistance. In addition, two
suggestively associated QTL for ESC resistance were identified on LG 12 and
LG 16. The nlrc3 duplicates were identified within all the three QTL, suggesting 
their importance in association with the QTL. Within the significant QTL on LG 1,
16 genes with known functions in immunity were identified. Of particular interest
is the nck1 gene nearby the most significantly associated SNP. Nck1 was known to 
function as an adaptor to facilitating the pathogenesis of enteropathogenic
Escherichia coli (EPEC) in humans. E. ictaluri and EPEC pathogens belong to the
same bacterial family and share many common characteristics. The fact that nck1
is mapped in the QTL and that it was significantly upregulated in channel catfish
intestine after ESC challenge suggested its candidacy of being involved in
resistance/susceptibility of ESC.

DOI: 10.1007/s00438-016-1269-x 
PMID: 27826737  [Indexed for MEDLINE]


72. Vet Microbiol. 2017 Feb;199:23-30. doi: 10.1016/j.vetmic.2016.11.017. Epub 2016
Nov 23.

Carriage of antimicrobial resistant Escherichia coli in dogs: Prevalence,
associated risk factors and molecular characteristics.

Wedley AL(1), Dawson S(2), Maddox TW(3), Coyne KP(1), Pinchbeck GL(1), Clegg
P(4), Nuttall T(2), Kirchner M(5), Williams NJ(6).

Author information: 
(1)Department of Epidemiology and Population Health, Institute of Infection and
Global Health, Leahurst Campus, University of Liverpool, CH64 7TE, UK.
(2)School of Veterinary Science, Leahurst Campus, University of Liverpool, CH64
7TE, UK.
(3)Small Animal Teaching Hospital, Leahurst Campus, University of Liverpool, CH64
7TE, UK.
(4)Department of Musculoskeletal Biology, Institute of Ageing and Chronic
Disease, Leahurst Campus, University of Liverpool, CH64 7TE, UK.
(5)Animal and Plant Health Agency, Department of Bacteriology, Woodham Lane, New 
Haw, Surrey, KT15 3NB, UK.
(6)Department of Epidemiology and Population Health, Institute of Infection and
Global Health, Leahurst Campus, University of Liverpool, CH64 7TE, UK. Electronic
address: njwillms@liverpool.ac.uk.

Resistance to antimicrobials, in particular that mediated by extended spectrum
β-lactamases (ESBL) and AmpC β-lactamases are frequently reported in bacteria
causing canine disease as well as in commensal bacteria, which could be a
potential health risk for humans they come into contact with. This
cross-sectional study aimed to estimate the prevalence and investigate the
molecular characteristics of ESBL and plasmid encoded AmpC (pAmpC)-producing E.
coli in the mainland UK vet-visiting canine population and, using responses from 
detailed questionnaires identify factors associated with their carriage. Faecal
samples were cultured for antimicrobial resistant (AMR), ESBL and pAmpC-producing
E. coli. A subset of ESBL and pAmpC-producing isolates were subjected to
multi-locus sequence typing and DNA microarray analyses. Multivariable logistic
regression analysis was used to construct models to identify risk factors
associated with multidrug resistant (MDR, resistance to three or more
antimicrobial classes), fluoroquinolone resistant, ESBL and AmpC-producing E.
coli. AMR E.coli were isolated from 44.8% (n=260) of samples, with 1.9% and 7.1% 
of samples carrying ESBL and pAmpC-producing E. coli, respectively. MDR E. coli
were identified in 18.3% of samples. Recent use of antimicrobials and being fed
raw poultry were both identified as risk factors in the outcomes investigated. A 
number of virulence and resistance genes were identified, including genes
associated with extra-intestinal and enteropathogenic E. coli genotypes.
Considering the close contact that people have with dogs, the high levels of AMR 
E. coli in canine faeces may be a potential reservoir of AMR bacteria or
resistance determinants.

Copyright © 2016. Published by Elsevier B.V.

DOI: 10.1016/j.vetmic.2016.11.017 
PMID: 28110781  [Indexed for MEDLINE]


73. Bioinformation. 2017 Jan 31;13(1):25-30. doi: 10.6026/97320630013025. eCollection
2017.

Elucidation of the sequential transcriptional activity in Escherichia coli using 
time-series RNA-seq data.

Wong PS(1), Tashiro K(2), Kuhara S(2), Aburatani S(1)(3).

Author information: 
(1)Biotechnology Research Institute for Drug Discovery, National Institute of
AIST, Tokyo, Japan.
(2)Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu
University, Fukuoka, Japan.
(3)Com. Bio Big Data Open Innovation Lab. (CBBD-OIL), National Institute of AIST,
Tokyo, Japan.

Functional genomics and gene regulation inference has readily expanded our
knowledge and understanding of gene interactions with regards to expression
regulation. With the advancement of transcriptome sequencing in time-series comes
the ability to study the sequential changes of the transcriptome. Here, we
present a new method to augment regulation networks accumulated in literature
with transcriptome data gathered from time-series experiments to construct a
sequential representation of transcription factor activity. We apply our method
on a time-series RNA-Seq data set of Escherichia coli as it transitions from
growth to stationary phase over five hours and investigate the various activity
in gene regulation process by taking advantage of the correlation between
regulatory gene pairs to examine their activity on a dynamic network. We analyse 
the changes in metabolic activity of the pagP gene and associated transcription
factors during phase transition, and visualize the sequential transcriptional
activity to describe the change in metabolic pathway activity originating from
the pagP transcription factor, phoP. We observe a shift from amino acid and
nucleic acid metabolism, to energy metabolism during the transition to stationary
phase in E. coli.

DOI: 10.6026/97320630013025 
PMCID: PMC5405090
PMID: 28479747 


74. Sci Rep. 2017 Jan 17;7:40599. doi: 10.1038/srep40599.

Differential Effects of Vitamins A and D on the Transcriptional Landscape of
Human Monocytes during Infection.

Klassert TE(1), Bräuer J(1), Hölzer M(2), Stock M(1), Riege K(2), Zubiría-Barrera
C(1), Müller MM(1)(3), Rummler S(4), Skerka C(5), Marz M(2), Slevogt H(1).

Author information: 
(1)Jena University Hospital, Septomics Research Center, Jena, 07745, Germany.
(2)Friedrich Schiller University, Bioinformatics/High Throughput Analysis, Jena, 
07743, Germany.
(3)Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger 
Allee 101, Jena, 07747, Germany.
(4)Jena University Hospital, Institute of Transfusion Medicine, Jena, 07747,
Germany.
(5)Leibniz Institute for Natural Product Research and Infection Biology, Hans
Knöll Institute, Department of Infection Biology, Jena, 07745, Germany.

Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic 
effects in humans. Beyond their well-documented roles in cellular
differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, 
both vitamins have attracted considerable attention due to their association
with-immunological traits. Nevertheless, our knowledge of their immunomodulatory 
potential during infection is restricted to single gene-centric studies, which do
not reflect the complexity of immune processes. In the present study, we
performed a comprehensive RNA-seq-based approach to define the whole
immunomodulatory role of vitamins A and D during infection. Using human monocytes
as host cells, we characterized the differential role of both vitamins upon
infection with three different pathogens: Aspergillus fumigatus, Candida albicans
and Escherichia coli. Both vitamins showed an unexpected ability to counteract
the pathogen-induced transcriptional responses. Upon infection, we identified 346
and 176 immune-relevant genes that were regulated by atRA and vitD, respectively.
This immunomodulatory activity was dependent on the inflammatory stimulus,
allowing us to distinguish regulatory patterns which were specific for each
stimulatory setting. Moreover, we explored possible direct and indirect
mechanisms of vitamin-mediated regulation of the immune response. Our findings
highlight the importance of vitamin-monitoring in critically ill patients.
Moreover, our results underpin the potential of atRA and vitD as therapeutic
options for anti-inflammatory treatment.

DOI: 10.1038/srep40599 
PMCID: PMC5240108
PMID: 28094291 


75. Mol Syst Biol. 2017 Jan 16;13(1):907. doi: 10.15252/msb.20167150.

Genomewide landscape of gene-metabolome associations in Escherichia coli.

Fuhrer T(1), Zampieri M(1), Sévin DC(1), Sauer U(2), Zamboni N(1).

Author information: 
(1)Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
(2)Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
sauer@imsb.biol.ethz.ch.

Metabolism is one of the best-understood cellular processes whose network
topology of enzymatic reactions is determined by an organism's genome. The
influence of genes on metabolite levels, however, remains largely unknown,
particularly for the many genes encoding non-enzymatic proteins. Serendipitously,
genomewide association studies explore the relationship between genetic variants 
and metabolite levels, but a comprehensive interaction network has remained
elusive even for the simplest single-celled organisms. Here, we systematically
mapped the association between > 3,800 single-gene deletions in the bacterium
Escherichia coli and relative concentrations of > 7,000 intracellular metabolite 
ions. Beyond expected metabolic changes in the proximity to abolished enzyme
activities, the association map reveals a largely unknown landscape of
gene-metabolite interactions that are not represented in metabolic models.
Therefore, the map provides a unique resource for assessing the genetic basis of 
metabolic changes and conversely hypothesizing metabolic consequences of genetic 
alterations. We illustrate this by predicting metabolism-related functions of 72 
so far not annotated genes and by identifying key genes mediating the cellular
response to environmental perturbations.

© 2017 The Authors. Published under the terms of the CC BY 4.0 license.


PMCID: PMC5293155
PMID: 28093455  [Indexed for MEDLINE]


76. Methods Mol Biol. 2017;1520:263-279. doi: 10.1007/978-1-4939-6634-9_16.

Expression Profiling of Antibiotic-Resistant Bacteria Obtained by Laboratory
Evolution.

Suzuki S(1), Horinouchi T(1), Furusawa C(2).

Author information: 
(1)Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka, 565-0874,
Japan.
(2)Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka, 565-0874,
Japan. chikara.furusawa@riken.jp.

To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and 
genotypic features in resistant strains is important. Here, we describe the
expression profiling of antibiotic-resistant Escherichia coli strains obtained by
laboratory evolution, and a method for extracting a small number of genes whose
expression changes can contribute to the acquisition of resistance.

DOI: 10.1007/978-1-4939-6634-9_16 
PMID: 27873258  [Indexed for MEDLINE]


77. PLoS One. 2016 Dec 22;11(12):e0163057. doi: 10.1371/journal.pone.0163057.
eCollection 2016.

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA
Composite Operon in Escherichia coli.

Takada H(1), Shimada T(1)(2), Dey D(3), Quyyum MZ(3), Nakano M(4), Ishiguro A(1),
Yoshida H(5), Yamamoto K(1)(6), Sen R(3), Ishihama A(1)(6).

Author information: 
(1)Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo,
Japan.
(2)Laboratory for Chemistry and Life Science, Tokyo Institute of Technology,
Nagatsuda, Yokohama, Japan.
(3)Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India.
(4)Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan.
(5)Department of Physics, Osaka Medical College, Takatsuki, Osaka, Japan.
(6)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

Escherichia coli contains seven rRNA operons, each consisting of the genes for
three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the
spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3'
proximal region. All of these rRNA and tRNA genes are transcribed from two
promoters, P1 and P2, into single large precursors that are afterward processed
to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX 
screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing 
RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of 
all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an
internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA
operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM
observation. Using this riRNA promoter within the rrnD operon as a
representative, transcription in vitro was detected with use of the purified RpoD
holoenzyme, confirming the presence of a constitutive promoter in this region.
LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The
location of riRNA promoter in vivo as identified using a set of reporter plasmids
agrees well with that identified in vitro. Based on transcription profile in
vitro and Northern blot analysis in vivo, the majority of transcript initiated
from this riRNA promoter was estimated to terminate near the beginning of 23S
rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under
starved conditions, transcription of the rRNA operon is markedly repressed to
reduce the intracellular level of ribosomes, but the levels of both riRNA and its
processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the
continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We
then propose that the tRNA genes organized within the spacers of rRNA-tRNA
composite operons are expressed independent of rRNA synthesis under specific
conditions where further synthesis of ribosomes is not needed.

DOI: 10.1371/journal.pone.0163057 
PMCID: PMC5179076
PMID: 28005933  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no competing
interests exist.


78. BMC Genomics. 2016 Dec 21;17(1):1049. doi: 10.1186/s12864-016-3374-y.

Potential immunosuppressive effects of Escherichia coli O157:H7 experimental
infection on the bovine host.

Kieckens E(1)(2), Rybarczyk J(3)(4), Li RW(5), Vanrompay D(4), Cox E(3).

Author information: 
(1)Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, 9820, Merelbeke, Belgium. evelien.kieckens@ugent.be.
(2)Laboratory of Immunology and Animal Biotechnology, Faculty of Bioscience
Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
evelien.kieckens@ugent.be.
(3)Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, 9820, Merelbeke, Belgium.
(4)Laboratory of Immunology and Animal Biotechnology, Faculty of Bioscience
Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
(5)USDA-ARS, Bovine Functional Genomics Laboratory, Beltsville, MD, USA.

BACKGROUND: Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are 
frequently detected in bovine faecal samples at slaughter. Cattle do not show
clinical symptoms upon infection, but for humans the consequences after consuming
contaminated beef can be severe. The immune response against EHEC in cattle
cannot always clear the infection as persistent colonization and shedding in
infected animals over a period of months often occurs. In previous infection
trials, we observed a primary immune response after infection which was unable to
protect cattle from re-infection. These results may reflect a suppression of
certain immune pathways, making cattle more prone to persistent colonization
after re-infection. To test this, RNA-Seq was used for transcriptome analysis of 
recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian
calves in response to a primary and secondary Escherichia coli O157:H7 infection 
with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served 
as controls.
RESULTS: In tissue of the recto-anal junction, only 15 genes were found to be
significantly affected by a first infection compared to 1159 genes in the ileal
Peyer's patches. Whereas, re-infection significantly changed the expression of 10
and 17 genes in the recto-anal junction tissue and the Peyer's patches,
respectively. A significant downregulation of 69 immunostimulatory genes and a
significant upregulation of seven immune suppressing genes was observed.
CONCLUSIONS: Although the recto-anal junction is a major site of colonization,
this area does not seem to be modulated upon infection to the same extent as
ileal Peyer's patches as the changes in gene expression were remarkably higher in
the ileal Peyer's patches than in the recto-anal junction during a primary but
not a secondary infection. We can conclude that the main effect on the
transcriptome was immunosuppression by E. coli O157:H7 (Stx-) due to an
upregulation of immune suppressive effects (7/12 genes) or a downregulation of
immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data 
might indicate that a primary infection promotes a re-infection with EHEC by
suppressing the immune function.

DOI: 10.1186/s12864-016-3374-y 
PMCID: PMC5178093
PMID: 28003017  [Indexed for MEDLINE]


79. Mol Cells. 2016 Nov 30;39(11):807-813. Epub 2016 Nov 18.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its
Use in Various Detection Formats.

Dua P(1), Ren S(2), Lee SW(2), Kim JK(1), Shin HS(1), Jeong OC(3), Kim S(2), Lee 
DK(1).

Author information: 
(1)Global Research Laboratory (GRL) for RNAi Medicine, Department of Chemistry,
Sungkyunkwan University (SKKU), Suwon 16419, Korea.
(2)Department of Bioengineering, Dongguk University, Seoul 04620, Korea.
(3)Department of Biomedical Engineering and School of Mechanical Engineering,
Inje University, Gimhae 50834, Korea.

Escherichia coli are important indicator organisms, used routinely for the
monitoring of water and food safety. For quick, sensitive and real-time detection
of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31
nucleotide truncated Ec3 demonstrated improved binding and low nano-molar
affinity to E. coli. The aptamer developed by us out-performs the commercial
antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli
detection was done using three different detection formats and the assay
sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic 
separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml.
Although, optical analytic technique, biolayer interferometry, did not improve
the sensitivity of detection for whole cells, a very significant improvement in
the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we 
developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor 
that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any
labeling and signal amplification techniques. We believe that our developed
method can step towards more complex and real sample application.

DOI: 10.14348/molcells.2016.0167 
PMCID: PMC5125936
PMID: 27871171  [Indexed for MEDLINE]


80. Sci Rep. 2016 Nov 30;6:38248. doi: 10.1038/srep38248.

Complete genome sequence and transcriptomic analysis of a novel marine strain
Bacillus weihaiensis reveals the mechanism of brown algae degradation.

Zhu Y(1), Chen P(1), Bao Y(1), Men Y(1), Zeng Y(1), Yang J(1), Sun J(1), Sun
Y(1).

Author information: 
(1)National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.

A novel marine strain representing efficient degradation ability toward brown
algae was isolated, identified, and assigned to Bacillus weihaiensis Alg07. The
alga-associated marine bacteria promote the nutrient cycle and perform important 
functions in the marine ecosystem. The de novo sequencing of the B. weihaiensis
Alg07 genome was carried out. Results of gene annotation and carbohydrate-active 
enzyme analysis showed that the strain harbored enzymes that can completely
degrade alginate and laminarin, which are the specific polysaccharides of brown
algae. We also found genes for the utilization of mannitol, the major storage
monosaccharide in the cell of brown algae. To understand the process of brown
algae decomposition by B. weihaiensis Alg07, RNA-seq transcriptome analysis and
qRT-PCR were performed. The genes involved in alginate metabolism were all
up-regulated in the initial stage of kelp degradation, suggesting that the strain
Alg07 first degrades alginate to destruct the cell wall so that the laminarin and
mannitol are released and subsequently decomposed. The key genes involved in
alginate and laminarin degradation were expressed in Escherichia coli and
characterized. Overall, the model of brown algae degradation by the marine strain
Alg07 was established, and novel alginate lyases and laminarinase were
discovered.

DOI: 10.1038/srep38248 
PMCID: PMC5128808
PMID: 27901120 


81. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2016 Nov 9;51(13):1100-6.
doi: 10.1080/10934529.2016.1199650. Epub 2016 Jul 15.

Detection of pepper mild mottle virus as an indicator for drinking water quality 
in Hanoi, Vietnam, in large volume of water after household treatment.

Sangsanont J(1), The Dan D(2), Thi Viet Nga T(3), Katayama H(2), Furumai H(4).

Author information: 
(1)a Graduate Program in Sustainability Science-Global Leadership Initiative ,
Graduate School of Frontier Sciences, The University of Tokyo , Chiba , Japan.
(2)b Department of Urban Engineering , The University of Tokyo , Tokyo , Japan.
(3)c Department of Environmental Engineering , National University of Civil
Engineering (NUCE) , Hanoi , Vietnam.
(4)d Research Center for Water Environment Technology , The University of Tokyo ,
Tokyo , Japan.

The aims of this study were to examine the removal of bacteria and viruses by
household point-of-use (POU) treatments and to apply a previously developed
large-volume virus concentration method (∼20 L). First, the removal of microbes
by household POU treatment was investigated in the laboratory. Second, the
prevalence of viruses in drinking water sources for households and the removal
efficiency of microbes by POU treatments in two suburban communities in Hanoi,
Vietnam, were investigated. Indigenous pepper mild mottle virus (PMMoV) was used 
as the main target together with adenovirus, Aichi virus, enterovirus, F-specific
bacteriophage genogroup 1, and Escherichia coli to investigate the removal
efficiency of household treatments. The results from laboratory and field survey 
were compared. From the laboratory study, ceramic membranes were not effective
for removing viruses and bacteria from water; pathogen reduction was less than
1.5 log10. By contrast, reverse osmosis (RO) devices reduced microbes by 3 to >
5 log10. In a field study, PMMoV was found to be the most prevalent waterborne
virus. Household sand filtration was ineffective for removing E. coli, total
coliforms and PMMoV; the reduction was less than 1 order of magnitude. Boiling
the water and then filtering it with a ceramic membrane reduced E. coli by 3
orders of magnitude, but this was not effective for removing PMMoV. RO filtration
was one of the promising methods for removing E. coli, total coliforms and PMMoV 
to below their detection limits in most of the samples studied. The removal of E.
coli, total coliforms and PMMoV was >2.3, >4 and >3 log10, respectively. The
laboratory results of virus removal efficiency by POU devices agreed with the
field study. Due to the prevalence and characteristics of PMMoV, it is a strong
candidate for an indigenous indicator to investigate the viral removal efficiency
of household POU treatments.

DOI: 10.1080/10934529.2016.1199650 
PMID: 27419274  [Indexed for MEDLINE]


82. Bioengineered. 2016 Nov;7(6):459-477. doi: 10.1080/21655979.2016.1208868. Epub
2016 Jul 26.

Agroinfiltration contributes to VP1 recombinant protein degradation.

Pillay P(1), Kunert KJ(1), van Wyk S(1), Makgopa ME(1), Cullis CA(2), Vorster
BJ(1).

Author information: 
(1)a Department of Plant and Soil Sciences , Forestry and Agricultural
Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria ,
South Africa.
(2)b Department of Biology , Case Western Reserve University , Cleveland , OH ,
USA.

There is a growing interest in applying tobacco agroinfiltration for recombinant 
protein production in a plant based system. However, in such a system, the action
of proteases might compromise recombinant protein production. Protease
sensitivity of model recombinant foot-and-mouth disease (FMD) virus
P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli
glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely 
degraded when treated with the serine protease trypsin than when treated with the
cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic
activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1
was degraded when incubated with such a protease-containing tobacco extract. In
silico analysis revealed potential protease cleavage sites within the P1, VP1 and
GOR sequences. The interaction modeling of the single VP1 protein with the
proteases papain and trypsin showed greater proximity to proteolytic active sites
compared to modeling with the entire P1-polyprotein fusion complex. Several plant
transcripts with differential expression were detected 24 hr
post-agroinfiltration when the RNA-seq technology was applied to identify changed
protease transcripts using the recently available tobacco draft genome. Three
candidate genes were identified coding for proteases which included the
Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing
enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested
recombinant proteins are sensitive to protease action and agroinfiltration
induces the expression of potential proteases that can compromise recombinant
protein production.

DOI: 10.1080/21655979.2016.1208868 
PMCID: PMC5094629
PMID: 27459147  [Indexed for MEDLINE]


83. Curr Genet. 2016 Nov;62(4):897-910. Epub 2016 Apr 1.

Development and validation of a custom microarray for global transcriptome
profiling of the fungus Aspergillus nidulans.

Deloménie C(1), Grentzmann G(2), Oestreicher N(3)(4), Mesnage R(5)(6), Vélot
C(3)(4)(6).

Author information: 
(1)UMS IPSIT, Plateforme Trans-Prot, Faculté de Pharmacie, Univ. Paris-Sud,
Université Paris-Saclay, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry,
France. claudine.delomenie@u-psud.fr.
(2)PharmaBioServices, 1 square du Cèdre, 91370, Verrières-Le-Buisson, France.
(3)Laboratoire VEAC, Faculté des Sciences, Univ. Paris-Sud, Université
Paris-Saclay, Bât. 360, Rue du Doyen André Guinier, 91405, Orsay, France.
(4)Pôle Risques MRSH-CNRS, Université de Caen, Esplanade de la Paix, 14032, Caen,
France.
(5)Gene Expression and Therapy Group, Department of Medical and Molecular
Genetics, Faculty of Life Sciences and Medicine, King's College London, 8th
Floor, Tower Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK.
(6)CRIIGEN, 81 rue Monceau, 75008, Paris, France.

Transcriptome profiling is a powerful tool for identifying gene networks from
whole genome expression analysis in many living species. Here is described the
first extensively characterized platform using Agilent microarray technology for 
transcriptome analysis in the filamentous fungus Aspergillus (Emericella)
nidulans. We developed and validated a reliable gene expression microarray in
8 × 15 K format, with predictive and experimental data establishing its
specificity and sensitivity. Either one or two 60-mer oligonucleotide probes were
selected for each of 10,550 nuclear as well as 20 mitochondrial coding sequences.
More than 99 % of probes were predicted to hybridize with 100 % identity to their
aimed specific A. nidulans target only. Probe sensitivity was supported by a
highly narrow distribution of melting temperatures together with thermodynamic
features, which strongly favored probe-target perfect match hybridization, in
comparison with predicted secondary structures. Array quality was evaluated
through transcriptome comparison of two A. nidulans strains, differing by the
presence or not of Escherichia coli LacZ transgene. High signal-to-noise ratios
were measured, and signal reproducibility was established at intra-probe and
inter-probe levels. Reproducibility of microarray performances was assessed by
high correlation between two-color dye signals and between technical replicates. 
Results were confirmed by RT-qPCR analysis on five genes. Though it covers 100 % 
of the A. nidulans targeted coding sequences, this low density array allows
limited experimental costs and simplified data analysis process, making it
suitable for studying gene expression in this model organism through large
numbers of experimental conditions, in basic, biomedical or industrial
microbiology research fields.

DOI: 10.1007/s00294-016-0597-z 
PMID: 27038308  [Indexed for MEDLINE]


84. Microb Pathog. 2016 Nov;100:1-9. doi: 10.1016/j.micpath.2016.08.032. Epub 2016
Aug 26.

DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia
coli O2 strain E058 during its infection of chicken.

Gao Q(1), Xia L(2), Liu J(3), Wang X(4), Gao S(5), Liu X(6).

Author information: 
(1)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
gq00888@sina.com.
(2)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
1406480277@qq.com.
(3)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
1473668159@qq.com.
(4)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
wangxb@yzu.edu.cn.
(5)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
gsong@yzu.edu.cn.
(6)Animal Infectious Disease Laboratory, Ministry of Agriculture, Jiangsu
Co-innovation Center for the Prevention and Control of Important Animal
Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, PR China. Electronic address:
xfliu@yzu.edu.cn.

Avian pathogenic Escherichia coli (APEC) cause typical extraintestinal infections
in poultry, including acute fatal septicemia, subacute pericarditis, and
airsacculitis. These bacteria most often infect chickens, turkeys, ducks, and
other avian species, and therefore pose a significant economic burden on the
poultry industry worldwide. Few studies have analyzed the genome-wide
transcriptional profile of APEC during infection in vivo. In this study, we
examined the genome-wide transcriptional response of APEC O2 strain E058 in an
in vivo chicken infection model to better understand the factors necessary for
APEC colonization, growth, and survival in vivo. An Affymetrix multigenome DNA
microarray, which contains most of the genomic open reading frames of E. coli
K-12 strain MG1655, uropathogenic E. coli strain CFT073, and E. coli O157:H7
strain EDL 933, was used to profile the gene expression in APEC E058. We
identified the in vivo transcriptional response of APEC E058 bacteria collected
directly from the blood of infected chickens. Significant differences in
expression levels were detected between the in vivo expression profile and the
in vitro expression profile in LB medium. The genes highly expressed during
infection were involved in metabolism, iron acquisition or transport, virulence, 
response to stress, and biological regulation. The reliability of the microarray 
data was confirmed by performing quantitative real-time PCR on 12 representative 
genes. Moreover, several significantly upregulated genes, including yjiY, sodA,
phoB and spy, were selected to study their role in APEC pathogenesis. The data
will help to better understand the mechanisms of APEC pathogenesis.

Copyright © 2016 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.micpath.2016.08.032 
PMID: 27569534  [Indexed for MEDLINE]


85. Plant Cell Rep. 2016 Nov;35(11):2403-2421. Epub 2016 Sep 3.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose
synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum
caudatum Ait.

Yin S(1), Kong JQ(2).

Author information: 
(1)State Key Laboratory of Bioactive Substance and Function of Natural Medicines 
and Ministry of Health Key Laboratory of Biosynthesis of Natural Products,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China.
(2)State Key Laboratory of Bioactive Substance and Function of Natural Medicines 
and Ministry of Health Key Laboratory of Biosynthesis of Natural Products,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China. jianqiangk@imm.ac.cn.

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and
OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose
synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of
converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer
xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum
caudatum Ait. However, the cDNA isolation and functional characterization of
genes encoding the two enzymes from O. caudatum has never been documented.
Previously, the transcriptome sequencing of O. caudatum was performed in our
laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS 
were first retrieved based on RNA-Seq data. The eight putative genes were then
successfully isolated from transcriptome of O. caudatum by reverse transcription 
polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six
putative UXS isoforms can be classified into three types, one soluble and two
distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were
predicted to be soluble forms. Subsequently, these candidate cDNAs were
characterized to be bona fide genes by functional expression in Escherichia coli 
individually. Although UXS and UAXS catalyzed the same reaction, their
biochemical properties varied significantly. It is worth noting that a ratio
switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to
be helpful for its biotechnological application. Furthermore, a series of mutants
were generated to test the function of NAD+ binding motif GxxGxxG. Most
importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3
in xylose-containing polysaccharides biosynthesis in O. caudatum. These data
provide a comprehensive knowledge for UXS and UAXS families in plants.

DOI: 10.1007/s00299-016-2044-5 
PMID: 27591771  [Indexed for MEDLINE]


86. Oncotarget. 2016 Oct 25;7(43):70685-70698. doi: 10.18632/oncotarget.12206.

Pseudoexons provide a mechanism for allele-specific expression of APC in familial
adenomatous polyposis.

Nieminen TT(1), Pavicic W(1)(2), Porkka N(1), Kankainen M(3), Järvinen HJ(4),
Lepistö A(5), Peltomäki P(1).

Author information: 
(1)University of Helsinki, Medical and Clinical Genetics, Helsinki, Finland.
(2)Laboratorio de Citogenética y Mutagénesis, Instituto Multidisciplinario de
Biología Celular (IMBICE-CONICET-CICPBA), La Plata, Argentina.
(3)University of Helsinki, Institute for Molecular Medicine Finland, Helsinki,
Finland.
(4)Second Department of Surgery, Helsinki University Central Hospital, Helsinki, 
Finland.
(5)Department of Colorectal Surgery, Abdominal Center, Helsinki University
Hospital, Helsinki, Finland.

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene
occurs in up to one-third of families with adenomatous polyposis (FAP) that have 
screened mutation-negative by conventional techniques. To advance our
understanding of the genomic basis of this phenomenon, 54 APC mutation-negative
families (21 with classical FAP and 33 with attenuated FAP, AFAP) were
investigated. We focused on four families with validated ASE and scrutinized
these families by sequencing of the blood transcriptomes (RNA-seq) and genomes
(WGS). Three families, two with classical FAP and one with AFAP, revealed deep
intronic mutations associated with pseudoexons. In all three families, intronic
mutations (c.646-1806T>G in intron 6, c.1408+729A>G in intron 11, and
c.1408+731C>T in intron 11) created new splice donor sites resulting in the
insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in
the APC transcript. The respective intronic mutations were absent in the
remaining polyposis families and the general population. Premature stop of
translation as the predicted consequence as well as co-segregation with polyposis
supported the pathogenicity of the pseudoexons. We conclude that next generation 
sequencing on RNA and genomic DNA is an effective strategy to reveal and validate
pseudoexons that are regularly missed by traditional screening methods and is
worth considering in apparent mutation-negative polyposis families.

DOI: 10.18632/oncotarget.12206 
PMCID: PMC5342583
PMID: 27683109  [Indexed for MEDLINE]


87. Animal. 2016 Oct;10(10):1602-8. doi: 10.1017/S1751731116000306. Epub 2016 Mar 3.

A genome-wide association analysis for susceptibility of pigs to enterotoxigenic 
Escherichia coli F41.

Ji HY(1), Yang B(1), Zhang ZY(1), Ouyang J(1), Yang M(1), Zhang XF(1), Zhang
WC(1), Su Y(1), Zhao KW(1), Xiao SJ(1), Yan XM(1), Ren J(1), Huang LS(1).

Author information: 
(1)1State Key Laboratory for Pig Genetic Improvement and Production
Science,Jiangxi Agricultural University,330045 Nanchang,China.

Enterotoxigenic Escherichia coli (ETEC) is a type of pathogenic bacteria that
cause diarrhea in piglets through colonizing pig small intestine epithelial cells
by their surface fimbriae. Different fimbriae type of ETEC including F4, F18, K99
and F41 have been isolated from diarrheal pigs. In this study, we performed a
genome-wide association study to map the loci associated with the susceptibility 
of pigs to ETEC F41 using 39454 single nucleotide polymorphisms (SNPs) in 667 F2 
pigs from a White Duroc×Erhualian F2 cross. The most significant SNP
(ALGA0022658, P=5.59×10-13) located at 6.95 Mb on chromosome 4. ALGA0022658 was
in high linkage disequilibrium (r 2>0.5) with surrounding SNPs that span a 1.21
Mb interval. Within this 1.21 Mb region, we investigated ZFAT as a positional
candidate gene. We re-sequenced cDNA of ZFAT in four pigs with different
susceptibility phenotypes, and identified seven coding variants. We genotyped
these seven variants in 287 unrelated pigs from 15 diverse breeds that were
measured with ETEC F41 susceptibility phenotype. Five variants showed nominal
significant association (P<0.05) with ETEC F41 susceptibility phenotype in
International commercial pigs. This study provided refined region associated with
susceptibility of pigs to ETEC F41 than that reported previously. Further works
are needed to uncover the underlying causal mutation(s).

DOI: 10.1017/S1751731116000306 
PMID: 26936422  [Indexed for MEDLINE]


88. Appl Environ Microbiol. 2016 Sep 23. pii: AEM.02479-16. [Epub ahead of print]

Two outer membrane proteins contribute to cellular fitness in Caulobacter
crescentus by preventing intracellular S-layer protein accumulation.

Overton KW(1), Park DM(1), Yung MC(1), Dohnalkova AC(2), Smit J(3), Jiao Y(4).

Author information: 
(1)Biosciences and Biotechnology Division, Physical and Life Sciences
Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
(2)Environmental Molecular Sciences Laboratory, Pacific Northwest National
Laboratory, Richland, Washington, USA.
(3)Department of Microbiology and Immunology, University of British Columbia,
Vancouver, British Columbia, Canada.
(4)Biosciences and Biotechnology Division, Physical and Life Sciences
Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA
jiao1@llnl.gov.

Surface layers, or S-layers, are two-dimensional protein arrays that form the
outermost layer of many bacteria and archaea. They serve several functions
including physical protection of the cell from environmental threats. The high
abundance of S-layer proteins necessitates a highly efficient export mechanism to
transport S-layer protein from the cytoplasm to the cell exterior. Caulobacter
crescentus is unique in that it has two homologous, seemingly redundant outer
membrane proteins, RsaFa and RsaFb, that, together with other components, form a 
type I protein translocation pathway for S-layer export. These proteins have
homology to E. coli TolC, the outer membrane channel of multidrug efflux pumps.
Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in
either the maintenance of membrane stability or the active export of
antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent
intracellular accumulation and aggregation of the S-layer protein RsaA; deletion 
of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness.
Using Western blotting, transmission electron microscopy, and RNA-seq, we show
that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the
cytoplasm, which in turn caused upregulation of a number of genes involved in
protein mis-folding and degradation pathways. These findings provide new insight 
into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to
antimicrobial agents and further our understanding of the S-layer export
mechanism on both the transcriptional and translational levels in C. crescentus
IMPORTANCE: Decreased growth rate and reduced cell fitness are common side
effects of protein production in overexpression systems. Inclusion bodies
typically form inside the cell largely due to lack of sufficient export machinery
to transport the overexpressed proteins to the extracellular environment. This
phenomenon can conceivably also occur in natural systems. As one example of a
system evolved to prevent intracellular protein accumulation, our study
demonstrates that Caulobacter crescentus has two homologous outer membrane
transporter proteins that are involved in S-layer export. This is an interesting 
case study that demonstrates how bacteria can evolve redundancy to ensure
adequate protein export functionality and maintain high cellular fitness.
Moreover, we provide evidence that these two outer membrane proteins, although
being the closest C. crescentus homologs to TolC in E. coli, do not process TolC 
functionality in C. crescentus.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AEM.02479-16 
PMCID: PMC5103095
PMID: 27663028 


89. Data Brief. 2016 Sep 7;9:314-7. doi: 10.1016/j.dib.2016.08.069. eCollection 2016 
Dec.

Data set for transcriptome analysis of Escherichia coli exposed to nickel.

Gault M(1), Rodrigue A(1).

Author information: 
(1)Microbiologie, Adaptation et Pathogénie, UMR5240, INSA Lyon, Université Lyon
1, CNRS, Université de Lyon, F-69621 Villeurbanne, France.

Ni is recognized as an element that is toxic to humans, acting as an allergen and
a carcinogenic agent, and it is also toxic to plants. The toxicity of Ni has been
understudied in microorganisms. The data presented here were obtained by
submitting the model bacterium Escherichia coli K-12 to nickel stress. To
identify expressed genes, RNA-Seq was performed. Bacteria were exposed to 50 µM
NiCl2 during 10 min. Exposure to Ni lead to the deregulation of 57% of the E.
coli transcripts. Further analysis using DAVID identified most affected
biological pathways. The list of differentially expressed genes and physiological
consequences of Ni stress are described in "Ni exposure impacts the pool of free 
Fe and modifies DNA supercoiling via metal-induced oxidative stress in
Escherichia coli K-12" (M. Gault, G. Effantin, A. Rodrigue, 2016) [1].

DOI: 10.1016/j.dib.2016.08.069 
PMCID: PMC5026706
PMID: 27668277 


90. Microbiology. 2016 Sep;162(9):1698-1707. doi: 10.1099/mic.0.000337. Epub 2016 Jul
19.

Transcription factor DecR (YbaO) controls detoxification of L-cysteine in
Escherichia coli.

Shimada T(1), Tanaka K(2), Ishihama A(3).

Author information: 
(1)1​Laboratory for Chemistry and Life Science, Institute of Innovative Research,
Tokyo Institute of Technology, Nagatsuta, 4259-R1-29, Yokohama 226-8503, Japan
2​Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo
184-8584, Japan.
(2)1​Laboratory for Chemistry and Life Science, Institute of Innovative Research,
Tokyo Institute of Technology, Nagatsuta, 4259-R1-29, Yokohama 226-8503, Japan.
(3)2​Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo 
184-8584, Japan.

YbaO is an uncharacterized AsnC-family transcription factor of Escherichia coli. 
In both Salmonella enterica and Pantoea ananatis, YbaO homologues were identified
to regulate the adjacent gene encoding cysteine desulfhydrase for detoxification 
of cysteine. Using the genomic SELEX (systematic evolution of ligands by
exponential enrichment) screening system, we identified the yhaOM operon, located
far from the ybaO gene on the E. coli genome, as a single regulatory target of
YbaO. In both gel shift assay in vitro and reporter and Northern blot assays in
vivo, YbaO was found to regulate the yhaOM promoter. The growth of mutants
lacking either ybaO or its targets yhaOM was delayed in the presence of cysteine,
indicating involvement of these genes in cysteine detoxification. In the major
pathway of cysteine degradation, hydrogen sulfide is produced in wild-type E.
coli, but its production was not observed in each of the ybaO, yhaO and yhaM
mutants. The yhaOM promoter was activated in the presence of cysteine, implying
the role of cysteine in activation of YbaO. Taken together, we propose that YbaO 
is the cysteine-sensing transcriptional activator of the yhaOM operon, which is
involved in the detoxification of cysteine. We then propose the naming of ybaO as
decR (regulator of detoxification of cysteine).

DOI: 10.1099/mic.0.000337 
PMID: 27435271  [Indexed for MEDLINE]


91. MBio. 2016 Aug 30;7(4). pii: e00961-16. doi: 10.1128/mBio.00961-16.

New Insights into the Formation of Viable but Nonculturable Escherichia coli
O157:H7 Induced by High-Pressure CO2.

Zhao F(1), Wang Y(1), An H(2), Hao Y(3), Hu X(1), Liao X(4).

Author information: 
(1)College of Food Science and Nutritional Engineering, China Agricultural
University, Beijing, China Key Lab of Fruit and Vegetable Processing, Ministry of
Agriculture, Beijing, China.
(2)College of Food Science and Nutritional Engineering, China Agricultural
University, Beijing, China.
(3)Beijing Advanced Innovation Center for Food Nutrition and Human Health,
Beijing, China College of Food Science and Nutritional Engineering, China
Agricultural University, Beijing, China haoyl@cau.edu.cn liaoxjun@hotmail.com.
(4)Beijing Advanced Innovation Center for Food Nutrition and Human Health,
Beijing, China College of Food Science and Nutritional Engineering, China
Agricultural University, Beijing, China Key Lab of Fruit and Vegetable
Processing, Ministry of Agriculture, Beijing, China haoyl@cau.edu.cn
liaoxjun@hotmail.com.

The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced
by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq)
transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ)
proteomic methods. The analyses revealed that 97 genes and 56 proteins were
significantly changed upon VBNC state entry. Genes and proteins related to
membrane transport, central metabolisms, DNA replication, and cell division were 
mainly downregulated in the VBNC cells. This caused low metabolic activity
concurrently with a division arrest in cells, which may be related to VBNC state 
formation. Cell division repression and outer membrane overexpression were
confirmed to be involved in VBNC state formation by homologous expression of
z2046 coding for transcriptional repressor and ompF encoding outer membrane
protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from
the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a
low level of ATP. Combating the low energy supply, ATP production in the VBNC
cells was compensated by the degradation of l-serine and l-threonine, the
increased AMP generation, and the enhanced electron transfer. Furthermore,
tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2
stresses was enhanced by promoting the production of ammonia and NADPH and by
reducing CO2 production during VBNC state formation. Most genes and proteins
related to pathogenicity were downregulated in the VBNC cells. This would
decrease the cell pathogenicity, which was confirmed by adhesion assays. In
conclusion, the decreased metabolic activity, repressed cell division, and
enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state 
formation.IMPORTANCE: Escherichia coli O157:H7 has been implicated in large
foodborne outbreaks worldwide. It has been reported that the presence of as few
as 10 cells in food could cause illness. However, the presence of only 0.73 to
1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in
Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable
(VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC
state are not well known. In a previous study, we demonstrated that high-pressure
CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In
this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ
proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by 
HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC
cells induced by HPCD, which may provide a theoretical foundation for controlling
the VBNC state entry induced by HPCD treatment.

Copyright © 2016 Zhao et al.

DOI: 10.1128/mBio.00961-16 
PMCID: PMC4999544
PMID: 27578754  [Indexed for MEDLINE]


92. BMC Genomics. 2016 Aug 23;17:671. doi: 10.1186/s12864-016-2988-4.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial 
ecosystems.

Abot A(1)(2)(3), Arnal G(1)(2)(3), Auer L(1)(2)(3), Lazuka A(1)(2)(3),
Labourdette D(1)(2)(3), Lamarre S(1)(2)(3), Trouilh L(1)(2)(3), Laville
E(1)(2)(3), Lombard V(4)(5)(6), Potocki-Veronese G(1)(2)(3), Henrissat
B(4)(5)(6)(7), O'Donohue M(1)(2)(3), Hernandez-Raquet G(1)(2)(3), Dumon
C(1)(2)(3), Leberre VA(8)(9)(10)(11).

Author information: 
(1)Université de Toulouse, INSA, UPS, INP; LISBP, 135 Avenue de Rangueil,
F-31077, Toulouse, France.
(2)INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400,
Toulouse, France.
(3)CNRS, UMR5504, F-31400, Toulouse, France.
(4)Centre National de la Recherche Scientifique, UMR 7257, F-13288, Marseille,
France.
(5)Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille
University, F-13288, Marseille, France.
(6)INRA, USC 1408 AFMB, F-13288, Marseille, France.
(7)Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi
Arabia.
(8)Université de Toulouse, INSA, UPS, INP; LISBP, 135 Avenue de Rangueil,
F-31077, Toulouse, France. leberre@insa-toulouse.fr.
(9)INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400,
Toulouse, France. leberre@insa-toulouse.fr.
(10)CNRS, UMR5504, F-31400, Toulouse, France. leberre@insa-toulouse.fr.
(11)Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP),
UMR INSA/CNRS 5504/INRA 792, INSA Batiment Bio 5, 135, avenue de Rangueil,
F-31077, Toulouse cedex 4, France. leberre@insa-toulouse.fr.

BACKGROUND: Microorganisms constitute a reservoir of enzymes involved in
environmental carbon cycling and degradation of plant polysaccharides through
their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was
developed to allow a rapid characterization at transcriptomic level of these GHs 
and to identify enzymes acting on hydrolysis of polysaccharides or glycans.
RESULTS: This DNA biochip contains the signature of 55,220 bacterial GHs
available in the CAZy database. Probes were designed using two softwares, and
microarrays were directly synthesized using the in situ ink-jet technology.
CAZyChip specificity and reproducibility was validated by hybridization of known 
GHs RNA extracted from recombinant E. coli strains, which were previously
identified by a functional metagenomic approach. The GHs arsenal was also studied
in bioprocess conditions using rumen derived microbiota.
CONCLUSIONS: The CAZyChip appears to be a user friendly tool for profiling the
expression of a large variety of GHs. It can be used to study temporal variations
of functional diversity, thereby facilitating the identification of new efficient
candidates for enzymatic conversions from various ecosystems.

DOI: 10.1186/s12864-016-2988-4 
PMCID: PMC4994258
PMID: 27552843  [Indexed for MEDLINE]


93. Nucleic Acids Res. 2016 Aug 19;44(14):6660-75. doi: 10.1093/nar/gkw115. Epub 2016
Feb 22.

The MazF-regulon: a toolbox for the post-transcriptional stress response in
Escherichia coli.

Sauert M(1), Wolfinger MT(2), Vesper O(1), Müller C(1), Byrgazov K(1), Moll I(3).

Author information: 
(1)Max F. Perutz Laboratories, Center for Molecular Biology, Department of
Microbiology, Immunobiology and Genetics, University of Vienna, Vienna Biocenter 
(VBC), Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria.
(2)Max F. Perutz Laboratories, Department of Biochemistry and Molecular Cell
Biology, University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9/5, A-1030
Vienna, Austria Max F. Perutz Laboratories, Center for Integrative Bioinformatics
Vienna, University of Vienna, Medical University of Vienna, Vienna Biocenter
(VBC), Dr. Bohr-Gasse 9, A-1030 Vienna, Austria Institute for Theoretical
Chemistry, University of Vienna, Währingerstraße 17, A-1090 Vienna, Austria.
(3)Max F. Perutz Laboratories, Center for Molecular Biology, Department of
Microbiology, Immunobiology and Genetics, University of Vienna, Vienna Biocenter 
(VBC), Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria Isabella.moll@univie.ac.at.

Flexible adaptation to environmental stress is vital for bacteria. An
energy-efficient post-transcriptional stress response mechanism in Escherichia
coli is governed by the toxin MazF. After stress-induced activation the
endoribonuclease MazF processes a distinct subset of transcripts as well as the
16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes'
are specific for the MazF-processed mRNAs, the translational program is changed. 
To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation
coupled with RNA-seq analysis) and analyzed alterations introduced into the
transcriptome and translatome after mazF overexpression. Unexpectedly, our
results reveal that the corresponding protein products are involved in all
cellular processes and do not particularly contribute to the general stress
response. Moreover, our findings suggest that translational reprogramming serves 
as a fast-track reaction to harsh stress and highlight the so far underestimated 
significance of selective translation as a global regulatory mechanism in gene
expression. Considering the reported implication of toxin-antitoxin (TA) systems 
in persistence, our results indicate that MazF acts as a prime effector during
harsh stress that potentially introduces translational heterogeneity within a
bacterial population thereby stimulating persister cell formation.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkw115 
PMCID: PMC5001579
PMID: 26908653  [Indexed for MEDLINE]


94. Fish Shellfish Immunol. 2016 Aug;55:315-22. doi: 10.1016/j.fsi.2016.06.004. Epub 
2016 Jun 6.

Transcriptome analysis of immune response genes induced by pathogen agonists in
the Antarctic bullhead notothen Notothenia coriiceps.

Ahn DH(1), Kang S(1), Park H(2).

Author information: 
(1)Division of Polar Life Sciences, Korea Polar Research Institute, Yeonsu-gu,
Incheon, 21990, South Korea.
(2)Division of Polar Life Sciences, Korea Polar Research Institute, Yeonsu-gu,
Incheon, 21990, South Korea; Polar Sciences, University of Science & Technology, 
Yuseong-gu, Daejeon, 34113, South Korea. Electronic address: hpark@kopri.re.kr.

Fish are a representative population of lower vertebrates that serve as an
essential link to early vertebrate evolution, and this has fueled academic
interest in studying ancient vertebrate immune defense mechanisms in teleosts.
Notothenia coriiceps, a typical Antarctic notothenioid teleost, has evolved to
adapt to the cold and thermally stable Antarctic sea. In this study, we examined 
adaptive signaling pathways and immune responses to bacterial and viral
pathogenic exposure in N. coriiceps. Using RNA sequencing, we investigated
transcriptional differences in the liver tissues of N. coriiceps challenged with 
two pathogen-mimicking agonists, a bacterial ligand (heat-killed Escherichia
coli, HKEB) and a viral ligand (polyinosinic:polycytidylic acid, Poly I:C). We
found that 567 unique genes were up-regulated two-fold in the HKEB-exposed group,
whereas 392 unique genes, including 124 immune-relevant genes, were up-regulated 
two-fold in the Poly I:C-exposed group. A KEGG pathway analysis of the 124
immune-relevant genes revealed that they exhibited major features of antigen
processing and presentation bacterial ligand exposure, but they were
down-regulated after viral ligand exposure. A quantitative real time RT-PCR
analysis revealed that TNFα and TNF2, major inducers of apoptosis, were highly
up-regulated after exposure to the viral ligand but not the bacterial ligand. The
results suggest that the bacterial and viral ligands up-regulate inducers of
different immune mechanisms in N. coriiceps liver tissue. N. coriiceps has an
immune response defense strategy that uses antigen presentation against bacterial
infection, but it may use a different defense, such as TNF-mediated apoptosis,
against viral infection. The specific immune responses of N. coriiceps may be
adaptations to the Antarctic environment and pathogens. These results will help
define the characteristics of Antarctic fish and increase our understanding of
their immune response mechanisms.

Copyright © 2016 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.fsi.2016.06.004 
PMID: 27276114  [Indexed for MEDLINE]


95. Free Radic Biol Med. 2016 Aug;97:351-361. doi:
10.1016/j.freeradbiomed.2016.06.030. Epub 2016 Jun 29.

Ni exposure impacts the pool of free Fe and modifies DNA supercoiling via
metal-induced oxidative stress in Escherichia coli K-12.

Gault M(1), Effantin G(1), Rodrigue A(2).

Author information: 
(1)Microbiologie, Adaptation et Pathogénie, UMR5240, INSA Lyon, Université Lyon
1, CNRS, Université de Lyon, F-69621 Villeurbanne, France.
(2)Microbiologie, Adaptation et Pathogénie, UMR5240, INSA Lyon, Université Lyon
1, CNRS, Université de Lyon, F-69621 Villeurbanne, France. Electronic address:
agnes.rodrigue@insa-lyon.fr.

The biology of nickel has been widely studied in mammals because of its
carcinogenic properties, whereas few studies have been performed in
microorganisms. In the present work, changes accompanying stress caused by nickel
were evaluated at the cellular level using RNA-Seq in Escherichia coli K-12.
Interestingly, a very large number of genes were found to be deregulated by Ni
stress. Iron and oxidative stress homeostasis maintenance were among the most
highly enriched functional categories, and genes involved in periplasmic copper
efflux were among the most highly upregulated. These results suggest that the
deregulation of Fe and Cu homeostatic genes is caused by a release of free Cu and
Fe ions in the cell which in turn activate the Cu and Fe homeostatic systems. The
content of Cu was not significantly affected upon the addition of Ni to the
growth medium, nor were the Cus and CopA Cu-efflux systems important for the
survival of bacteria under Ni stress In contrast the addition of Ni slightly
decreased the amount of cellular Fe and activated the transcription of Fur
regulated genes in a Fur-dependent manner. Cu or Fe imbalance together with
oxidative stress might affect the structure of DNA. Further experiments revealed 
that Ni alters the state of DNA folding by causing a relaxed conformation, a
phenomenon that is reversible by addition of the antioxidant Tiron or the Fe
chelator Dip. The Tiron-reversible DNA relaxation was also observed for Fe and to
a lesser extent with Cu but not with Co. DNA supercoiling is well recognized as
an integral aspect of gene regulation. Moreover our results show that Ni modifies
the expression of several nucleoid-associated proteins (NAPs), important agents
of DNA topology and global gene regulation. This is the first report describing
the impact of metal-induced oxidative on global regulatory networks.

Copyright © 2016 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.freeradbiomed.2016.06.030 
PMID: 27375130  [Indexed for MEDLINE]


96. J Bacteriol. 2016 Jul 28;198(16):2166-79. doi: 10.1128/JB.00144-16. Print 2016
Aug 15.

The Bacterial iprA Gene Is Conserved across Enterobacteriaceae, Is Involved in
Oxidative Stress Resistance, and Influences Gene Expression in Salmonella
enterica Serovar Typhimurium.

Herman A(1), Serfecz J(1), Kinnally A(1), Crosby K(1), Youngman M(1), Wykoff
D(1), Wilson JW(2).

Author information: 
(1)Villanova University, Department of Biology, Villanova, Pennsylvania, USA.
(2)Villanova University, Department of Biology, Villanova, Pennsylvania, USA
james.w.wilson@villanova.edu.

The iprA gene (formerly known as yaiV or STM0374) is located in a two-gene operon
in the Salmonella enterica serovar Typhimurium genome and is associated with
altered expression during spaceflight and rotating-wall-vessel culture conditions
that increase virulence. However, iprA is uncharacterized in the literature. In
this report, we present the first targeted characterization of this gene, which
revealed that iprA is highly conserved across Enterobacteriaceae We found that S 
Typhimurium, Escherichia coli, and Enterobacter cloacae ΔiprA mutant strains
display a multi-log-fold increase in oxidative stress resistance that is
complemented using a plasmid-borne wild-type (WT) copy of the S Typhimurium iprA 
gene. This observation was also associated with increased catalase activity,
increased S Typhimurium survival in macrophages, and partial dependence on the
katE gene and full dependence on the rpoS gene. Our results indicate that IprA
protein activity is sensitive to deletion of the N- and C-terminal 10 amino
acids, while a region that includes amino acids 56 to 80 is dispensable for
activity. RNA sequencing (RNA-Seq) analysis revealed several genes altered in
expression in the S Typhimurium ΔiprA mutant strain compared to the WT, including
those involved in fimbria formation, spvABCD-mediated virulence, ethanolamine
utilization, the phosphotransferase system (PTS) transport, and flagellin phase
switching from FlgB to FliC (likely a stochastic event) and several genes of
hypothetical or putative function.IMPORTANCE: Overall, this work reveals that the
conserved iprA gene measurably influences bacterial biology and highlights the
pool of currently uncharacterized genes that are conserved across bacterial
genomes. These genes represent potentially useful targets for bacterial
engineering, vaccine design, and other possible applications.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.00144-16 
PMCID: PMC4966445
PMID: 27246569  [Indexed for MEDLINE]


97. Crit Rev Food Sci Nutr. 2016 Jul 3;56(9):1519-31. doi:
10.1080/10408398.2013.775567.

Commercially Available Rapid Methods for Detection of Selected Food-borne
Pathogens.

Valderrama WB(1), Dudley EG(1), Doores S(1), Cutter CN(1).

Author information: 
(1)a Department of Food Science , Pennsylvania State University , University Park
, Pennsylvania , USA.

Generally, the enumeration and isolation of food-borne pathogens is performed
using culture-dependent methods. These methods are sensitive, inexpensive, and
provide both qualitative and quantitative assessment of the microorganisms
present in a sample, but these are time-consuming. For this reason, researchers
are developing new techniques that allow detection of food pathogens in shorter
period of time. This review identifies commercially available methods for rapid
detection and quantification of Listeria monocytogenes, Salmonella spp.,
Staphylococcus aureus, and Shiga toxin-producing Escherichia coli in food
samples. Three categories are discussed: immunologically based methods, nucleic
acid-based assays, and biosensors. This review describes the basic mechanism and 
capabilities of each method, discusses the difficulties of choosing the most
convenient method, and provides an overview of the future challenges for the
technology for rapid detection of microorganisms.

DOI: 10.1080/10408398.2013.775567 
PMID: 25749054  [Indexed for MEDLINE]


98. FEMS Microbiol Lett. 2016 Jul;363(13). pii: fnw131. doi: 10.1093/femsle/fnw131.
Epub 2016 May 17.

A new custom microarray for sRNA profiling in Escherichia coli.

Ruiz-Larrabeiti O(1), Plágaro AH(1), Gracia C(2), Sevillano E(1), Gallego L(1),
Hajnsdorf E(2), Kaberdin VR(3).

Author information: 
(1)Department of Immunology, Microbiology and Parasitology, University of the
Basque Country UPV/EHU, Leioa, Spain.
(2)CNRS UMR8261 (previously FRE3630), University Paris Diderot, Sorbonne Paris
Cité, Institut de Biologie Physico-Chimique, 75005 Paris, France.
(3)Department of Immunology, Microbiology and Parasitology, University of the
Basque Country UPV/EHU, Leioa, Spain IKERBASQUE, Basque Foundation for Science,
48011 Bilbao, Spain vladimir.kaberdin@ehu.eus.

Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional
control of gene expression. To improve their detection by conventional
microarrays, we designed a custom microarray containing a group of probes
targeting known and some putative Escherichia coli sRNAs. To assess its potential
in detection of sRNAs, RNA profiling experiments were performed with total RNA
extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani)
and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably
strong and statistically significant signals corresponding to nearly all sRNAs
annotated in the EcoCyc database. Besides differential expression of two sRNAs
(GcvB and RydB), expression of other sRNAs was less affected by the composition
of the growth media. Other examples of the differentially expressed sRNAs were
revealed by comparing gene expression of the wild-type strain and its isogenic
mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot
analysis was employed to validate these data and to assess the existence of new
putative sRNAs. Our results suggest that the use of custom microarrays with
improved capacities for detection of sRNAs can offer an attractive opportunity
for efficient gene expression profiling of sRNAs and their target mRNAs at the
whole transcriptome level.

© FEMS 2016. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.

DOI: 10.1093/femsle/fnw131 
PMID: 27190161  [Indexed for MEDLINE]


99. Microbiology. 2016 Jul;162(7):1253-64. doi: 10.1099/mic.0.000292. Epub 2016 Apr
25.

Transcription factor CecR (YbiH) regulates a set of genes affecting the
sensitivity of Escherichia coli against cefoperazone and chloramphenicol.

Yamanaka Y(1), Shimada T(2), Yamamoto K(3), Ishihama A(1).

Author information: 
(1)1​ Research Center for Micro-Nano Technology, Nano Technology, Koganei, Tokyo 
184-8584, Japan.
(2)2​ Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda,
Yokohama, Japan.
(3)3​ Department of Frontier Bioscience, Hosei University, Koganei, Tokyo
184-8584, Japan.

Genomic SELEX (systematic evolution of ligands by exponential enrichment)
screening was performed for identification of the binding site of YbiH, an as yet
uncharacterized TetR-family transcription factor, on the Escherichia coli genome.
YbiH was found to be a unique single-target regulator that binds in vitro within 
the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR
and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a
membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a
ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses
indicated one clear binding site of YbiH, including a complete palindromic
sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of 
the ybiH operon and activation of the rhlE operon by YbiH. After phenotype
microarray screening, YbiH was indicated to confer resistance to chloramphenicol 
and cefazoline (a first-generation cephalosporin). A systematic survey of the
participation of each of the predicted YbiH-regulated genes in the antibiotic
sensitivity indicated involvement of the YbhFSR ABC-type transporter in the
sensitivity to cefoperazone (a third-generation cephalosporin) and of the
membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken
together with the growth test in the presence of these two antibiotics and in
vitro transcription assay, it was concluded that the hitherto uncharacterized
YbiH regulates transcription of both the bidirectional transcription units, the
ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the
control of sensitivity to cefoperazone and chloramphenicol. We thus propose to
rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity).

DOI: 10.1099/mic.0.000292 
PMID: 27112147  [Indexed for MEDLINE]


100. Poult Sci. 2016 Jul 1;95(7):1543-54. doi: 10.3382/ps/pew088. Epub 2016 Mar 14.

Long-term effects of early life microbiota disturbance on adaptive immunity in
laying hens.

Simon K(1), Verwoolde MB(1), Zhang J(2), Smidt H(3), de Vries Reilingh G(1), Kemp
B(1), Lammers A(1).

Author information: 
(1)Adaptation Physiology Group, Department of Animal Sciences, Wageningen
University, De Elst 1, 6708 WD Wageningen, The Netherlands.
(2)Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB
Wageningen, The Netherlands Kristina.Simon@wur.nl.
(3)Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB
Wageningen, The Netherlands.

Due to an interplay between intestinal microbiota and immune system, disruption
of intestinal microbiota composition during immune development may have
consequences for immune responses later in life. The present study investigated
the effects of antibiotic treatment in the first weeks of life on the specific
antibody response later in life in chickens. Layer chicks received an antibiotic 
cocktail consisting of vancomycin, neomycin, metronidazole, and amphotericin-B by
oral gavage every 12 h, and ampicillin and colistin in drinking water for the
first week of life. After the first week of life, chicks received ampicillin and 
colistin in drinking water for two more weeks. Control birds received no
antibiotic cocktail and plain drinking water. Fecal microbiota composition was
determined during antibiotic treatment (d 8 and 22), two weeks after cessation of
antibiotic treatment (d 36), and at the end of the experimental period at d 175
using a 16S ribosomal RNA gene targeted microarray, the Chicken Intestinal Tract 
Chip (ChickChip). During antibiotic treatment fecal microbiota composition
differed strongly between treatment groups. Fecal microbiota of antibiotic
treated birds consisted mainly of Proteobacteria, and in particular E.coli,
whereas fecal microbiota of control birds consisted mainly of Firmicutes, such as
lactobacilli and clostridia. Two weeks after cessation of antibiotic treatment
fecal microbiota composition of antibiotic treated birds had recovered and was
similar to that of control birds. On d 105, 12 weeks after cessation of
antibiotic treatment, chicks of both treatment groups received an intra-tracheal 
lipopolysaccharide (LPS)/human serum albumin (HuSA) challenge. Antibody titers
against LPS and HuSA were measured 10 days after administration of the challenge.
While T cell independent antibody titers (LPS) were not affected by antibiotic
treatment, antibiotic treated birds showed lower T cell dependent antibody titers
(HuSA) compared with control birds. In conclusion, intestinal microbial dysbiosis
early in life may still have effects on the specific antibody response months
after cessation of antibiotic treatment and despite an apparent recovery in
microbiota composition.

© 2016 Poultry Science Association Inc.

DOI: 10.3382/ps/pew088 
PMID: 26976906  [Indexed for MEDLINE]


101. BMC Microbiol. 2016 Jun 27;16(1):130. doi: 10.1186/s12866-016-0741-4.

5-azacytidine induces transcriptome changes in Escherichia coli via DNA
methylation-dependent and DNA methylation-independent mechanisms.

Militello KT(1), Simon RD(2), Mandarano AH(2)(3), DiNatale A(2), Hennick SM(2),
Lazatin JC(2), Cantatore S(2).

Author information: 
(1)State University of New York at Geneseo, ISC 357, 1 College Circle, Geneseo,
NY, 14454, USA. militello@geneseo.edu.
(2)State University of New York at Geneseo, ISC 357, 1 College Circle, Geneseo,
NY, 14454, USA.
(3)Cornell University, Ithaca, NY, 14853, USA.

BACKGROUND: Escherichia coli K-12 strains contain DNA cytosine methyltransferase 
(Dcm), which generates 5-methylcytosine at 5'CCWGG3' sites. Although the role of 
5-methylcytosine in eukaryotic gene expression is relatively well described, the 
role of 5-methylcytosine in bacterial gene expression is largely unknown.
RESULTS: To identify genes that are controlled by 5-methylcytosine in E. coli, we
compared the transcriptomes of cells grown in the absence and presence of the DNA
methylation inhibitor 5-azacytidine. We observed expression changes for 63 genes.
The majority of the gene expression changes occurred at early stationary phase
and were up-regulations. To identify gene expression changes due to a loss of DNA
methylation, we compared the expression of selected genes in a wild-type and dcm 
knockout strain via reverse transcription quantitative PCR.
CONCLUSIONS: Our data indicate that 5-azacytidine can influence gene expression
by at least two distinct mechanisms: DNA methylation loss and a mechanism that is
independent of DNA methylation loss. In addition, we have identified new targets 
of 5-methylcytosine-mediated regulation of gene expression. In summary, our data 
indicate that 5-azacytidine impacts the composition of the bacterial
transcriptome, and the primary effect is increased gene expression at early
stationary phase.

DOI: 10.1186/s12866-016-0741-4 
PMCID: PMC4924334
PMID: 27349222  [Indexed for MEDLINE]


102. BMC Res Notes. 2016 Jun 13;9:305. doi: 10.1186/s13104-016-2113-4.

Thermodynamically optimal whole-genome tiling microarray design and validation.

Cho H(1), Chou HH(2)(3).

Author information: 
(1)Department of Genetics, Development and Cell Biology, Iowa State University,
Ames, IA, 50011, USA.
(2)Department of Genetics, Development and Cell Biology, Iowa State University,
Ames, IA, 50011, USA. hhchou@iastate.edu.
(3)Department of Computer Science, Iowa State University, Ames, IA, 50011, USA.
hhchou@iastate.edu.

BACKGROUND: Microarray is an efficient apparatus to interrogate the whole
transcriptome of species. Microarray can be designed according to annotated gene 
sets, but the resulted microarrays cannot be used to identify novel transcripts
and this design method is not applicable to unannotated species. Alternatively, a
whole-genome tiling microarray can be designed using only genomic sequences
without gene annotations, and it can be used to detect novel RNA transcripts as
well as known genes. The difficulty with tiling microarray design lies in the
tradeoff between probe-specificity and coverage of the genome. Sequence
comparison methods based on BLAST or similar software are commonly employed in
microarray design, but they cannot precisely determine the subtle thermodynamic
competition between probe targets and partially matched probe nontargets during
hybridizations.
FINDINGS: Using the whole-genome thermodynamic analysis software PICKY to design 
tiling microarrays, we can achieve maximum whole-genome coverage allowable under 
the thermodynamic constraints of each target genome. The resulted tiling
microarrays are thermodynamically optimal in the sense that all selected probes
share the same melting temperature separation range between their targets and
closest nontargets, and no additional probes can be added without violating the
specificity of the microarray to the target genome.
CONCLUSIONS: This new design method was used to create two whole-genome tiling
microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the
experiment results validated the design.

DOI: 10.1186/s13104-016-2113-4 
PMCID: PMC4906886
PMID: 27295952  [Indexed for MEDLINE]


103. Antimicrob Agents Chemother. 2016 May 23;60(6):3462-9. doi: 10.1128/AAC.00098-16.
Print 2016 Jun.

Analysis of β-Lactamase Resistance Determinants in Enterobacteriaceae from
Chicago Children: a Multicenter Survey.

Logan LK(1), Hujer AM(2), Marshall SH(3), Domitrovic TN(2), Rudin SD(2), Zheng
X(4), Qureshi NK(5), Hayden MK(6), Scaggs FA(7), Karadkhele A(7), Bonomo RA(8).

Author information: 
(1)Department of Pediatrics, Rush University Medical Center, Chicago, Illinois,
USA Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Case Western Reserve School of Medicine, Cleveland, Ohio, USA
latania_logan@rush.edu.
(2)Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Case Western Reserve School of Medicine, Cleveland, Ohio, USA
Department of Medicine, Case Western Reserve School of Medicine, Cleveland, Ohio,
USA.
(3)Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Case Western Reserve School of Medicine, Cleveland, Ohio, USA.
(4)Department of Microbiology, Ann and Robert H. Lurie Children's Hospital of
Chicago, Chicago, Illinois, USA Department of Pathology, Northwestern University 
Feinberg School of Medicine, Chicago, Illinois, USA.
(5)Department of Pediatrics, Loyola University Medical Center, Maywood, Illinois,
USA.
(6)Departments of Medicine and Pathology, Rush University Medical Center,
Chicago, Illinois, USA.
(7)Department of Pediatrics, Rush University Medical Center, Chicago, Illinois,
USA.
(8)Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Case Western Reserve School of Medicine, Cleveland, Ohio, USA
Department of Medicine, Case Western Reserve School of Medicine, Cleveland, Ohio,
USA Department of Pharmacology, Case Western Reserve School of Medicine,
Cleveland, Ohio, USA Departments of Molecular Biology and Microbiology, Case
Western Reserve School of Medicine, Cleveland, Ohio, USA.

Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S.
children; however, there is a paucity of multicentered analyses of antibiotic
resistance genes responsible for MDR phenotypes among pediatric
Enterobacteriaceae isolates. In this study, 225 isolates phenotypically
identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers,
recovered from children ages 0 to 18 years hospitalized between January 2011 and 
April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray
platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type
β-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence
typing (MLST) were performed to assess isolate similarity. Plasmid replicon
typing was conducted to classify plasmids. The median patient age was 4.2 years, 
56% were female, and 44% presented in the outpatient setting. The majority
(60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of
225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7%
contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 
1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). 
Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. 
coli phylogenetic group was the virulent B2 group (67.6%) associated with
ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%),
and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were
non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR
contained plasmid replicon FIIA. We found that β-lactam resistance in children is
diverse and that certain resistance mechanisms differ from known circulating
genotypes in adults in an endemic area. The potential impact of complex molecular
types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable
population needs to be studied further.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AAC.00098-16 
PMCID: PMC4879402
PMID: 27021322  [Indexed for MEDLINE]


104. BMC Evol Biol. 2016 May 23;16(1):114. doi: 10.1186/s12862-016-0683-3.

Do novel genes drive morphological novelty? An investigation of the nematosomes
in the sea anemone Nematostella vectensis.

Babonis LS(1), Martindale MQ(2)(3), Ryan JF(2)(3).

Author information: 
(1)Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean
Shore Blvd, St. Augustine, FL, 32080, USA. babonis@whitney.ufl.edu.
(2)Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean
Shore Blvd, St. Augustine, FL, 32080, USA.
(3)Department of Biology, University of Florida, Gainesville, FL, 32611, USA.

BACKGROUND: The evolution of novel genes is thought to be a critical component of
morphological innovation but few studies have explicitly examined the
contribution of novel genes to the evolution of novel tissues. Nematosomes, the
free-floating cellular masses that circulate through the body cavity of the sea
anemone Nematostella vectensis, are the defining apomorphy of the genus
Nematostella and are a useful model for understanding the evolution of novel
tissues. Although many hypotheses have been proposed, the function of nematosomes
is unknown. To gain insight into their putative function and to test hypotheses
about the role of lineage-specific genes in the evolution of novel structures, we
have re-examined the cellular and molecular biology of nematosomes.
RESULTS: Using behavioral assays, we demonstrate that nematosomes are capable of 
immobilizing live brine shrimp (Artemia salina) by discharging their abundant
cnidocytes. Additionally, the ability of nematosomes to engulf fluorescently
labeled bacteria (E. coli) reveals the presence of phagocytes in this tissue.
Using RNA-Seq, we show that the gene expression profile of nematosomes is
distinct from that of the tentacles and the mesenteries (their tissue of origin) 
and, further, that nematosomes (a Nematostella-specific tissue) are enriched in
Nematostella-specific genes.
CONCLUSIONS: Despite the small number of cell types they contain, nematosomes are
distinct among tissues, both functionally and molecularly. We provide the first
evidence that nematosomes comprise part of the innate immune system in N.
vectensis, and suggest that this tissue is potentially an important place to look
for genes associated with pathogen stress. Finally, we demonstrate that
Nematostella-specific genes comprise a significant proportion of the
differentially expressed genes in all three of the tissues we examined and may
play an important role in novel cell functions.

DOI: 10.1186/s12862-016-0683-3 
PMCID: PMC4877951
PMID: 27216622  [Indexed for MEDLINE]


105. Front Microbiol. 2016 May 19;7:724. doi: 10.3389/fmicb.2016.00724. eCollection
2016.

Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic 
Escherichia coli PCN033.

Chen J(1), Su L(2), Wang X(1), Zhang T(1), Liu F(1), Chen H(3), Tan C(3).

Author information: 
(1)State Key Laboratory of Agricultural Microbiology, College of Veterinary
Medicine, Huazhong Agricultural University Wuhan, China.
(2)School of Public Health, Guangzhou Medical University Guangzhou, China.
(3)State Key Laboratory of Agricultural Microbiology, College of Veterinary
Medicine, Huazhong Agricultural UniversityWuhan, China; Key Laboratory for the
Development of Veterinary Diagnostic Products, The Cooperative Innovation Center 
for Sustainable Pig Production, Ministry of Agriculture, Huazhong Agricultural
UniversityWuhan, China.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of acute
infections in its hosts, and multidrug-resistant strains present significant
challenges to public health and animal husbandry. Therefore, it is necessary to
explore new drug targets to control E. coli epidemics. Previous studies have
reported that ppk mutants of Burkholderia pseudomallei and Mycobacterium
tuberculosis are more susceptible than the wild types (WTs) to stress. Therefore,
we investigated the stress response to antibiotics mediated by polyphosphate
kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a ppk
knockout strain (Δppk) were more susceptible to antibiotics than was WT. However,
biofilm-grown Δppk cells showed similar susceptibility to that of the WT and were
more tolerant than the planktonic cells. During the planktonic lifestyle, the
expression of genes involved in antibiotic tolerance (including
resistance-conferring genes, and antibiotic influx, and efflux genes) did not
change in the Δppk mutant without antibiotic treatment. However, the
resistance-conferring gene bla and efflux genes were upregulated more in the WT
than in the Δppk mutant by treatment with tazobactam. After treatment with
gentamycin, the efflux genes and influx genes were upregulated and downregulated,
respectively, more in the WT than in the Δppk mutant. The expression of genes
involved in biofilm regulation also changed after treatment with tazobactam or
gentamycin, and which is consistent with the results of the biofilm formation.
Together, these observations indicate that PPK is important for the antibiotic
stress response during the planktonic growth of ExPEC and might be a potential
drug target in bacteria.

DOI: 10.3389/fmicb.2016.00724 
PMCID: PMC4871857
PMID: 27242742 


106. PLoS One. 2016 May 19;11(5):e0155740. doi: 10.1371/journal.pone.0155740.
eCollection 2016.

Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight
into Transcriptional Regulation.

Junier I(1)(2), Rivoire O(3)(4).

Author information: 
(1)CNRS, TIMC-IMAG, F-38000 Grenoble, France.
(2)Univ. Grenoble Alpes, TIMC-IMAG, F-38000 Grenoble, France.
(3)CNRS, LIPhy, F-38000 Grenoble, France.
(4)Univ. Grenoble Alpes, LIPhy, F-38000 Grenoble, France.

Genome-wide measurements of transcriptional activity in bacteria indicate that
the transcription of successive genes is strongly correlated beyond the scale of 
operons. Here, we analyze hundreds of bacterial genomes to identify
supra-operonic segments of genes that are proximal in a large number of genomes. 
We show that these synteny segments correspond to genomic units of strong
transcriptional co-expression. Structurally, the segments contain operons with
specific relative orientations (co-directional or divergent) and
nucleoid-associated proteins are found to bind at their boundaries. Functionally,
operons inside a same segment are highly co-expressed even in the apparent
absence of regulatory factors at their promoter regions. Remote operons along DNA
can also be co-expressed if their corresponding segments share a transcriptional 
or sigma factor, without requiring these factors to bind directly to the
promoters of the operons. As evidence that these results apply across the
bacterial kingdom, we demonstrate them both in the Gram-negative bacterium
Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The
underlying process that we propose involves only RNA-polymerases and DNA: it
implies that the transcription of an operon mechanically enhances the
transcription of adjacent operons. In support of a primary role of this
regulation by facilitated co-transcription, we show that the transcription en
bloc of successive operons as a result of transcriptional read-through is
strongly and specifically enhanced in synteny segments. Finally, our analysis
indicates that facilitated co-transcription may be evolutionary primitive and may
apply beyond bacteria.

DOI: 10.1371/journal.pone.0155740 
PMCID: PMC4873041
PMID: 27195891  [Indexed for MEDLINE]


107. PLoS Pathog. 2016 May 9;12(5):e1005616. doi: 10.1371/journal.ppat.1005616.
eCollection 2016 May.

Age-Dependent Susceptibility to Enteropathogenic Escherichia coli (EPEC)
Infection in Mice.

Dupont A(1)(2), Sommer F(3), Zhang K(1)(2), Repnik U(4), Basic M(5), Bleich A(5),
Kühnel M(6), Bäckhed F(3), Litvak Y(7), Fulde M(2), Rosenshine I(7), Hornef
MW(1)(2).

Author information: 
(1)Institute for Medical Microbiology, RWTH Aachen University Hospital, Aachen,
Germany.
(2)Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical 
School, Hannover, Germany.
(3)The Wallenberg Laboratory, Department of Molecular and Clinical Medicine,
University of Gothenburg, Gothenburg, Sweden.
(4)Department of Biosciences, University of Oslo, Oslo, Norway.
(5)Institute for Laboratory Animal Science, Hannover Medical School, Hannover,
Germany.
(6)Institute for Functional and Applied Anatomy, Hannover Medical School,
Hannover, Germany.
(7)Department for Microbiology and Molecular Genetics, Institute of Medical
Research Israel-Canada, Faculty of Medicine, The Hebrew University of Jerusalem, 
Jerusalem, Israel.

Enteropathogenic Escherichia coli (EPEC) represents a major causative agent of
infant diarrhea associated with significant morbidity and mortality in developing
countries. Although studied extensively in vitro, the investigation of the
host-pathogen interaction in vivo has been hampered by the lack of a suitable
small animal model. Using RT-PCR and global transcriptome analysis, high
throughput 16S rDNA sequencing as well as immunofluorescence and electron
microscopy, we characterize the EPEC-host interaction following oral challenge of
newborn mice. Spontaneous colonization of the small intestine and colon of
neonate mice that lasted until weaning was observed. Intimate attachment to the
epithelial plasma membrane and microcolony formation were visualized only in the 
presence of a functional bundle forming pili (BFP) and type III secretion system 
(T3SS). Similarly, a T3SS-dependent EPEC-induced innate immune response, mediated
via MyD88, TLR5 and TLR9 led to the induction of a distinct set of genes in
infected intestinal epithelial cells. Infection-induced alterations of the
microbiota composition remained restricted to the postnatal period. Although EPEC
colonized the adult intestine in the absence of a competing microbiota, no
microcolonies were observed at the small intestinal epithelium. Here, we
introduce the first suitable mouse infection model and describe an age-dependent,
virulence factor-dependent attachment of EPEC to enterocytes in vivo.

DOI: 10.1371/journal.ppat.1005616 
PMCID: PMC4861285
PMID: 27159323  [Indexed for MEDLINE]


108. Arch Microbiol. 2016 May;198(4):353-62. doi: 10.1007/s00203-016-1191-y. Epub 2016
Feb 2.

Understanding the host-adapted state of Citrobacter rodentium by transcriptomic
analysis.

Smith AD(1), Yan X(2), Chen C(3), Dawson HD(3), Bhagwat AA(2).

Author information: 
(1)Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research 
Center, USDA-ARS, 10300 Baltimore Ave., B307C, Rm. 228, BARC-E, Beltsville, MD,
20705, USA. allen.smith@ars.usda.gov.
(2)Environmental, Microbial, and Food Safety Laboratory, Beltsville Agriculture
Research Center, USDA-ARS, Beltsville, MD, USA.
(3)Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research 
Center, USDA-ARS, 10300 Baltimore Ave., B307C, Rm. 228, BARC-E, Beltsville, MD,
20705, USA.

Citrobacter rodentium (Cr) is a mouse pathogen that mimics many aspects of
enteropathogenic Escherichia coli infections including producing attaching and
effacing (A/E) lesions. Host-adapted (HA) Cr cells that are shed at the peak of
infection have been reported to be hyper-infective. The exact mechanism
underlying this phenomenon has remained elusive since the pathogen loses its HA
'status' immediately upon subculturing in laboratory media. We sequenced the
entire transcriptome of Cr directly from the feces of infected mice and analyzed 
the gene expression pattern. We observed that the entire transcriptional
machinery as well as several transcriptional regulators to be differentially
expressed when compared with the transcriptome of cells grown on laboratory
media. Major adhesion and effector genes, tir and eae, were highly expressed in
HA along with many genes located on all five loci of enterocyte effacement
regions (LEE 1-5). Notable absent among the HA expressed genes were 19 fimbrial
operons and non-fimbrial adhesions and several non-LEE encoded effectors. These
results demonstrate that host-adapted Cr has a unique transcriptome that is
associated with increased host transmission.

DOI: 10.1007/s00203-016-1191-y 
PMID: 26837900  [Indexed for MEDLINE]


109. J Gastroenterol. 2016 May;51(5):447-57. doi: 10.1007/s00535-015-1121-9. Epub 2015
Sep 11.

Mouse model of proximal colon-specific tumorigenesis driven by microsatellite
instability-induced Cre-mediated inactivation of Apc and activation of Kras.

Kawaguchi Y(1), Hinoi T(2), Saito Y(1), Adachi T(1), Miguchi M(1), Niitsu H(1),
Sasada T(1), Shimomura M(1), Egi H(1), Oka S(3), Tanaka S(3), Chayama K(4),
Sentani K(5), Oue N(5), Yasui W(5), Ohdan H(1).

Author information: 
(1)Department of Gastroenterological and Transplant Surgery, Applied Life
Sciences, Institute of Biomedical and Health Sciences, Hiroshima University,
1-2-3 Kasumi Minami-ku, Hiroshima, 7348551, Japan.
(2)Department of Gastroenterological and Transplant Surgery, Applied Life
Sciences, Institute of Biomedical and Health Sciences, Hiroshima University,
1-2-3 Kasumi Minami-ku, Hiroshima, 7348551, Japan. thinoi@hiroshima-u.ac.jp.
(3)Department of Endoscopy and Medicine, Graduate School of Biomedical and Health
Sciences, Hiroshima University, Hiroshima, Japan.
(4)Department of Gastroenterology and Metabolism, Applied life sciences,
Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi
Minami-ku, Hiroshima, 7348551, Japan.
(5)Department of Molecular Pathology, Applied life sciences, Institute of
Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi Minami-ku,
Hiroshima, 7348551, Japan.

BACKGROUND: KRAS gene mutations are found in 40-50% of colorectal cancer cases,
but their functional contribution is not fully understood. To address this issue,
we generated genetically engineered mice with colon tumors expressing an
oncogenic Kras(G12D) allele in the context of the Adenomatous polyposis coli
(Apc) deficiency to compare them to tumors harboring Apc deficiency alone.
METHODS: CDX2P9.5-G22Cre (referred to as G22Cre) mice showing inducible Cre
recombinase transgene expression in the proximal colon controlled under the CDX2 
gene promoter were intercrossed with Apc (flox/flox) mice and LSL-Kras (G12D)
mice carrying loxP-flanked Apc and Lox-Stop-Lox oncogenic Kras(G12D) alleles,
respectively, to generate G22Cre; Apc(flox/flox); Kras(G12D) and G22Cre;
Apc(flox/flox); KrasWT mice. Gene expression profiles of the tumors were analyzed
using high-density oligonucleotide arrays.
RESULTS: Morphologically, minimal difference in proximal colon tumor was observed
between the two mouse models. Consistent with previous findings in vitro, Glut1
transcript and protein expression was up-regulated in the tumors of G22Cre;Apc
(flox/flox) ; Kras(G12D) mice. Immunohistochemical staining analysis revealed
that GLUT1 protein expression correlated with KRAS mutations in human colorectal 
cancer. Microarray analysis identified 11 candidate genes upregulated more than
fivefold and quantitative PCR analysis confirmed that Aqp8, Ttr, Qpct, and
Slc26a3 genes were upregulated 3.7- to 30.2-fold in tumors with mutant Kras.
CONCLUSIONS: These results demonstrated the validity of the G22Cre;
Apc(flox/flox) ;Kras (G12D) mice as a new mouse model with oncogenic Kras
activation. We believe that this model can facilitate efforts to define novel
factors that contribute to the pathogenesis of human colorectal cancer with KRAS 
mutations.

DOI: 10.1007/s00535-015-1121-9 
PMID: 26361962  [Indexed for MEDLINE]


110. J Infect Dis. 2016 May 1;213(9):1495-504. doi: 10.1093/infdis/jiv593. Epub 2016
Jan 18.

Transcriptomic Analysis of the Host Response and Innate Resilience to
Enterotoxigenic Escherichia coli Infection in Humans.

Yang WE(1), Suchindran S(2), Nicholson BP(3), McClain MT(4), Burke T(2), Ginsburg
GS(2), Harro CD(5), Chakraborty S(5), Sack DA(5), Woods CW(4), Tsalik EL(6).

Author information: 
(1)Duke University School of Medicine, Department of Medicine, Duke University
Center for Applied Genomics & Precision Medicine, Department of Medicine, Duke
University.
(2)Center for Applied Genomics & Precision Medicine, Department of Medicine, Duke
University.
(3)Center for Applied Genomics & Precision Medicine, Department of Medicine, Duke
University Internal Medicine Service, Durham VA Medical Center, Duke University
Medical Center, North Carolina.
(4)Center for Applied Genomics & Precision Medicine, Department of Medicine, Duke
University Internal Medicine Service, Durham VA Medical Center, Duke University
Medical Center, North Carolina Division of Infectious Diseases, Department of
Medicine, Duke University Medical Center, North Carolina.
(5)Department of International Health, Johns Hopkins Bloomberg School of Public
Health, Baltimore, Maryland.
(6)Center for Applied Genomics & Precision Medicine, Department of Medicine, Duke
University Division of Infectious Diseases, Department of Medicine, Duke
University Medical Center, North Carolina Emergency Medicine Service, Durham VA
Medical Center, North Carolina.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause
of diarrhea. Though usually self-limited, it can be severe and debilitating.
Little is known about the host transcriptional response to infection. We report
the first gene expression analysis of the human host response to experimental
challenge with ETEC.
METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and
collected serial blood samples shortly after inoculation and daily for 8 days. We
performed gene expression analysis on whole peripheral blood RNA samples from
subjects in whom severe symptoms developed (n = 6) and a subset of those who
remained asymptomatic (n = 6) despite shedding.
RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly 
different expression of 406 genes highlighting increased immune response and
decreased protein synthesis. Compared with asymptomatic subjects, symptomatic
subjects differentially expressed 254 genes primarily associated with immune
response. This comparison also revealed 29 genes differentially expressed between
groups at baseline, suggesting innate resilience to infection. Drug repositioning
analysis identified several drug classes with potential utility in augmenting
immune response or mitigating symptoms.
CONCLUSIONS: There are statistically significant and biologically plausible
differences in host gene expression induced by ETEC infection. Differential
baseline expression of some genes may indicate resilience to infection.

© The Author 2016. Published by Oxford University Press for the Infectious
Diseases Society of America. All rights reserved. For permissions, e-mail
journals.permissions@oup.com.

DOI: 10.1093/infdis/jiv593 
PMCID: PMC5007733
PMID: 26787651  [Indexed for MEDLINE]


111. Mol Biol Rep. 2016 May;43(5):371-9. doi: 10.1007/s11033-016-3967-1. Epub 2016 Mar
12.

cDNA cloning and molecular characterization of a defensin-like antimicrobial
peptide from larvae of Protaetia brevitarsis seulensis (Kolbe).

Lee J(1), Bang K(1), Hwang S(1), Cho S(2)(3).

Author information: 
(1)Department of Applied Biology, Division of Bioresource Science, College of
Agriculture and Life Science, Kangwon National University, Kangwon-do, Chuncheon,
200-701, Republic of Korea.
(2)Department of Applied Biology, Division of Bioresource Science, College of
Agriculture and Life Science, Kangwon National University, Kangwon-do, Chuncheon,
200-701, Republic of Korea. saeyoullcho@kangwon.ac.kr.
(3)Environmental Friendly Agriculture Center, Kangwon National University,
Chuncheon, Republic of Korea. saeyoullcho@kangwon.ac.kr.

We identified new defensin-like cDNA (called Psdefensin) by searching data set of
high-throughput RNA sequencing (RNA-seq) expression profiling of immunized larva 
of white-spotted flower chafers, Protaetia brevitarsis seulensis. The length of
the analyzed new defensin-like sequences were 240 base pair (bp) and encoded the 
deduced polypeptide of 79 amino acid residues with signal peptides (amino acids
1-20), pro-peptide region (amino acids 21-36), and mature peptide region (amino
acids 37-79). The Psdefensin transcript levels were slightly up-regulated at 4 h 
post-infection and were highly expressed at 8 h post-infection compared to
control larvae injected with sterile water. In addition, the Psdefensin did have 
antimicrobial activity against both Gram-negative bacteria, E. coli and
Gram-positive bacteria, B. subtilis suggesting potentially pharmacologic agent.

DOI: 10.1007/s11033-016-3967-1 
PMID: 26970764  [Indexed for MEDLINE]


112. PLoS One. 2016 Apr 22;11(4):e0153637. doi: 10.1371/journal.pone.0153637.
eCollection 2016.

Isolation of an Aptamer that Binds Specifically to E. coli.

Marton S(1), Cleto F(1), Krieger MA(1)(2), Cardoso J(1).

Author information: 
(1)Instituto de Biologia Molecular do Paraná, Department of Research and
Development, 3375 Professor Algacyr Munhoz Mader Street, Curitiba, Brazil.
(2)Instituto Carlos Chagas, Laboratório de Genomica Functional, 3375 Professor
Algacyr Munhoz Mader Street, Curitiba, Brazil.

Escherichia coli is a bacterial species found ubiquitously in the intestinal
flora of animals, although pathogenic variants cause major public health
problems. Aptamers are short oligonucleotides that bind to targets with high
affinity and specificity, and have great potential for use in diagnostics and
therapy. We used cell-based Systematic Evolution of Ligands by EXponential
enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that
bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the
nanomolar range. Fluorescently labeled aptamers label the surface of E. coli
cells, as viewed by fluorescent microscopy. Specificity tests with twelve
different bacterial species showed that one of the aptamers-called P12-31-is
highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis
associated E. coli (MNEC) clinical isolates, and is the first aptamer described
with potential for use in the diagnosis of MNEC-borne pathologies.

DOI: 10.1371/journal.pone.0153637 
PMCID: PMC4841571
PMID: 27104834  [Indexed for MEDLINE]


113. J Biotechnol. 2016 Apr 20;224:55-63. doi: 10.1016/j.jbiotec.2016.03.012. Epub
2016 Mar 10.

Tailoring of global transcription sigma D factor by random mutagenesis to improve
Escherichia coli tolerance towards low-pHs.

Gao X(1), Jiang L(2), Zhu L(3), Xu Q(4), Xu X(5), Huang H(6).

Author information: 
(1)College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Nanjing 210009, PR China. Electronic address: melindag@yeah.net.
(2)College of Food Science and Light Industry, Nanjing Tech University, Nanjing
210009, PR China. Electronic address: jiangling@njtech.edu.cn.
(3)College of Sciences, Nanjing Tech University, Nanjing 210009, PR China.
Electronic address: zlyhappy@njtech.edu.cn.
(4)College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Nanjing 210009, PR China. Electronic address:
xuqing357@njtech.edu.cn.
(5)College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Nanjing 210009, PR China. Electronic address: xuxian@njtech.edu.cn.
(6)College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, Nanjing 210009, PR China. Electronic address: biotech@njtech.edu.cn.

Bioconversion processes of organic acid or acid hydrolysis of raw material for
microbial metabolism often suffer limitations as a result of microbial
sensitivity in low-pH conditions. We adopted a three-step method called RAndom
Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the
components of global regulator Sigma D factor (RpoD) of Escherichia coli to
improve its acid tolerance. The best strain Mutant VII was identified from random
mutagenesis libraries based on the growth performance, which exhibited much
higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as
3.17. Combined transcriptome and phenome analysis of E. coli was carried out to
better understand the global effects of RpoD on the regulatory networks. Our
analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%)
genes were repressed, including those for trehalose biosynthesis, nucleotides
biosynthesis, carbon metabolism, amino acid utilization, except for acid
resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS)
and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR).
These results demonstrated that RpoD acts as global regulator in the growth phase
of E. coli and consequently improves acid tolerances.

Copyright © 2016 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jbiotec.2016.03.012 
PMID: 26971973  [Indexed for MEDLINE]


114. Nat Microbiol. 2016 Apr 4;1:16041. doi: 10.1038/nmicrobiol.2016.41.

Identifying lineage effects when controlling for population structure improves
power in bacterial association studies.

Earle SG(#)(1), Wu CH(#)(1), Charlesworth J(#)(1), Stoesser N(1), Gordon NC(1),
Walker TM(1), Spencer CCA(2), Iqbal Z(2), Clifton DA(3), Hopkins KL(4), Woodford 
N(4), Smith EG(5), Ismail N(6)(7), Llewelyn MJ(8), Peto TE(1), Crook DW(1),
McVean G(2)(9), Walker AS(1), Wilson DJ(1)(2).

Author information: 
(1)Nuffield Department of Medicine, University of Oxford, John Radcliffe
Hospital, Oxford OX3 9DU, UK.
(2)Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt
Drive, Oxford OX3 7BN, UK.
(3)Institute of Biomedical Engineering, Department of Engineering Science,
University of Oxford, Oxford OX3 7DQ, UK.
(4)Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, 
Public Health England, London NW9 5EQ, UK.
(5)Public Health England, West Midlands Public Health Laboratory, Heartlands
Hospital, Birmingham B9 5SS, UK.
(6)Centre for Tuberculosis, National Institute for Communicable Diseases,
Johannesburg 2131 South Africa.
(7)Department of Medical Microbiology, University of Pretoria, Pretoria, South
Africa.
(8)Department of Infectious Diseases and Microbiology, Royal Sussex County
Hospital, Brighton BN2 5BE, UK.
(9)The Li Ka Shing Centre for Health Information and Discovery, University of
Oxford, Oxford OX3 7FZ, UK.
(#)Contributed equally

Bacteria pose unique challenges for genome-wide association studies because of
strong structuring into distinct strains and substantial linkage disequilibrium
across the genome(1,2). Although methods developed for human studies can correct 
for strain structure(3,4), this risks considerable loss-of-power because genetic 
differences between strains often contribute substantial phenotypic
variability(5). Here, we propose a new method that captures lineage-level
associations even when locus-specific associations cannot be fine-mapped. We
demonstrate its ability to detect genes and genetic variants underlying
resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse
clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus
aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection,
recombination and penetrance confer high power to recover known antimicrobial
resistance mechanisms and reveal a candidate association between the outer
membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method
pinpoints locus-specific effects where possible and boosts power by detecting
lineage-level differences when fine-mapping is intractable.

DOI: 10.1038/nmicrobiol.2016.41 
PMCID: PMC5049680
PMID: 27572646  [Indexed for MEDLINE]

Conflict of interest statement: The authors declare no competing financial
interests.


115. Biotechnol Biofuels. 2016 Mar 22;9:71. doi: 10.1186/s13068-016-0487-0.
eCollection 2016.

Crystal structure and biochemical characterization of the recombinant ThBgl, a
GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass
degradation conditions.

Santos CA(#)(1), Zanphorlin LM(#)(2), Crucello A(1), Tonoli CCC(3), Ruller R(2), 
Horta MAC(1), Murakami MT(3), de Souza AP(1)(4).

Author information: 
(1)Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de
Campinas, Campinas, SP Brazil.
(2)Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de 
Pesquisa em Energia e Materiais, Campinas, SP Brazil.
(3)Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e 
Materiais, Campinas, SP Brazil.
(4)Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual
de Campinas, Campinas, SP Brazil.
(#)Contributed equally

BACKGROUND: The conversion of biomass-derived sugars via enzymatic hydrolysis for
biofuel production is a challenge. Therefore, the search for microorganisms and
key enzymes that increase the efficiency of the saccharification of cellulosic
substrates remains an important and high-priority area of study. Trichoderma
harzianum is an important fungus known for producing high levels of cellulolytic 
enzymes that can be used for cellulosic ethanol production. In this context,
β-glucosidases, which act synergistically with cellobiohydrolases and
endo-β-1,4-glucanases in the saccharification process, are potential biocatalysts
for the conversion of plant biomass to free glucose residues.
RESULTS: In the present study, we used RNA-Seq and genomic data to identify the
major β-glucosidase expressed by T. harzianum under biomass degradation
conditions. We mapped and quantified the expression of all of the β-glucosidases 
from glycoside hydrolase families 1 and 3, and we identified the enzyme with the 
highest expression under these conditions. The target gene was cloned and
heterologously expressed in Escherichia coli, and the recombinant protein
(rThBgl) was purified with high yields. rThBgl was characterized using a
comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques.
Finally, we determined the crystallographic structure of the recombinant protein 
at a resolution of 2.6 Å.
CONCLUSIONS: Using a rational approach, we investigated the biochemical
characteristics and determined the three-dimensional protein structure of a
β-glucosidase that is highly expressed by T. harzianum under biomass degradation 
conditions. The methodology described in this manuscript will be useful for the
bio-prospection of key enzymes, including cellulases and other accessory enzymes,
for the development and/or improvement of enzymatic cocktails designed to produce
ethanol from plant biomass.

DOI: 10.1186/s13068-016-0487-0 
PMCID: PMC4802607
PMID: 27006690 


116. Nucleic Acids Res. 2016 Mar 18;44(5):2058-74. doi: 10.1093/nar/gkw051. Epub 2016 
Feb 3.

Transcription profile of Escherichia coli: genomic SELEX search for regulatory
targets of transcription factors.

Ishihama A(1), Shimada T(2), Yamazaki Y(3).

Author information: 
(1)Micro-Nano Technology Research Center, Hosei University, Koganei, Tokyo,
184-8584, Japan aishiham@hosei.ac.jp.
(2)Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda,
Yokohama 226-8503, Japan.
(3)National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
yyamazak@nig.ac.jp.

Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which
achieves gene selectivity through interaction with sigma factors that recognize
promoters, and transcription factors (TFs) that control the activity and
specificity of RNAP holoenzyme. To understand the molecular mechanisms of
transcriptional regulation, the identification of regulatory targets is needed
for all these factors. We then performed genomic SELEX screenings of targets
under the control of each sigma factor and each TF. Here we describe the assembly
of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence
of effector ligands. The results reveal several novel concepts: (i) each TF
regulates more targets than hitherto recognized; (ii) each promoter is regulated 
by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are
located within operons and even inside open reading frames. The binding sites of 
a set of global regulators, including cAMP receptor protein, LeuO and Lrp,
overlap with those of the silencer H-NS, suggesting that certain global
regulators play an anti-silencing role. To facilitate sharing of these
accumulated SELEX datasets with the research community, we compiled a database,
'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/).

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkw051 
PMCID: PMC4797297
PMID: 26843427  [Indexed for MEDLINE]


117. Database (Oxford). 2016 Mar 15;2016. pii: baw003. doi: 10.1093/database/baw003.
Print 2016.

Effect of database drift on network topology and enrichment analyses: a case
study for RegulonDB.

Beber ME(1), Muskhelishvili G(2), Hütt MT(3).

Author information: 
(1)Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1,
Bremen 28759, Germany and Bioinformatics Group, Max Planck Institute for
Molecular Genetics, Ihnestraße 63-73, Berlin 14195, Germany
m.huett@jacobs-university.de.
(2)Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1,
Bremen 28759, Germany and.
(3)Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1,
Bremen 28759, Germany and m.huett@jacobs-university.de.

RegulonDB is a database storing the biological information behind the
transcriptional regulatory network (TRN) of the bacterium Escherichia coli. It is
one of the key bioinformatics resources for Systems Biology investigations of
bacterial gene regulation. Like most biological databases, the content drifts
with time, both due to the accumulation of new information and due to refinements
in the underlying biological concepts. Conclusions based on previous database
versions may no longer hold. Here, we study the change of some topological
properties of the TRN of E. coli, as provided by RegulonDB across 16 versions, as
well as a simple index, digital control strength, quantifying the match between
gene expression profiles and the transcriptional regulatory networks. While many 
of network characteristics change dramatically across the different versions, the
digital control strength remains rather robust and in tune with previous results 
for this index. Our study shows that: (i) results derived from network topology
should, when possible, be studied across a range of database versions, before
detailed biological conclusions are derived, and (ii) resorting to simple
indices, when interpreting high-throughput data from a network perspective, may
help achieving a robustness of the findings against variation of the underlying
biological information. Database URL: www.regulondb.ccg.unam.mx.

© The Author(s) 2016. Published by Oxford University Press.

DOI: 10.1093/database/baw003 
PMCID: PMC4792529
PMID: 26980514  [Indexed for MEDLINE]


118. PLoS One. 2016 Mar 9;11(3):e0151284. doi: 10.1371/journal.pone.0151284.
eCollection 2016.

Comparative Transcriptomic Analysis of Rectal Tissue from Beef Steers Revealed
Reduced Host Immunity in Escherichia coli O157:H7 Super-Shedders.

Wang O(1), Liang G(1), McAllister TA(2), Plastow G(1), Stanford K(3), Selinger
B(4), Guan le L(1).

Author information: 
(1)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, Canada.
(2)Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, 
Canada.
(3)Alberta Agriculture and Forestry, Lethbridge, AB, Canada.
(4)Biological Sciences Department, University of Lethbridge, Lethbridge, AB,
Canada.

Super-shedder cattle are a major disseminator of E. coli O157:H7 into the
environment, and the terminal rectum has been proposed as the primary E. coli
O157:H7 colonization site. This study aimed to identify host factors that are
associated with the super-shedding process by comparing transcriptomic profiles
in rectal tissue collected from 5 super-shedder cattle and 4 non-shedder cattle
using RNA-Seq. In total, 17,859 ± 354 genes and 399 ± 16 miRNAs were detected,
and 11,773 genes were expressed in all animals. Fifty-eight differentially
expressed (DE) genes (false discovery rate < 0.05) including 11 up-regulated and 
47 down-regulated (log 2 (fold change) ranged from -5.5 to 4.2), and 2
up-regulated DE miRNAs (log 2 (fold change) = 2.1 and 2.5, respectively) were
identified in super-shedders compared to non-shedders. Functional analysis of DE 
genes revealed that 31 down-regulated genes were potentially associated with
reduced innate and adaptive immune functions in super-shedders, including 13
lymphocytes membrane receptors, 3 transcription factors and 5 cytokines,
suggesting the decreased key host immune functions in the rectal tissue of
super-shedders, including decreased quantity and migration of immune cells such
as lymphocytes, neutrophils and dendritic cells. The up-regulation of
bta-miR-29d-3p and the down regulation of its predicted target gene, regulator of
G-protein signaling 13, suggested a potential regulatory role of this miRNA in
decreased migration of lymphocytes in super-shedders. Based on these findings,
the rectal tissue of super-shedders may inherently exhibit less effective innate 
and adaptive immune protection. Further study is required to confirm if such
effect on host immunity is due to the nature of the host itself or due to actions
mediated by E. coli O157:H7.

DOI: 10.1371/journal.pone.0151284 
PMCID: PMC4784738
PMID: 26959367  [Indexed for MEDLINE]


119. Pediatr Res. 2016 Mar;79(3):473-81. doi: 10.1038/pr.2015.233. Epub 2015 Nov 5.

Umbilical cord gene expression reveals the molecular architecture of the fetal
inflammatory response in extremely preterm newborns.

Costa D(1), Castelo R(2)(3).

Author information: 
(1)Department of Pediatrics, Hospital de Figueres, Figueres, Spain.
(2)Department of Experimental and Health Sciences, Universitat Pompeu Fabra,
Barcelona, Spain.
(3)Research Program on Biomedical Informatics, Institut Hospital del Mar
d'Investigacions Mèdiques, Barcelona, Spain.

BACKGROUND: The fetal inflammatory response (FIR) in placental membranes to an
intrauterine infection often precedes premature birth raising neonatal mortality 
and morbidity. However, the precise molecular events behind FIR still remain
largely unknown, and little has been investigated at gene expression level.
METHODS: We collected publicly available microarray expression data profiling
umbilical cord (UC) tissue derived from the cohort of extremely low gestational
age newborns (ELGANs) and interrogate them for differentially expressed (DE)
genes between FIR and non-FIR-affected ELGANs.
RESULTS: We found a broad and complex FIR UC gene expression signature, changing 
up to 19% (3,896/20,155) of all human genes at 1% false discovery rate.
Significant changes of a minimum 50% magnitude (1,097/3,896) affect the
upregulation of many inflammatory pathways and molecules, such as cytokines,
toll-like receptors, and calgranulins. Remarkably, they also include the
downregulation of neurodevelopmental pathways and genes, such as Fragile-X mental
retardation 1 (FMR1), contactin 1 (CNTN1), and adenomatous polyposis coli (APC).
CONCLUSION: The FIR expression signature in UC tissue contains molecular clues
about signaling pathways that trigger FIR, and it is consistent with an acute
inflammatory response by fetal innate and adaptive immune systems, which
participate in the pathogenesis of neonatal brain damage.

DOI: 10.1038/pr.2015.233 
PMCID: PMC4823644
PMID: 26539667  [Indexed for MEDLINE]


120. Front Microbiol. 2016 Feb 19;7:193. doi: 10.3389/fmicb.2016.00193. eCollection
2016.

RNA-seq Profiling Reveals Novel Target Genes of LexA in the Cyanobacterium
Synechocystis sp. PCC 6803.

Kizawa A(1), Kawahara A(2), Takimura Y(2), Nishiyama Y(1), Hihara Y(3).

Author information: 
(1)Department of Biochemistry and Molecular Biology, Graduate School of Science
and Engineering, Saitama University Saitama, Japan.
(2)Biological Science Laboratories, KAO Corporation Wakayama, Japan.
(3)Department of Biochemistry and Molecular Biology, Graduate School of Science
and Engineering, Saitama UniversitySaitama, Japan; Core Research of Evolutional
Science and Technology, Japan Science and Technology AgencySaitama, Japan.

LexA is a well-established transcriptional repressor of SOS genes induced by DNA 
damage in Escherichia coli and other bacterial species. However, LexA in the
cyanobacterium Synechocystis sp. PCC 6803 has been suggested not to be involved
in SOS response. In this study, we performed RNA-seq analysis of the wild-type
strain and the lexA-disrupted mutant to obtain the comprehensive view of
LexA-regulated genes in Synechocystis. Disruption of lexA positively or
negatively affected expression of genes related to various cellular functions
such as phototactic motility, accumulation of the major compatible solute
glucosylglycerol and subunits of bidirectional hydrogenase, photosystem I, and
phycobilisome complexes. We also observed increase in the expression level of
genes related to iron and manganese uptake in the mutant at the later stage of
cultivation. However, none of the genes related to DNA metabolism were affected
by disruption of lexA. DNA gel mobility shift assay using the recombinant LexA
protein suggested that LexA binds to the upstream region of pilA7, pilA9, ggpS,
and slr1670 to directly regulate their expression, but changes in the expression 
level of photosystem I genes by disruption of lexA is likely a secondary effect.

DOI: 10.3389/fmicb.2016.00193 
PMCID: PMC4759255
PMID: 26925056 


121. Nucleic Acids Res. 2016 Feb 18;44(3):1256-70. doi: 10.1093/nar/gkv1370. Epub 2016
Jan 5.

tRNA is a new target for cleavage by a MazF toxin.

Schifano JM(1), Cruz JW(1), Vvedenskaya IO(2), Edifor R(3), Ouyang M(4), Husson
RN(3), Nickels BE(5), Woychik NA(6).

Author information: 
(1)Department of Biochemistry and Molecular Biology, Rutgers University, Robert
Wood Johnson Medical School, Piscataway, NJ, USA.
(2)Waksman Institute, Rutgers University, Piscataway, NJ, USA Department of
Genetics, Rutgers University, Piscataway, NJ, USA.
(3)Division of Infectious Diseases, Boston Children's Hospital/Harvard Medical
School, Boston, MA, USA.
(4)Department of Computer Science, University of Massachusetts Boston, Boston,
MA, USA.
(5)Waksman Institute, Rutgers University, Piscataway, NJ, USA Department of
Genetics, Rutgers University, Piscataway, NJ, USA Member, Rutgers Cancer
Institute of New Jersey, NJ, USA.
(6)Department of Biochemistry and Molecular Biology, Rutgers University, Robert
Wood Johnson Medical School, Piscataway, NJ, USA Member, Rutgers Cancer Institute
of New Jersey, NJ, USA nancy.woychik@rutgers.edu.

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm
formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA
system is a sequence- and single-strand-specific endoribonuclease, and many
studies have led to the proposal that MazF family members exclusively target
mRNA. However, recent data indicate some MazF toxins can cleave specific sites
within rRNA in concert with mRNA. In this report, we identified the repertoire of
RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based 
approach. This analysis revealed that two tRNAs were the principal targets of
MazF-mt9, and each was cleaved at a single site in either the tRNA(Pro14) D-loop 
or within the tRNA(Lys43) anticodon. This highly selective target discrimination 
occurs through recognition of not only sequence but also structural determinants.
Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to
require RNA structure for recognition and cleavage. Interestingly, the tRNase
activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also
generate stable tRNA-derived fragments that can inhibit translation in response
to stress. Our data also suggest a role for tRNA distinct from its canonical
adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates
bacterial growth.

© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkv1370 
PMCID: PMC4756823
PMID: 26740583  [Indexed for MEDLINE]


122. Microb Cell Fact. 2016 Feb 6;15:30. doi: 10.1186/s12934-016-0431-9.

Effect of cerulenin on fatty acid composition and gene expression pattern of
DHA-producing strain Colwellia psychrerythraea strain 34H.

Wan X(1)(2), Peng YF(3), Zhou XR(4)(5), Gong YM(6), Huang FH(7)(8), Moncalián
G(9).

Author information: 
(1)Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of
Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural
Sciences, Wuhan, 430062, China. wanxia@oilcrops.cn.
(2)Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan, 430062, China.
wanxia@oilcrops.cn.
(3)Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of
Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural
Sciences, Wuhan, 430062, China. 1171418260@qq.com.
(4)CSIRO Agriculture, Canberra, ACT, 2601, Australia. Xue-Rong.Zhou@csiro.au.
(5)CSIRO Food and Nutrition, Canberra, ACT, 2601, Australia.
Xue-Rong.Zhou@csiro.au.
(6)Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of
Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural
Sciences, Wuhan, 430062, China. gongym@oilcrops.cn.
(7)Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of
Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural
Sciences, Wuhan, 430062, China. huangfh@oilcrops.cn.
(8)Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan, 430062, China.
huangfh@oilcrops.cn.
(9)Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología 
de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, C/Albert Einstein 22,
39011, Santander, Spain. gabriel.moncalian@unican.es.

BACKGROUND: Colwellia psychrerythraea 34H is a psychrophilic bacterium able to
produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be 
responsible for DHA production in marine bacteria.
RESULTS: Five pfa genes from strain 34H were confirmed to be responsible for DHA 
formation by heterogeneous expression in Escherichia coli. The complexity of
fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of
cells with cerulenin resulted in significantly reduced level of C16
monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of
saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and
3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA
sequencing (RNA-Seq) revealed the altered gene expression pattern when C.
psychrerythraea cells were treated with cerulenin. Genes involved in polyketide
synthase pathway and fatty acid biosynthesis pathway were not obviously affected 
by cerulenin treatment. In contrast, several genes involved in fatty acid
degradation or β-oxidation pathway were dramatically reduced at the
transcriptional level.
CONCLUSIONS: Genes responsible for DHA formation in C. psychrerythraea was first 
cloned and characterized. We revealed the complexity of fatty acid profile in
this DHA-producing strain. Cerulenin could substantially change the fatty acid
composition by affecting the fatty acid degradation at transcriptional level.
Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation
pathway may be important to the selectivity of degraded fatty acids. In addition,
inhibition of FabB protein by cerulenin may lead to the accumulation of
malonyl-CoA, which is the substrate for DHA formation.

DOI: 10.1186/s12934-016-0431-9 
PMCID: PMC4744452
PMID: 26852325  [Indexed for MEDLINE]


123. Genom Data. 2016 Feb 3;7:293-6. doi: 10.1016/j.gdata.2016.02.001. eCollection
2016 Mar.

Differential RNA-seq analysis comparing APC-defective and APC-restored SW480
colorectal cancer cells.

King LE(1), Love CG(2), Sieber OM(3), Faux MC(1), Burgess AW(4).

Author information: 
(1)Structural Biology Division, The Walter and Eliza Hall Institute of Medical
Research, Parkville, Victoria, Australia; Department of Medical Biology, The
University of Melbourne, Parkville, Victoria, Australia; Tumour Biology Branch,
Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
(2)Department of Medical Biology, The University of Melbourne, Parkville,
Victoria, Australia; Tumour Biology Branch, Ludwig Institute for Cancer Research,
Parkville, Victoria, Australia; Systems Biology and Personalised Medicine
Division, The Walter and Eliza Hall Institute of Medical Research, Parkville,
Victoria, Australia.
(3)Department of Medical Biology, The University of Melbourne, Parkville,
Victoria, Australia; Tumour Biology Branch, Ludwig Institute for Cancer Research,
Parkville, Victoria, Australia; Systems Biology and Personalised Medicine
Division, The Walter and Eliza Hall Institute of Medical Research, Parkville,
Victoria, Australia; School of Biomedical Sciences, Monash University, Australia;
Department Surgery, RMH, University of Melbourne, Parkville, Victoria, Australia.
(4)Structural Biology Division, The Walter and Eliza Hall Institute of Medical
Research, Parkville, Victoria, Australia; Department of Medical Biology, The
University of Melbourne, Parkville, Victoria, Australia; Tumour Biology Branch,
Ludwig Institute for Cancer Research, Parkville, Victoria, Australia; Department 
Surgery, RMH, University of Melbourne, Parkville, Victoria, Australia.

The adenomatous polyposis coli (APC) tumour suppressor gene is mutated in about
80% of colorectal cancers (CRC) Brannon et al. (2014) [1]. APC is a large
multifunctional protein that regulates many biological functions including Wnt
signalling (through the regulation of beta-catenin stability) Reya and Clevers
(2005) [2], cell migration Kroboth et al. (2007), Sansom et al. (2004) [3], [4], 
mitosis Kaplan et al. (2001) [5], cell adhesion Faux et al. (2004), Carothers et 
al. (2001) [6], [7] and differentiation Sansom et al. (2004) [4]. Although the
role of APC in CRC is often described as the deregulation of Wnt signalling, its 
other biological functions suggest that there are other factors at play that
contribute to the onset of adenomas and the progression of CRC upon the
truncation of APC. To identify genes and pathways that are dysregulated as a
consequence of loss of function of APC, we compared the gene expression profiles 
of the APC mutated human CRC cell line SW480 following reintroduction of
wild-type APC (SW480 + APC) or empty control vector (SW480 + vector control) Faux
et al. (2004) . Here we describe the RNA-seq data derived for three biological
replicates of parental SW480, SW480 + vector control and SW480 + APC cells, and
present the bioinformatics pipeline used to test for differential gene expression
and pathway enrichment analysis. A total of 1735 genes showed significant
differential expression when APC was restored and were enriched for genes
associated with cell polarity, Wnt signalling and the epithelial to mesenchymal
transition. There was additional enrichment for genes involved in cell-cell
adhesion, cell-matrix junctions, angiogenesis, axon morphogenesis and cell
movement. The raw and analysed RNA-seq data have been deposited in the Gene
Expression Omnibus (GEO) database under accession number GSE76307. This dataset
is useful for further investigations of the impact of APC mutation on the
properties of colorectal cancer cells.

DOI: 10.1016/j.gdata.2016.02.001 
PMCID: PMC4778681
PMID: 26981430 


124. Appl Biochem Biotechnol. 2016 Feb;178(4):849-64. doi: 10.1007/s12010-015-1913-7. 
Epub 2015 Nov 5.

Selection of Nucleic Acid Aptamers Specific for Mycobacterium tuberculosis.

Mozioglu E(1)(2), Gokmen O(3), Tamerler C(4)(5), Kocagoz ZT(6)(7), Akgoz M(8).

Author information: 
(1)Molecular Biology-Biotechnology & Genetics Research Center, Istanbul Technical
University, Istanbul, Turkey. erkanmozioglu@yahoo.com.
(2)Bioanalysis Laboratory, TÜBİTAK UME (National Metrology Institute), Kocaeli,
Turkey. erkanmozioglu@yahoo.com.
(3)Chemistry Department, Gebze Institute of Technology, Kocaeli, Turkey.
ozgurgokmen75@hotmail.com.
(4)Molecular Biology-Biotechnology & Genetics Research Center, Istanbul Technical
University, Istanbul, Turkey. candan.tamerler@gmail.com.
(5)Mechanical Engineering and Bioengineering Research Center, University of
Kansas, Lawrence, KS, USA. candan.tamerler@gmail.com.
(6)Department of Microbiology and Clinical Microbiology, Acıbadem University,
Istanbul, Turkey. tanilkocagoz@gmail.com.
(7)Trends in Innovative Biotechnology Organization, Istanbul, Turkey.
tanilkocagoz@gmail.com.
(8)Bioanalysis Laboratory, TÜBİTAK UME (National Metrology Institute), Kocaeli,
Turkey. muslum.akgoz@tubitak.gov.tr.

Tuberculosis (TB) remains to be a major global health problem, with about 9
million new cases and 1.4 million deaths in 2011. For the control of tuberculosis
as well as other infectious diseases, WHO recommended "ASSURED" (Affordable,
Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and
Deliverable to the end user) diagnostic tools that can easily be maintained and
used in developing countries. Aptamers are promising tools for developing
point-of-care diagnostic assays for TB. In this study, ssDNA aptamers that
recognize Mycobacterium tuberculosis H37Ra were selected by systematic evolution 
of ligands by exponential enrichment (SELEX). For this purpose, two different
selection protocols, ultrafiltration and centrifugation, were applied. A total of
21 TB specific aptamers were selected. These aptamers exhibited "G-rich" regions 
on the 3' terminus of the aptamers, including a motif of "TGGGG," "GTGG," or
"CTGG." Binding capability of selected aptamers were investigated by quantitative
PCR and Mtb36 DNA aptamer was found the most specific aptamer to M. tuberculosis 
H37Ra. The dissociation constant (K d) of Mtb36 aptamer was calculated as
5.09 ± 1.43 nM in 95% confidence interval. Relative binding ratio of Mtb36
aptamer to M. tuberculosis H37Ra over Mycobacterium bovis and Escherichia coli
was also determined about 4 times and 70 times more, respectively. Mtb36 aptamer 
is highly selective for M. tuberculosis, and it can be used in an aptamer-based
biosensor for the detection of M. tuberculosis.

DOI: 10.1007/s12010-015-1913-7 
PMID: 26541162  [Indexed for MEDLINE]


125. Appl Biochem Biotechnol. 2016 Feb;178(3):527-43. doi: 10.1007/s12010-015-1892-8. 
Epub 2015 Oct 15.

Transcriptomic Analysis of 3-Hydroxypropanoic Acid Stress in Escherichia coli.

Yung TW(1), Jonnalagadda S(2), Balagurunathan B(2), Zhao H(3).

Author information: 
(1)Industrial Biotechnology Division, Institute of Chemical and Engineering
Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road,
Jurong Island, Singapore, 627833, Singapore.
(2)Process Science and Modelling Division, Institute of Chemical and Engineering 
Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road,
Jurong Island, Singapore, 627833, Singapore.
(3)Industrial Biotechnology Division, Institute of Chemical and Engineering
Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road,
Jurong Island, Singapore, 627833, Singapore. zhao_hua@ices.a-star.edu.sg.

The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was
elucidated through global transcriptomic analysis. Around 375 genes showed
difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed
that the toxicity effect of 3-HP was due to the cation and anion components of
this acid and some effects-specific to 3-HP. Genes related to the oxidative
stress, DNA protection, and repair were upregulated in treated cells due to the
lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used
the arginine acid tolerance mechanism to increase the cytoplasmic pH.
Additionally, the anion effects were manifested as imbalance in the osmotic
pressure. Analysis of top ten highly upregulated genes suggests the formation of 
3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed
light on the global genetic reprogramming due to 3-HP stress and suggests
strategies for increasing the tolerance of E. coli toward 3-HP.

DOI: 10.1007/s12010-015-1892-8 
PMID: 26472673  [Indexed for MEDLINE]


126. J Biol Chem. 2016 Jan 29;291(5):2357-70. doi: 10.1074/jbc.M115.696815. Epub 2015 
Dec 3.

Interaction of the RcsB Response Regulator with Auxiliary Transcription
Regulators in Escherichia coli.

Pannen D(1), Fabisch M(1), Gausling L(1), Schnetz K(2).

Author information: 
(1)From the Institute for Genetics, University of Cologne, Zülpicher Strasse 47a,
50674 Cologne, Germany.
(2)From the Institute for Genetics, University of Cologne, Zülpicher Strasse 47a,
50674 Cologne, Germany schnetz@uni-koeln.de.

The Rcs phosphorelay is a two-component signal transduction system that is
induced by cell envelope stress. RcsB, the response regulator of this signaling
system, is a pleiotropic transcription regulator, which is involved in the
control of various stress responses, cell division, motility, and biofilm
formation. RcsB regulates transcription either as a homodimer or together with
auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli. In this
study, we show that RcsB in addition forms heterodimers with MatA (also known as 
EcpR) and with DctR. Our data suggest that the MatA-dependent transcription
regulation is mediated by the MatA-RcsB heterodimer and is independent of RcsB
phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of
the active quintet of conserved residues, and of surface-exposed residues for
activity of RcsB. The data suggest that the activity of the
phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by
mutation of residues in the vicinity of the phosphorylation site, suggesting that
a phosphorylation-induced structural change modulates their activity. In
contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB
are affected by only very few mutations. Heterodimerization of RcsB with various 
auxiliary regulators and their differential dependence on phosphorylation add an 
additional level of control to the Rcs system that is operating at the output
level.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI: 10.1074/jbc.M115.696815 
PMCID: PMC4732218
PMID: 26635367  [Indexed for MEDLINE]


127. Pathogens. 2016 Jan 15;5(1). pii: E7. doi: 10.3390/pathogens5010007.

Measuring Escherichia coli Gene Expression during Human Urinary Tract Infections.

Mobley HL(1).

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109, USA. hmobley@umich.edu.

Extraintestinal Escherichia coli (E. coli) evolved by acquisition of
pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene
transfer. Strains are heterogeneous but virulent uropathogenic isolates more
often have specific fimbriae, toxins, and iron receptors than commensal strains. 
One may ask whether it is the virulence factors alone that are required to
establish infection. While these virulence factors clearly contribute strongly to
pathogenesis, bacteria must survive by metabolizing nutrients available to them. 
By constructing mutants in all major metabolic pathways and co-challenging mice
transurethrally with each mutant and the wild type strain, we identified which
major metabolic pathways are required to infect the urinary tract. We must also
ask what else is E. coli doing in vivo? To answer this question, we examined the 
transcriptome of E. coli CFT073 in the murine model of urinary tract infection
(UTI) as well as for E. coli strains collected and analyzed directly from the
urine of patients attending either a urology clinic or a university health clinic
for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene
expression for these uropathogenic E. coli strains, identifying genes upregulated
during murine and human UTI. Our findings allow us to propose a new definition of
bacterial virulence.

DOI: 10.3390/pathogens5010007 
PMCID: PMC4810128
PMID: 26784237 


128. BMC Plant Biol. 2016 Jan 5;16:4. doi: 10.1186/s12870-015-0698-8.

Identification and functional analysis of the geranylgeranyl pyrophosphate
synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid
biosynthesis in Euglena gracilis.

Kato S(1)(2), Takaichi S(3), Ishikawa T(4), Asahina M(5), Takahashi S(6),
Shinomura T(7)(8).

Author information: 
(1)Department of Biosciences, School of Science and Engineering, Teikyo
University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
shota.kato.680@gmail.com.
(2)Plant Molecular and Cellular Biology Laboratory, Department of Biosciences,
School of Science and Engineering, Teikyo University, 1-1 Toyosatodai,
Utsunomiya, Tochigi, 320-8551, Japan. shota.kato.680@gmail.com.
(3)Department of Biology, Nippon Medical School, 1-7-1 Kyonan-cho, Musashino,
Tokyo, 180-0023, Japan. takaichi@nms.ac.jp.
(4)Department of Life Science and Biotechnology, Faculty of Life and
Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane,
690-8504, Japan. ishikawa@life.shimane-u.ac.jp.
(5)Department of Biosciences, School of Science and Engineering, Teikyo
University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
asahina@nasu.bio.teikyo-u.ac.jp.
(6)Department of Biosciences, School of Science and Engineering, Teikyo
University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
takahasi@nasu.bio.teikyo-u.ac.jp.
(7)Department of Biosciences, School of Science and Engineering, Teikyo
University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
shinomura@nasu.bio.teikyo-u.ac.jp.
(8)Plant Molecular and Cellular Biology Laboratory, Department of Biosciences,
School of Science and Engineering, Teikyo University, 1-1 Toyosatodai,
Utsunomiya, Tochigi, 320-8551, Japan. shinomura@nasu.bio.teikyo-u.ac.jp.

BACKGROUND: Euglena gracilis, a unicellular phytoflagellate within Euglenida, has
attracted much attention as a potential feedstock for renewable energy
production. In outdoor open-pond cultivation for biofuel production, excess
direct sunlight can inhibit photosynthesis in this alga and decrease its
productivity. Carotenoids play important roles in light harvesting during
photosynthesis and offer photoprotection for certain non-photosynthetic and
photosynthetic organisms including cyanobacteria, algae, and higher plants.
Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin,
diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid
biosynthesis has not been elucidated.
RESULTS: To clarify the carotenoid biosynthetic pathway in E. gracilis, we
searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP)
synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from
RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia
coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E.
gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene
synthase, respectively. Phylogenetic analyses indicated that the predicted
proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP
synthase and phytoene synthase proteins, respectively, of algae and higher
plants. In addition, we investigated the effects of light stress on the
expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920
μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 %
and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2)
s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal
cells turned reddish-orange and showed a 1.3-fold increase in the crtB
expression. In contrast, EgcrtE expression was not significantly affected by the 
light-stress treatments examined.
CONCLUSIONS: We identified genes encoding CrtE and CrtB in E. gracilis and found 
that their protein products catalyze the early steps of carotenoid biosynthesis. 
Further, we found that the response of the carotenoid biosynthetic pathway to
light stress in E. gracilis is controlled, at least in part, by the level of crtB
transcription. This is the first functional analysis of crtE and crtB in Euglena.

DOI: 10.1186/s12870-015-0698-8 
PMCID: PMC4702402
PMID: 26733341  [Indexed for MEDLINE]


129. Anal Sci. 2016;32(2):215-8. doi: 10.2116/analsci.32.215.

Establishment and Application of a Visual DNA Microarray for the Detection of
Food-borne Pathogens.

Li Y(1).

Author information: 
(1)Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and
Development, Key Laboratory of Aquatic Animal Genetic Breeding and Nutrition,
Chinese Academy of Fishery Sciences, Huzhou University.

The accurate detection and identification of food-borne pathogenic microorganisms
is critical for food safety nowadays. In the present work, a visual DNA
microarray was established and applied to detect pathogens commonly found in
food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and
Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to
simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for
Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively.
Biotinylated PCR amplicons annealed to the microarray probes were then reacted
with a streptavidin-alkaline phosphatase conjugate and nitro blue
tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP);
the positive results were easily visualized as blue dots formatted on the
microarray surface. The performance of a DNA microarray was tested against 14
representative collection strains and mock-contamination food samples. The
combination of mPCR and a visual micro-plate chip specifically and sensitively
detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria
monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) 
CFU/mL of bacterial culture. Thus, the developed method is advantageous because
of its high throughput, cost-effectiveness and ease of use.

DOI: 10.2116/analsci.32.215 
PMID: 26860568  [Indexed for MEDLINE]


130. Avian Pathol. 2016;45(1):94-105. doi: 10.1080/03079457.2015.1125995.

In vivo influence of in vitro up-regulated genes in the virulence of an APEC
strain associated with swollen head syndrome.

de Paiva JB(1), da Silva LP(1), Casas MR(2), Conceição RA(1), Nakazato G(3), de
Pace F(4), Sperandio V(5), da Silveira WD(1).

Author information: 
(1)a Department of Genetics, Evolution and Bioagents , Institute of Biology,
University of Campinas , Campinas , SP , Brazil.
(2)b Adolfo Lutz Institute , São Paulo , SP , Brazil.
(3)c Department of Microbiology , Londrina State University , Londrina , PR ,
Brazil.
(4)d Department of Clinical Medicine, Faculty of Medical Sciences , University of
Campinas , Campinas , SP , Brazil.
(5)e Department of Microbiology and Biochemistry, University of Texas
Southwestern Medical Center , Dallas , TX , USA.

Avian Pathogenic Escherichia coli is responsible for significant economic losses 
in the poultry industry by causing a range of systemic or localized diseases
collectively termed colibacillosis. The virulence mechanisms of these strains
that are pathogenic in poultry and possibly pathogenic in humans have not yet
been fully elucidated. This work was developed to study if over-expressed genes
in a microarray assay could be potentially involved in the pathogenicity of an
Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome
case. For this study, five over-expressed genes were selected for the
construction of null mutants [flgE (flagellar hook), tyrR (transcriptional
regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr
(bacterioferritin)]. The constructed mutants were evaluated for their capacity
for the adhesion and invasion of in vitro cultured cells, their motility
capacity, and their pathogenic potential in one-day-old chickens compared with
the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity
on avian fibroblasts compared with WT, in the presence and absence of
alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in
the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to
invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The
mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The
motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the
ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain 
caused the same mortality of the WT in one-day-old chickens, showing attenuation 
to different degrees.

DOI: 10.1080/03079457.2015.1125995 
PMID: 26926136  [Indexed for MEDLINE]


131. Plant Physiol Biochem. 2016 Jan;98:112-8. doi: 10.1016/j.plaphy.2015.11.016. Epub
2015 Nov 27.

CYP74B24 is the 13-hydroperoxide lyase involved in biosynthesis of green leaf
volatiles in tea (Camellia sinensis).

Ono E(1), Handa T(2), Koeduka T(3), Toyonaga H(4), Tawfik MM(5), Shiraishi A(6), 
Murata J(7), Matsui K(8).

Author information: 
(1)Research Institute, Suntory Global Innovation Center Ltd, 8-1-1 Seika-dai,
Seika, Soraku, Kyoto 619-0238, Japan. Electronic address:
eiichiro_ono@suntory.co.jp.
(2)Graduate School of Medicine and Department of Biological Chemistry, Faculty of
Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address:
r038ge@yamaguchi-u.ac.jp.
(3)Graduate School of Medicine and Department of Biological Chemistry, Faculty of
Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address:
takaori@yamaguchi-u.ac.jp.
(4)Research Institute, Suntory Global Innovation Center Ltd, 8-1-1 Seika-dai,
Seika, Soraku, Kyoto 619-0238, Japan. Electronic address:
hiromi_toyonaga@skal.suntory.co.jp.
(5)Graduate School of Medicine and Department of Biological Chemistry, Faculty of
Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address:
t014uj@yamaguchi-u.ac.jp.
(6)Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1
Seika-dai, Seika, Soraku, Kyoto 619-0238, Japan. Electronic address:
shiraishi@sunbor.or.jp.
(7)Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1
Seika-dai, Seika, Soraku, Kyoto 619-0238, Japan. Electronic address:
murata-j@sunbor.or.jp.
(8)Graduate School of Medicine and Department of Biological Chemistry, Faculty of
Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address:
matsui@yamaguchi-u.ac.jp.

Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and
biosynthesized from the central precursor fatty acid 13-hydroperoxides by
13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been
implicated as defense compounds in plants, GLVs give characteristic grassy note
to a bouquet of aroma in green tea, which is manufactured from young leaves of
Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via 
RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate
gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein
heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal 
from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed
throughout the aerial organs in a rather constitutive manner and further induced 
by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea
leaves might account for low but substantial and constitutive formation of a
subset of GLVs, some of which are stored as glycosides. Our results not only
provide novel insights into the biological roles that GLVs play in tea plants,
but also serve as basis for the improvement of aroma quality in tea manufacturing
processes.

Copyright © 2015 Elsevier Masson SAS. All rights reserved.

DOI: 10.1016/j.plaphy.2015.11.016 
PMID: 26686283  [Indexed for MEDLINE]


132. Nucleic Acids Res. 2015 Dec 2;43(21):e144. doi: 10.1093/nar/gkv718. Epub 2015 Jul
14.

Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers.

Arora A(1), Sunbul M(1), Jäschke A(2).

Author information: 
(1)Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im
Neuenheimer Feld 364, Heidelberg 69120, Germany.
(2)Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im
Neuenheimer Feld 364, Heidelberg 69120, Germany jaeschke@uni-hd.de.

In order to gain deeper insight into the functions and dynamics of RNA in cells, 
the development of methods for imaging multiple RNAs simultaneously is of
paramount importance. Here, we describe a modular approach to image RNA in living
cells using an RNA aptamer that binds to dinitroaniline, an efficient general
contact quencher. Dinitroaniline quenches the fluorescence of different
fluorophores when directly conjugated to them via ethylene glycol linkers by
forming a non-fluorescent intramolecular complex. Since the binding of the RNA
aptamer to the quencher destroys the fluorophore-quencher complex, fluorescence
increases dramatically upon binding. Using this principle, a series of
fluorophores were turned into fluorescent turn-on probes by conjugating them to
dinitroaniline. These probes ranged from fluorescein-dinitroaniline (green) to
TexasRed-dinitroaniline (red) spanning across the visible spectrum. The
dinitroaniline-binding aptamer (DNB) was generated by in vitro selection, and was
found to bind all probes, leading to fluorescence increase in vitro and in living
cells. When expressed in E. coli, the DNB aptamer could be labelled and
visualized with different-coloured fluorophores and therefore it can be used as a
genetically encoded tag to image target RNAs. Furthermore, combining
contact-quenched fluorogenic probes with orthogonal DNB (the quencher-binding RNA
aptamer) and SRB-2 aptamers (a fluorophore-binding RNA aptamer) allowed
dual-colour imaging of two different fluorescence-enhancing RNA tags in living
cells, opening new avenues for studying RNA co-localization and trafficking.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkv718 
PMCID: PMC4666381
PMID: 26175046  [Indexed for MEDLINE]


133. J Bacteriol. 2015 Dec;197(24):3751-9. doi: 10.1128/JB.00721-15. Epub 2015 Oct 5.

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing 
Translation of pnp Transcripts That Have Been Previously Processed by RNase III
and PNPase.

Park H(1), Yakhnin H(1), Connolly M(1), Romeo T(2), Babitzke P(3).

Author information: 
(1)Department of Biochemistry and Molecular Biology, Center for RNA Molecular
Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
(2)Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida, USA.
(3)Department of Biochemistry and Molecular Biology, Center for RNA Molecular
Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
pxb28@psu.edu.

Comment in
    J Bacteriol. 2015 Dec;197(24):3748-50.

Csr is a conserved global regulatory system that represses or activates gene
expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA
binding protein that regulates transcription elongation, translation initiation, 
and mRNA stability by binding to the 5' untranslated leader or initial coding
sequence of target transcripts. pnp mRNA, encoding the 3' to 5' exoribonuclease
polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target
by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase
III and PNPase participate in a pnp autoregulatory mechanism in which RNase III
cleavage of the untranslated leader, followed by PNPase degradation of the
resulting 5' fragment, leads to pnp repression by an undefined translational
repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp
leader RNA but only after the transcript is fully processed by RNase III and
PNPase. In the absence of processing, both of the binding sites are sequestered
in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer
bridges the upstream high-affinity site to the downstream site that overlaps the 
pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp 
translation. CsrA-mediated translational repression also leads to a small
increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate
translation and mRNA stability of numerous genes in a variety of organisms, this 
is the first example in which prior mRNA processing is required for CsrA-mediated
regulation.IMPORTANCE: CsrA protein represses translation of numerous mRNA
targets, typically by binding to multiple sites in the untranslated leader region
preceding the coding sequence. We found that CsrA represses translation of pnp by
binding to two sites in the pnp leader transcript but only after it is processed 
by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA
secondary structure such that it becomes accessible to the ribosome for
translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp
ribosome binding site, bound CsrA prevents ribosome binding. This is the first
example in which regulation by CsrA requires prior mRNA processing and should
link pnp expression to conditions affecting CsrA activity.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.00721-15 
PMCID: PMC4652041
PMID: 26438818  [Indexed for MEDLINE]


134. J Mol Evol. 2015 Dec;81(5-6):172-8. doi: 10.1007/s00239-015-9718-4. Epub 2015 Nov
14.

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein
Using Fluorescence-Activated Cell Sorting.

Zou J(1), Huang X(1), Wu L(1), Chen G(1), Dong J(1), Cui X(1), Tang Z(2).

Author information: 
(1)Natural Products Research Center, Chengdu Institute of Biology, Chinese
Academy of Sciences, Chengdu, 610041, People's Republic of China.
(2)Natural Products Research Center, Chengdu Institute of Biology, Chinese
Academy of Sciences, Chengdu, 610041, People's Republic of China.
tangzhuo@cib.ac.cn.

Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia
coli cells that were highly fluorescent due to the expression of RNA aptamers
that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two 
different aptamers, named ZT-26 and ZT-324, were identified by this method and
compared to the fluorescence-signaling properties of Spinach, a previously
reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence 
over Spinach only in vitro. However, aptamer ZT-324 is 36% brighter than Spinach 
when expressed in E. coli. The FACS-based selection strategy presented here is
attractive for deriving fluorescent RNA aptamers that function in cells as it
directly selects for cells with a high level of fluorescence due to the
expression of the RNA aptamer.

DOI: 10.1007/s00239-015-9718-4 
PMID: 26573804  [Indexed for MEDLINE]


135. Int J Food Microbiol. 2015 Nov 20;213:130-8. doi:
10.1016/j.ijfoodmicro.2015.04.009. Epub 2015 Apr 10.

First step in using molecular data for microbial food safety risk assessment;
hazard identification of Escherichia coli O157:H7 by coupling genomic data with
in vitro adherence to human epithelial cells.

Pielaat A(1), Boer MP(2), Wijnands LM(3), van Hoek AH(3), Bouw E(3), Barker
GC(4), Teunis PF(5), Aarts HJ(3), Franz E(3).

Author information: 
(1)National Institute for Public Health and the Environment (RIVM), Centre for
Infectious Disease Control, A. van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The
Netherlands. Electronic address: annemarie.pielaat@rivm.nl.
(2)Wageningen UR Biometris, Droevendaalsesteeg 1, 6708 PB Wageningen, The
Netherlands.
(3)National Institute for Public Health and the Environment (RIVM), Centre for
Infectious Disease Control, A. van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The
Netherlands.
(4)IFR, Institute of Food Research, Norwich Research Park, Norwich, UK.
(5)National Institute for Public Health and the Environment (RIVM), Centre for
Infectious Disease Control, A. van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The
Netherlands; Rollins School of Public Health, Emory University, Atlanta, GA, USA.

The potential for using whole genome sequencing (WGS) data in microbiological
risk assessment (MRA) has been discussed on several occasions since the beginning
of this century. Still, the proposed heuristic approaches have never been applied
in a practical framework. This is due to the non-trivial problem of mapping
microbial information consisting of thousands of loci onto a probabilistic scale 
for risks. The paradigm change for MRA involves translation of multidimensional
microbial genotypic information to much reduced (integrated) phenotypic
information and onwards to a single measure of human risk (i.e. probability of
illness). In this paper a first approach in methodology development is described 
for the application of WGS data in MRA; this is supported by a practical example.
That is, combining genetic data (single nucleotide polymorphisms; SNPs) for Shiga
toxin-producing Escherichia coli (STEC) O157 with phenotypic data (in vitro
adherence to epithelial cells as a proxy for virulence) leads to hazard
identification in a Genome Wide Association Study (GWAS). This application
revealed practical implications when using SNP data for MRA. These can be
summarized by considering the following main issues: optimum sample size for
valid inference on population level, correction for population structure,
quantification and calibration of results, reproducibility of the analysis, links
with epidemiological data, anchoring and integration of results into a systems
biology approach for the translation of molecular studies to human health risk.
Future developments in genetic data analysis for MRA should aim at resolving the 
mapping problem of processing genetic sequences to come to a quantitative
description of risk. The development of a clustering scheme focusing on
biologically relevant information of the microbe involved would be a useful
approach in molecular data reduction for risk assessment.

Copyright © 2015. Published by Elsevier B.V.

DOI: 10.1016/j.ijfoodmicro.2015.04.009 
PMCID: PMC4613885
PMID: 25910947  [Indexed for MEDLINE]


136. BMC Biotechnol. 2015 Nov 16;15:105. doi: 10.1186/s12896-015-0221-1.

Mouse Resistin (mRetn): cloning, expression and purification in Escherichia coli 
and the potential regulative effects on murine bone marrow hematopoiesis.

Wang F(1)(2), Gao J(3)(4), Malisani A(5)(6), Xi X(7), Han W(8), Wan X(9).

Author information: 
(1)The Center of Research Laboratory, The International Peace Maternity and Child
Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai,
200030, China. sunflowerwang@hotmail.com.
(2)Department of Obstetrics and Gynecology, Shanghai Jiao Tong University
Affiliated First People's Hospital, Shanghai, 200080, China.
sunflowerwang@hotmail.com.
(3)School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
g_jin@sjtu.edu.cn.
(4)College of Pharmacy, Washington State University, Spokane, WA, 99202, USA.
g_jin@sjtu.edu.cn.
(5)College of Pharmacy, Washington State University, Spokane, WA, 99202, USA.
122603326@qq.com.
(6)College of Arts and Sciences, Gonzaga University, Spokane, 99258, USA.
122603326@qq.com.
(7)Department of Obstetrics and Gynecology, Shanghai Jiao Tong University
Affiliated First People's Hospital, Shanghai, 200080, China.
xixiaowei1966@126.com.
(8)School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
weihan@sjtu.edu.cn.
(9)Department of Obstetrics and Gynecology, Shanghai First Maternity and Infant
Hospital, Tong Ji University School of Medicine, No.536, Changle Road, Shanghai, 
200080, China. wanxp@sjtu.edu.cn.

BACKGROUND: Resistin (Retn) is a cytokine which has a controversial physiological
role regarding its involvement with obesity and type II diabetes mellitus.
Recently, murine Retn was found to be a possibly potential regulator of
hematopoiesis in mice shown in the screening results of a set of gene chips which
mapped the expression level of murine genes during regeneration of impaired bone 
marrow (BM) by 5-fluorouracil.
RESULTS: Recombinant mice Retn was expressed in Escherichia coli and purified
using ion exchange chromatography. Totally 11.4 mg rmRetn was obtained from 500
ml culture with endotoxin level less than 1.0 EU/ug. The purity of recombinant
murine Resistin reached to at least 97.6% via SDS-PAGE analysis and HPLC. The
protein possessed chemotaxis effects in the mouse aortic endothelial cells in
vitro in transwell analysis. In vitro, rmRetn could up regulate the CFU number of
mice BM and after rmRetn was administered, the cell number of murine bone marrow 
was significantly increased in vivo after chemotherapy. Finally, rmRetn was found
able to protect mice from the chemotoxicity of 5-fluorouracil.
CONCLUSIONS: The discovery demonstrated a new function of murine Retn and
suggested that it could potentially accelerate bone marrow regeneration post
chemotherapy.

DOI: 10.1186/s12896-015-0221-1 
PMCID: PMC4647653
PMID: 26572487  [Indexed for MEDLINE]


137. BMC Genomics. 2015 Nov 14;16:934. doi: 10.1186/s12864-015-2139-3.

Deep sequencing of the uterine immune response to bacteria during the equine
oestrous cycle.

Marth CD(1), Young ND(2), Glenton LY(3), Noden DM(4), Browning GF(5), Krekeler
N(6).

Author information: 
(1)Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural 
Sciences, The University of Melbourne, 250 Princes Highway, Werribee, VIC, 3030, 
Australia. christina.marth@unimelb.edu.au.
(2)Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural 
Sciences, The University of Melbourne, 250 Princes Highway, Werribee, VIC, 3030, 
Australia. nyoung@unimelb.edu.au.
(3)Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural 
Sciences, The University of Melbourne, 250 Princes Highway, Werribee, VIC, 3030, 
Australia. l.glenton@student.unimelb.edu.au.
(4)Department of Biomedical Sciences, College of Veterinary Medicine, Cornell
University, Ithaca, NY, 14853-6401, USA. drewnoden@gmail.com.
(5)Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural 
Sciences, The University of Melbourne, 250 Princes Highway, Werribee, VIC, 3030, 
Australia. glenfb@unimelb.edu.au.
(6)Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural 
Sciences, The University of Melbourne, 250 Princes Highway, Werribee, VIC, 3030, 
Australia. krekeler@unimelb.edu.au.

BACKGROUND: The steroid hormone environment in healthy horses seems to have a
significant impact on the efficiency of their uterine immune response. The
objective of this study was to characterize the changes in gene expression in the
equine endometrium in response to the introduction of bacterial pathogens and the
influence of steroid hormone concentrations on this expression.
METHODS: Endometrial biopsies were collected from five horses before and 3 h
after the inoculation of Escherichia coli once in oestrus (follicle >35 mm in
diameter) and once in dioestrus (5 days after ovulation) and analysed using
high-throughput RNA sequencing techniques (RNA-Seq).
RESULTS: Comparison between time points revealed that 2422 genes were expressed
at significantly higher levels and 2191 genes at significantly lower levels 3 h
post inoculation in oestrus in comparison to pre-inoculation levels. In
dioestrus, the expression of 1476 genes was up-regulated and 383 genes were
down-regulated post inoculation. Many immune related genes were found to be
up-regulated after the introduction of E. coli. These include pathogen
recognition receptors, particularly toll-like receptors TLR2 and 4 and NOD-like
receptor NLRC5. In addition, several interleukins including IL1B, IL6, IL8 and
IL1ra were significantly up-regulated. Genes for chemokines, including CCL 2,
CXCL 6, 9, 10, 11 and 16 and those for antimicrobial peptides, including
secretory phospholipase sPLA 2, lipocalin 2, lysozyme and equine β-defensin 1, as
well as the gene for tissue inhibitor for metalloproteinases TIMP-1 were also
up-regulated post inoculation.
CONCLUSION: The results of this study emphasize the complexity of an effective
uterine immune response during acute endometritis and the tight balance between
pro- and anti-inflammatory factors required for efficient elimination of
bacteria. It is one of the first high-throughput analyses of the uterine
inflammatory response in any species and several new potential targets for
treatment of inflammatory diseases of the equine uterus have been identified.

DOI: 10.1186/s12864-015-2139-3 
PMCID: PMC4647707
PMID: 26572250  [Indexed for MEDLINE]


138. Sci Rep. 2015 Nov 13;5:16505. doi: 10.1038/srep16505.

Dynamic interplay of multidrug transporters with TolC for isoprenol tolerance in 
Escherichia coli.

Wang C(1), Yang L(1), Shah AA(1), Choi ES(2), Kim SW(1).

Author information: 
(1)Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National
University, Jinju 660-701, Republic of Korea.
(2)Industrial Biotechnology Research Center, KRIBB, Daejeon 305-806, Republic of 
Korea.

Engineering of efflux pumps is a promising way to improve host's tolerance to
biofuels such as medium-chain alcohols (CmOHs); however, this strategy is
restricted by poor understanding of the efflux pumps engaged in extrusion of
solvents. In this study, several Escherichia coli mutants of multidrug
transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were 
observed in the mutants with individual deletion of six transporters, AcrD,
EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter
resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the
major transporter forming tripartite transperiplasmic complex with outer membrane
channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB
inactivation enables to enhance TolC availability for the multidrug transporters 
associated with extrusion of CmOHs and increase the tolerance to CmOHs including 
isoprenol. It is assumed that outer membrane channel TolC plays an important role
in extrusion of isoprenol and other CmOHs.

DOI: 10.1038/srep16505 
PMCID: PMC4643228
PMID: 26563610  [Indexed for MEDLINE]


139. Antimicrob Agents Chemother. 2015 Nov;59(11):6873-81. doi: 10.1128/AAC.01341-15. 
Epub 2015 Aug 24.

Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis
resistance to an imidazopyridine-based agent.

Winglee K(1), Lun S(1), Pieroni M(2), Kozikowski A(3), Bishai W(4).

Author information: 
(1)Center for Tuberculosis Research, Department of Medicine, Johns Hopkins
University, Baltimore, Maryland, USA.
(2)Dipartimento di Farmacia, P4T group, University of Parma, Parma, Italy.
(3)Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois at Chicago, Chicago, Illinois, USA.
(4)Center for Tuberculosis Research, Department of Medicine, Johns Hopkins
University, Baltimore, Maryland, USA wbishai1@jhmi.edu.

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it 
is critical to identify novel drug targets and new antimycobacterial compounds.
We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based
agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we
evaluated mechanisms of resistance to MP-III-71. We derived three independent M. 
tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing
of these mutants. Loss-of-function mutations in Rv2887 were common to all three
MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene
required for MP-III-71 susceptibility using complementation. The Rv2887 protein
was previously unannotated, but domain and homology analyses suggested it to be a
transcriptional regulator in the MarR (multiple antibiotic resistance repressor) 
family, a group of proteins first identified in Escherichia coli to negatively
regulate efflux pumps and other mechanisms of multidrug resistance. We found that
two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action
of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used
transcriptome sequencing (RNA-seq) to identify genes which are differentially
expressed in the presence and absence of a functional Rv2887 protein. We found
that genes involved in benzoquinone and menaquinone biosynthesis were repressed
by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative
MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective
antimycobacterial compound that shows no cross-resistance to existing
antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887
mutants may involve efflux pump upregulation and also drug methylation.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AAC.01341-15 
PMCID: PMC4604386
PMID: 26303802  [Indexed for MEDLINE]


140. Genes Cells. 2015 Nov;20(11):915-31. doi: 10.1111/gtc.12282. Epub 2015 Sep 2.

Expanded roles of two-component response regulator OmpR in Escherichia coli:
genomic SELEX search for novel regulation targets.

Shimada T(1)(2), Takada H(1), Yamamoto K(1)(3), Ishihama A(1)(3).

Author information: 
(1)Micro-Nano Technology Research Center, Hosei University, Koganai, Tokyo,
184-8584, Japan.
(2)Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda,
Yokohama, 226-8503, Japan.
(3)Department of Frontier Bioscience, Hosei University, Koganai, Tokyo, 184-8584,
Japan.

The two-component system (TCS) is a sophisticated bacterial signal transduction
system for regulation of genome transcription in response to environmental
conditions. The EnvZ-OmpR system is one of the well-characterized TCS of
Escherichia coli, responding to changes in environmental osmolality. Regulation
has largely focused on the differential expression of two porins, OmpF and OmpC, 
which transport small molecules across the outer membrane. Recently, it has
become apparent that OmpR serves a more global regulatory role and regulates
additional targets. To identify the entire set of regulatory targets of OmpR, we 
performed the genomic SELEX screening of OmpR-binding sites along the E. coli
genome. As a result, more than 30 novel genes have been identified to be under
the direct control of OmpR. One abundant group includes the genes encoding a
variety of membrane-associated transporters that mediate uptake or efflux of
small molecules, while another group encodes a set of transcription regulators,
raising a concept that OmpR is poised to control a diverse set of responses by
altering downstream transcriptional regulators.

© 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

DOI: 10.1111/gtc.12282 
PMID: 26332955  [Indexed for MEDLINE]


141. J Biochem. 2015 Nov;158(5):393-401. doi: 10.1093/jb/mvv052. Epub 2015 May 21.

Characterization, validation and application of a DNA microarray for the
detection of mandatory and other waterborne pathogens.

Gomes M(1), Vieira H(2), Vale FF(3).

Author information: 
(1)Faculdade de Engenharia, Universidade Católica Portuguesa, 2635-631 Rio de
Mouro, Portugal;
(2)University of Lisboa, Faculty of Sciences, BioISI-Biosystems & Integrative
Sciences Institute, Campo Grande, 1749-106 Lisboa, Portugal;
(3)Host-Pathogen Interactions Unit, Research Institute for Medicines
(iMed-ULisboa), Instituto de Medicina Molecular, Faculdade de Farmácia da
Universidade de Lisboa, Lisboa, Portugal f.vale@ff.ul.pt vale.filipa@gmail.com.

Culture methods for the detection of indicator bacteria are currently used for
detection of waterborne bacteria. The need for an increased range of analyzed
bacteria coupled with the obtainment of rapid and early results justify the
development of a DNA microarray for the identification of waterborne pathogens.
This DNA microarray has 16 implanted probes with a median size of 147 bases,
targeting 12 different parameters, including all mandatory indicator
microorganisms, such as Escherichia coli, Clostridium perfringens, Pseudomonas
aeruginosa, Staphylococcus aureus, total and fecal coliforms and enterococci. The
validation performed with DNA extracted from pure microbial cultures showed the
suitability of the probes for detection of the target microorganism. To overcome 
the high dilution of water samples it was included either a prior culture step of
bacterial contaminants retained after filtering 100 ml of water, or a 10-fold
increase in the volume of filtered water, that resulted in the increase of the
detected bacteria. The analysis of complex environmental water samples using
culture methods and the DNA microarray revealed that the latter detected the same
parameters plus other bacteria tested only in the DNA microarray. The results
show that this DNA microarray may be a useful tool for water microbiological
surveillance.

© The Authors 2015. Published by Oxford University Press on behalf of the
Japanese Biochemical Society. All rights reserved.

DOI: 10.1093/jb/mvv052 
PMID: 25998249  [Indexed for MEDLINE]


142. J Chromatogr A. 2015 Oct 30;1418:130-139. doi: 10.1016/j.chroma.2015.09.055. Epub
2015 Sep 25.

Imidazole-free purification of His3-tagged recombinant proteins using ssDNA
aptamer-based affinity chromatography.

Bartnicki F(1), Kowalska E(1), Pels K(1), Strzalka W(2).

Author information: 
(1)Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
(2)Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
Electronic address: wojciech.strzalka@uj.edu.pl.

Immobilized metal ion affinity chromatography (IMAC) is widely used for the
purification of many different His6-tagged recombinant proteins. On the one hand,
it is a powerful technique but on the other hand it has its disadvantages. In
this report, we present the development of a unique ssDNA aptamer for the
purification of His3-tagged recombinant proteins. Our study shows that stability 
of the His3-tag/H3T aptamer complex can be controlled by the sodium ion
concentration. Based on this feature, we demonstrate that H3T aptamer resin was
successfully employed for the purification of three out of four tested
His3-tagged recombinant proteins from an E. coli total protein extract using
imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins 
is superior when purified with the help of the H3T aptamer in comparison with
Ni-NTA resin.

Copyright © 2015 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.chroma.2015.09.055 
PMID: 26427325  [Indexed for MEDLINE]


143. Appl Environ Microbiol. 2015 Oct;81(19):6857-63. doi: 10.1128/AEM.01349-15. Epub 
2015 Jul 24.

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and
Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Ng AH(1), Berla BM(2), Pakrasi HB(3).

Author information: 
(1)Department of Biology, Washington University, St. Louis, Missouri, USA.
(2)Department of Energy, Environmental, and Chemical Engineering, Washington
University, St. Louis, Missouri, USA.
(3)Department of Biology, Washington University, St. Louis, Missouri, USA
Department of Energy, Environmental, and Chemical Engineering, Washington
University, St. Louis, Missouri, USA Pakrasi@wustl.edu.

Cyanobacteria are photosynthetic cell factories that use solar energy to convert 
CO2 into useful products. Despite this attractive feature, the development of
tools for engineering cyanobacterial chassis has lagged behind that for
heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous
genes in cyanobacteria are often integrated at presumptively "neutral"
chromosomal sites, with unknown effects. We used transcriptome sequencing
(RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to 
identify neutral sites from which no transcripts are expressed. We characterized 
the two largest such sites on the chromosome, a site on an endogenous plasmid,
and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP)
expression cassette expressed from either the Pcpc560 or the Ptrc1O promoter into
each locus. Expression from the endogenous plasmid was as much as 14-fold higher 
than that from the chromosome, with intermediate expression from the shuttle
vector. The expression characteristics of each locus correlated predictably with 
the promoters used. These findings provide novel, characterized tools for
synthetic biology and metabolic engineering in cyanobacteria.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AEM.01349-15 
PMCID: PMC4561709
PMID: 26209663  [Indexed for MEDLINE]


144. Mol Med Rep. 2015 Oct;12(4):5807-15. doi: 10.3892/mmr.2015.4126. Epub 2015 Jul
27.

Histopathology of melanosis coli and determination of its associated genes by
comparative analysis of expression microarrays.

Li XΑ(1), Zhou Y(1), Zhou SΧ(1), Liu HR(1), Xu JM(1), Gao L(1), Yu XJ(1), Li
XH(1).

Author information: 
(1)The Gastroenterology Tumor and Microenvironment Laboratory, Department of
Gastroenterology, The First Affiliated Hospital of Chengdu Medical College,
Chengdu Medical College, Chengdu, Sichuan 610041, P.R. China.

Melanosis coli (MC) refers to the condition characterized by abnormal brown or
black pigmentation deposits on the colonic mucosa. However, the histopathological
findings and genes associated with the pathogenesis of melanosis coli remain to
be fully elucidated. The present study aimed to examine the histopathological
features and differentially expressed genes of MC. This involved performing
hematoxylin and eosin staining, specific staining and immunohistochemistry on
tissues sections, which were isolated from patients diagnosed with MC. DNA
expression microarray analysis, western blotting and immunofluorescence assays
were performed to analyze the differentially expressed genes of melanosis coli.
The results demonstrated that the pigment deposits in MC consisted of lipofuscin.
A TUNEL assay revealed that a substantial number of apoptotic cells were present 
within the macrophages and superficial lamina propria of the colonic epithelium. 
Expression microarray analysis revealed that the significantly downregulated
genes were CYP3A4, CYP3A7, UGT2B11 and UGT2B15 in melanosis coli. Western
blotting and immunofluorescence assays indicated that the expression of CYP3A4 in
the normal tissue was higher than in the MC tissue. The results of the present
study provided a comprehensive description of the histopathological
characteristics and pathogenesis of MC and for the first time, to the best of our
knowledge, demonstrated that the cytochrome P450‑associated genes were
significantly downregulated in melanosis coli. This novel information can be used
to assist in further investigations of melanosis coli.

DOI: 10.3892/mmr.2015.4126 
PMCID: PMC4581826
PMID: 26238215  [Indexed for MEDLINE]


145. Talanta. 2015 Oct 1;143:19-26. doi: 10.1016/j.talanta.2015.04.041. Epub 2015 May 
8.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a
microarray using a biotinylated intercalator as coupling site for an enzyme
label.

Zimdars A(1), Gebala M(2), Hartwich G(1), Neugebauer S(2), Schuhmann W(3).

Author information: 
(1)FRIZ Biochem, Floriansbogen 2-4, D-82061 Neuried, Germany.
(2)Analytical Chemistry - Center for Electrochemical Sciences (CES),
Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
(3)Analytical Chemistry - Center for Electrochemical Sciences (CES),
Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
Electronic address: wolfgang.schuhmann@rub.de.

The direct electrochemical detection of synthetic DNA and native 16S rRNA
fragments isolated from Escherichia coli is described. Oligonucleotides are
detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes 
with a biotinylated intercalator that enables high-specific binding of a
streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes
formation of p-aminophenol that is subsequently oxidized at the underlying gold
electrode and hence enables the detection of complementary hybridization of the
DNA capture strands due to the enzymatic signal amplification. The hybridization 
assay was performed on microarrays consisting of 32 individually addressable gold
microelectrodes. Synthetic DNA strands with sequences representing six different 
pathogens which are important for the diagnosis of urinary tract infections could
be detected at concentrations of 60 nM. Native 16S rRNA isolated from the
different pathogens could be detected at a concentration of 30 fM. Optimization
of the sensing surface is described and influences on the assay performance are
discussed.

Copyright © 2015 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.talanta.2015.04.041 
PMID: 26078123  [Indexed for MEDLINE]


146. Biosens Bioelectron. 2015 Sep 15;71:186-193. doi: 10.1016/j.bios.2015.04.034.
Epub 2015 Apr 14.

Ultrasensitive detection and rapid identification of multiple foodborne pathogens
with the naked eyes.

Zhang H(1), Zhang Y(2), Lin Y(3), Liang T(3), Chen Z(2), Li J(4), Yue Z(3), Lv
J(3), Jiang Q(2), Yi C(5).

Author information: 
(1)Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong
Province), School of Engineering, Sun Yat-Sen University, Guangzhou, China;
Shenzhen Key Research Laboratory of Detection Technology R&D on Food Safety,
Technical Centre for Food Inspection and Quarantine, Shenzhen Entry-Exit
Inspection and Quarantine Bureau, Shenzhen, China; Shenzhen Academy of Inspection
and Quarantine, Shenzhen, China.
(2)Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong
Province), School of Engineering, Sun Yat-Sen University, Guangzhou, China.
(3)Shenzhen Key Research Laboratory of Detection Technology R&D on Food Safety,
Technical Centre for Food Inspection and Quarantine, Shenzhen Entry-Exit
Inspection and Quarantine Bureau, Shenzhen, China; Shenzhen Academy of Inspection
and Quarantine, Shenzhen, China.
(4)Department of Transfusion Medicine, Southern Medical University, Guangzhou,
China.
(5)Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong
Province), School of Engineering, Sun Yat-Sen University, Guangzhou, China.
Electronic address: yichq@mail.sysu.edu.cn.

In this study, a novel approach for ultrasensitive detection and rapid
high-throughput identification of a panel of common foodborne pathogens with the 
naked eyes is presented. As a proof-of-concept application, a multiple pathogen
analysis array is fabricated through immobilizing three specific polyT-capture
probes which can respectively recognize rfbE gene (Escherichia coli O157:H7),
invA gene (Salmonella enterica), inlA gene (Listeria monocytogenes) on the
plastic substrates. PCR has been developed for amplification and labeling target 
genes of rfbE, invA, inlA with biotin. The biotinated target DNA is then captured
onto the surface of plastic strips through specific DNA hybridization. The
succeeding staining of biotinated DNA duplexes with avidin-horseradish peroxidise
(AV-HRP) and biotinated anti-HRP antibody greatly amplifies the detectable signal
through the multiple cycle signal amplification strategy, and thus realizing
ultrasensitive and specific detection of the above three pathogens in food
samples with the naked eyes. Results showed approximately 5 copies target
pathogenic DNA could be detected with the naked eyes. This simple but very
efficient colorimetric assay also show excellent anti-interference capability and
good stability, and can be readily applied to point-of-care diagnosis.

Copyright © 2015 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.bios.2015.04.034 
PMID: 25909338  [Indexed for MEDLINE]


147. J Biotechnol. 2015 Sep 10;209:41-9. doi: 10.1016/j.jbiotec.2015.06.389. Epub 2015
Jun 12.

Generation and characterization of nucleic acid aptamers targeting the capsid P
domain of a human norovirus GII.4 strain.

Moore MD(1), Escudero-Abarca BI(2), Suh SH(2), Jaykus LA(2).

Author information: 
(1)Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State
University, 315 Schaub Hall, 400 Dan Allen Drive, Raleigh, NC 27695, USA.
Electronic address: mdmoore5@ncsu.edu.
(2)Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State
University, 315 Schaub Hall, 400 Dan Allen Drive, Raleigh, NC 27695, USA.

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis
worldwide. Significant antigenic diversity of NoV strains has limited the
availability of broadly reactive ligands for design of detection assays. The
purpose of this work was to produce and characterize single stranded (ss)DNA
aptamers with binding specificity to human NoV using an easily produced NoV
target-the P domain protein. Aptamer selection was done using SELEX (Systematic
Evolution of Ligands by EXponential enrichment) directed against an Escherichia
coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique
aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity
testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of
14 virus-like particles (VLPs) showed these aptamers had broad reactivity and
exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs.
Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested.
Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New
Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic
capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P
domain protein as a functional target for the selection of nucleic acid aptamers 
that specifically bind and broadly recognize diverse human NoV strains.

Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jbiotec.2015.06.389 
PMID: 26080079  [Indexed for MEDLINE]


148. Anal Bioanal Chem. 2015 Sep;407(24):7285-94. doi: 10.1007/s00216-015-8890-0. Epub
2015 Jul 22.

Microarray on digital versatile disc for identification and genotyping of
Salmonella and Campylobacter in meat products.

Tortajada-Genaro LA(1), Rodrigo A(2), Hevia E(1), Mena S(1), Niñoles R(1),
Maquieira Á(3).

Author information: 
(1)Instituto Int. Reconocimiento Molecular (IDM), Departamento de Química,
Universitat Politècnica de València, Camino de Vera s/n, 46022, Valencia, Spain.
(2)Asociación de Investigación de la Industria Agroalimentaria (AINIA), Benjamín 
Franklin 5-11, 46980, Paterna, Valencia, Spain.
(3)Instituto Int. Reconocimiento Molecular (IDM), Departamento de Química,
Universitat Politècnica de València, Camino de Vera s/n, 46022, Valencia, Spain. 
amaquieira@qim.upv.es.

Highly portable, cost-effective, and rapid-response devices are required for the 
subtyping of the most frequent food-borne bacteria; thereby the sample rejection 
strategies and hygienization techniques along the food chain can be
tailor-designed. Here, a novel biosensor is presented for the generic detection
of Salmonella and Campylobacter and the discrimination between their most
prevalent serovars (Salmonella Enteritidis, Salmonella Typhimurium) and species
(Campylobacter jejuni, Campylobacter coli), respectively. The method is based on 
DNA microarray developed on a standard digital versatile disc (DVD) as support
for a hybridization assay and a DVD driver as scanner. This approach was found to
be highly sensitive (detection limit down to 0.2 pg of genomic DNA), reproducible
(relative standard deviation 4-19 %), and high working capacity (20 samples per
disc). The inclusivity and exclusivity assays indicated that designed
oligonucleotides (primers and probes) were able to discriminate targeted
pathogens from other Salmonella serovars, Campylobacter species, or common
food-borne pathogens potentially present in the indigenous microflora. One
hundred isolates from meat samples, collected in a poultry factory, were analyzed
by the DVD microarraying and fluorescent real-time PCR. An excellent correlation 
was observed for both generic and specific detection (relative sensitivity
93-99 % and relative specificity 93-100 %). Therefore, the developed assay has
been shown to be a reliable tool to be used in routine food safety analysis,
especially in settings with limited infrastructure due to the excellent
efficiency-cost ratio of compact disc technology. Graphical Abstract DNA
microarray performed by DVD technology for pathogen genotyping.

DOI: 10.1007/s00216-015-8890-0 
PMID: 26198111  [Indexed for MEDLINE]


149. Infect Immun. 2015 Sep;83(9):3545-54. doi: 10.1128/IAI.00312-15. Epub 2015 Jun
22.

ArcA Controls Metabolism, Chemotaxis, and Motility Contributing to the
Pathogenicity of Avian Pathogenic Escherichia coli.

Jiang F(1), An C(1), Bao Y(1), Zhao X(2), Jernigan RL(2), Lithio A(3), Nettleton 
D(3), Li L(4), Wurtele ES(4), Nolan LK(5), Lu C(1), Li G(6).

Author information: 
(1)Department of Veterinary Preventive Medicine, College of Veterinary Medicine, 
Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
(2)Department of Molecular Biology, Biochemistry and Biophysics, Iowa State
University, Ames, Iowa, USA.
(3)Department of Statistics, Iowa State University, Ames, Iowa, USA.
(4)Department of Genetics, Development, and Cellular Biology, Iowa State
University, Ames, Iowa, USA Center for Metabolic Biology, Iowa State University, 
Ames, Iowa, USA.
(5)Department of Veterinary Diagnostic and Production Animal Medicine, College of
Veterinary Medicine, Iowa State University, Ames, Iowa, USA.
(6)Department of Veterinary Preventive Medicine, College of Veterinary Medicine, 
Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China
Department of Veterinary Diagnostic and Production Animal Medicine, College of
Veterinary Medicine, Iowa State University, Ames, Iowa, USA liganwu@iastate.edu.

Avian pathogenic Escherichia coli (APEC) strains cause one of the three most
significant infectious diseases in the poultry industry and are also potential
food-borne pathogens threating human health. In this study, we showed that ArcA
(aerobic respiratory control), a global regulator important for E. coli's
adaptation from anaerobic to aerobic conditions and control of that bacterium's
enzymatic defenses against reactive oxygen species (ROS), is involved in the
virulence of APEC. Deletion of arcA significantly attenuates the virulence of
APEC in the duck model. Transcriptome sequencing (RNA-Seq) analyses comparing the
APEC wild type and the arcA mutant indicate that ArcA regulates the expression of
129 genes, including genes involved in citrate transport and metabolism,
flagellum synthesis, and chemotaxis. Further investigations revealed that
citCEFXG contributed to APEC's microaerobic growth at the lag and log phases when
cultured in duck serum and that ArcA played a dual role in the control of citrate
metabolism and transportation. In addition, deletion of flagellar genes motA and 
motB and chemotaxis gene cheA significantly attenuated the virulence of APEC, and
ArcA was shown to directly regulate the expression of motA, motB, and cheA. The
combined results indicate that ArcA controls metabolism, chemotaxis, and motility
contributing to the pathogenicity of APEC.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/IAI.00312-15 
PMCID: PMC4534672
PMID: 26099584  [Indexed for MEDLINE]


150. Nat Methods. 2015 Sep;12(9):879-84. doi: 10.1038/nmeth.3508. Epub 2015 Aug 3.

ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape 
of modified tRNA fragments.

Cozen AE(1), Quartley E(2), Holmes AD(1), Hrabeta-Robinson E(1), Phizicky
EM(2)(3), Lowe TM(1).

Author information: 
(1)Department of Biomolecular Engineering, University of California Santa Cruz,
Santa Cruz, California, USA.
(2)Department of Biochemistry &Biophysics, University of Rochester School of
Medicine, Rochester, New York, USA.
(3)Center for RNA Biology, University of Rochester School of Medicine, Rochester,
New York, USA.

Comment in
    Nat Methods. 2015 Sep;12(9):821-2.

High-throughput RNA sequencing has accelerated discovery of the complex
regulatory roles of small RNAs, but RNAs containing modified nucleosides may
escape detection when those modifications interfere with reverse transcription
during RNA-seq library preparation. Here we describe AlkB-facilitated RNA
methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli
AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and
N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative
methylation analysis using ARM-seq provides the first detailed,
transcriptome-scale map of these modifications and reveals an abundance of
previously undetected, methylated small RNAs derived from tRNAs. ARM-seq
demonstrates that tRNA fragments accurately recapitulate the m(1)A modification
state for well-characterized yeast tRNAs and generates new predictions for a
large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs.
Thus, ARM-seq provides broad utility for identifying previously overlooked
methyl-modified RNAs, can efficiently monitor methylation state and may reveal
new roles for tRNA fragments as biomarkers or signaling molecules.

DOI: 10.1038/nmeth.3508 
PMCID: PMC4553111
PMID: 26237225  [Indexed for MEDLINE]


151. Microb Genom. 2015 Jul 15;1(1):e000001. doi: 10.1099/mgen.0.000001. eCollection
2015 Jul.

Expanded roles of leucine-responsive regulatory protein in transcription
regulation of the Escherichia coli genome: Genomic SELEX screening of the
regulation targets.

Shimada T(1), Saito N(2), Maeda M(3), Tanaka K(4), Ishihama A(5).

Author information: 
(1)Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta,
Yokohama, Japan; Department of Frontier Bioscience, Hosei University, Koganei,
Tokyo, Japan; Research Center for Micro-Nano Technology, Hosei University,
Koganei, Tokyo, Japan.
(2)Department of Chemistry and Material Engineering, Tsuruoka National College of
Technology, Yamagata, Japan; Institute for Advanced Biosciences, Keio University,
Yamagata, Japan.
(3)School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.
(4)Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta,
Yokohama, Japan.
(5)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan;
Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo,
Japan.

Leucine-responsive regulatory protein (Lrp) is a transcriptional regulator for
the genes involved in transport, biosynthesis and catabolism of amino acids in
Escherichia coli. In order to identify the whole set of genes under the direct
control of Lrp, we performed Genomic SELEX screening and identified a total of
314 Lrp-binding sites on the E. coli genome. As a result, the regulation target
of Lrp was predicted to expand from the hitherto identified genes for amino acid 
metabolism to a set of novel target genes for utilization of amino acids for
protein synthesis, including tRNAs, aminoacyl-tRNA synthases and rRNAs. Northern 
blot analysis indicated alteration of mRNA levels for at least some novel
targets, including the aminoacyl-tRNA synthetase genes. Phenotype MicroArray of
the lrp mutant indicated significant alteration in utilization of amino acids and
peptides, whilst metabolome analysis showed variations in the concentration of
amino acids in the lrp mutant. From these two datasets we realized a reverse
correlation between amino acid levels and cell growth rate: fast-growing cells
contain low-level amino acids, whilst a high level of amino acids exists in
slow-growing cells. Taken together, we propose that Lrp is a global regulator of 
transcription of a large number of the genes involved in not only amino acid
transport and metabolism, but also amino acid utilization.

DOI: 10.1099/mgen.0.000001 
PMCID: PMC5320599
PMID: 28348809 


152. J Wildl Dis. 2015 Jul;51(3):555-63. doi: 10.7589/2014-08-200. Epub 2015 Apr 28.

MOLECULAR DETECTION OF ANTIBIOTIC-RESISTANCE DETERMINANTS IN ESCHERICHIA COLI
ISOLATED FROM THE ENDANGERED AUSTRALIAN SEA LION (NEOPHOCA CINEREA).

Delport TC(1), Harcourt RG(1), Beaumont LJ(1), Webster KN(1), Power ML(1).

Author information: 
(1)1  Department of Biological Sciences, Faculty of Science, Macquarie
University, Sydney, NSW 2109, Australia.

Greater interaction between humans and wildlife populations poses significant
risks of anthropogenic impact to natural ecosystems, especially in the marine
environment. Understanding the spread of microorganisms at the marine interface
is therefore important if we are to mitigate adverse effects on marine wildlife. 
We investigated the establishment of Escherichia coli in the endangered
Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and
captive sea lion populations. Fecal samples were collected from wild colonies
March 2009-September 2010 and from captive individuals March 2011-May 2013. Using
molecular screening, we assigned a phylotype to E. coli isolates and determined
the presence of integrons, mobile genetic elements that capture gene cassettes
conferring resistance to antimicrobial agents common in fecal coliforms. Group B2
was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, 
B1, and D also identified. Integrons were not observed in E. coli (n = 21)
isolated from wild sea lions, but were identified in E. coli from captive animals
(n = 16), from which class I integrases were detected in eight isolates.
Sequencing of gene cassette arrays identified genes conferring resistance to
streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II
integrases were not detected in the E. coli isolates. The frequent detection in
captive sea lions of E. coli with resistance genes commonly identified in human
clinical cases suggests that conditions experienced in captivity may contribute
to establishment. Identification of antibiotic resistance in the microbiota of
Australian sea lions provides crucial information for disease management. Our
data will inform conservation management strategies and provide a mechanism to
monitor microorganism dissemination to sensitive pinniped populations.

DOI: 10.7589/2014-08-200 
PMID: 25919463  [Indexed for MEDLINE]


153. PLoS Genet. 2015 Jul 1;11(7):e1005348. doi: 10.1371/journal.pgen.1005348.
eCollection 2015 Jul.

A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia
coli and Vibrio cholerae Revealed by 5' RNA-seq.

Druzhinin SY(1), Tran NT(2), Skalenko KS(1), Goldman SR(1), Knoblauch JG(1), Dove
SL(2), Nickels BE(1).

Author information: 
(1)Department of Genetics and Waksman Institute, Rutgers University, Piscataway, 
New Jersey, United States of America.
(2)Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical
School, Boston, Massachusetts, United States of America.

Transcription initiation that involves the use of a 2- to ~4-nt
oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown
to be widely prevalent at promoters of genes expressed during the stationary
phase of growth in Escherichia coli. However, the extent to which PDI impacts E. 
coli physiology, and the extent to which PDI occurs in other bacteria is not
known. Here we establish a physiological role for PDI in E. coli as a regulatory 
mechanism that modulates biofilm formation. We further demonstrate using
high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the
pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. 
cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the
two organisms. In particular, PDI is detected in stationary phase, is not
detected in exponential phase, and is preferentially apparent at promoters
carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the
position of de novo initiation). Our findings demonstrate a physiological role
for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose
that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent
process that leads to the preferential generation of the linear dinucleotides
5´-UA-3´ and 5´-GG-3´.

DOI: 10.1371/journal.pgen.1005348 
PMCID: PMC4488433
PMID: 26131907  [Indexed for MEDLINE]


154. Front Bioeng Biotechnol. 2015 Jun 24;3:91. doi: 10.3389/fbioe.2015.00091.
eCollection 2015.

Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore
Device.

Smith AM(1), Abu-Shumays R(2), Akeson M(3), Bernick DL(2).

Author information: 
(1)Department of Chemistry and Biochemistry, University of California Santa Cruz 
, Santa Cruz, CA , USA ; Department of Biomolecular Engineering, University of
California Santa Cruz , Santa Cruz, CA , USA.
(2)Department of Biomolecular Engineering, University of California Santa Cruz , 
Santa Cruz, CA , USA.
(3)Department of Biomolecular Engineering, University of California Santa Cruz , 
Santa Cruz, CA , USA ; Genomics Institute, University of California Santa Cruz , 
Santa Cruz, CA , USA.

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical
roles as both translators of the genetic code and regulators of protein
synthesis. As such, numerous methods have focused on studying tRNA abundance and 
regulation, with the most widely used methods being RNA-seq and microarrays.
Though revolutionary to transcriptomics, these assays are limited by an inability
to encode tRNA modifications in the requisite cDNA. These modifications are
abundant in tRNA and critical to their function. Here, we describe
proof-of-concept experiments where individual tRNA molecules are examined as
linear strands using a biological nanopore. This method utilizes an enzymatically
ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the
nanopore device and efficiently denature individual tRNA molecules, as they are
pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter
provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake 
on the translocating tRNA. This increases the dwell time of adapted tRNA in the
nanopore, allowing us to identify the region of the nanopore signal that is
produced by the translocating tRNA itself. Using adapter-modified Escherichia
coli tRNA(fMet) and tRNA(Lys), we show that the nanopore signal during controlled
translocation is dependent on the identity of the tRNA. This confirms that
adapter-modified tRNA can translocate end-to-end through nanopores and provide
the foundation for future work in direct sequencing of individual transfer RNA
with a nanopore-based device.

DOI: 10.3389/fbioe.2015.00091 
PMCID: PMC4478443
PMID: 26157798 


155. Biosens Bioelectron. 2015 Jun 15;68:272-80. doi: 10.1016/j.bios.2015.01.009. Epub
2015 Jan 3.

Analytical bioconjugates, aptamers, enable specific quantitative detection of
Listeria monocytogenes.

Lee SH(1), Ahn JY(1), Lee KA(1), Um HJ(1), Sekhon SS(1), Sun Park T(2), Min J(3),
Kim YH(4).

Author information: 
(1)Department of Microbiology, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 362-763, South Korea.
(2)Division of Endocrinology & Metabolism, Department of Internal Medicine,
Chonbuk National University Medical School, 634-18 Geumam-Dong, Duckjin-Gu,
Jeonju 561-712, South Korea.
(3)Graduate School of Semiconductor and Chemical Engineering, Chonbuk National
University, 664-14 Deokjin-dong, 1Ga Deokjin-Gu, Jeonju 561-756, South Korea.
Electronic address: jihomin@jbnu.ac.kr.
(4)Department of Microbiology, Chungbuk National University, 1 Chungdae-Ro,
Seowon-Gu, Cheongju 362-763, South Korea. Electronic address: kyh@chungbuk.ac.kr.

As a major human pathogen in the Listeria genus, Listeria monocytogenes causes
the bacterial disease listeriosis, which is a serious infection caused by eating 
food contaminated with the bacteria. We have developed an aptamer-based sandwich 
assay (ABSA) platform that demonstrates a promising potential for use in pathogen
detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX
(WB-SELEX) strategy was adopted to generate aptamers with high affinity and
specificity against live L. monocytogenes. Of the 35 aptamer candidates tested,
LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were
consequently chosen as sensing probes. The ABSA platform can significantly
enhance the sensitivity by employing a very specific aptamer pair for the
sandwich complex. The ABSA platform exhibited a linear response over a wide
concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was
closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99).
Our proposed ABSA platform also provided excellent specificity for the tests to
distinguish L. monocytogenes from other Listeria species and other bacterial
genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and
3 Shigella spp.). Improvements in the sensitivity and specificity have not only
facilitated the reliable detection of L. monocytogenes at extremely low
concentrations, but also allowed for the development of a 96-well plate-based
routine assay platform for multivalent diagnostics.

Copyright © 2015 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.bios.2015.01.009 
PMID: 25590973  [Indexed for MEDLINE]


156. Front Microbiol. 2015 Jun 12;6:569. doi: 10.3389/fmicb.2015.00569. eCollection
2015.

RNA-Seq analysis of isolate- and growth phase-specific differences in the global 
transcriptomes of enteropathogenic Escherichia coli prototype isolates.

Hazen TH(1), Daugherty SC(2), Shetty A(2), Mahurkar AA(2), White O(2), Kaper
JB(3), Rasko DA(1).

Author information: 
(1)Institute for Genome Sciences, University of Maryland School of Medicine
Baltimore, MD, USA ; Department of Microbiology and Immunology, University of
Maryland School of Medicine Baltimore, MD, USA.
(2)Institute for Genome Sciences, University of Maryland School of Medicine
Baltimore, MD, USA.
(3)Department of Microbiology and Immunology, University of Maryland School of
Medicine Baltimore, MD, USA.

Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness
among infants in developing countries. E. coli isolates classified as typical
EPEC are identified by the presence of the locus of enterocyte effacement (LEE)
and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while
the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin.
Comparative genomic analyses have demonstrated that EPEC isolates belong to
diverse evolutionary lineages and possess lineage- and isolate-specific genomic
content. To investigate whether this genomic diversity results in significant
differences in global gene expression, we used an RNA sequencing (RNA-Seq)
approach to characterize the global transcriptomes of the prototype typical EPEC 
isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate
E110019. The global transcriptomes were characterized during laboratory growth in
two different media and three different growth phases, as well as during
adherence of the EPEC isolates to human cells using in vitro tissue culture
assays. Comparison of the global transcriptomes during these conditions was used 
to identify isolate- and growth phase-specific differences in EPEC gene
expression. These analyses resulted in the identification of genes that encode
proteins involved in survival and metabolism that were coordinately expressed
with virulence factors. These findings demonstrate there are isolate- and growth 
phase-specific differences in the global transcriptomes of EPEC prototype
isolates, and highlight the utility of comparative transcriptomics for
identifying additional factors that are directly or indirectly involved in EPEC
pathogenesis.

DOI: 10.3389/fmicb.2015.00569 
PMCID: PMC4464170
PMID: 26124752 


157. Plant Cell Physiol. 2015 Jun;56(6):1172-82. doi: 10.1093/pcp/pcv043. Epub 2015
Mar 9.

Functional Characterization of Cucurbitadienol Synthase and Triterpene
Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia
grosvenorii.

Dai L(1), Liu C(1), Zhu Y(1), Zhang J(1), Men Y(1), Zeng Y(1), Sun Y(2).

Author information: 
(1)National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
(2)National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
syx0430@hotmail.com.

Mogrosides, the major bioactive components isolated from the fruits of Siraitia
grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins
that are used worldwide as high-potency sweeteners and possess a variety of
notable pharmacological activities. Mogrosides are synthesized from
2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase
(CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However,
the relevant genes have not been characterized to date. In this study, we report 
successful identification of SgCbQ and UGT74AC1, which were previously predicted 
via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis
of the fruits of S. grosvenorii. SgCbQ was functionally characterized by
expression in the lanosterol synthase-deficient yeast strain GIL77 and was found 
to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously
expressed in Escherichia coli as a His-tag protein and it showed specificity for 
mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE
by in vitro enzymatic activity assays. This study reports the identification of
CbQ and glycosyltransferase from S. grosvenorii for the first time. The results
also suggest that RNA-seq, combined with DGE profile analysis, is a promising
approach for discovery of candidate genes involved in biosynthesis of triterpene 
saponins.

© The Author 2015. Published by Oxford University Press on behalf of Japanese
Society of Plant Physiologists. All rights reserved. For permissions, please
email: journals.permissions@oup.com.

DOI: 10.1093/pcp/pcv043 
PMID: 25759326  [Indexed for MEDLINE]


158. PLoS One. 2015 May 28;10(5):e0124106. doi: 10.1371/journal.pone.0124106.
eCollection 2015.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional
Characterization of the Three First Steps of the Pathway in Salvia fruticosa and 
Rosmarinus officinalis.

Božić D(1), Papaefthimiou D(1), Brückner K(2), de Vos RC(3), Tsoleridis CA(4),
Katsarou D(1), Papanikolaou A(1), Pateraki I(5), Chatzopoulou FM(1), Dimitriadou 
E(1), Kostas S(6), Manzano D(5), Scheler U(2), Ferrer A(7), Tissier A(2), Makris 
AM(8), Kampranis SC(9), Kanellis AK(1).

Author information: 
(1)Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, 
Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 
24 Thessaloniki, Greece.
(2)Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic
Biology, Halle (Saale), Germany.
(3)Plant Research International, Wageningen University and Research Centre, The
Netherlands; Netherlands Metabolomics Centre, Leiden, The Netherlands.
(4)Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University
of Thessaloniki, 541 24 Thessaloniki, Greece.
(5)Department of Molecular Genetics, Centre for Research in Agricultural Genomics
(CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain.
(6)Laboratory of Floriculture, School of Agriculture, Aristotle University of
Thessaloniki, 541 24 Thessaloniki, Greece.
(7)Department of Molecular Genetics, Centre for Research in Agricultural Genomics
(CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Vallés, 08193 Barcelona, Spain;
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University
of Barcelona, 08028 Barcelona, Spain.
(8)Institute of Applied Biosciences, Centre for Research and Technology Hellas,
Thermi Thessaloniki, Greece.
(9)Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 
2208, 710 03 Heraklion, Greece.

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic,
antibacterial and neuroprotective properties that is produced by a number of
species from several genera of the Lamiaceae family, including Salvia fruticosa
(Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA
biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined
for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS
and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene
synthase-like genes, respectively, were isolated and functionally characterized. 
Recombinant expression in Escherichia coli followed by in vitro enzyme activity
assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS 
with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene,
as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). 
Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana
further confirmed production of miltiradiene in planta. To elucidate the
subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis
were searched for cytochrome P450 (CYP) encoding genes potentially involved in
the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three
candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N.
benthamiana expression systems, all three where confirmed to be coding for
ferruginol synthases, thus revealing the enzymatic activities responsible for the
first three steps leading to CA in two Lamiaceae genera.

DOI: 10.1371/journal.pone.0124106 
PMCID: PMC4447455
PMID: 26020634  [Indexed for MEDLINE]


159. Nucleic Acids Res. 2015 May 26;43(10):e67. doi: 10.1093/nar/gkv177. Epub 2015 Mar
12.

Analysis of strand-specific RNA-seq data using machine learning reveals the
structures of transcription units in Clostridium thermocellum.

Chou WC(1), Ma Q(1), Yang S(2), Cao S(3), Klingeman DM(4), Brown SD(4), Xu Y(5).

Author information: 
(1)Computational Systems Biology Lab, Department of Biochemistry and Molecular
Biology, and Institute of Bioinformatics, University of Georgia, GA 30602, USA
BioEnergy Science Center, TN 37831, USA.
(2)BioEnergy Science Center, TN 37831, USA Biosciences Division, Oak Ridge
National Laboratory, TN 37831, USA National Bioenergy Center, National Renewable 
Energy Laboratory, Golden, CO 80401, USA.
(3)Computational Systems Biology Lab, Department of Biochemistry and Molecular
Biology, and Institute of Bioinformatics, University of Georgia, GA 30602, USA.
(4)BioEnergy Science Center, TN 37831, USA Biosciences Division, Oak Ridge
National Laboratory, TN 37831, USA.
(5)Computational Systems Biology Lab, Department of Biochemistry and Molecular
Biology, and Institute of Bioinformatics, University of Georgia, GA 30602, USA
BioEnergy Science Center, TN 37831, USA College of Computer Science and
Technology and School of Public Health, Jilin University, Changchun, Jilin
130012, China xyn@bmb.uga.edu.

Identification of transcription units (TUs) encoded in a bacterial genome is
essential to elucidation of transcriptional regulation of the organism. To gain a
detailed understanding of the dynamically composed TU structures, we have used
four strand-specific RNA-seq (ssRNA-seq) datasets collected under two
experimental conditions to derive the genomic TU organization of Clostridium
thermocellum using a machine-learning approach. Our method accurately predicted
the genomic boundaries of individual TUs based on two sets of parameters
measuring the RNA-seq expression patterns across the genome: expression-level
continuity and variance. A total of 2590 distinct TUs are predicted based on the 
four RNA-seq datasets. Among the predicted TUs, 44% have multiple genes. We
assessed our prediction method on an independent set of RNA-seq data with longer 
reads. The evaluation confirmed the high quality of the predicted TUs. Functional
enrichment analyses on a selected subset of the predicted TUs revealed
interesting biology. To demonstrate the generality of the prediction method, we
have also applied the method to RNA-seq data collected on Escherichia coli and
achieved high prediction accuracies. The TU prediction program named SeqTU is
publicly available at https://code.google.com/p/seqtu/. We expect that the
predicted TUs can serve as the baseline information for studying transcriptional 
and post-transcriptional regulation in C. thermocellum and other bacteria.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gkv177 
PMCID: PMC4446414
PMID: 25765651  [Indexed for MEDLINE]


160. PLoS One. 2015 May 19;10(5):e0121911. doi: 10.1371/journal.pone.0121911.
eCollection 2015.

Repositioning of Memantine as a Potential Novel Therapeutic Agent against
Meningitic E. coli-Induced Pathogenicities through Disease-Associated Alpha7
Cholinergic Pathway and RNA Sequencing-Based Transcriptome Analysis of Host
Inflammatory Responses.

Yu JY(1), Zhang B(1), Peng L(2), Wu CH(3), Cao H(4), Zhong JF(5), Hoffman J(3),
Huang SH(1).

Author information: 
(1)Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical
Disease Research, School of Public Health and Tropical Medicine, Southern Medical
University, Guangzhou 510515, China; Saban Research Institute of Children's
Hospital Los Angeles, Department of Pediatrics, Keck School of Medicine,
University of Southern California, Los Angeles, CA, 90027, United States of
America.
(2)Saban Research Institute of Children's Hospital Los Angeles, Department of
Pediatrics, Keck School of Medicine, University of Southern California, Los
Angeles, CA, 90027, United States of America; Department of Clinic Laboratory,
the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260,
China.
(3)Saban Research Institute of Children's Hospital Los Angeles, Department of
Pediatrics, Keck School of Medicine, University of Southern California, Los
Angeles, CA, 90027, United States of America.
(4)Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical
Disease Research, School of Public Health and Tropical Medicine, Southern Medical
University, Guangzhou 510515, China.
(5)Department of Pathology, Keck School of Medicine, University of Southern
California, Los Angeles, CA, 90033, United States of America; Department of
Perio, Diagnostic Sciences & Biomedical Sciences, School of Dentistry, University
of Southern California, Los Angeles, CA, 93003, United States of America;
Department of Pediatrics, School of Medicine, University of Southern California, 
Los Angeles, CA, 93003, United States of America.

Neonatal sepsis and meningitis (NSM) remains a leading cause worldwide of
mortality and morbidity in newborn infants despite the availability of
antibiotics over the last several decades. E. coli is the most common
gram-negative pathogen causing NSM. Our previous studies show that α7 nicotinic
receptor (α7 nAChR), an essential regulator of inflammation, plays a detrimental 
role in the host defense against NSM. Despite notable successes, there still
exists an unmet need for new effective therapeutic approaches to treat this
disease. Using the in vitro/in vivo models of the blood-brain barrier (BBB) and
RNA-seq, we undertook a drug repositioning study to identify unknown
antimicrobial activities for known drugs. We have demonstrated for the first time
that memantine (MEM), a FDA-approved drug for treatment of Alzheimer's disease,
could very efficiently block E. coli-caused bacteremia and meningitis in a mouse 
model of NSM in a manner dependent on α7 nAChR. MEM was able to synergistically
enhance the antibacterial activity of ampicillin in HBMEC infected with E. coli
K1 (E44) and in neonatal mice with E44-caused bacteremia and meningitis.
Differential gene expression analysis of RNA-Seq data from mouse BMEC infected
with E. coli K1 showed that several E44-increased inflammatory factors, including
IL33, IL18rap, MMP10 and Irs1, were significantly reduced by MEM compared to the 
infected cells without drug treatment. MEM could also significantly up-regulate
anti-inflammatory factors, including Tnfaip3, CISH, Ptgds and Zfp36. Most
interestingly, these factors may positively and negatively contribute to
regulation of NF-κB, which is a hallmark feature of bacterial meningitis.
Furthermore, we have demonstrated that circulating BMEC (cBMEC) are the potential
novel biomarkers for NSM. MEM could significantly reduce E44-increased blood
level of cBMEC in mice. Taken together, our data suggest that memantine can
efficiently block host inflammatory responses to bacterial infection through
modulation of both inflammatory and anti-inflammatory pathways.

DOI: 10.1371/journal.pone.0121911 
PMCID: PMC4437645
PMID: 25993608  [Indexed for MEDLINE]


161. Antimicrob Agents Chemother. 2015 May;59(5):2508-14. doi: 10.1128/AAC.05180-14.
Epub 2015 Feb 9.

Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia
sericata show combinatorial activity against bacteria.

Pöppel AK(1), Vogel H(2), Wiesner J(1), Vilcinskas A(3).

Author information: 
(1)Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of 
Bioresources, Giessen, Germany.
(2)Max Planck Institute for Chemical Ecology, Department of Entomology, Jena,
Germany.
(3)Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of 
Bioresources, Giessen, Germany Institute of Phytopathology and Applied Zoology,
Justus-Liebig-University of Giessen, Giessen, Germany
andreas.vilcinskas@agrar.uni-giessen.de.

The larvae of the common green bottle fly (Lucilia sericata) produce
antibacterial secretions that have a therapeutic effect on chronic and nonhealing
wounds. Recent developments in insect biotechnology have made it possible to use 
these larvae as a source of novel anti-infectives. Here, we report the
application of next-generation RNA sequencing (RNA-Seq) to characterize the
transcriptomes of the larval glands, crop, and gut, which contribute to the
synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our
data confirm that L. sericata larvae have adapted in order to colonize
microbially contaminated habitats, such as carrion and necrotic wounds, and are
protected against infection by a diverse spectrum of AMPs. L. sericata AMPs
include not only lucifensin and lucimycin but also novel attacins, cecropins,
diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes
encoding putative AMPs and produced 23 as synthetic analogs, among which some
displayed activities against a broad spectrum of microbial pathogens, including
Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against
Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found
mostly additive effects but also synergistic activity when selected AMPs were
tested in combination. The AMPs that are easy to synthesize are currently being
produced in bulk to allow their evaluation as novel anti-infectives that can be
formulated in hydrogels to produce therapeutic wound dressings and adhesive
bandages.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AAC.05180-14 
PMCID: PMC4394815
PMID: 25666157  [Indexed for MEDLINE]


162. ISME J. 2015 May;9(5):1130-40. doi: 10.1038/ismej.2014.204. Epub 2014 Oct 24.

Gene expression analysis of E. coli strains provides insights into the role of
gene regulation in diversification.

Vital M(1), Chai B(1), Østman B(2), Cole J(1), Konstantinidis KT(3), Tiedje
JM(1).

Author information: 
(1)Center for Microbial Ecology, Michigan State University, East Lansing, MI,
USA.
(2)BEACON Center for the Study of Evolution in Action, Michigan State University,
East Lansing, MI, USA.
(3)School of Civil and Environmental Engineering and School of Biology, Georgia
Institute of Technology, Atlanta, GA, USA.

Escherichia coli spans a genetic continuum from enteric strains to several
phylogenetically distinct, atypical lineages that are rare in humans, but more
common in extra-intestinal environments. To investigate the link between gene
regulation, phylogeny and diversification in this species, we analyzed global
gene expression profiles of four strains representing distinct evolutionary
lineages, including a well-studied laboratory strain, a typical commensal
(enteric) strain and two environmental strains. RNA-Seq was employed to compare
the whole transcriptomes of strains grown under batch, chemostat and starvation
conditions. Highly differentially expressed genes showed a significantly lower
nucleotide sequence identity compared with other genes, indicating that gene
regulation and coding sequence conservation are directly connected. Overall,
distances between the strains based on gene expression profiles were largely
dependent on the culture condition and did not reflect phylogenetic relatedness. 
Expression differences of commonly shared genes (all four strains) and E. coli
core genes were consistently smaller between strains characterized by more
similar primary habitats. For instance, environmental strains exhibited increased
expression of stress defense genes under carbon-limited growth and entered a more
pronounced survival-like phenotype during starvation compared with other strains,
which stayed more alert for substrate scavenging and catabolism during no-growth 
conditions. Since those environmental strains show similar genetic distance to
each other and to the other two strains, these findings cannot be simply
attributed to genetic relatedness but suggest physiological adaptations. Our
study provides new insights into ecologically relevant gene-expression and
underscores the role of (differential) gene regulation for the diversification of
the model bacterial species.

DOI: 10.1038/ismej.2014.204 
PMCID: PMC4409157
PMID: 25343512  [Indexed for MEDLINE]


163. Microbiology. 2015 May;161(Pt 5):980-8. doi: 10.1099/mic.0.000063. Epub 2015 Feb 
24.

Guide to the various phylogenetic classification schemes for Escherichia coli and
the correspondence among schemes.

Clermont O(1), Gordon D(2), Denamur E(3).

Author information: 
(1)INSERM, IAME, UMR 1137, F-75018 Paris, France Université Paris Diderot, IAME, 
UMR 1137, Sorbonne Paris Cité, F-75018 Paris, France.
(2)Division of Evolution, Ecology & Genetics, Research School of Biology,
Australian National University, Canberra, ACT 0200 Australia.
(3)INSERM, IAME, UMR 1137, F-75018 Paris, France Université Paris Diderot, IAME, 
UMR 1137, Sorbonne Paris Cité, F-75018 Paris, France erick.denamur@inserm.fr.

Numerous tools allowing the rapid and universal identification of the
clones/clonal complexes/phylogroups of Escherichia coli have been developed, as
it is a commensal of the vertebrate gut, a major pathogen in veterinary and human
medicine, and a bacterial indicator of faecal contamination. The ability to
identify clones/clonal complexes/phylogroups is crucial, as a strain's ecological
niche, lifestyle and propensity to cause disease vary with its phylogenetic
origins. There are currently three multi-locus sequence typing (MLST) schemes for
E. coli, as well as several PCR-based assays for determining a strain's
phylogroup or clonal complex. In this work, we present data that will enable
investigators to determine the correspondence between the PCR-based assays and
the three MLST schemes, and provide the means for assigning a sequence type (ST) 
to a phylogroup when no other data on the strain phylogroup membership are
available. Such information will help the scientific community to accurately
identify the E. coli clones reported in various publications. Although
whole-genome sequencing will replace classical MLST and most alternative
PCR-based methods, the ST nomenclature of the MLST scheme hosted at the
University of Warwick will largely persist.

© 2015 The Authors.

DOI: 10.1099/mic.0.000063 
PMID: 25714816  [Indexed for MEDLINE]


164. Mol Biosyst. 2015 May;11(5):1434-42. doi: 10.1039/c4mb00600c.

Comparative transcriptome analysis between csrA-disruption Clostridium
acetobutylicum and its parent strain.

Tan Y(1), Liu ZY, Liu Z, Zheng HJ, Li FL.

Author information: 
(1)Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess
Technology, Chinese Academy of Sciences, Songling Road No. 189, Qingdao 266101,
China. lifl@qibebt.ac.cn.

The genome of Clostridium acetobutylicum contains the gene encoding CsrA, a
carbon storage regulator. We investigated the function of CsrA in C.
acetobutylicum by insertionally inactivating the encoding gene, CA_C2209 using
the ClosTron. Disruption of csrA obviously decreases the growth of the organism
and reduces the yield of acetone, butanol and ethanol (ABEs). Like the csrA in
Escherichia coli, RNA-seq and β-galactosidase analysis revealed that csrA in C.
acetobutylicum was closely involved in regulating multiple pathways including
flagella assembly, oligopeptide transporting, iron uptake, and central carbon
metabolism. It has also been newly demonstrated that csrA in C. acetobutylicum is
related to the regulation of pathways involved in the phosphotransferase
transporting systems, synthesis of riboflavin, and stage III sporulation. This
research represented the first investigation of global regulation by CsrA in the 
strain belonging to Gram-positive bacteria through transcriptome analysis and
provided the important theoretical evidence for improving solvent production by
transcriptor engineering in C. acetobutylicum.

DOI: 10.1039/c4mb00600c 
PMID: 25832359  [Indexed for MEDLINE]


165. RNA. 2015 May;21(5):1018-30. doi: 10.1261/rna.048470.114. Epub 2015 Mar 3.

Whole-genome mapping of 5' RNA ends in bacteria by tagged sequencing: a
comprehensive view in Enterococcus faecalis.

Innocenti N(1), Golumbeanu M(2), Fouquier d'Hérouël A(3), Lacoux C(4), Bonnin
RA(5), Kennedy SP(6), Wessner F(4), Serror P(4), Bouloc P(5), Repoila F(4),
Aurell E(7).

Author information: 
(1)Department of Computational Biology, KTH Royal Institute of Technology,
AlbaNova University Center, SE-10691 Stockholm, Sweden INRA, UMR1319 Micalis,
Domaine de Vilvert, F-78352, Jouy-en-Josas, France AgroParisTech, UMR Micalis,
Domaine de Vilvert, F-78350, Jouy-en-Josas, France.
(2)Department of Biosystems Science and Engineering, ETH Zürich, CH-4058, Basel, 
Switzerland SIB Swiss Institute of Bioinformatics, University of Basel, CH-4056, 
Basel, Switzerland.
(3)Department of Computational Biology, KTH Royal Institute of Technology,
AlbaNova University Center, SE-10691 Stockholm, Sweden Luxembourg Centre for
Systems Biomedicine, University of Luxembourg, L-4362, Esch-sur-Alzette,
Luxembourg.
(4)INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France
AgroParisTech, UMR Micalis, Domaine de Vilvert, F-78350, Jouy-en-Josas, France.
(5)Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, UMR8621,
F-91405, Orsay, France.
(6)INRA, MetaGenoPolis US1367, Domaine de Vilvert, F-78350, Jouy-en-Josas,
France.
(7)Department of Computational Biology, KTH Royal Institute of Technology,
AlbaNova University Center, SE-10691 Stockholm, Sweden Department of Information 
and Computer Science, Aalto University, FI-02150 Espoo, Finland.

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the 
first snapshot of transcriptional organizations in this bacterium, we used a
modified RNA-seq approach enabling to discriminate primary from processed 5' RNA 
ends. We also validated our approach by confirming known features in Escherichia 
coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites
(PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-
and SigN-dependent promoters preceding transcription start sites mapped. We
discovered 85 novel putative regulatory RNAs, small- and antisense RNAs, and 72
transcriptional antisense organizations. Presented data constitute a significant 
insight into bacterial RNA landscapes and a step toward the inference of
regulatory processes at transcriptional and post-transcriptional levels in a
comprehensive manner.

© 2015 Innocenti et al.; Published by Cold Spring Harbor Laboratory Press for the
RNA Society.

DOI: 10.1261/rna.048470.114 
PMCID: PMC4408782
PMID: 25737579  [Indexed for MEDLINE]


166. Infect Immun. 2015 Apr;83(4):1443-50. doi: 10.1128/IAI.02904-14. Epub 2015 Jan
26.

Blocking yersiniabactin import attenuates extraintestinal pathogenic Escherichia 
coli in cystitis and pyelonephritis and represents a novel target to prevent
urinary tract infection.

Brumbaugh AR(1), Smith SN(1), Subashchandrabose S(1), Himpsl SD(1), Hazen TH(2), 
Rasko DA(2), Mobley HL(3).

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, Michigan, USA.
(2)Department of Microbiology and Immunology, Institute for Genome Sciences,
School of Medicine, University of Maryland, Baltimore, Maryland, USA.
(3)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, Michigan, USA hmobley@umich.edu.

The emergence and spread of extended-spectrum beta-lactamases and carbapenemases 
among common bacterial pathogens are threatening our ability to treat routine
hospital- and community-acquired infections. With the pipeline for new
antibiotics virtually empty, there is an urgent need to develop novel
therapeutics. Bacteria require iron to establish infection, and specialized
pathogen-associated iron acquisition systems like yersiniabactin, common among
pathogenic species in the family Enterobacteriaceae, including
multidrug-resistant Klebsiella pneumoniae and pathogenic Escherichia coli,
represent potentially novel therapeutic targets. Although the yersiniabactin
system was recently identified as a vaccine target for uropathogenic E. coli
(UPEC)-mediated urinary tract infection (UTI), its contribution to UPEC
pathogenesis is unknown. Using an E. coli mutant (strain 536ΔfyuA) unable to
acquire yersiniabactin during infection, we established the yersiniabactin
receptor as a UPEC virulence factor during cystitis and pyelonephritis, a fitness
factor during bacteremia, and a surface-accessible target of the experimental
FyuA vaccine. In addition, we determined through transcriptome sequencing
(RNA-seq) analyses of RNA from E. coli causing cystitis in women that iron
acquisition systems, including the yersiniabactin system, are highly expressed by
bacteria during natural uncomplicated UTI. Given that yersiniabactin contributes 
to the virulence of several pathogenic species in the family Enterobacteriaceae, 
including UPEC, and is frequently associated with multidrug-resistant strains, it
represents a promising novel target to combat antibiotic-resistant infections.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/IAI.02904-14 
PMCID: PMC4363403
PMID: 25624354  [Indexed for MEDLINE]


167. Microbiology. 2015 Apr;161(Pt 4):729-38. doi: 10.1099/mic.0.000026. Epub 2015 Jan
7.

Cooperative regulation of the common target genes between H₂O₂-sensing YedVW and 
Cu²⁺-sensing CusSR in Escherichia coli.

Urano H(1), Umezawa Y(2), Yamamoto K(3), Ishihama A(3), Ogasawara H(4).

Author information: 
(1)Research Center for Human and Environmental Sciences, Shinshu University,
Ueda, Nagano 386-8567, Japan.
(2)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan.
(3)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo
184-8584, Japan.
(4)Research Center for Human and Environmental Sciences, Shinshu University,
Ueda, Nagano 386-8567, Japan hogasawara@shinshu-u.ac.jp.

YedVW is one of the uncharacterized two-component systems (TCSs) of Escherichia
coli. In order to identify the regulation targets of YedVW, we performed genomic 
SELEX (systematic evolution of ligands by exponential enrichment) screening using
phosphorylated YedW and an E. coli DNA library, and identified YedW-binding sites
within three intergenic spacers, yedW-hiuH, cyoA-ampG and cusR-cusC, along the E.
coli genome. Using a reporter assay system, we found that transcription of hiuH, 
encoding 5-hydroxyisourate hydrolase, was induced at high concentrations of
either Cu(2+) or H₂O₂. Cu(2+)-dependent expression of hiuH was observed in the
yedWV knockout mutant, but was reduced markedly in the cusRS-null mutant.
However, H₂O₂-induced hiuH expression was observed in the cusRS-null mutant, but 
not in the yedWV-null mutant. Gel mobility shift and DNase I footprinting
analyses showed binding of both YedW and CusR to essentially the same sequence
within the hiuH promoter region. Taken together, we concluded that YedVW and
CusSR formed a unique cooperative TCS pair by recognizing and regulating the same
targets, but under different environmental conditions - YedVW played a role in
H₂O₂ response regulation, whilst CusSR played a role in Cu(2+) response
regulation.

DOI: 10.1099/mic.0.000026 
PMID: 25568260  [Indexed for MEDLINE]


168. PLoS One. 2015 Mar 19;10(3):e0119927. doi: 10.1371/journal.pone.0119927.
eCollection 2015.

A method for multiplex gene synthesis employing error correction based on
expression.

Hsiau TH(1), Sukovich D(1), Elms P(1), Prince RN(1), Strittmatter T, Ruan P(2),
Curry B(3), Anderson P(3), Sampson J(3), Anderson JC(4).

Author information: 
(1)Department of Bioengineering, University of California, Berkeley, CA, United
States of America.
(2)Department of Electrical Engineering and Computer Science, University of
California, Berkeley, CA, United States of America.
(3)Agilent Technologies, Santa Clara, CA, United States of America.
(4)Department of Bioengineering, University of California, Berkeley, CA, United
States of America; Synthetic Biology Institute, University of California,
Berkeley, CA, United States of America.

Erratum in
    PLoS One. 2015;10(5):e0126078. Stritmatter, Tobias [corrected to Strittmatter
Tobias].

Our ability to engineer organisms with new biosynthetic pathways and genetic
circuits is limited by the availability of protein characterization data and the 
cost of synthetic DNA. With new tools for reading and writing DNA, there are
opportunities for scalable assays that more efficiently and cost effectively mine
for biochemical protein characteristics. To that end, we have developed the
Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid
assembly, error correction, and expression characterization of many genes as a
pooled library. This methodology enables gene synthesis from
microarray-synthesized oligonucleotide pools with a one-pot technique,
eliminating the need for robotic liquid handling. Post assembly, the gene library
is subjected to an ampicillin based quality control selection, which serves as
both an error correction step and a selection for proteins that are properly
expressed and folded in E. coli. Next generation sequencing of post selection DNA
enables quantitative analysis of gene expression characteristics. We demonstrate 
the feasibility of this approach by building and testing over 90 genes for
empirical evidence of soluble expression. This technique reduces the problem of
part characterization to multiplex oligonucleotide synthesis and deep sequencing,
two technologies under extensive development with projected cost reduction.

DOI: 10.1371/journal.pone.0119927 
PMCID: PMC4366238
PMID: 25790188  [Indexed for MEDLINE]


169. MBio. 2015 Mar 10;6(2):e00025. doi: 10.1128/mBio.00025-15.

Bacterial secretions of nonpathogenic Escherichia coli elicit inflammatory
pathways: a closer investigation of interkingdom signaling.

Zargar A, Quan DN, Carter KK, Guo M(1), Sintim HO(1), Payne GF, Bentley WE(2).

Author information: 
(1)Department of Chemistry and Biochemistry, University of Maryland, College
Park, Maryland, USA.
(2)bentley@umd.edu.

There have been many studies on the relationship between nonpathogenic bacteria
and human epithelial cells; however, the bidirectional effects of the secretomes 
(secreted substances in which there is no direct bacterium-cell contact) have yet
to be fully investigated. In this study, we use a transwell model to explore the 
transcriptomic effects of bacterial secretions from two different nonpathogenic
Escherichia coli strains on the human colonic cell line HCT-8 using
next-generation transcriptome sequencing (RNA-Seq). E. coli BL21 and W3110, while
genetically very similar (99.1% homology), exhibit key phenotypic differences,
including differences in their production of macromolecular structures (e.g.,
flagella and lipopolysaccharide) and in their secretion of metabolic byproducts
(e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2
[AI-2]). After analysis of differential epithelial responses to the respective
secretomes, this study shows for the first time that a nonpathogenic bacterial
secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while 
also upregulating negative-feedback components, including the NOD-like signaling 
pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule 
and the differences in its secretion rates between these strains, we investigated
its role in HCT-8 cells. We found that the expression of the inflammatory
cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid
upregulation before subsequent downregulation after 24 h. Collectively, these
data demonstrate that secreted products from nonpathogenic bacteria stimulate the
transcription of immune-related biological pathways, followed by the upregulation
of negative-feedback elements that may serve to temper the inflammatory
response.IMPORTANCE: The symbiotic relationship between the microbiome and the
host is important in the maintenance of human health. There is a growing need to 
further understand the nature of these relationships to aid in the development of
homeostatic probiotics and also in the design of novel antimicrobial
therapeutics. To our knowledge, this is the first global-transcriptome study of
bacteria cocultured with human epithelial cells in a model to determine the
transcriptional effects of epithelial cells in which epithelial and bacterial
cells are allowed to "communicate" with each other only through diffusible small 
molecules and proteins. By beginning to demarcate the direct and indirect effects
of bacteria on the gastrointestinal (GI) tract, two-way interkingdom
communication can potentially be mediated between host and microbe.

Copyright © 2015 Zargar et al.

DOI: 10.1128/mBio.00025-15 
PMCID: PMC4453519
PMID: 25759496  [Indexed for MEDLINE]


170. BMC Genomics. 2015 Feb 26;16:134. doi: 10.1186/s12864-015-1315-9.

IPred - integrating ab initio and evidence based gene predictions to improve
prediction accuracy.

Zickmann F(1), Renard BY(2).

Author information: 
(1)Research Group Bioinformatics (NG4), Robert Koch-Institute, Berlin, Germany.
zickmannf@rki.de.
(2)Research Group Bioinformatics (NG4), Robert Koch-Institute, Berlin, Germany.
renardb@rki.de.

BACKGROUND: Gene prediction is a challenging but crucial part in most genome
analysis pipelines. Various methods have evolved that predict genes ab initio on 
reference sequences or evidence based with the help of additional information,
such as RNA-Seq reads or EST libraries. However, none of these strategies is
bias-free and one method alone does not necessarily provide a complete set of
accurate predictions.
RESULTS: We present IPred (Integrative gene Prediction), a method to integrate ab
initio and evidence based gene identifications to complement the advantages of
different prediction strategies. IPred builds on the output of gene finders and
generates a new combined set of gene identifications, representing the integrated
evidence of the single method predictions.
CONCLUSION: We evaluate IPred in simulations and real data experiments on
Escherichia Coli and human data. We show that IPred improves the prediction
accuracy in comparison to single method predictions and to existing methods for
prediction combination.

DOI: 10.1186/s12864-015-1315-9 
PMCID: PMC4345001
PMID: 25766582  [Indexed for MEDLINE]


171. PLoS One. 2015 Feb 23;10(2):e0117261. doi: 10.1371/journal.pone.0117261.
eCollection 2015.

CD14 and complement crosstalk and largely mediate the transcriptional response to
Escherichia coli in human whole blood as revealed by DNA microarray.

Lau C(1), Nygård S(2), Fure H(1), Olstad OK(3), Holden M(4), Lappegård KT(5),
Brekke OL(6), Espevik T(7), Hovig E(8), Mollnes TE(9).

Author information: 
(1)Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, 
Bodø, Norway.
(2)Department of Informatics, University of Oslo, Oslo, Norway; Bioinformatics
Core Facility and Institute for Medical Informatics, Oslo University Hospital,
Oslo, Norway.
(3)Department of Medical Biochemistry, OUS, Ullevaal, Oslo, Norway.
(4)Norwegian Computing Center, Oslo, Norway.
(5)Faculty of Health Sciences, University of Tromsø, Tromsø, Norway; Division of 
Medicine, Nordland Hospital, Bodø, Norway.
(6)Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, 
Bodø, Norway; Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.
(7)Center of Molecular Inflammation Research, Department of Cancer Research and
Molecular Medicine, Norwegian University of Science and Technology, Trondheim,
Norway.
(8)Department of Informatics, University of Oslo, Oslo, Norway; Bioinformatics
Core Facility and Institute for Medical Informatics, Oslo University Hospital,
Oslo, Norway; Department of Tumor Biology, Institute for Cancer Research, Oslo
University Hospital, Oslo, Norway; Department of Cancer Genetics and Informatics,
Oslo University Hospital, Oslo, Norway.
(9)Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, 
Bodø, Norway; Faculty of Health Sciences, University of Tromsø, Tromsø, Norway;
Center of Molecular Inflammation Research, Department of Cancer Research and
Molecular Medicine, Norwegian University of Science and Technology, Trondheim,
Norway; Institute of Immunology, Oslo University Hospital Rikshospitalet and
University of Oslo, Oslo, Norway.

Systemic inflammation like in sepsis is still lacking specific diagnostic markers
and effective therapeutics. The first line of defense against intruding pathogens
and endogenous damage signals is pattern recognition by e.g., complement and
Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 
and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic
inflammatory responses. Using DNA microarray and gene annotation analyses, we
aimed to decipher the effect of combined inhibition of C3 and CD14 on the
transcriptional response to bacterial challenge in human whole blood.
Importantly, combined inhibition reversed the transcriptional changes of 70% of
the 2335 genes which significantly responded to heat-inactivated Escherichia coli
by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a 
suppressive effect of C3 on 21% of the responding genes which was partially
counteracted by CD14. Furthermore, CD14 dependency of the Escherichia
coli-induced response was increased in C5-deficient compared to C5-sufficient
blood. The observed crucial distinct and synergistic roles for complement and
CD14 on the transcriptional level correspond to their broad impact on the
inflammatory response in human blood, and their combined inhibition may become
inevitable in the early treatment of acute systemic inflammation.

DOI: 10.1371/journal.pone.0117261 
PMCID: PMC4338229
PMID: 25706641  [Indexed for MEDLINE]


172. BMC Genomics. 2015 Feb 14;16:72. doi: 10.1186/s12864-015-1237-6.

Next generation sequencing analysis reveals that the ribonucleases RNase II,
RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

Pobre V(1), Arraiano CM(2).

Author information: 
(1)Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova 
de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. vaniapobre@itqb.unl.pt.
(2)Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova 
de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. cecilia@itqb.unl.pt.

BACKGROUND: The RNA steady-state levels in the cell are a balance between
synthesis and degradation rates. Although transcription is important, RNA
processing and turnover are also key factors in the regulation of gene
expression. In Escherichia coli there are three main exoribonucleases (RNase II, 
RNase R and PNPase) involved in RNA degradation. Although there are many studies 
about these exoribonucleases not much is known about their global effect in the
transcriptome.
RESULTS: In order to study the effects of the exoribonucleases on the
transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from
mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each 
of the mutant transcriptome with the wild-type to determine the global effects of
the deletion of each exoribonucleases in exponential phase. We determined that
the deletion of RNase II significantly affected 187 transcripts, while deletion
of RNase R affects 202 transcripts and deletion of PNPase affected 226
transcripts. Surprisingly, many of the transcripts are actually down-regulated in
the exoribonuclease mutants when compared to the wild-type control. The results
obtained from the transcriptomic analysis pointed to the fact that these enzymes 
were changing the expression of genes related with flagellum assembly, motility
and biofilm formation. The three exoribonucleases affected some stable RNAs, but 
PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by
qPCR some fold-change values obtained from the RNA-Seq data, we also observed
that all the exoribonuclease mutants were significantly less motile than the
wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce
more biofilm than the wild-type control while the PNPase mutant did not form
biofilms.
CONCLUSIONS: In this work we demonstrate how deep sequencing can be used to
discover new and relevant functions of the exoribonucleases. We were able to
obtain valuable information about the transcripts affected by each of the
exoribonucleases and compare the roles of the three enzymes. Our results show
that the three exoribonucleases affect cell motility and biofilm formation that
are two very important factors for cell survival, especially for pathogenic
cells.

DOI: 10.1186/s12864-015-1237-6 
PMCID: PMC4335698
PMID: 25757888  [Indexed for MEDLINE]


173. Vet Microbiol. 2015 Jan 30;175(1):123-31. doi: 10.1016/j.vetmic.2014.11.015. Epub
2014 Nov 26.

Influence of the major nitrite transporter NirC on the virulence of a Swollen
Head Syndrome avian pathogenic E. coli (APEC) strain.

de Paiva JB(1), Leite JL(2), da Silva LP(2), Rojas TC(2), de Pace F(3), Conceição
RA(2), Sperandio V(4), da Silveira WD(5).

Author information: 
(1)Department of Genetics, Evolution and Bioagents, Institute of Biology, State
University of Campinas-UNICAMP, PO Box 6109, Campinas, SP, Brazil. Electronic
address: jackboldrin@hotmail.com.
(2)Department of Genetics, Evolution and Bioagents, Institute of Biology, State
University of Campinas-UNICAMP, PO Box 6109, Campinas, SP, Brazil.
(3)Department of Clinical Medicine, Faculty of Medical Sciences, State University
of Campinas-UNICAMP, Campinas, SP, Brazil.
(4)University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.
(5)Department of Genetics, Evolution and Bioagents, Institute of Biology, State
University of Campinas-UNICAMP, PO Box 6109, Campinas, SP, Brazil. Electronic
address: wds@unicamp.br.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli
that infect poultry and cause diseases. Nitrite is a central branch-point in
bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike
nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The
NirC protein acts as a specific channel to facilitate the transport of nitrite
into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic
detoxification. NirC is also required for the pathogenicity of Salmonella by
downregulating the production of NO by the host macrophages. Based on an in vitro
microarray that revealed the overexpression of the nirC gene in APEC strain
SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its
virulence potential using in vivo and in vitro assays. The final cumulative
mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC
caused a gradual increase in the mortality rate during the seven days recorded,
the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells
in the spleen, lung and liver were higher than those of the WT after 48 hpi but
similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in 
macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell 
adhesion ability of the ΔnirC strain was about half the WT level in the presence 
and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene
influences the pathogenicity of SCI-07 strain.

Copyright © 2014 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.vetmic.2014.11.015 
PMID: 25487442  [Indexed for MEDLINE]


174. ACS Synth Biol. 2015 Jan 16;4(1):17-22. doi: 10.1021/sb5001565. Epub 2014 Jun 20.

Direct mutagenesis of thousands of genomic targets using microarray-derived
oligonucleotides.

Bonde MT(1), Kosuri S, Genee HJ, Sarup-Lytzen K, Church GM, Sommer MO, Wang HH.

Author information: 
(1)Novo Nordisk Foundation Center for Biosustainability, Technical University of 
Denmark , DK-2970 Hørsholm, Denmark.

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of 
multiple target sites in bacterial genomes using short oligonucleotides. However,
large-scale mutagenesis requires hundreds to thousands of unique oligos, which
are costly to synthesize and impossible to scale-up by traditional
phosphoramidite column-based approaches. Here, we describe a novel method to
amplify oligos from microarray chips for direct use in MAGE to perturb thousands 
of genomic sites simultaneously. We demonstrated the feasibility of large-scale
mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using
this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting
mutant library was characterized by high-throughput sequencing to show that all
attempted insertions were estimated to have occurred at an average frequency of
0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables
cost-effective large-scale targeted genome engineering that should be useful for 
a variety of applications in synthetic biology and metabolic engineering.

DOI: 10.1021/sb5001565 
PMCID: PMC4304438
PMID: 24856730  [Indexed for MEDLINE]


175. Mol Syst Biol. 2015 Jan 12;11(1):781. doi: 10.15252/msb.20145794.

Genome-wide study of mRNA degradation and transcript elongation in Escherichia
coli.

Chen H(1), Shiroguchi K(2), Ge H(3), Xie XS(4).

Author information: 
(1)Department of Molecular and Cellular Biology, Harvard University, Cambridge,
MA, USA Department of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA, USA.
(2)Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
MA, USA.
(3)Biodynamic Optical Imaging Center (BIOPIC), School of Life Science Peking
University, Beijing, China Beijing International Center for Mathematical Research
(BICMR) Peking University, Beijing, China haoge@pku.edu.cn
xie@chemistry.harvard.edu.
(4)Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
MA, USA Biodynamic Optical Imaging Center (BIOPIC), School of Life Science Peking
University, Beijing, China haoge@pku.edu.cn xie@chemistry.harvard.edu.

Erratum in
    Mol Syst Biol. 2015 May;11(5):808.

An essential part of gene expression is the coordination of RNA synthesis and
degradation, which occurs in the same cellular compartment in bacteria. Here, we 
report a genome-wide RNA degradation study in Escherichia coli using RNA-seq, and
present evidence that the stereotypical exponential RNA decay curve obtained
using initiation inhibitor, rifampicin, consists of two phases: residual RNA
synthesis, a delay in the interruption of steady state that is dependent on
distance relative to the mRNA's 5' end, and the exponential decay. This gives a
more accurate RNA lifetime and RNA polymerase elongation rate simultaneously
genome-wide. Transcripts typically have a single RNA decay constant along all
positions, which is distinct between different operons, indicating that RNA
stability is unlikely determined by local sequences. These measurements allowed
us to establish a model for RNA processing involving co-transcriptional
degradation, providing quantitative description of the macromolecular
coordination in gene expression in bacteria on a system-wide level.

© 2015 The Authors. Published under the terms of the CC BY 4.0 license.


PMCID: PMC4332155
PMID: 25583150  [Indexed for MEDLINE]


176. Antimicrob Agents Chemother. 2015 Jan;59(1):536-43. doi: 10.1128/AAC.04037-14.
Epub 2014 Nov 10.

Genomic and transcriptomic analyses of colistin-resistant clinical isolates of
Klebsiella pneumoniae reveal multiple pathways of resistance.

Wright MS(1), Suzuki Y(1), Jones MB(1), Marshall SH(2), Rudin SD(2), van Duin
D(3), Kaye K(4), Jacobs MR(5), Bonomo RA(6), Adams MD(7).

Author information: 
(1)J. Craig Venter Institute, La Jolla, California, USA.
(2)Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Cleveland, Ohio, USA.
(3)Division of Infectious Diseases, University of North Carolina, Chapel Hill,
North Carolina.
(4)Detroit Medical Center, Detroit, Michigan, USA.
(5)Department of Pathology, Case Western Reserve University and University
Hospitals Case Medical Center, Cleveland, Ohio, USA.
(6)Research Service, Louis Stokes Cleveland Department of Veterans Affairs
Medical Center, Cleveland, Ohio, USA Departments of Medicine, Pharmacology,
Molecular Biology and Microbiology, Case Western Reserve University, Cleveland,
Ohio, USA Robert.Bonomo@va.gov madams@jcvi.org.
(7)J. Craig Venter Institute, La Jolla, California, USA Robert.Bonomo@va.gov
madams@jcvi.org.

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in 
a more frequent reliance on treatment using colistin. However, resistance to
colistin (Col(r)) is increasingly reported from clinical settings. The genetic
mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized.
Using a combination of genome sequencing and transcriptional profiling by RNA
sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine 
Col(r) clinical isolates. Col(r) was related to mutations in three different
genes in K. pneumoniae strains, with distinct impacts on gene expression.
Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N)
modification of lipid A was found in all Col(r) strains. Alteration of the mgrB
gene was observed in six strains. One strain had a mutation in phoQ. Common among
these seven strains was elevated expression of phoPQ and unaltered expression of 
pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide
(LPS). In two strains, separate mutations were found in a previously
uncharacterized histidine kinase gene that is part of a two-component regulatory 
system (TCRS) now designated crrAB. In these strains, expression of pmrCAB,
crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was
elevated. Complementation with the wild-type allele restored colistin
susceptibility in both strains. The crrAB genes are present in most K. pneumoniae
genomes, but not in Escherichia coli. Additional upregulated genes in all strains
include those involved in cation transport and maintenance of membrane integrity.
Because the crrAB genes are present in only some strains, Col(r) mechanisms may
be dependent on the genetic background.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AAC.04037-14 
PMCID: PMC4291396
PMID: 25385117  [Indexed for MEDLINE]


177. Euro Surveill. 2015;20(47). doi: 10.2807/1560-7917.ES.2015.20.47.30073.

The utility of multiple molecular methods including whole genome sequencing as
tools to differentiate Escherichia coli O157:H7 outbreaks.

Berenger BM(1), Berry C, Peterson T, Fach P, Delannoy S, Li V, Tschetter L, Nadon
C, Honish L, Louie M, Chui L.

Author information: 
(1)Alberta Provincial Laboratory for Public Health, Alberta, Canada.

A standardised method for determining Escherichia coli O157:H7 strain relatedness
using whole genome sequencing or virulence gene profiling is not yet established.
We sought to assess the capacity of either high-throughput polymerase chain
reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or
k-mer clustering to discriminate between outbreak-associated and sporadic E. coli
O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the
province of Alberta, Canada were included in the study. Two of the outbreaks
occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel
electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis
(MLVA) were employed as comparator typing methods. The virulence gene profiles of
isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic
isolates were mostly identical; therefore the set of virulence genes chosen in
this study were not discriminatory enough to distinguish between outbreak
clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer
phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct
events. k-mer phylogenies demonstrated increased discriminatory power compared
with core SNV phylogenies. Prior to the widespread implementation of whole genome
sequencing for routine public health use, issues surrounding cost, technical
expertise, software standardisation, and data sharing/comparisons must be
addressed.

DOI: 10.2807/1560-7917.ES.2015.20.47.30073 
PMID: 26625187  [Indexed for MEDLINE]


178. FEMS Microbiol Lett. 2015 Jan;362(1):1-8. doi: 10.1093/femsle/fnu013. Epub 2014
Dec 4.

Role of transcription factor NimR (YeaM) in sensitivity control of Escherichia
coli to 2-nitroimidazole.

Ogasawara H(1), Ohe S(2), Ishihama A(3).

Author information: 
(1)Research Center for Human and Environmental Sciences, Shinshu University,
Ueda, Nagano 386-8567, Japan.
(2)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan.
(3)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo
184-8584, Japan aishiham@hosei.ac.jp.

The binding site(s) on the Escherichia coli genome was determined for an
uncharacterized AraC/XylS superfamily transcription factor YeaM by using the in
vitro genomic SELEX screening system. The only one clear binding target of YeaM
was found to locate in the spacer between the divergently transcribed yeaM and
yeaN genes. After the phenotype microarray analysis, the major facilitator
superfamily transporter YeaN was found to confer E. coli the resistance to
2-nitroimidazole, the antibacterial and antifungal antibiotic, suggesting that
YeaN plays a role in 2-nitromidazole efflux. Purified YeaM bound to three sites
within this yeaM-yeaN spacer region. Several lines of in vitro and in vivo
evidence indicate that YeaM regulates transcription of both the yeaM gene itself 
and the yeaNO operon. Taken together we propose to rename yeaN to nimT
(nitroimidazole transporter) and yeaM to nimR (regulator of nimT).

© FEMS 2014. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.

DOI: 10.1093/femsle/fnu013 
PMID: 25790494  [Indexed for MEDLINE]


179. Int J Biol Macromol. 2015;77:293-302. doi: 10.1016/j.ijbiomac.2015.03.043. Epub
2015 Apr 1.

Development of receptor-based inhibitory RNA aptamers for anthrax toxin
neutralization.

Lee SC(1), Gedi V(1), Ha NR(1), Cho JH(1), Park HC(2), Yoon MY(3).

Author information: 
(1)Department of Chemistry and Research Institute of Natural Sciences, Hanyang
University, Seoul 133-791, Republic of Korea.
(2)Veterinary Drugs & Biologics Division, Animal and Plant Quarantine Agency
(QIA), Anyang 430-757, Republic of Korea.
(3)Department of Chemistry and Research Institute of Natural Sciences, Hanyang
University, Seoul 133-791, Republic of Korea. Electronic address:
myyoon@hanyang.ac.kr.

Anthrax toxin excreted by Bacillus anthracis is the key causative agent of
infectious anthrax disease. In the present study, we targeted the binding of PA
to the ATR/TEM8 Von Willebrand factor type A (VWA) domain, which we cloned into
Escherichia coli and purified to homogeneity under denaturing conditions. To
develop an anthrax toxin inhibitor, we selected and identified short single
strand RNA aptamers (approximately 30mer) consisting of different sequences of
nucleic acids with a high binding affinity in the 100 nanomolar range against the
recombinant ATR/TEM8 VWA domain using systematic evolution of ligands by
exponential enrichment (SELEX). Five candidate aptamers were further
characterized by several techniques including secondary structural analysis. The 
inhibitor efficiency (IC50) of one of the aptamers toward anthrax toxin was
approximately 5μM in macrophage RAW 264.7 cells, as determined from cytotoxicity 
analysis by MTT assay. We believe that the candidate aptamers should be useful
for blocking the binding of PA to its receptor in order to neutralize anthrax
toxin.

Copyright © 2015 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.ijbiomac.2015.03.043 
PMID: 25841381  [Indexed for MEDLINE]


180. J Bacteriol. 2015 Jan 1;197(1):18-28. doi: 10.1128/JB.02096-14. Epub 2014 Sep 29.

Global transcriptional start site mapping using differential RNA sequencing
reveals novel antisense RNAs in Escherichia coli.

Thomason MK(1), Bischler T(2), Eisenbart SK(3), Förstner KU(2), Zhang A(1),
Herbig A(4), Nieselt K(4), Sharma CM(5), Storz G(6).

Author information: 
(1)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland, USA.
(2)Research Center for Infectious Diseases (ZINF), University of Würzburg,
Würzburg, Germany.
(3)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland, USA Research Center for Infectious Diseases (ZINF), University of
Würzburg, Würzburg, Germany.
(4)Center for Bioinformatics Tübingen (ZBIT), University of Tübingen, Tübingen,
Germany.
(5)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland, USA Research Center for Infectious Diseases (ZINF), University of
Würzburg, Würzburg, Germany cynthia.sharma@uni-wuerzburg.de storzg@mail.nih.gov.
(6)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland, USA cynthia.sharma@uni-wuerzburg.de storzg@mail.nih.gov.

Comment in
    J Bacteriol. 2015 Jan 1;197(1):4-6.

While the model organism Escherichia coli has been the subject of intense study
for decades, the full complement of its RNAs is only now being examined. Here we 
describe a survey of the E. coli transcriptome carried out using a differential
RNA sequencing (dRNA-seq) approach, which can distinguish between primary and
processed transcripts, and an automated prediction algorithm for transcriptional 
start sites (TSS). With the criterion of expression under at least one of three
growth conditions examined, we predicted 14,868 TSS candidates, including 5,574
internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential
antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by
Northern analysis using RNA from wild-type E. coli and from strains defective for
RNases III and E, two RNases reported to be involved in asRNA processing.
Interestingly, nine asRNAs detected as distinct bands by Northern analysis were
differentially affected by the rnc and rne mutations. We also compared our asRNA 
candidates with previously published asRNA annotations from RNA-seq data and
discuss the challenges associated with these cross-comparisons. Our global
transcriptional start site map represents a valuable resource for identification 
of transcription start sites, promoters, and novel transcripts in E. coli and is 
easily accessible, together with the cDNA coverage plots, in an online genome
browser.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.02096-14 
PMCID: PMC4288677
PMID: 25266388  [Indexed for MEDLINE]


181. Microbiology. 2015 Jan;161(Pt 1):99-111. doi: 10.1099/mic.0.083550-0. Epub 2014
Nov 18.

Regulatory role of transcription factor SutR (YdcN) in sulfur utilization in
Escherichia coli.

Yamamoto K(1)(2), Nakano M(2), Ishihama A(1)(2).

Author information: 
(1)1Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 185-8584,
Japan.
(2)2Research Institute of Micro-Nano Technology, Hosei University, Koganei, Tokyo
185-8584, Japan.

Sulfur makes up 1 % of the dry mass of bacteria, and it is an abundant element
(0.1 %) on earth. Sulfur in the environment is, however, mostly in oxidized forms
and inaccessible to living organisms. At present, the entire assimilation pathway
of external sulfur to sulfur-containing biomolecules and its regulation in
Escherichia coli remain poorly understood, except for the metabolic pathway of
cysteine synthesis, the first-step metabolite of sulfur assembly. During the
search for regulation targets of uncharacterized transcription factors by Genomic
SELEX screening, we found that the hitherto uncharacterized YdcN regulates a set 
of genes involved in the utilization of sulfur, including the generation of
sulfate and its reduction, the synthesis of cysteine, the synthesis of enzymes
containing Fe-S as cofactors, and the modification of tRNA with use of
sulfur-containing substrates. Taking these findings together, we propose renaming
YdcN as SutR (regulator of sulfur utilization).

© 2015 The Authors.

DOI: 10.1099/mic.0.083550-0 
PMID: 25406449  [Indexed for MEDLINE]


182. Vestn Ross Akad Med Nauk. 2015;(1):70-81.

[Molecular-genetic characterization of shiga-toxin producing Escherichia coli
isolated during a food-borne outbreak in St. Petersburg in 2013].

[Article in Russian]

Onishchenko GG, Dyatlov IA, Svetoch EA, Volozhantsev NV, Bannov VA, Kartsev NN,
Borzenkov VN, Fursova NK, Shemyakin IG, Bogun AG, Kislichkina AA, Popova AV,
Myakinina VP, Teimurazov MG, Polosenko OV, Kaftyreva LA, Makarova MA, Matveeva
ZN, Grechaninova TA, Grigor'eva NS, Kicha EV, Zabalueva GV, Kutasova TB, Korzhaev
YN, Bashketova NS, Bushmanova ON, Stalevskaya AV, Tchinjeria IG, Zhebrun FB.

Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported 
worldwide and represent a serious problem for public healthcare. In the Russian
Federation there is little information on epidemiology and etiology of
STEC-infections as well as on molecular-genetic peculiarities of STEC
pathogens.OBJECTIVE: Our aim was to describe a food-borne outbreak as hemorrhagic
colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute
gastroenteritis in children in St. Petersburg in 2013.
METHODS: Epidemiological, microbiological, molecular-genetic and bioinformatic
methods were applied.
RESULTS: Objects to study were clinical specimens, milk and food samples, as well
as STEC strains isolated during the outbreak. The outbreak of food-borne
infection was found to be caused by STEC-contaminated raw milk as confirmed by
epidemiological analysis, detection of STEC DNA and isolation of relevant
pathogens in milk and sick children fecal specimens. The whole-genome sequencing 
revealed two groups ofpathogens, E. coli O157:H7 and E. coli O101:H33 among
collected strains. Group I strains were attributed to the previously known
sequence type ST24, while group II strains belonged to the previously
non-described sequence type ST145. In strain genomes of both groups there were
identified nucleotide sequences of VT2-like prophage carrying stx2c gene, plasmid
enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of 
intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. 
coli O101:H33 strains. The latter previously was identified only in
enteropathogenic E. coli (EPEC) strains.
CONCLUSION: The additional knowledge of epidemiology and biology of STEC
pathogens would assist clinicians and epidemiologists in diagnosing, treating and
preventing hemorrhagic colitis.


PMID: 26027274  [Indexed for MEDLINE]


183. Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):18327-32. doi:
10.1073/pnas.1415959112. Epub 2014 Dec 8.

Host-specific induction of Escherichia coli fitness genes during human urinary
tract infection.

Subashchandrabose S(1), Hazen TH(2), Brumbaugh AR(1), Himpsl SD(1), Smith SN(1), 
Ernst RD(3), Rasko DA(2), Mobley HL(4).

Author information: 
(1)Department of Microbiology and Immunology and.
(2)Department of Microbiology and Immunology, Institute for Genome Sciences,
School of Medicine, University of Maryland, Baltimore, MD 21201.
(3)University Health Service, University of Michigan Medical School, Ann Arbor,
MI 48109; and.
(4)Department of Microbiology and Immunology and hmobley@umich.edu.

Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of
uncomplicated urinary tract infection (UTI), manifested by inflammation of the
urinary bladder, in humans and is a major global public health concern. Molecular
pathogenesis of UPEC has been primarily examined using murine models of UTI.
Translational research to develop novel therapeutics against this major pathogen,
which is becoming increasingly antibiotic resistant, requires a thorough
understanding of mechanisms involved in pathogenesis during human UTIs. Total
RNA-sequencing (RNA-seq) and comparative transcriptional analysis of UTI samples 
to the UPEC isolates cultured in human urine and laboratory medium were used to
identify novel fitness genes that were specifically expressed during human
infection. Evidence for UPEC genes involved in ion transport, including copper
efflux, nickel and potassium import systems, as key fitness factors in
uropathogenesis were generated using an experimental model of UTI. Translational 
application of this study was investigated by targeting Cus, a bacterial copper
efflux system. Copper supplementation in drinking water reduces E. coli
colonization in the urinary bladder of mice. Additionally, our results suggest
that anaerobic processes in UPEC are involved in promoting fitness during UTI in 
humans. In summary, RNA-seq was used to establish the transcriptional signature
in UPEC during naturally occurring, community acquired UTI in women and multiple 
novel fitness genes used by UPEC during human infection were identified. The
repertoire of UPEC genes involved in UTI presented here will facilitate further
translational studies to develop innovative strategies against UTI caused by
UPEC.

DOI: 10.1073/pnas.1415959112 
PMCID: PMC4280598
PMID: 25489107  [Indexed for MEDLINE]


184. J Biotechnol. 2014 Dec 20;192 Pt A:231-9.

Transcriptome analyses to understand effects of the Fusarium deoxynivalenol and
nivalenol mycotoxins on Escherichia coli.

Park J, Lee HH, Youn K, Kim S, Jung B, Lee J, Seo YS.

Fusarium spp. cause many diseases in farming systems and can produce diverse
mycotoxins that can easily impact humans and animals through the ingestion of
food and feed. Among these mycotoxins, deoxynivalenol (DON) and nivalenol (NIV)
are considered the most important hazards because they can rapidly diffuse into
cells and block eukaryotic ribosomes, leading to inhibition of the translation
system. Conversely, the effects of DON and NIV mycotoxins on bacteria remain
unclear. We employed RNA-seq technology to obtain information regarding the
biological responses of bacteria and putative bacterial mechanisms of resistance 
to DON and NIV mycotoxins. Most differentially expressed genes down-regulated in 
response to these mycotoxins were commonly involved in phenylalanine metabolism, 
glyoxylate cycle, and cytochrome o ubiquinol oxidase systems. In addition, we
generated an overall network of 1028 up-regulated genes to identify core genes
under DON and NIV conditions. The results of our study provide a snapshot view of
the transcriptome of Escherichia coli K-12 under DON and NIV conditions.
Furthermore, the information provided herein will be useful for development of
methods to detect DON and NIV.

DOI: 10.1016/j.jbiotec.2014.10.018 
PMID: 25456064  [Indexed for MEDLINE]


185. BMC Genomics. 2014 Dec 12;15:1099. doi: 10.1186/1471-2164-15-1099.

Microarray analysis of the Escherichia coli response to CdTe-GSH Quantum Dots:
understanding the bacterial toxicity of semiconductor nanoparticles.

Monrás JP, Collao B, Molina-Quiroz RC, Pradenas GA, Saona LA, Durán-Toro V,
Ordenes-Aenishanslins N, Venegas FA, Loyola DE, Bravo D, Calderón PF, Calderón
IL, Vásquez CC, Chasteen TG, Lopez DA, Pérez-Donoso JM(1).

Author information: 
(1)Bionanotechnology and Microbiology Lab, Center for Bioinformatics and
Integrative Biology (CBIB), Universidad Andres Bello, Santiago, Chile.
jose.perez@unab.cl.

BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are 
made of heavy metals and involve synthetic methods that require organic solvents 
and high temperatures. This issue makes the development of water-soluble
nanoparticles with lower toxicity a major topic of interest. In a previous work
our group described a biomimetic method for the aqueous synthesis of CdTe-GSH
Quantum Dots (QDs) using biomolecules present in cells as reducing and
stabilizing agents. This protocol produces nanoparticles with good fluorescent
properties and less toxicity than those synthesized by regular chemical methods. 
Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so
it is important to know in detail the effects of these semiconductor
nanoparticles on cells, their levels of toxicity and the strategies that cells
develop to overcome it.
RESULTS: In this work, the response of E. coli exposed to different
sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through
transcriptomic, biochemical, microbiological and genetic approaches. It was
determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii)
QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA)
and tellurium does not contribute significantly to QDs-mediated toxicity since
cells incorporate low levels of Te, iii) red QDs also induce genes related to
oxidative stress response and membrane proteins, iv) Cd2+ release is higher in
red QDs, and v) QDs render the cells more sensitive to polymyxin B.
CONCLUSION: Based on the results obtained in this work, a general model of
CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial
toxicity of QDs is mainly associated with cadmium release, oxidative stress and
loss of membrane integrity. The higher toxicity of red QDs is most probably due
to higher cadmium content and release from the nanoparticle as compared to green 
QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making
these biomimetic QDs candidates for adjuvant therapies against bacterial
infections.

DOI: 10.1186/1471-2164-15-1099 
PMCID: PMC4300170
PMID: 25496196  [Indexed for MEDLINE]


186. PLoS Biol. 2014 Dec 2;12(12):e1002009. doi: 10.1371/journal.pbio.1002009.
eCollection 2014 Dec.

Genome-wide association mapping identifies a new arsenate reductase enzyme
critical for limiting arsenic accumulation in plants.

Chao DY(1), Chen Y(2), Chen J(3), Shi S(4), Chen Z(5), Wang C(4), Danku JM(6),
Zhao FJ(7), Salt DE(6).

Author information: 
(1)National Key Laboratory of Plant Molecular Genetics (NKLPMG), Institute of
Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, China; Institute of Biological and
Environmental Sciences, University of Aberdeen, Aberdeen, United Kingdom.
(2)Sustainable Soils and Grassland Systems Department, Rothamsted Research,
Harpenden, Hertfordshire, United Kingdom.
(3)National Key Laboratory of Plant Molecular Genetics (NKLPMG), Institute of
Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, China.
(4)State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of
Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing,
China.
(5)National Key Laboratory of Plant Molecular Genetics (NKLPMG), Institute of
Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, China; University of Chinese Academy of
Sciences, Beijing, China.
(6)Institute of Biological and Environmental Sciences, University of Aberdeen,
Aberdeen, United Kingdom.
(7)State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of
Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing,
China; Rothamsted Research, Harpenden, Hertfordshire, United Kingdom.

Comment in
    PLoS Biol. 2014 Dec;12(12):e1002008.

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice
presents a significant risk to human health. Plants chemically reduce arsenate to
arsenite. Using genome-wide association (GWA) mapping of loci controlling natural
variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify 
the arsenate reductase required for this reduction, which we named High Arsenic
Content 1 (HAC1). Complementation verified the identity of HAC1, and expression
in Escherichia coli lacking a functional arsenate reductase confirmed the
arsenate reductase activity of HAC1. The HAC1 protein accumulates in the
epidermis, the outer cell layer of the root, and also in the pericycle cells
surrounding the central vascular tissue. Plants lacking HAC1 lose their ability
to efflux arsenite from roots, leading to both increased transport of arsenic
into the central vascular tissue and on into the shoot. HAC1 therefore functions 
to reduce arsenate to arsenite in the outer cell layer of the root, facilitating 
efflux of arsenic as arsenite back into the soil to limit both its accumulation
in the root and transport to the shoot. Arsenate reduction by HAC1 in the
pericycle may play a role in limiting arsenic loading into the xylem. Loss of
HAC1-encoded arsenic reduction leads to a significant increase in arsenic
accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We
also confirmed the previous observation that the ACR2 arsenate reductase in A.
thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does
not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1
double mutant is disrupted in an identical manner to that described for the hac1 
single mutant. Our identification of HAC1 and its associated natural variation
provides an important new resource for the development of low arsenic-containing 
food such as rice.

DOI: 10.1371/journal.pbio.1002009 
PMCID: PMC4251824
PMID: 25464340  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no competing
interests exist.


187. Appl Biochem Biotechnol. 2014 Dec;174(7):2548-56. doi: 10.1007/s12010-014-1206-6.
Epub 2014 Sep 4.

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

Ferreira IM(1), de Souza Lacerda CM, de Faria LS, Corrêa CR, de Andrade AS.

Author information: 
(1)Centro de Desenvolvimento da Tecnologia Nuclear/Comissão Nacional de Energia
Nuclear (CDTN/CNEN), Rua Professor Mário Werneck S/N°, Cidade
Universitária-Campus da UFMG, CEP:31120-970, Belo Horizonte, MG, Brasil,
imendesf@yahoo.com.br.

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer 
cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by
peptides. Peptidoglycan has a particular composition which makes it a possible
target for specific bacterial recognition. Aptamers are single-stranded DNA or
RNA oligonucleotides that bind to target molecules with high affinity and
specificity. Aptamers can be labeled with different radioisotopes and possess
several properties that make them suitable for molecular imaging. The purpose of 
this study was to obtain aptamers for use as radiopharmaceutical in bacterial
infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan
were selected through the Systematic Evolution of Ligands by Exponential
Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was
0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled
with (32)P showed high affinity for Staphylococcus aureus cells. The binding to
S. aureus and Escherichia coli in vitro were significantly higher than for
Candida albicans and human fibroblasts, demonstrating their specificity for
bacterial cells. These results point Antibac1 and Antibac2 as promising tools for
bacterial infections identification.

DOI: 10.1007/s12010-014-1206-6 
PMID: 25185503  [Indexed for MEDLINE]


188. Mol Syst Biol. 2014 Nov 27;10:762. doi: 10.15252/msb.20145227.

Protein acetylation affects acetate metabolism, motility and acid stress response
in Escherichia coli.

Castaño-Cerezo S(1), Bernal V(2), Post H(3), Fuhrer T(4), Cappadona S(5),
Sánchez-Díaz NC(1), Sauer U(4), Heck AJ(3), Altelaar AF(3), Cánovas M(2).

Author information: 
(1)Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de
Química, Universidad de Murcia Campus de Excelencia Mare Nostrum, Murcia, Spain.
(2)Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de
Química, Universidad de Murcia Campus de Excelencia Mare Nostrum, Murcia, Spain
vicente.bernal@gmail.com mcanovas@um.es.
(3)Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht 
University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The 
Netherlands.
(4)Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
(5)Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht 
University, Utrecht, The Netherlands.

Although protein acetylation is widely observed, it has been associated with few 
specific regulatory functions making it poorly understood. To interrogate its
functionality, we analyzed the acetylome in Escherichia coli knockout mutants of 
cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein
acetyltransferase. For four growth conditions, more than 2,000 unique acetylated 
peptides, belonging to 809 proteins, were identified and differentially
quantified. Nearly 65% of these proteins are related to metabolism. The global
activity of CobB contributes to the deacetylation of a large number of substrates
and has a major impact on physiology. Apart from the regulation of acetyl-CoA
synthetase, we found that CobB-controlled acetylation of isocitrate lyase
contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the
transcription factor RcsB prevents DNA binding, activating flagella biosynthesis 
and motility, and increases acid stress susceptibility. Surprisingly, deletion of
patZ increased acetylation in acetate cultures, which suggests that it regulates 
the levels of acetylating agents. The results presented offer new insights into
functional roles of protein acetylation in metabolic fitness and global cell
regulation.

© 2014 The Authors. Published under the terms of the CC BY 4.0 license.


PMCID: PMC4299603
PMID: 25518064  [Indexed for MEDLINE]


189. J Am Chem Soc. 2014 Nov 19;136(46):16299-308. doi: 10.1021/ja508478x. Epub 2014
Nov 5.

Broccoli: rapid selection of an RNA mimic of green fluorescent protein by
fluorescence-based selection and directed evolution.

Filonov GS(1), Moon JD, Svensen N, Jaffrey SR.

Author information: 
(1)Department of Pharmacology, Weill Cornell Medical College , New York, New York
10065, United States.

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse
applications, including imaging RNA trafficking and as a component of RNA-based
sensors that exhibit fluorescence upon binding small molecules in live cells.
These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the
fluorescence of fluorophores similar to that found in green fluorescent protein. 
Although additional highly fluorescent RNA-fluorophore complexes would extend the
utility of this technology, the identification of novel RNA-fluorophore complexes
is difficult. Current approaches select aptamers on the basis of their ability to
bind fluorophores, even though fluorophore binding alone is not sufficient to
activate fluorescence. Additionally, aptamers require extensive mutagenesis to
efficiently fold and exhibit fluorescence in living cells. Here we describe a
platform for rapid generation of highly fluorescent RNA-fluorophore complexes
that are optimized for function in cells. This procedure involves selection of
aptamers on the basis of their binding to fluorophores, coupled with
fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in
Escherichia coli. Promising aptamers are then further optimized using a
FACS-based directed evolution approach. Using this approach, we identified
several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and
activates the fluorescence of
(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one.
Broccoli shows robust folding and green fluorescence in cells, and increased
fluorescence relative to Spinach2. This reflects, in part, improved folding in
the presence of low cytosolic magnesium concentrations. Thus, this novel
fluorescence-based selection approach simplifies the generation of aptamers that 
are optimized for expression and performance in living cells.

DOI: 10.1021/ja508478x 
PMCID: PMC4244833
PMID: 25337688  [Indexed for MEDLINE]


190. PLoS One. 2014 Nov 12;9(11):e106319. doi: 10.1371/journal.pone.0106319.
eCollection 2014.

Ensemble-based network aggregation improves the accuracy of gene network
reconstruction.

Zhong R(1), Allen JD(2), Xiao G(1), Xie Y(2).

Author information: 
(1)Quantitative Biomedical Research Center, Department of Clinical Sciences,
University of Texas Southwestern Medical Center, Dallas, Texas, United States of 
America.
(2)Quantitative Biomedical Research Center, Department of Clinical Sciences,
University of Texas Southwestern Medical Center, Dallas, Texas, United States of 
America; Harold C. Simmons Comprehensive Cancer Center, University of Texas
Southwestern Medical Center, Dallas, Texas, United States of America.

Reverse engineering approaches to constructing gene regulatory networks (GRNs)
based on genome-wide mRNA expression data have led to significant biological
findings, such as the discovery of novel drug targets. However, the reliability
of the reconstructed GRNs needs to be improved. Here, we propose an
ensemble-based network aggregation approach to improving the accuracy of network 
topologies constructed from mRNA expression data. To evaluate the performances of
different approaches, we created dozens of simulated networks from combinations
of gene-set sizes and sample sizes and also tested our methods on three
Escherichia coli datasets. We demonstrate that the ensemble-based network
aggregation approach can be used to effectively integrate GRNs constructed from
different studies - producing more accurate networks. We also apply this approach
to building a network from epithelial mesenchymal transition (EMT) signature
microarray data and identify hub genes that might be potential drug targets. The 
R code used to perform all of the analyses is available in an R package entitled 
"ENA", accessible on CRAN (http://cran.r-project.org/web/packages/ENA/).

DOI: 10.1371/journal.pone.0106319 
PMCID: PMC4229114
PMID: 25390635  [Indexed for MEDLINE]


191. PLoS One. 2014 Nov 3;9(11):e111800. doi: 10.1371/journal.pone.0111800.
eCollection 2014.

Genome-wide analyses and functional classification of proline repeat-rich
proteins: potential role of eIF5A in eukaryotic evolution.

Mandal A(1), Mandal S(1), Park MH(1).

Author information: 
(1)Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health,
Bethesda, Maryland, United States of America.

The eukaryotic translation factor, eIF5A has been recently reported as a
sequence-specific elongation factor that facilitates peptide bond formation at
consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation
factor P (EF-P) does in bacteria. We have searched the genome databases of 35
representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or
PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A.
We have made detailed analyses of proteome data of 5 selected species,
Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus
and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes.
However, their frequencies markedly increase with the biological complexity of
eukaryotic organisms, and are higher in newly derived proteins than in those
orthologous proteins commonly shared in all species. Ontology classifications of 
S. cerevisiae and human genes encoding the highest level of polyprolines reveal
their strong association with several specific biological processes, including
actin/cytoskeletal associated functions, RNA splicing/turnover, DNA
binding/transcription and cell signaling. Previously reported phenotypic defects 
in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the 
proposed role for eIF5A in the translation of the polyproline-containing
proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline
repeat frequency correlates with functional complexity of the five organisms
examined. Taken together, these findings suggest the importance of proline
repeat-rich proteins and a potential role for eIF5A and its hypusine modification
pathway in the course of eukaryotic evolution.

DOI: 10.1371/journal.pone.0111800 
PMCID: PMC4218817
PMID: 25364902  [Indexed for MEDLINE]


192. Inflamm Bowel Dis. 2014 Nov;20(11):1919-32. doi: 10.1097/MIB.0000000000000183.

Inflammation-associated adherent-invasive Escherichia coli are enriched in
pathways for use of propanediol and iron and M-cell translocation.

Dogan B(1), Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, Stewart K, 
Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B, Schukken YH, Stanhope MJ,
Simpson KW.

Author information: 
(1)Departments of *Clinical Sciences, and †Population Medicine and Diagnostic
Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York;
‡Department of Environmental Science and Engineering, Graduate School of Science 
and Engineering, Yamaguchi University, Yamaguchi, Japan; §Departments of
Medicine, and ‖Microbiology and Immunology, University of North Carolina at
Chapel Hill School of Medicine, Chapel Hill, North Carolina; ¶Department of
Gastroenterology, Institute of Translational Medicine, University of Liverpool,
Liverpool, United Kingdom; and **Department of Medicine, Jill Roberts Center for 
Inflammatory Bowel Disease, Weill Medical College of Cornell University, New
York, New York. Dr. T. Lefébure is now with the Université de Lyon and Université
Lyon 1, Centre National de la Recherche Scientifique, Ecologie des Hydrosystèmes 
Naturels et Anthropisés, Villeurbanne, France.

BACKGROUND: Perturbations of the intestinal microbiome, termed dysbiosis, are
linked to intestinal inflammation. Isolation of adherent-invasive Escherichia
coli (AIEC) from intestines of patients with Crohn's disease (CD), dogs with
granulomatous colitis, and mice with acute ileitis suggests these bacteria share 
pathoadaptive virulence factors that promote inflammation.
METHODS: To identify genes associated with AIEC, we sequenced the genomes of
phylogenetically diverse AIEC strains isolated from people with CD (4), dogs with
granulomatous colitis (2), and mice with ileitis (2) and 1 non-AIEC strain from
CD ileum and compared them with 38 genome sequences of E. coli and Shigella. We
then determined the prevalence of AIEC-associated genes in 49 E. coli strains
from patients with CD and controls and correlated genotype with invasion of
intestinal epithelial cells, persistence within macrophages, AIEC pathotype, and 
growth in standardized conditions.
RESULTS: Genes encoding propanediol utilization (pdu operon) and iron acquisition
(yersiniabactin, chu operon) were overrepresented in AIEC relative to
nonpathogenic E. coli. PduC (propanediol dehydratase) was enriched in CD-derived 
AIEC, correlated with increased cellular invasion, and persistence in vitro and
was increasingly expressed in fucose-containing media. Growth of AIEC required
iron, and the presence of chuA (heme acquisition) correlated with persistence in 
macrophages. CD-associated AIEC with lpfA 154 (long polar fimbriae) demonstrated 
increased invasion of epithelial cells and translocation across M cells.
CONCLUSIONS: Our findings provide novel insights into the genetic basis of the
AIEC pathotype, supporting the concept that AIEC are equipped to exploit and
promote intestinal inflammation and reveal potential targets for intervention
against AIEC and inflammation-associated dysbiosis.

DOI: 10.1097/MIB.0000000000000183 
PMID: 25230163  [Indexed for MEDLINE]


193. Sci China Life Sci. 2014 Nov;57(11):1121-30. doi: 10.1007/s11427-014-4734-y. Epub
2014 Sep 18.

Understanding the commonalities and differences in genomic organizations across
closely related bacteria from an energy perspective.

Ma Q(1), Chen X, Liu C, Mao X, Zhang H, Ji F, Wu C, Xu Y.

Author information: 
(1)Computational Systems Biology Laboratory, Department of Biochemistry and
Molecular Biology and Institute of Bioinformatics, University of Georgia, Athens,
GA, 30602, USA.

The availability of a large number of sequenced bacterial genomes facilitates
in-depth studies about why genes (operons) in a bacterial genome are globally
organized the way they are. We have previously discovered that (the relative)
transcription- activation frequencies among different biological pathways encoded
in a genome have a dominating role in the global arrangement of operons. One
complicating factor in such a study is that some operons may be involved in
multiple pathways with different activation frequencies. A quantitative model has
been developed that captures this information, which tends to be minimized by the
current global arrangement of operons in a bacterial (and archaeal) genome
compared to possible alternative arrangements. A study is carried out here using 
this model on a collection of 52 closely related E. coli genomes, which revealed 
interesting new insights about how bacterial genomes evolve to optimally adapt to
their environments through adjusting the (relative) genomic locations of the
encoding operons of biological pathways once their utilization and hence
transcription activation frequencies change, to maintain the above
energy-efficiency property. More specifically we observed that it is the
frequencies of the transcription activation of pathways relative to those of the 
other encoded pathways in an organism as well as the variation in the activation 
frequencies of a specific pathway across the related genomes that play a key role
in the observed commonalities and differences in the genomic organizations of
genes (and operons) encoding specific pathways across different genomes.

DOI: 10.1007/s11427-014-4734-y 
PMID: 25234108  [Indexed for MEDLINE]


194. J Biotechnol. 2014 Oct 20;188:61-71. doi: 10.1016/j.jbiotec.2014.08.005. Epub
2014 Aug 14.

Selection of Escherichia coli heat shock promoters toward their application as
stress probes.

Rodrigues JL(1), Sousa M(2), Prather KL(3), Kluskens LD(2), Rodrigues LR(4).

Author information: 
(1)Centre of Biological Engineering, University of Minho, 4710-057 Braga,
Portugal; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon,
Portugal.
(2)Centre of Biological Engineering, University of Minho, 4710-057 Braga,
Portugal.
(3)Department of Chemical Engineering, Synthetic Biology Engineering Research
Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139,
USA; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon,
Portugal.
(4)Centre of Biological Engineering, University of Minho, 4710-057 Braga,
Portugal; MIT-Portugal Program, Cambridge, MA, USA; MIT-Portugal Program, Lisbon,
Portugal. Electronic address: lrmr@deb.uminho.pt.

The mechanism of heat shock response of Escherichia coli can be explored to
program novel biological functions. In this study, the strongest heat shock
promoters were identified by microarray experiments conducted at different
temperatures (37°C and 45°C, 5min). The promoters of the genes ibpA, dnaK and
fxsA were selected and validated by RT-qPCR. These promoters were used to
construct and characterize stress probes using green fluorescence protein (GFP). 
Cellular stress levels were evaluated in experiments conducted at different shock
temperatures during several exposure times. It was concluded that the strength of
the promoter is not the only relevant factor in the construction of an efficient 
stress probe. Furthermore, it was found to be crucial to test and optimize the
ribosome binding site (RBS) in order to obtain translational efficiency that
balances the transcription levels previously verified by microarrays and RT-qPCR.
These heat shock promoters can be used to trigger in situ gene expression of
newly constructed biosynthetic pathways.

Copyright © 2014 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jbiotec.2014.08.005 
PMID: 25128614  [Indexed for MEDLINE]


195. Beijing Da Xue Xue Bao Yi Xue Ban. 2014 Oct 18;46(5):698-702.

[Screening and structure analysis of the aptamer target to Escherichia coli tolC 
protein].

[Article in Chinese]

Chen LL(1), Li J(1), Zhang XQ(1), Song L(1), Qian C(1), Ge JW(1).

Author information: 
(1)Medical school of Hunan University of Chinese Medicine, Changsha 410208,
China.

OBJECTIVE: To screen and characterize the aptamer of Escherichia coli outer
member protein tolC.
METHODS: By using the recombinant E.coli outer member protein tolC for the
screening target, oligonucleotides which were capable of specifically binding to 
the protein were screened from a random oligonucleotide library through the
stematic evolution of ligand by exponential enrichment (SELEX) technique. The
binding capacity of ssDNA to the targeted protein from each round was detected by
the FITC fluorescence labeling technique.The ssDNA from the last cycle was cloned
and sequenced,and the second structure was further analyzed by the DNAMan
program.
RESULTS: After 12 cycles of selection, 40 clones were selected randomly and
sequenced. Although a unique conserved sequence was not obtained among the 23
obtained aptamers by the primary structure analysis,three pairs of aptamers and
two pairs of aptamers were found to be identical.Analysis of the secondary
structure revealed that the stem-loop and bulge loop were the main
motifs,indicating that they might play a key role in the binding of aptamers to
the target protein. According to the characteristic of the second structure,23
aptamers were divided into four families,and aptamer 20 bore the greatest
affinity.
CONCLUSION: Aptamers against E.coli outer member protein tolC were successfully
identified by the SELEX method. The results laid a foundation for the
investigation of the interference to the drug resistance of E. coli and the
underlying mechanisms.


PMID: 25331389  [Indexed for MEDLINE]


196. Appl Environ Microbiol. 2014 Oct;80(19):5955-64. doi: 10.1128/AEM.01913-14. Epub 
2014 Jul 25.

Polyamine transporters and polyamines increase furfural tolerance during xylose
fermentation with ethanologenic Escherichia coli strain LY180.

Geddes RD(1), Wang X(1), Yomano LP(1), Miller EN(1), Zheng H(1), Shanmugam KT(1),
Ingram LO(2).

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida, USA.
(2)Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida, USA ingram@ufl.edu.

Expression of genes encoding polyamine transporters from plasmids and polyamine
supplements increased furfural tolerance (growth and ethanol production) in
ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing
xylose). This represents a new approach to increase furfural tolerance and may be
useful for other organisms. Microarray comparisons of two furfural-resistant
mutants (EMFR9 and EMFR35) provided initial evidence for the importance of
polyamine transporters. Each mutant contained a single polyamine transporter gene
that was upregulated over 100-fold (microarrays) compared to that in the parent
LY180, as well as a mutation that silenced the expression of yqhD. Based on these
genetic changes, furfural tolerance was substantially reconstructed in the
parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of
the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance,
indicating functional importance of the native genes. Of the 8 polyamine
transporters (18 genes) cloned and tested, half were beneficial for furfural
tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium
with individual polyamines (agmatine, putrescine, and cadaverine) also increased 
furfural tolerance but to a smaller extent. In pH-controlled fermentations,
polyamine transporter plasmids were shown to promote the metabolism of furfural
and substantially reduce the time required to complete xylose fermentation. This 
increase in furfural tolerance is proposed to result from polyamine binding to
negatively charged cellular constituents such as nucleic acids and phospholipids,
providing protection from damage by furfural.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/AEM.01913-14 
PMCID: PMC4178697
PMID: 25063650  [Indexed for MEDLINE]


197. Biotechnol Bioeng. 2014 Oct;111(10):2067-75. doi: 10.1002/bit.25271. Epub 2014
Jun 4.

NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA
deletion mutant: influence on global gene expression and role of oxygen supply.

Siedler S(1), Bringer S, Polen T, Bott M.

Author information: 
(1)Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie,
Forschungszentrum Jülich, D-52425, Jülich, Germany.

An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses 
a partially cyclized pentose phosphate pathway leading to an increased NADPH per 
glucose ratio. This effect decreases the amount of glucose required for NADPH
regeneration in reductive biotransformations, such as the conversion of methyl
acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol
dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses
were performed to study regulatory responses during reductive biotransformation. 
DNA microarray analysis revealed amongst other things increased expression of
soxS, supporting previous results indicating that a high NADPH demand contributes
to the activation of SoxR, the transcriptional activator of soxS. Furthermore,
several target genes of the ArcAB two-component system showed a lower mRNA level 
in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q
ratio in the reference strain. This prompted us to analyze yields and
productivities of MAA reduction to MHB under different oxygen regimes in a
bioreactor. Under anaerobic conditions, the specific MHB production rates of both
strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than
under conditions of 15% dissolved oxygen, where those of the reference strain
(12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1)
) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference 
strain increased after the addition of MAA, presumably due to the oxidation of
the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly
decreased, indicating a very low respiration rate despite sufficient oxygen
supply. The latter effect can likely be attributed to a restricted conversion of 
NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is
outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol
dehydrogenase. The differences in respiration rates can explain the suggested
higher ArcAB activity in the reference strain.

© 2014 Wiley Periodicals, Inc.

DOI: 10.1002/bit.25271 
PMID: 24771245  [Indexed for MEDLINE]


198. Gen Comp Endocrinol. 2014 Oct 1;207:74-85. doi: 10.1016/j.ygcen.2014.05.006. Epub
2014 May 20.

Production, gene structure and characterization of two orthologs of leptin and a 
leptin receptor in tilapia.

Shpilman M(1), Hollander-Cohen L(1), Ventura T(2), Gertler A(3), Levavi-Sivan
B(4).

Author information: 
(1)Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture,
Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel.
(2)Faculty of Science, Health, Education and Engineering, GeneCology Research
Centre, University of the Sunshine Coast, Queensland, Australia.
(3)Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith
Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem,
Rehovot 76100, Israel.
(4)Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture,
Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel.
Electronic address: berta.sivan@mail.huji.ac.il.

Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin
receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The
full-length cDNA of tLepR was 3423bp, encoding a protein of 1140 amino acid (aa) 
which contained all functionally important domains conserved among vertebrate
leptin receptors. The cDNAs of tLepA and tLepB were 486bp and 459bp in length,
encoding proteins of 161 aa and 152 aa, respectively. Modeling the
three-dimensional structures of tLepA and tLepB predicted strong conservation of 
tertiary structure with that of human leptin, comprised of four helixes. Using
synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The
cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression
in Escherichia coli was prepared according to the cloned sequence. The tLepA- and
tLepB-expressing plasmids were transformed into E. coli and expressed as
recombinant proteins upon induction with nalidixic acid, found almost entirely in
insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and
purified to homogeneity by anion-exchange chromatography. In the case of tLepA,
the fraction eluted contained a mixture of monomers and dimers. The purified
tLepA and tLepB monomers and tLepA dimer showed a single band of ∼15kDa on an
SDS-polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer
showed one band of ∼30kDa in the absence of reducing agent, indicating its
formation by S-S bonds. The three tLeps were biologically active in promoting
proliferation of BAF/3 cells stably transfected with the long form of human
leptin receptor (hLepR), but their activity was four orders of magnitude lower
than that of mammalian leptin. Furthermore, the three tLeps were biologically
active in promoting STAT-LUC activation in COS7 cells transfected with the
identified tLepR but not in cells transfected with hLepR. tLepA was more active
than tLepB. Low or no activity likely resulted from low identity (9-22%) to
mammalian leptins. In an in vivo experiment in which tilapia were fed ad libitum 
or fasted, there was no significant difference in the expressions of tLepA, tLepB
or tLepR in the brain between the two groups examined both by real-time PCR and
RNA next generation sequencing. In conclusion, in the present report we show
novel, previously unknown sequences of tilapia leptin receptor and two leptins
and prepare two biologically active recombinant leptin proteins.

Copyright © 2014 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ygcen.2014.05.006 
PMID: 24852346  [Indexed for MEDLINE]


199. Immunogenetics. 2014 Oct;66(9-10):563-73. doi: 10.1007/s00251-014-0792-3. Epub
2014 Jul 31.

Characterization of Toll-like receptor gene expression and the pathogen agonist
response in the antarctic bullhead notothen Notothenia coriiceps.

Ahn DH(1), Shin SC, Park H.

Author information: 
(1)Division of Polar Life Sciences, Korea Polar Research Institute, Yeonsu-gu,
Incheon, 406-840, South Korea.

Notothenia coriiceps, a typical Antarctic notothenioid teleost, has evolved to
adapt to the extreme Antarctic marine environment. We previously reported an
extensive analysis of the Antarctic notothenioid transcriptome. In this study, we
focused on a key component of the innate immune system, the Toll-like receptors
(TLRs). We cloned the full-length sequence of 12 TLRs of N. coriiceps. The N.
coriiceps transcriptome for TLR homologue (ncTLR) genes encode a typical TLR
structure, with multiple extracellular leucine-rich regions and an intracellular 
Toll/IL-1 receptor (TIR) domain. Using phylogenetic analysis, we established that
all of the cloned ncTLR genes could be classified into the same orthologous clade
with other teleost TLRs. ncTLRs were widely expressed in various organs, with the
highest expression levels observed in immune-related tissues, such as the skin,
spleen, and kidney. A subset of the ncTLR genes was expressed at higher levels in
fish exposed to pathogen-mimicking agonists, heat-killed Escherichia coli, and
polyinosinic-polycytidylic acid (poly(I:C)). However, the mechanism involved in
the upregulation of TLR expression following pathogen exposure in fish is
currently unknown. Further research is required to elucidate these mechanisms and
to thereby increase our understanding of vertebrate immune system evolution.

DOI: 10.1007/s00251-014-0792-3 
PMID: 25073429  [Indexed for MEDLINE]


200. Nucleic Acids Res. 2014 Oct;42(18):11733-51. doi: 10.1093/nar/gku808. Epub 2014
Sep 18.

Direct entry by RNase E is a major pathway for the degradation and processing of 
RNA in Escherichia coli.

Clarke JE(1), Kime L(1), Romero A D(1), McDowall KJ(2).

Author information: 
(1)Astbury Centre for Structural Molecular Biology, School of Molecular and
Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2
9JT, UK.
(2)Astbury Centre for Structural Molecular Biology, School of Molecular and
Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2
9JT, UK k.j.mcdowall@leeds.ac.uk.

Escherichia coli endoribonuclease E has a major influence on gene expression. It 
is essential for the maturation of ribosomal and transfer RNA as well as the
rapid degradation of messenger RNA. The latter ensures that translation closely
follows programming at the level of transcription. Recently, one of the hallmarks
of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown
to be unnecessary for the initial cleavage of some polycistronic tRNA precursors.
Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and
a 5'-sensor mutant of RNase E in vitro that, contrary to current models,
5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for
degrading and processing RNA. Moreover, we present further evidence that direct
entry is facilitated by RNase E binding simultaneously to multiple unpaired
regions. These simple requirements may maximize the rate of degradation and
processing by permitting multiple sites to be surveyed directly without being
constrained by 5'-end tethering. Cleavage was detected at a multitude of sites
previously undescribed for RNase E, including ones that regulate the activity and
specificity of ribosomes. A potentially broad role for RNase G, an RNase E
paralogue, in the trimming of 5'-monophosphorylated ends was also revealed.

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gku808 
PMCID: PMC4191395
PMID: 25237058  [Indexed for MEDLINE]


201. J Chromatogr A. 2014 Sep 5;1358:269-76. doi: 10.1016/j.chroma.2014.06.079. Epub
2014 Jul 1.

Interaction evaluation of bacteria and protoplasts with single-stranded
deoxyribonucleic acid library based on capillary electrophoresis.

Meng C(1), Zhao X(2), Qu F(3), Mei F(4), Gu L(4).

Author information: 
(1)School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun 
Street, Beijing 100081, China; State Key Laboratory of Reproductive Biology,
Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
(2)Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China.
(3)School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun 
Street, Beijing 100081, China. Electronic address: qufengqu@bit.edu.cn.
(4)School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun 
Street, Beijing 100081, China.

For whole-cell aptamers selection, cells surface situation has great impact on
single-stranded (ssDNA) binding and aptamers selection. In this work, both
Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were 
as cells targets, their interaction with ssDNA library were evaluated based on
capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE)
with UV and LIF detection. Our results demonstrated that protoplasts without
cells wall had apparently stronger interaction with ssDNA library than bacteria, 
the protoplasts-ssDNA complex could be observed clearly with CZE-LIF.
Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol,
formaldehyde and glutaraldehyde) showed binding difference with ssDNA library,
which could be identified with ACE-UV. Binding constants indicated the
interaction of E. coli with ssDNA library were in the order of E. coli
protoplasts>methanol (ethanol) treated E. coli>formaldehyde (glutaraldehyde)
treated E. coli≈E. coli. Above results suggest that cells surface situation
determines their binding affinity with ssDNA, which should be considered in
whole-cell aptamers selection and aptamers further application. Capillary
electrophoresis is a preferable technique for interaction evaluation of composite
targets binding with ssDNA library.

Copyright © 2014 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.chroma.2014.06.079 
PMID: 25035238  [Indexed for MEDLINE]


202. Appl Biochem Biotechnol. 2014 Sep;174(2):793-802. doi: 10.1007/s12010-014-1103-z.
Epub 2014 Aug 6.

Development of ssDNA aptamers for the sensitive detection of Salmonella
typhimurium and Salmonella enteritidis.

Park HC(1), Baig IA, Lee SC, Moon JY, Yoon MY.

Author information: 
(1)Department of Chemistry, College of Natural Science, Hanyang University,
Seoul, 133-791, Republic of Korea.

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica
subsp. enterica ser. typhimurium are the most common and severe food-borne
pathogens responsible for causing salmonellosis in humans and animals. The
development of an early and ultra-sensitive detection system is the first
critical step in controlling this disease. To accomplish this, we used the cell
systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique 
to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes
that can specifically bind to S. enteritidis and S. typhimurium. A total of 12
target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX
under stringent selection conditions, and negative selection further enhanced the
selectivity among these aptamers. Aptamer specificity was investigated using the 
gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher 
towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers 
demonstrated higher binding affinities and the dissociation constants (Kd) were
found to be in the range of nanomolar to submicromolar levels. Furthermore,
individual aptamers were conjugated onto polyvalent directed aptamer polymer,
which led to 100-fold increase in binding affinity compared to the individual
aptamers alone. Taken together, this study reports the identification of higher
affinity and specificity ssDNA aptamers (30mer), which may be useful as capture
and detection probes in biosensor-based detection systems for salmonellosis.

DOI: 10.1007/s12010-014-1103-z 
PMID: 25096391  [Indexed for MEDLINE]


203. J Microbiol Methods. 2014 Sep;104:94-100. doi: 10.1016/j.mimet.2014.06.016. Epub 
2014 Jul 4.

Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by
quantitative PCR controlled Cell-SELEX.

Savory N(1), Nzakizwanayo J(2), Abe K(3), Yoshida W(4), Ferri S(5), Dedi C(6),
Jones BV(7), Ikebukuro K(8).

Author information: 
(1)Department of Biotechnology and Life Science, Tokyo University of Agriculture 
and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic
address: 50012831103@st.tuat.ac.jp.
(2)School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley
Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom. Electronic 
address: J.Nzakizwanayo@brighton.ac.uk.
(3)Department of Biotechnology and Life Science, Tokyo University of Agriculture 
and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic
address: abekou@cc.tuat.ac.jp.
(4)School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1
Katakuramachi, Hachioji, Tokyo 192-0982, Japan. Electronic address:
yoshidawtr@stf.teu.ac.jp.
(5)Department of Biotechnology and Life Science, Tokyo University of Agriculture 
and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic
address: stefano@cc.tuat.ac.jp.
(6)School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley
Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom. Electronic 
address: C.G.Dedi@brighton.ac.uk.
(7)School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley
Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom; Queen
Victoria Hospital NHS Foundation Trust, Holtye Road, East Grinstead, West Sussex,
RD19 3DZ, United Kingdom. Electronic address: B.V.Jones@brighton.ac.uk.
(8)Department of Biotechnology and Life Science, Tokyo University of Agriculture 
and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic
address: ikebu@cc.tuat.ac.jp.

In order to better control nosocomial infections, and facilitate the most prudent
and effective use of antibiotics, improved strategies for the rapid detection and
identification of problematic bacterial pathogens are required. DNA aptamers have
much potential in the development of diagnostic assays and biosensors to address 
this important healthcare need, but further development of aptamers targeting
common pathogens, and the strategies used to obtain specific aptamers are
required. Here we demonstrate the application of a quantitative PCR (qPCR)
controlled Cell-SELEX process, coupled with downstream
secondary-conformation-based aptamer profiling. We used this approach to identify
and select DNA aptamers targeted against uropathogenic Escherichia coli, for
which specific aptamers are currently lacking, despite the prevalence of these
infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal
number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation
of standard PCR amplification of recovered aptamer pools at each round.
Identification of useful aptamer candidates was also facilitated by profiling of 
secondary conformations and selection based on putative aptamer secondary
structure. One aptamer selected this way (designated EcA5-27), displaying a
guanine-quadruplex sequence motif, was shown to have high affinity and
specificity for target cells, and the potential to discriminate between distinct 
strains of E. coli, highlighting the possibility for development of aptamers
selectively recognising pathogenic strains. Overall, the identified aptamers hold
much potential for the development of rapid diagnostic assays for nosocomial
urinary tract infections caused by E. coli.

Copyright © 2014. Published by Elsevier B.V.

DOI: 10.1016/j.mimet.2014.06.016 
PMID: 25008464  [Indexed for MEDLINE]


204. PLoS One. 2014 Aug 27;9(8):e106181. doi: 10.1371/journal.pone.0106181.
eCollection 2014.

RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

Naseer N(1), Shapiro JA(1), Chander M(1).

Author information: 
(1)Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania, United
States of America.

The redox-regulated transcription factor SoxR is conserved in diverse bacteria,
but emerging studies suggest that this protein plays distinct physiological roles
in different bacteria. SoxR regulates a global oxidative stress response
(involving > 100 genes) against exogenous redox-cycling drugs in Escherichia coli
and related enterics. In the antibiotic producers Streptomyces coelicolor and
Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that
encode membrane transporters and proteins with homology to antibiotic-tailoring
enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are
expressed in stationary phase during the production of endogenously-produced
redox-active antibiotics. These observations suggest that SoxR evolved to sense
endogenous secondary metabolites and activate machinery to process and transport 
them in antibiotic-producing bacteria. Previous bioinformatics analysis that
searched the genome for SoxR-binding sites in putative promoters defined a
five-gene SoxR regulon in S. coelicolor including an ABC transporter, two
oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in
silico screen may have missed potential SoxR-targets, we conducted a whole genome
transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant
in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated
gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge
of the full complement of genes regulated by SoxR will facilitate studies to
elucidate the function of this regulatory molecule in antibiotic producers.

DOI: 10.1371/journal.pone.0106181 
PMCID: PMC4146615
PMID: 25162599  [Indexed for MEDLINE]


205. MBio. 2014 Aug 5;5(4):e01075-14. doi: 10.1128/mBio.01075-14.

In vivo mRNA profiling of uropathogenic Escherichia coli from diverse phylogroups
reveals common and group-specific gene expression profiles.

Bielecki P(1), Muthukumarasamy U, Eckweiler D, Bielecka A, Pohl S, Schanz A(2),
Niemeyer U(3), Oumeraci T(4), von Neuhoff N(4), Ghigo JM(5), Häussler S(6).

Author information: 
(1)Institute for Molecular Bacteriology, Twincore, Centre for Clinical and
Experimental Infection Research, A Joint Venture of the Helmholtz Centre for
Infection Research and the Hannover Medical School, Hannover, Germany.
(2)Private Practice, Salzgitter, Germany.
(3)Private Practice, Wolfenbuettel, Germany.
(4)Institute of Cell and Molecular Pathology, Hannover Medical School (MHH),
Hannover, Germany.
(5)Institute Pasteur, Unité de Génétique des Biofilms, Paris, France.
(6)susanne.haeussler@twincore.de.

mRNA profiling of pathogens during the course of human infections gives detailed 
information on the expression levels of relevant genes that drive pathogenicity
and adaptation and at the same time allows for the delineation of phylogenetic
relatedness of pathogens that cause specific diseases. In this study, we used
mRNA sequencing to acquire information on the expression of Escherichia coli
pathogenicity genes during urinary tract infections (UTI) in humans and to assign
the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the
in vivo gene expression profiles of the majority of genes were conserved among 21
E. coli strains in the urine of elderly patients suffering from an acute UTI, the
specific gene expression profiles of the flexible genomes was diverse and
reflected phylogenetic relationships. Furthermore, genes transcribed in vivo
relative to laboratory media included well-described virulence factors, small
regulatory RNAs, as well as genes not previously linked to bacterial virulence.
Knowledge on relevant transcriptional responses that drive pathogenicity and
adaptation of isolates to the human host might lead to the introduction of a
virulence typing strategy into clinical microbiology, potentially facilitating
management and prevention of the disease. Importance: Urinary tract infections
(UTI) are very common; at least half of all women experience UTI, most of which
are caused by pathogenic Escherichia coli strains. In this study, we applied
massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and
accurate insight into the nature and the dimension of the uropathogenic E. coli
gene expression profile during an acute UTI within the human host. This work was 
undertaken to identify key players in physiological adaptation processes and,
hence, potential targets for new infection prevention and therapy interventions
specifically aimed at sabotaging bacterial adaptation to the human host.

Copyright © 2014 Bielecki et al.

DOI: 10.1128/mBio.01075-14 
PMCID: PMC4128348
PMID: 25096872  [Indexed for MEDLINE]


206. Cell Biol Toxicol. 2014 Aug;30(4):207-32. doi: 10.1007/s10565-014-9281-6. Epub
2014 Jun 22.

cDNA microarray assessment of early gene expression profiles in Escherichia coli 
cells exposed to a mixture of heavy metals.

Gómez-Sagasti MT(1), Becerril JM, Martín I, Epelde L, Garbisu C.

Author information: 
(1)NEIKER-Tecnalia, Department of Ecology and Natural Resources, Soil Microbial
Ecology Group, c/Berreaga 1, E-48160, Derio, Spain.

Many contaminated sites are characterized by the presence of different metals,
thus increasing the complexity of toxic responses in exposed organisms. Within
toxicogenomics, transcriptomics can be approached through the use of microarrays 
aimed at producing a genetic fingerprint for the response of model organisms to
the presence of chemicals. We studied temporal changes in the early gene
expression profiles of Escherichia coli cells exposed to three metal doses of a
polymetallic solution over three exposure times, through the application of cDNA 
microarray technology. In the absence of metals, many genes belonging to a
variety of cellular functions were up- and down-regulated over time. At the
lowest metal dose, an activation of metal-specific transporters (Cus and ZraP
proteins) and a mobilization of glutathione transporters involved in metal
sequestration and trafficking was observed over time; this metal dose resulted in
the generation of ROS capable of stimulating the transcription of Mn-superoxide
dismutase, the assembly of Fe-S clusters and the synthesis of cysteine. At the
intermediate dose, an overexpression of ROS scavengers (AhpF, KatG, and YaaA) and
heat shock proteins (ClpP, HslV, DnaK, and IbpAB) was observed. Finally, at the
highest dose, E. coli cells showed a repression of genes related with DNA
mutation correctors (MutY glycopeptidases).

DOI: 10.1007/s10565-014-9281-6 
PMID: 24952731  [Indexed for MEDLINE]


207. J Bacteriol. 2014 Aug;196(15):2718-27. doi: 10.1128/JB.01579-14. Epub 2014 May
16.

Intracellular concentrations of 65 species of transcription factors with known
regulatory functions in Escherichia coli.

Ishihama A(1), Kori A(2), Koshio E(3), Yamada K(2), Maeda H(3), Shimada T(2),
Makinoshima H(4), Iwata A(4), Fujita N(3).

Author information: 
(1)Department of Frontier Bioscience and Micro-Nano Technology Research Center,
Hosei University, Koganei, Tokyo, Japan Nippon Institute for Biological Science, 
Ome, Tokyo, Japan Department of Molecular Genetics, National Institute of
Genetics, Mishima, Shizuoka, Japan aishiham@hosei.ac.jp.
(2)Department of Frontier Bioscience and Micro-Nano Technology Research Center,
Hosei University, Koganei, Tokyo, Japan Nippon Institute for Biological Science, 
Ome, Tokyo, Japan.
(3)Department of Molecular Genetics, National Institute of Genetics, Mishima,
Shizuoka, Japan.
(4)Nippon Institute for Biological Science, Ome, Tokyo, Japan.

The expression pattern of the Escherichia coli genome is controlled in part by
regulating the utilization of a limited number of RNA polymerases among a total
of its approximately 4,600 genes. The distribution pattern of RNA polymerase
changes from modulation of two types of protein-protein interactions: the
interaction of core RNA polymerase with seven species of the sigma subunit for
differential promoter recognition and the interaction of RNA polymerase
holoenzyme with about 300 different species of transcription factors (TFs) with
regulatory functions. We have been involved in the systematic search for the
target promoters recognized by each sigma factor and each TF using the newly
developed Genomic SELEX system. In parallel, we developed the promoter-specific
(PS)-TF screening system for identification of the whole set of TFs involved in
regulation of each promoter. Understanding the regulation of genome transcription
also requires knowing the intracellular concentrations of the sigma subunits and 
TFs under various growth conditions. This report describes the intracellular
levels of 65 species of TF with known function in E. coli K-12 W3110 at various
phases of cell growth and at various temperatures. The list of intracellular
concentrations of the sigma factors and TFs provides a community resource for
understanding the transcription regulation of E. coli under various stressful
conditions in nature.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.01579-14 
PMCID: PMC4135669
PMID: 24837290  [Indexed for MEDLINE]


208. Stat Appl Genet Mol Biol. 2014 Aug;13(4):403-22. doi: 10.1515/sagmb-2013-0017.

Comparison of statistical methods for finding network motifs.

Albieri V, Didelez V.

There has been much recent interest in systems biology for investigating the
structure of gene regulatory systems. Such networks are often formed of specific 
patterns, or network motifs, that are interesting from a biological point of
view. Our aim in the present paper is to compare statistical methods specifically
with regard to the question of how well they can detect such motifs. One popular 
approach is by network analysis with Gaussian graphical models (GGMs), which are 
statistical models associated with undirected graphs, where vertices of the graph
represent genes and edges indicate regulatory interactions. Gene expression
microarray data allow us to observe the amount of mRNA simultaneously for a large
number of genes p under different experimental conditions n, where p is usually
much larger than n prohibiting the use of standard methods. We therefore compare 
the performance of a number of procedures that have been specifically designed to
address this large p-small n issue: G-Lasso estimation, Neighbourhood selection, 
Shrinkage estimation using empirical Bayes for model selection, and PC-algorithm.
We found that all approaches performed poorly on the benchmark E. coli network.
Hence we systematically studied their ability to detect specific network motifs, 
pairs, hubs and cascades, in extensive simulations. We conclude that all methods 
have difficulty detecting hubs, but the PC-algorithm is most promising.

DOI: 10.1515/sagmb-2013-0017 
PMID: 24933631  [Indexed for MEDLINE]


209. PLoS One. 2014 Jul 28;9(7):e102232. doi: 10.1371/journal.pone.0102232.
eCollection 2014.

Rapid identification of carbapenemase genes in gram-negative bacteria with an
oligonucleotide microarray-based assay.

Braun SD(1), Monecke S(2), Thürmer A(3), Ruppelt A(3), Makarewicz O(4), Pletz
M(4), Reiβig A(1), Slickers P(1), Ehricht R(1).

Author information: 
(1)Alere Technologies GmbH, Jena, Germany.
(2)Alere Technologies GmbH, Jena, Germany; Technische Universität Dresden,
Medizinische Fakultät "Carl Gustav Carus", Dresden, Germany.
(3)Technische Universität Dresden, Medizinische Fakultät "Carl Gustav Carus",
Dresden, Germany.
(4)Center for Infectious Diseases and Infection Control and Center for Sepsis
Care and Control, Jena University Hospital, Jena, Germany; Center for Sepsis Care
and Control, Jena University Hospital, Jena, Germany.

Erratum in
    PLoS One. 2014;9(9):e107079.

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria
is crucial for infection control and prevention, surveillance and for
epidemiological purposes. Furthermore, it may have a significant impact upon
determining the appropriate initial treatment and greatly benefit for critically 
ill patients. A novel oligonucleotide microarray-based assay was developed to
simultaneously detect genes encoding clinically important carbapenemases as well 
as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly
from clonal culture material within few hours. Additionally, a panel of species
specific markers was included to identify Escherichia coli, Pseudomonas
aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter
baumannii. The assay was tested using a panel of 117 isolates collected from
urinary, blood and stool samples. For these isolates, phenotypic identifications 
and susceptibility tests were available. An independent detection of
carbapenemase, ESBL and NSBL genes was carried out by various external reference 
laboratories using PCR methods. In direct comparison, the microarray correctly
identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out
of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31
(10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33),
blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified
additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33),
OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM
(1/1)]. In direct comparison to phenotypical identification obtained by VITEK or 
MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii
(28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae 
(62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter
freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a
panel of species specific probes. This assay might be easily extended, adapted
and transferred to point of care platforms enabling fast surveillance, rapid
detection and appropriate early treatment of infections caused by multiresistant 
Gram-negative bacteria.

DOI: 10.1371/journal.pone.0102232 
PMCID: PMC4113224
PMID: 25068267  [Indexed for MEDLINE]


210. Cell. 2014 Jul 17;158(2):368-382. doi: 10.1016/j.cell.2014.05.042.

APC is an RNA-binding protein, and its interactome provides a link to neural
development and microtubule assembly.

Preitner N(1), Quan J(1), Nowakowski DW(1), Hancock ML(1), Shi J(1), Tcherkezian 
J(2), Young-Pearse TL(3), Flanagan JG(4).

Author information: 
(1)Department of Cell Biology and Program in Neuroscience, Harvard Medical
School, Boston, MA 02115, USA.
(2)Laboratory for Therapeutic Development, Rosalind and Morris Goodman Cancer
Research Centre, McGill University, Montreal, QC H3G 1Y6, Canada.
(3)Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical 
School, Boston, MA 02115, USA.
(4)Department of Cell Biology and Program in Neuroscience, Harvard Medical
School, Boston, MA 02115, USA. Electronic address: flanagan@hms.harvard.edu.

Comment in
    Cell. 2014 Jul 17;158(2):245-7.

Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein
important in biology and disease. APC is implicated in RNA localization, although
the mechanisms and functional significance remain unclear. We show APC is an
RNA-binding protein and identify an RNA interactome by HITS-CLIP. Targets were
highly enriched for APC-related functions, including microtubule organization,
cell motility, cancer, and neurologic disease. Among the targets is β2B-tubulin, 
known to be required in human neuron and axon migration. We show β2B-tubulin is
synthesized in axons and localizes preferentially to dynamic microtubules in the 
growth cone periphery. APC binds the β2B-tubulin 3' UTR; experiments interfering 
with this interaction reduced β2B-tubulin mRNA axonal localization and
expression, depleted dynamic microtubules and the growth cone periphery, and
impaired neuron migration. These results identify APC as a platform binding
functionally related protein and RNA networks, and suggest a self-organizing
model for the microtubule to localize synthesis of its own subunits.

Copyright © 2014 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.cell.2014.05.042 
PMCID: PMC4133101
PMID: 25036633  [Indexed for MEDLINE]


211. Infect Immun. 2014 Jul;82(7):3023-32. doi: 10.1128/IAI.01836-14. Epub 2014 May 5.

Identification and characterization of a peculiar vtx2-converting phage
frequently present in verocytotoxin-producing Escherichia coli O157 isolated from
human infections.

Tozzoli R(1), Grande L(2), Michelacci V(2), Fioravanti R(2), Gally D(3), Xu X(3),
La Ragione R(4), Anjum M(5), Wu G(5), Caprioli A(2), Morabito S(2).

Author information: 
(1)EU Reference Laboratory for Escherichia coli, Istituto Superiore di Sanità,
Rome, Italy rosangela.tozzoli@iss.it.
(2)EU Reference Laboratory for Escherichia coli, Istituto Superiore di Sanità,
Rome, Italy.
(3)Immunity and Infection Division, Roslin Institute and R(D)SVS, University of
Edinburgh, Easter Bush, Midlothian, United Kingdom.
(4)Department of Bacteriology and Food Safety, Animal Health and Veterinary
Laboratories Agency, Weybridge, Addlestone, Surrey, United Kingdom School of
Veterinary Medicine, University of Surrey, Guildford, Surrey, United Kingdom.
(5)Department of Bacteriology and Food Safety, Animal Health and Veterinary
Laboratories Agency, Weybridge, Addlestone, Surrey, United Kingdom.

Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs),
such as PT8 and PT2, are associated with severe human infections, while others,
such as PT21, seem to be restricted to cattle. In an attempt to delve into the
mechanisms underlying such a differential distribution of PTs, we performed
microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main
differences observed were in the vtx2-converting phages, with the PT21 strains
bearing a phage identical to that present in the reference strain EDL933, BP933W,
and all the PT8 isolates displaying lack of hybridization in some regions of the 
phage genome. We focused on the region spanning the gam and cII genes and
developed a PCR tool to investigate the presence of PT8-like phages in a panel of
VTEC O157 strains belonging to different PTs and determined that a vtx2 phage
reacting with the primers deployed, which we named Φ8, was more frequent in VTEC 
O157 strains from human disease than in bovine strains. No differences were
observed in the production of the VT2 mRNA when Φ8-positive strains were compared
with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region
of phage Φ8 might carry genetic determinants downregulating the transcription of 
the genes encoding the components of the type III secretion system borne on the
locus of enterocyte effacement pathogenicity island.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/IAI.01836-14 
PMCID: PMC4097640
PMID: 24799627  [Indexed for MEDLINE]


212. J Antimicrob Chemother. 2014 Jul;69(7):2005-7. doi: 10.1093/jac/dku042. Epub 2014
Mar 4.

Detection of Shiga toxin- and extended-spectrum β-lactamase-producing Escherichia
coli O145:NM and Ont:NM from calves with diarrhoea.

Ewers C(1), Stamm I(2), Stolle I(3), Guenther S(4), Kopp PA(2), Fruth A(5),
Wieler LH(4), Scheufen S(3), Bauerfeind R(3), Bethe A(4), Prenger-Berninghoff
E(3).

Author information: 
(1)Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig
University Giessen, Giessen, Germany christa.ewers@vetmed.uni-giessen.de.
(2)Vet Med Labor GmbH, Division of IDEXX Laboratories, Ludwigsburg, Germany.
(3)Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig
University Giessen, Giessen, Germany.
(4)Institute of Microbiology and Epizootics, Free University Berlin, Berlin,
Germany.
(5)Robert Koch Institute, Wernigerode, Germany.

DOI: 10.1093/jac/dku042 
PMID: 24595804  [Indexed for MEDLINE]


213. J Fluoresc. 2014 Jul;24(4):1159-68. doi: 10.1007/s10895-014-1396-x. Epub 2014 Apr
25.

Rapid fluorescent detection of Escherichia coli K88 based on DNA aptamer library 
as direct and specific reporter combined with immuno-magnetic separation.

Peng Z(1), Ling M, Ning Y, Deng L.

Author information: 
(1)The Co-construction Laboratory of Microbial Molecular Biology of Province and 
Ministry of Science and Technology, College of Life Science, Hunan Normal
University, Changsha, 410081, Hunan, China.

Nucleic acid aptamers have long demonstrated the capacity to bind cells with high
affinity so that they have been utilized to diagnose various important pathogens.
In this study, a DNA aptamer library was on initial efforts developed to act as a
specific reporter for rapid detection of enter toxigenic Escherichia coli (ETEC) 
K88 combined with immuno-magnetic separation (IMS). During a Whole-cell
Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX) procedure,
the last selection pool against ETEC K88, which is named "DNA aptamer library"
here, was selected and subsequently identified by flow cytometric analysis and
confocal imaging. A K88 monoclonal antibody (mAb) with high affinity (K(aff):
1.616 ± 0.033 × 10(8) M(-1)) against K88 fimbrial protein was prepared,
biotinylated and conjugated to streptavidin-coated magnetic beads (MBs). After
the bacteria were effectively captured and enriched from the complex sample by
immuno-magnetic beads (IMBs), 5'-FITC modified aptamer library was directly bound
to target cells as a specific reporter for its detection. The detection system
showed clearly high specificity and sensitivity with the detection limit of
1.1 × 10(3) CFU/ml in pure culture and 2.2 × 10(3) CFU/g in artificially
contaminated fecal sample. The results also indicated that fluorophore-lablled
DNA aptamer library as specific reporter could generate more reliable signals
than individual aptamer with best affinity against target cells and implied it
would have great applied potential in directly reporting bacteria from complex
samples combined with IMS technology.

DOI: 10.1007/s10895-014-1396-x 
PMID: 24763818  [Indexed for MEDLINE]


214. BMC Bioinformatics. 2014 Jun 17;15:195. doi: 10.1186/1471-2105-15-195.

Quantitative group testing-based overlapping pool sequencing to identify rare
variant carriers.

Cao CC, Li C, Sun X(1).

Author information: 
(1)State Key Laboratory of Bioelectronics, School of Biological Science and
Medical Engineering, Southeast University, Nanjing, China. xsun@seu.edu.cn.

BACKGROUND: Genome-wide association studies have revealed that rare variants are 
responsible for a large portion of the heritability of some complex human
diseases. This highlights the increasing importance of detecting and screening
for rare variants. Although the massively parallel sequencing technologies have
greatly reduced the cost of DNA sequencing, the identification of rare variant
carriers by large-scale re-sequencing remains prohibitively expensive because of 
the huge challenge of constructing libraries for thousands of samples. Recently, 
several studies have reported that techniques from group testing theory and
compressed sensing could help identify rare variant carriers in large-scale
samples with few pooled sequencing experiments and a dramatically reduced cost.
RESULTS: Based on quantitative group testing, we propose an efficient overlapping
pool sequencing strategy that allows the efficient recovery of variant carriers
in numerous individuals with much lower costs than conventional methods. We used 
random k-set pool designs to mix samples, and optimized the design parameters
according to an indicative probability. Based on a mathematical model of
sequencing depth distribution, an optimal threshold was selected to declare a
pool positive or negative. Then, using the quantitative information contained in 
the sequencing results, we designed a heuristic Bayesian probability decoding
algorithm to identify variant carriers. Finally, we conducted in silico
experiments to find variant carriers among 200 simulated Escherichia coli
strains. With the simulated pools and publicly available Illumina sequencing
data, our method correctly identified the variant carriers for 91.5-97.9%
variants with the variant frequency ranging from 0.5 to 1.5%.
CONCLUSIONS: Using the number of reads, variant carriers could be identified
precisely even though samples were randomly selected and pooled. Our method
performed better than the published DNA Sudoku design and compressed sequencing, 
especially in reducing the required data throughput and cost.

DOI: 10.1186/1471-2105-15-195 
PMCID: PMC4229885
PMID: 24934981  [Indexed for MEDLINE]


215. J Proteomics. 2014 Jun 13;105:5-18. doi: 10.1016/j.jprot.2014.01.007. Epub 2014
Jan 16.

Non-model organisms, a species endangered by proteogenomics.

Armengaud J(1), Trapp J(2), Pible O(3), Geffard O(4), Chaumot A(4), Hartmann
EM(3).

Author information: 
(1)CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze F-30207, France. 
Electronic address: jean.armengaud@cea.fr.
(2)CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze F-30207, France; 
Irstea, UR MALY, F-69626 Villeurbanne, France.
(3)CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze F-30207, France.
(4)Irstea, UR MALY, F-69626 Villeurbanne, France.

Previously, large-scale proteomics was possible only for organisms whose genomes 
were sequenced, meaning the most common model organisms. The use of
next-generation sequencers is now changing the deal. With "proteogenomics", the
use of experimental proteomics data to refine genome annotations, a higher
integration of omics data is gaining ground. By extension, combining genomic and 
proteomic data is becoming routine in many research projects.
"Proteogenomic"-flavored approaches are currently expanding, enabling the
molecular studies of non-model organisms at an unprecedented depth. Today draft
genomes can be obtained using next-generation sequencers in a rather
straightforward way and at a reasonable cost for any organism. Unfinished genome 
sequences can be used to interpret tandem mass spectrometry proteomics data
without the need for time-consuming genome annotation, and the use of RNA-seq to 
establish nucleotide sequences that are directly translated into protein
sequences appears promising. There are, however, certain drawbacks that deserve
further attention for RNA-seq to become more efficient. Here, we discuss the
opportunities of working with non-model organisms, the proteomic methods that
have been used until now, and the dramatic improvements proffered by
proteogenomics. These put the distinction between model and non-model organisms
in great danger, at least in terms of proteomics!BIOLOGICAL SIGNIFICANCE: Model
organisms have been crucial for in-depth analysis of cellular and molecular
processes of life. Focusing the efforts of thousands of researchers on the
Escherichia coli bacterium, Saccharomyces cerevisiae yeast, Arabidopsis thaliana 
plant, Danio rerio fish and other models for which genetic manipulation was
possible was certainly worthwhile in terms of fundamental and invaluable
biological insights. Until recently, proteomics of non-model organisms was
limited to tedious, homology-based techniques, but today draft genomes or RNA-seq
data can be straightforwardly obtained using next-generation sequencers, allowing
the establishment of a draft protein database for any organism. Thus,
proteogenomics opens new perspectives for molecular studies of non-model
organisms, although they are still difficult experimental organisms. This article
is part of a Special Issue entitled: Proteomics of non-model organisms.

Copyright © 2014 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jprot.2014.01.007 
PMID: 24440519  [Indexed for MEDLINE]


216. PLoS One. 2014 Jun 13;9(6):e99627. doi: 10.1371/journal.pone.0099627. eCollection
2014.

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes
in growth kinetics induced by an abrupt temperature downshift.

King T(1), Kocharunchitt C(2), Gobius K(3), Bowman JP(2), Ross T(2).

Author information: 
(1)Commonwealth Scientific and Industrial Research Organisation, Animal, Food and
Health Sciences, North Ryde, New South Wales, Australia.
(2)Food Safety Centre, Tasmanian Institute of Agriculture, University of
Tasmania, Hobart, Tasmania, Australia.
(3)Commonwealth Scientific and Industrial Research Organisation, Animal, Food and
Health Sciences, Werribee, Victoria, Australia.

Escherichia coli O157∶H7 is a mesophilic food-borne pathogen. We investigated the
growth kinetics of E. coli O157∶H7 Sakai during an abrupt temperature downshift
from 35°C to either 20°C, 17°C, 14°C or 10°C; as well as the molecular mechanisms
enabling growth after cold stress upon an abrupt downshift from 35°C to 14°C in
an integrated transcriptomic and proteomic analysis. All downshifts caused a lag 
period of growth before growth resumed at a rate typical of the post-shift
temperature. Lag and generation time increased with the magnitude of the shift or
with the final temperature, while relative lag time displayed little variation
across the test range. Analysis of time-dependent molecular changes revealed, in 
keeping with a decreased growth rate at lower temperature, repression of genes
and proteins involved in DNA replication, protein synthesis and carbohydrate
catabolism. Consistent with cold-induced remodelling of the bacterial cell
envelope, alterations occurred in the expression of genes and proteins involved
in transport and binding. The RpoS regulon exhibited sustained induction
confirming its importance in adaptation and growth at 14°C. The RpoE regulon was 
transiently induced, indicating a potential role for this extracytoplasmic stress
response system in the early phase of low temperature adaptation during lag
phase. Interestingly, genes previously reported to be amongst the most highly
up-regulated under oxidative stress were consistently down-regulated. This
comprehensive analysis provides insight into the molecular mechanisms operating
during adaptation of E. coli to growth at low temperature and is relevant to its 
physiological state during chilling in foods, such as carcasses.

DOI: 10.1371/journal.pone.0099627 
PMCID: PMC4057180
PMID: 24926786  [Indexed for MEDLINE]


217. Front Microbiol. 2014 Jun 10;5:286. doi: 10.3389/fmicb.2014.00286. eCollection
2014.

An optimized method for the extraction of bacterial mRNA from plant roots
infected with Escherichia coli O157:H7.

Holmes A(1), Birse L(1), Jackson RW(2), Holden NJ(1).

Author information: 
(1)Cell and Molecular Sciences, The James Hutton Institute Invergowrie, Dundee,
UK.
(2)School of Biological Sciences, The University of Reading Knight Building,
Whiteknights, Reading, UK.

Analysis of microbial gene expression during host colonization provides valuable 
information on the nature of interaction, beneficial or pathogenic, and the
adaptive processes involved. Isolation of bacterial mRNA for in planta analysis
can be challenging where host nucleic acid may dominate the preparation, or
inhibitory compounds affect downstream analysis, e.g., quantitative reverse
transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to
optimize the isolation of bacterial mRNA of food-borne pathogens from living
plants. Reported methods for recovery of phytopathogen-infected plant material,
using hot phenol extraction and high concentration of bacterial inoculation or
large amounts of infected tissues, were found to be inappropriate for plant roots
inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low
and increased plant material resulted in a dominance of plant RNA in the sample. 
To improve the yield of bacterial RNA and reduce the number of plants required,
an optimized method was developed which combines bead beating with directed
bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as
phenolics and polysaccharides, were counteracted with the addition of
high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium
bromide. The new method increased the total yield of bacterial mRNA substantially
and allowed assessment of gene expression by qPCR. This method can be applied to 
other bacterial species associated with plant roots, and also in the wider
context of food safety.

DOI: 10.3389/fmicb.2014.00286 
PMCID: PMC4071639
PMID: 25018749 


218. FEMS Microbiol Lett. 2014 Jun;355(2):93-9. doi: 10.1111/1574-6968.12466. Epub
2014 May 27.

Involvement of cAMP-CRP in transcription activation and repression of the pck
gene encoding PEP carboxykinase, the key enzyme of gluconeogenesis.

Nakano M(1), Ogasawara H, Shimada T, Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience and Research Center for Micro-Nano
Technology, Hosei University, Koganei, Tokyo, Japan.

cAMP receptor protein (CRP) is the best characterized global regulator of
Escherichia coli. After genomic SELEX screening, a total of minimum 378 promoters
have been identified as its regulation targets on the E. coli genome. Among a
number of promoters carrying two CRP-binding sites, several promoters carry two
CRP-binding sites, one upstream but another downstream of transcription
initiation sites. The regulatory role of downstream CRP site remains unsolved.
Using the pck gene encoding phosphoenolpyruvate carboxykinase as a model
promoter, we analyzed the role of CRP-associated downstream of the transcription 
initiation site. Gel shift assay and AFM observation indicate that CRP binds to
both the promoter-distal site (CRP box-1) at -90.5 and the site (CRP box-2) at
+13.5 downstream of transcription initiation site. The binding affinity is higher
for CRP box-1. Roles of two CRP sites were examined using in vitro transcription 
assay and in vivo reporter assay. In both cases, transcription repression was
observed in the presence of high concentrations of CRP. Taken together, we
propose that cAMP-CRP associated at downstream CRP box-2 plays as a repressor for
pck transcription only in the presence of high levels of cAMP-CRP.

© 2014 Federation of European Microbiological Societies. Published by John Wiley 
& Sons Ltd. All rights reserved.

DOI: 10.1111/1574-6968.12466 
PMID: 24814025  [Indexed for MEDLINE]


219. J Bacteriol. 2014 Jun;196(11):2053-66. doi: 10.1128/JB.01370-13. Epub 2014 Mar
21.

The highly conserved MraZ protein is a transcriptional regulator in Escherichia
coli.

Eraso JM(1), Markillie LM, Mitchell HD, Taylor RC, Orr G, Margolin W.

Author information: 
(1)Department of Microbiology & Molecular Genetics, University of Texas Medical
School at Houston, Houston, Texas, USA.

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in
their position at the head of the division and cell wall (dcw) gene cluster.
Located directly upstream of the mraZ gene, the Pmra promoter drives the
transcription of mraZ and mraW, as well as many essential cell division and cell 
wall genes, but no regulator of Pmra has been found to date. Although MraZ has
structural similarity to the AbrB transition state regulator and the MazE
antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants
have no detectable phenotypes. Here we show that overproduction of Escherichia
coli MraZ inhibited cell division and was lethal in rich medium at high induction
levels and in minimal medium at low induction levels. Co-overproduction of MraW
suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting
that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein
localized to the nucleoid, suggesting that it binds DNA. Consistent with this
idea, purified MraZ directly bound a region of DNA containing three direct
repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an 
mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a
DNA-binding mutant form of MraZ failed to repress expression. Transcriptome
sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression
of genes outside the dcw cluster. In support of this, purified MraZ could
directly bind to a putative operator site upstream of mioC, one of the repressed 
genes identified by RNA-seq.

DOI: 10.1128/JB.01370-13 
PMCID: PMC4010979
PMID: 24659771  [Indexed for MEDLINE]


220. J Oral Sci. 2014 Jun;56(2):157-64.

MicroRNA-146a and microRNA-155 show tissue-dependent expression in dental pulp,
gingival and periodontal ligament fibroblasts in vitro.

Sipert CR(1), Morandini AC, Dionísio TJ, Trachtenberg AJ, Kuo WP, Santos CF.

Author information: 
(1)Department of Biological Sciences, Bauru School of Dentistry, University of
São Paulo.

MicroRNAs (miRNAs) are small non-coding RNAs showing a tissue-specific expression
pattern, and whose function is to suppress protein synthesis. In this study, we
hypothesized that expression of miRNAs would differ among fibroblasts from dental
pulp (DPF), gingiva (GF) and periodontal ligament (PLF) in vitro. Once
established by an explant technique, DPF, GF and PLF were collected for RNA
isolation and subjected to a miRNA microarray. Next, cells were stimulated with
E. coli lipopolysaccharide (LPS) for 24 h and then collected for RNA isolation.
Expression of miR-146a and miR-155 was investigated by qPCR. Microarray screening
revealed several miRNAs that showed specifically high expression in at least one 
of the fibroblast subtypes. These molecules are potentially involved in the
regulation of extracellular matrix turnover and production of inflammatory
mediators. Microarray analysis showed that both miR-146a and miR-155 were among
the miRNAs expressed exclusively in GF. qPCR demonstrated significant
upregulation of miR-146a only in GF after LPS stimulation, whereas basal
expression of miR-155 was higher in GF than in the other cell subtypes. LPS
downregulated the expression of miR-155 only in GF. Our results suggest that the 
expression and regulation of miR-146a and miR-155 are more pronounced in GF than 
in DPF and PLF.


PMID: 24930753  [Indexed for MEDLINE]


221. RETRACTED ARTICLE

Eur J Med Res. 2014 May 29;19:31. doi: 10.1186/2047-783X-19-31.

Reanalysis of the gene expression profile in chronic pancreatitis via
bioinformatics methods.

Yuan H, Wu B, Ma S, Yang X, Yin L, Li A(1).

Author information: 
(1)Department of the 2nd Special Treatment, Eastern Hepatobiliary Surgery
Hospital, Second Military Medical University, No 225, Changhai Road, Shanghai
200438, China. ajli62@gmail.com.

Retraction in
    Eur J Med Res. 2015;20:36.

BACKGROUND: Diagnosis at an early stage of chronic pancreatitis (CP) is
challenging. It has been reported that microRNAs (miRNAs) are increasingly found 
and applied as targets for the diagnosis and treatment of various cancers.
However, to the best of our knowledge, few published papers have described the
role of miRNAs in the diagnosis of CP.
METHOD: We downloaded gene expression profile data from the Gene Expression
Omnibus and identified differentially expressed genes (DEGs) between CP and
normal samples of Harlan mice and Jackson Laboratory mice. Common DEGs were
filtered out, and the semantic similarities of gene classes were calculated using
the GOSemSim software package. The gene class with the highest functional
consistency was selected, and then the Lists2Networks web-based system was used
to analyse regulatory relationships between miRNAs and gene classes. The
functional enrichment of the gene classes was assessed based on Gene Ontology
(GO) and Kyoto Encyclopedia of Genes and Genomes pathway annotation terms.
RESULTS: A total of 405 common upregulated DEGs and 7 common downregulated DEGs
were extracted from the two kinds of mice. Gene cluster D was selected from the
common upregulated DEGs because it had the highest semantic similarity. miRNA
124a (miR-124a) was found to have a significant regulatory relationship with
cluster D, and DEGs such as CHSY1 and ABCC4 were found to be regulated by
miR-124a. The GO term of response to DNA damage stimulus and the pathway of
Escherichia coli infection were significantly enriched in cluster D.
CONCLUSION: DNA damage and E. coli infection might play important roles in CP
pathogenesis. In addition, miR-124a might be a potential target for the diagnosis
and treatment of CP.

DOI: 10.1186/2047-783X-19-31 
PMCID: PMC4091748
PMID: 24886751  [Indexed for MEDLINE]


222. BMC Bioinformatics. 2014 May 16;15:145. doi: 10.1186/1471-2105-15-145.

Transcriptome dynamics-based operon prediction in prokaryotes.

Fortino V(1), Smolander OP, Auvinen P, Tagliaferri R, Greco D.

Author information: 
(1)Department of Computer Science (DI), NeuRoNe Lab, University of Salerno, via
ponte don Melillo 84084, Fisciano, (SA), Italy. vittorio.fortino@ttl.fi.

BACKGROUND: Inferring operon maps is crucial to understanding the regulatory
networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies
revealed that in many bacterial species the operon structure vary with the change
of environmental conditions. Therefore, new computational solutions that use both
static and dynamic data are necessary to create condition specific operon
predictions.
RESULTS: In this work, we propose a novel classification method that integrates
RNA-seq based transcriptome profiles with genomic sequence features to accurately
identify the operons that are expressed under a measured condition. The
classifiers are trained on a small set of confirmed operons and then used to
classify the remaining gene pairs of the organism studied. Finally, by linking
consecutive gene pairs classified as operons, our computational approach produces
condition-dependent operon maps. We evaluated our approach on various RNA-seq
expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, 
Escherichia coli and Salmonella enterica. Our results demonstrate that, using
features depending on both transcriptome dynamics and genome sequence
characteristics, we can identify operon pairs with high accuracy. Moreover, the
combination of DNA sequence and expression data results in more accurate
predictions than each one alone.
CONCLUSION: We present a computational strategy for the comprehensive analysis of
condition-dependent operon maps in prokaryotes. Our method can be used to
generate condition specific operon maps of many bacterial organisms for which
high-resolution transcriptome data is available.

DOI: 10.1186/1471-2105-15-145 
PMCID: PMC4235196
PMID: 24884724  [Indexed for MEDLINE]


223. BMC Syst Biol. 2014 May 16;8:56. doi: 10.1186/1752-0509-8-56.

Network topology-based detection of differential gene regulation and regulatory
switches in cell metabolism and signaling.

Piro RM, Wiesberg S, Schramm G, Rebel N, Oswald M, Eils R, Reinelt G, König R(1).

Author information: 
(1)Division of Theoretical Bioinformatics, German Cancer Research Center
(Deutsches Krebsforschungszentrum, DKFZ), Heidelberg, Germany. r.koenig@dkfz.de.

BACKGROUND: Common approaches to pathway analysis treat pathways merely as lists 
of genes disregarding their topological structures, that is, ignoring the genes' 
interactions on which a pathway's cellular function depends. In contrast,
PathWave has been developed for the analysis of high-throughput gene expression
data that explicitly takes the topology of networks into account to identify both
global dysregulation of and localized (switch-like) regulatory shifts within
metabolic and signaling pathways. For this purpose, it applies adjusted wavelet
transforms on optimized 2D grid representations of curated pathway maps.
RESULTS: Here, we present the new version of PathWave with several substantial
improvements including a new method for optimally mapping pathway networks unto
compact 2D lattice grids, a more flexible and user-friendly interface, and
pre-arranged 2D grid representations. These pathway representations are assembled
for several species now comprising H. sapiens, M. musculus, D. melanogaster, D.
rerio, C. elegans, and E. coli. We show that PathWave is more sensitive than
common approaches and apply it to RNA-seq expression data, identifying crucial
metabolic pathways in lung adenocarcinoma, as well as microarray expression data,
identifying pathways involved in longevity of Drosophila.
CONCLUSIONS: PathWave is a generic method for pathway analysis complementing
established tools like GSEA, and the update comprises efficient new features. In 
contrast to the tested commonly applied approaches which do not take network
topology into account, PathWave enables identifying pathways that are either
known be involved in or very likely associated with such diverse conditions as
human lung cancer or aging of D. melanogaster. The PathWave R package is freely
available at http://www.ichip.de/software/pathwave.html.

DOI: 10.1186/1752-0509-8-56 
PMCID: PMC4031158
PMID: 24886210  [Indexed for MEDLINE]


224. BMC Bioinformatics. 2014 May 13;15:140. doi: 10.1186/1471-2105-15-140.

A predictor for predicting Escherichia coli transcriptome and the effects of gene
perturbations.

Ling MH(1), Poh CL.

Author information: 
(1)School of Chemical and Biomedical Engineering, Nanyang Technological
University, Nanyang Ave, Singapore, Singapore. mauriceling@acm.org.

BACKGROUND: A means to predict the effects of gene over-expression, knockouts,
and environmental stimuli in silico is useful for system biologists to develop
and test hypotheses. Several studies had predicted the expression of all
Escherichia coli genes from sequences and reported a correlation of 0.301 between
predicted and actual expression. However, these do not allow biologists to study 
the effects of gene perturbations on the native transcriptome.
RESULTS: We developed a predictor to predict transcriptome-scale gene expression 
from a small number (n = 59) of known gene expressions using gene co-expression
network, which can be used to predict the effects of over-expressions and
knockdowns on E. coli transcriptome. In terms of transcriptome prediction, our
results show that the correlation between predicted and actual expression value
is 0.467, which is similar to the microarray intra-array variation
(p-value = 0.348), suggesting that intra-array variation accounts for a
substantial portion of the transcriptome prediction error. In terms of predicting
the effects of gene perturbation(s), our results suggest that the expression of
83% of the genes affected by perturbation can be predicted within 40% of error
and the correlation between predicted and actual expression values among the
affected genes to be 0.698. With the ability to predict the effects of gene
perturbations, we demonstrated that our predictor has the potential to estimate
the effects of varying gene expression level on the native transcriptome.
CONCLUSION: We present a potential means to predict an entire transcriptome and a
tool to estimate the effects of gene perturbations for E. coli, which will aid
biologists in hypothesis development. This study forms the baseline for future
work in using gene co-expression network for gene expression prediction.

DOI: 10.1186/1471-2105-15-140 
PMCID: PMC4038595
PMID: 24884349  [Indexed for MEDLINE]


225. Biotechnol Bioeng. 2014 May;111(5):980-99. doi: 10.1002/bit.25169. Epub 2014 Jan 
30.

Dynamic transcriptional response of Escherichia coli to inclusion body formation.

Baig F(1), Fernando LP, Salazar MA, Powell RR, Bruce TF, Harcum SW.

Author information: 
(1)Department of Bioengineering, Clemson University, 301 Rhodes Research Center, 
Clemson, South Carolina, 29634.

Escherichia coli is used intensively for recombinant protein production, but one 
key challenge with recombinant E. coli is the tendency of recombinant proteins to
misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high
concentrations of inactive recombinant protein that require recovery steps to
salvage a functional recombinant protein. Currently, no universally effective
method exists to prevent IB formation in recombinant E. coli. In this study, DNA 
microarrays were used to compare the E. coli gene expression response dynamics to
soluble and insoluble recombinant protein production. As expected and previously 
reported, the classical heat-shock genes had increased expression due to IB
formation, including protein folding chaperones and proteases. Gene expression
levels for protein synthesis-related and energy-synthesis pathways were also
increased. Many transmembrane transporter and corresponding catabolic pathways
genes had decreased expression for substrates not present in the culture medium. 
Additionally, putative genes represented over one-third of the genes identified
to have significant expression changes due to IB formation, indicating many
important cellular responses to IB formation still need to be characterized.
Interestingly, cells grown in 3% ethanol had significantly reduced gene
expression responses due to IB formation. Taken together, these results indicate 
that IB formation is complex, stimulates the heat-shock response, increases
protein and energy synthesis needs, and streamlines transport and catabolic
processes, while ethanol diminished all of these responses.

© 2014 Wiley Periodicals, Inc.

DOI: 10.1002/bit.25169 
PMCID: PMC3969792
PMID: 24338599  [Indexed for MEDLINE]


226. Genes Cells. 2014 May;19(5):405-18. doi: 10.1111/gtc.12139. Epub 2014 Mar 19.

Roles of cell division control factor SdiA: recognition of quorum sensing signals
and modulation of transcription regulation targets.

Shimada T(1), Shimada K, Matsui M, Kitai Y, Igarashi J, Suga H, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, 184-8584,
Japan; Research Center for Micro-Nano Technology, Hosei University, Koganei,
Tokyo, 184-8584, Japan; Chemical Resources Laboratory, Tokyo Institute of
Technology, Nagatsuda, Yokohama, 226-8503, Japan.

In Gram-negative bacteria, N-acylhomoserine lactone (HSL) is used as a signal in 
cell-cell communication and quorum sensing (QS). The model prokaryote Escherichia
coli lacks the system of HSL synthesis, but is capable of monitoring HSL signals 
in environment. Transcription factor SdiA for cell division control is believed
to play a role as a HSL sensor. Using a collection of 477 species of chemically
synthesized HSL analogues, we identified three synthetic signal molecules (SSMs) 
that bind in vitro to purified SdiA. After SELEX-chip screening of SdiA-binding
DNA sequences, a striking difference was found between these SSMs in the pattern 
of regulation target genes on the E. coli genome. Based on Northern blot analysis
in vivo, a set of target genes were found to be repressed by SdiA in the absence 
of effectors and derepressed by the addition of SSMs. Another set of genes were, 
however, expressed in the absence of effector ligands but repressed by the
addition of SSMs. Taken together, we propose that the spectrum of taget gene
selection by SdiA is modulated in multiple modes depending on the interacting
HSL-like signal molecules.

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of
Japan and Wiley Publishing Asia Pty Ltd.

DOI: 10.1111/gtc.12139 
PMID: 24645791  [Indexed for MEDLINE]


227. J Anim Sci. 2014 May;92(5):2050-62. doi: 10.2527/jas.2013-6422. Epub 2014 Mar 18.

Dietary plant extracts modulate gene expression profiles in ileal mucosa of
weaned pigs after an Escherichia coli infection.

Liu Y(1), Song M, Che TM, Lee JJ, Bravo D, Maddox CW, Pettigrew JE.

Author information: 
(1)Department of Animal Sciences and.

This study was conducted to characterize the effects of infection with a
pathogenic F-18 Escherichia coli and 3 different plant extracts on gene
expression of ileal mucosa in weaned pigs. Weaned pigs (total = 64, 6.3 ± 0.2 kg 
BW, and 21-d old) were housed in individual pens for 15 d, 4 d before and 11 d
after the first inoculation (d 0). Treatments were in a 2 × 4 factorial
arrangement: with or without an F-18 E. coli challenge and 4 diets (a nursery
basal, control diet [CON], 10 ppm of capsicum oleoresin [CAP], garlic botanical
[GAR], or turmeric oleoresin [TUR]). Results reported elsewhere showed that the
plant extracts reduced diarrhea in challenged pigs. Total RNA (4 pigs/treatment) 
was extracted from ileal mucosa of pigs at d 5 post inoculation. Double-stranded 
cDNA was amplified, labeled, and further hybridized to the microarray, and data
were analyzed in R. Differential gene expression was tested by fitting a mixed
linear model in a 2 × 4 factorial ANOVA. Bioinformatics analysis was conducted by
DAVID Bioinformatics Resources 6.7 (DAVID; National Institute of Allergy and
Infectious Diseases [NIAID, NIH], http://david.abcc.ncifcrf.gov). The E. coli
infection altered (P < 0.05) the expression of 240 genes in pigs fed the CON (148
up- and 92 down-regulated). Compared with the infected CON, feeding CAP, GAR, or 
TUR altered (P < 0.05) the expression of 52 genes (18 up, 34 down), 117 genes (34
up- and 83 down-regulated), or 84 genes (16 up- and 68 down-regulated),
respectively, often counteracting the effects of E. coli. The E. coli infection
up-regulated (P < 0.05) the expression of genes related to the activation of
immune response and complement and coagulation cascades, but down-regulated (P < 
0.05) the expression of genes involved in protein synthesis and accumulation.
Compared with the CON, feeding CAP and GAR increased (P < 0.05) the expression of
genes related to integrity of membranes in infected pigs, indicating enhanced gut
mucosa health. Moreover, feeding all 3 plant extracts reduced (P < 0.05) the
expression of genes associated with antigen presentation or other biological
processes of immune responses, indicating they attenuated overstimulation of
immune responses caused by E. coli. These findings may explain why diarrhea was
reduced and clinical immune responses were ameliorated in infected pigs fed plant
extracts. In conclusion, plant extracts altered the expression of genes in ileal 
mucosa of E. coli-infected pigs, perhaps leading to the reduction in diarrhea
reported previously.

DOI: 10.2527/jas.2013-6422 
PMID: 24663182  [Indexed for MEDLINE]


228. J Clin Microbiol. 2014 May;52(5):1501-10. doi: 10.1128/JCM.03617-13. Epub 2014
Feb 26.

Real-time whole-genome sequencing for routine typing, surveillance, and outbreak 
detection of verotoxigenic Escherichia coli.

Joensen KG(1), Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, Aarestrup FM.

Author information: 
(1)National Food Institute, Division for Epidemiology and Microbial Genomics,
Technical University of Denmark, Kongens Lyngby, Denmark.

Fast and accurate identification and typing of pathogens are essential for
effective surveillance and outbreak detection. The current routine procedure is
based on a variety of techniques, making the procedure laborious, time-consuming,
and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge
potential in both diagnostics and routine surveillance. The aim of this study was
to perform a real-time evaluation of WGS for routine typing and surveillance of
verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum
Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week
period in the fall of 2012, all incoming isolates were concurrently subjected to 
WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using
web-tools (www.genomicepidemiology.org) for species determination, multilocus
sequence type (MLST) typing, and determination of phylogenetic relationship, and 
a specific VirulenceFinder for detection of E. coli virulence genes was developed
as part of this study. In total, 46 suspected VTEC isolates were characterized in
parallel during the study. VirulenceFinder proved successful in detecting
virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1),
verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence
genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) 
subtypes. A real-time clustering of isolates in agreement with the epidemiology
was established from WGS, enabling discrimination between sporadic and outbreak
isolates. Overall, WGS typing produced results faster and at a lower cost than
the current routine. Therefore, WGS typing is a superior alternative to
conventional typing strategies. This approach may also be applied to typing and
surveillance of other pathogens.

DOI: 10.1128/JCM.03617-13 
PMCID: PMC3993690
PMID: 24574290  [Indexed for MEDLINE]


229. Cell. 2014 Apr 24;157(3):624-35. doi: 10.1016/j.cell.2014.02.033.

Quantifying absolute protein synthesis rates reveals principles underlying
allocation of cellular resources.

Li GW(1), Burkhardt D(2), Gross C(3), Weissman JS(4).

Author information: 
(1)Department of Cellular and Molecular Pharmacology, Howard Hughes Medical
Institute, University of California, San Francisco, San Francisco, CA 94158, USA;
California Institute of Quantitative Biosciences, University of California, San
Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology,
University of California, San Francisco, San Francisco, CA 94158, USA. Electronic
address: gene-wei.li@ucsf.edu.
(2)California Institute of Quantitative Biosciences, University of California,
San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and
Immunology, University of California, San Francisco, San Francisco, CA 94158,
USA.
(3)California Institute of Quantitative Biosciences, University of California,
San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and
Immunology, University of California, San Francisco, San Francisco, CA 94158,
USA; Department of Cell and Tissue Biology, University of California, San
Francisco, San Francisco, CA 94158, USA.
(4)Department of Cellular and Molecular Pharmacology, Howard Hughes Medical
Institute, University of California, San Francisco, San Francisco, CA 94158, USA;
California Institute of Quantitative Biosciences, University of California, San
Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology,
University of California, San Francisco, San Francisco, CA 94158, USA. Electronic
address: weissman@cmp.ucsf.edu.

Comment in
    Cell. 2014 Apr 24;157(3):529-31.

Quantitative views of cellular functions require precise measures of rates of
biomolecule production, especially proteins-the direct effectors of biological
processes. Here, we present a genome-wide approach, based on ribosome profiling, 
for measuring absolute protein synthesis rates. The resultant E. coli data set
transforms our understanding of the extent to which protein synthesis is
precisely controlled to optimize function and efficiency. Members of multiprotein
complexes are made in precise proportion to their stoichiometry, whereas
components of functional modules are produced differentially according to their
hierarchical role. Estimates of absolute protein abundance also reveal principles
for optimizing design. These include how the level of different types of
transcription factors is optimized for rapid response and how a metabolic pathway
(methionine biosynthesis) balances production cost with activity requirements.
Our studies reveal how general principles, important both for understanding
natural systems and for synthesizing new ones, emerge from quantitative analyses 
of protein synthesis.

Copyright © 2014 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.cell.2014.02.033 
PMCID: PMC4006352
PMID: 24766808  [Indexed for MEDLINE]


230. J Am Coll Cardiol. 2014 Apr 22;63(15):1542-55. doi: 10.1016/j.jacc.2014.01.007.
Epub 2014 Feb 5.

Genetic implication of a novel thiamine transporter in human hypertension.

Zhang K(1), Huentelman MJ(2), Rao F(1), Sun EI(3), Corneveaux JJ(2), Schork
AJ(1), Wei Z(1), Waalen J(4), Miramontes-Gonzalez JP(1), Hightower CM(1),
Maihofer AX(5), Mahata M(1), Pastinen T(6), Ehret GB(7); International Consortium
for Blood Pressure Genome-Wide Association Studies, Schork NJ(8), Eskin E(6),
Nievergelt CM(5), Saier MH Jr(3), O'Connor DT(9).

Author information: 
(1)Department of Medicine, University of California at San Diego (UCSD), La
Jolla, California.
(2)Division of Neurogenomics, Translational Genomics Research Institute, Phoenix,
Arizona.
(3)Department of Biology, UCSD, La Jolla, California.
(4)Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, California.
(5)Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, California; Departments of Human and Medical Genetics,
McGill University and Génome Québec Innovation Centre, Montréal, Québec, Canada.
(6)Departments of Human and Medical Genetics, McGill University and Génome Québec
Innovation Centre, Montréal, Québec, Canada.
(7)Center for Complex Disease Genomics, McKusick-Nathans, Johns Hopkins
University School of Medicine, Baltimore, Maryland.
(8)Department of Psychiatry, UCSD, La Jolla, California; Departments of Computer 
Science and Human Genetics, University of California at Los Angeles, Los Angeles,
California.
(9)Department of Medicine, University of California at San Diego (UCSD), La
Jolla, California; Veterans Affairs San Diego Healthcare System, San Diego,
California; Department of Pharmacology and the Institute for Genomic Medicine,
UCSD, La Jolla, California. Electronic address: doconnor@ucsd.edu.

OBJECTIVES: This study coupled 2 strategies-trait extremes and genome-wide
pooling-to discover a novel blood pressure (BP) locus that encodes a previously
uncharacterized thiamine transporter.
BACKGROUND: Hypertension is a heritable trait that remains the most potent and
widespread cardiovascular risk factor, although details of its genetic
determination are poorly understood.
METHODS: Representative genomic deoxyribonucleic acid (DNA) pools were created
from male and female subjects in the highest- and lowest-fifth percentiles of BP 
in a primary care population of >50,000 patients. The peak associated
single-nucleotide polymorphisms were typed in individual DNA samples, as well as 
in twins/siblings phenotyped for cardiovascular and autonomic traits. Biochemical
properties of the associated transporter were evaluated in cellular assays.
RESULTS: After chip hybridization and calculation of relative allele scores, the 
peak associations were typed in individual samples, revealing an association
between hypertension, systolic BP, and diastolic BP and the previously
uncharacterized solute carrier SLC35F3. The BP genetic association at SLC35F3 was
validated by meta-analysis in an independent sample from the original source
population, as well as the International Consortium for Blood Pressure
Genome-Wide Association Studies (across North America and western Europe).
Sequence homology to a putative yeast thiamine (vitamin B1) transporter prompted 
us to express human SLC35F3 in Escherichia coli, which catalyzed [(3)H]-thiamine 
uptake. SLC35F3 risk-allele homozygotes (T/T) displayed decreased erythrocyte
thiamine content on microbiological assay. In twin pairs, the SLC35F3 risk allele
predicted heritable cardiovascular traits previously associated with thiamine
deficiency, including elevated cardiac stroke volume with decreased vascular
resistance, and elevated pressor responses to environmental (cold) stress.
Allelic expression imbalance confirmed that cis variation at the human SLC35F3
locus influenced expression of that gene, and the allelic expression imbalance
peak coincided with the hypertension peak.
CONCLUSIONS: Novel strategies were coupled to position a new
hypertension-susceptibility locus, uncovering a previously unsuspected thiamine
transporter whose genetic variants predicted several disturbances in cardiac and 
autonomic function. The results have implications for the pathogenesis and
treatment of systemic hypertension.

Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier
Inc. All rights reserved.

DOI: 10.1016/j.jacc.2014.01.007 
PMCID: PMC3992204
PMID: 24509276  [Indexed for MEDLINE]


231. Biosens Bioelectron. 2014 Apr 15;54:195-8. doi: 10.1016/j.bios.2013.11.003. Epub 
2013 Nov 12.

Aptamer cocktails: enhancement of sensing signals compared to single use of
aptamers for detection of bacteria.

Kim YS(1), Chung J(1), Song MY(2), Jurng J(1), Kim BC(3).

Author information: 
(1)Center for Environment, Health and Welfare Research, Korea Institute of
Science and Technology, Seoul 136-701, Republic of Korea.
(2)Center for Environment, Health and Welfare Research, Korea Institute of
Science and Technology, Seoul 136-701, Republic of Korea; Graduate School of
Energy and Environmental System Engineering, University of Seoul, Seoul 130-743, 
Republic of Korea.
(3)Center for Environment, Health and Welfare Research, Korea Institute of
Science and Technology, Seoul 136-701, Republic of Korea. Electronic address:
bchankim@kist.re.kr.

Microbial cells have many binding moieties on their surface for binding to their 
specific bioreceptors. The whole-cell SELEX process enables the isolation of
various aptamers that can bind to different components on the cell surface such
as proteins, polysaccharides, or flagella with high affinity and specificity.
Here, we examine the binding capacity of an aptamer mixture (aptamer cocktail)
composed of various combinations of 3 different DNA aptamers isolated from
Escherichia coli and compare it with one of the single aptamers using
fluorescence-tagged aptamers. The aptamer mixtures showed higher fluorescence
signal than did any single aptamer used, which suggests that use of aptamer
mixtures can enhance the sensitivity of detection of microbial cells. To further 
evaluate this effect, the signal enhancement and improvement of sensitivity
provided by combinatorial use of aptamers were examined in an electrochemical
detection system. With regard to current decreases, the aptamer cocktail
immobilized on gold electrodes performed better than a single aptamer immobilized
on gold electrodes did. Consequently, the detection limit achieved using the
aptamers individually was approximately 18 times that when the 3 aptamers were
used in combination. These results support the use of aptamer cocktails for
detection of complex targets such as E. coli with enhanced sensitivity.

© 2013 Published by Elsevier B.V.

DOI: 10.1016/j.bios.2013.11.003 
PMID: 24280049  [Indexed for MEDLINE]


232. Nat Commun. 2014 Apr 8;5:3538. doi: 10.1038/ncomms4538.

An RNA-seq method for defining endoribonuclease cleavage specificity identifies
dual rRNA substrates for toxin MazF-mt3.

Schifano JM(1), Vvedenskaya IO(2), Knoblauch JG(2), Ouyang M(3), Nickels BE(2),
Woychik NA(1).

Author information: 
(1)Department of Biochemistry and Molecular Biology, Rutgers University, Robert
Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
(2)1] Waksman Institute, Rutgers University, Piscataway, New Jersey 08854, USA
[2] Department of Genetics, Rutgers University, Piscataway, New Jersey 08854,
USA.
(3)Department of Computer Science, University of Massachusetts Boston, Boston,
Massachusetts 02125, USA.

Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the
mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth 
by sequence-specific cleavage of single-stranded RNA. Defining the physiological 
targets of a MazF toxin first requires the identification of its cleavage
specificity, yet the current toolkit is antiquated and limited. We describe a
rapid genome-scale approach, MORE (mapping by overexpression of an RNase in
Escherichia coli) RNA-seq, for defining the cleavage specificity of
endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from
Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, 
evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno
(aSD) sequence of 16S rRNA. Our findings support an emerging model where both
ribosomal and messenger RNAs are principal targets of MazF toxins and suggest
that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies
ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.

DOI: 10.1038/ncomms4538 
PMCID: PMC4090939
PMID: 24709835  [Indexed for MEDLINE]


233. Emerg Infect Dis. 2014 Apr;20(4):732-3. doi: 10.3201/eid2004.131782.

Whole-genome sequencing for risk assessment of long-term Shiga toxin-producing
Escherichia coli.

Knobloch JK, Niemann S, Kohl TA, Lindner U, Nitschke M, Sayk F, Solbach W.

DOI: 10.3201/eid2004.131782 
PMCID: PMC3966390
PMID: 24655930  [Indexed for MEDLINE]


234. Microb Drug Resist. 2014 Apr;20(2):91-103. doi: 10.1089/mdr.2013.0063. Epub 2013 
Oct 18.

Transcriptomic analysis of triclosan-susceptible and -tolerant Escherichia coli
O157:H19 in response to triclosan exposure.

Lenahan M(1), Sheridan Á, Morris D, Duffy G, Fanning S, Burgess CM.

Author information: 
(1)1 Food Safety Department, Teagasc Food Research Centre , Dublin, Ireland .

Triclosan is an active agent that is commonly found in biocide formulations which
are used by the food industry to control microbial contamination. The aim of this
study was to use microarray analysis to compare gene expression between a
triclosan-susceptible Escherichia coli O157:H19 isolate (minimum inhibitory
concentration [MIC] 6.25 μg/ml) and its in vitro generated triclosan-tolerant
mutant (MIC >8,000 μg/ml). Gene expression profiling was performed on the
wild-type and mutant isogenic pairs after 30 min exposure to the parent MIC for
triclosan and an untreated control. Microarray analysis was carried out using the
Affymetrix GeneChip E. coli Genome 2.0 Array, and differential expression of
genes was analyzed using the pumaDE method in Bioconductor R software. Wild-type 
gene expression was found to be significantly different from the
triclosan-tolerant mutant for a large number of genes, even in the absence of
triclosan exposure. Significant differences were observed in the expression of a 
number of pathway genes involved in metabolism, transport, and chemotaxis. In
particular, gene expression in the triclosan-tolerant mutant was highly
up-regulated for 33 of 38 genes belonging to the flagellar assembly pathway. The 
presence of extended flagella in the mutant isolate was confirmed visually by
transmission electron microscopy, although no significant difference was observed
in the motility of the parent and mutant at low levels of triclosan. Data from
this study show that at a transcriptomic level, a triclosan-tolerant E. coli
O157:H19 mutant is significantly different from the wild-type strain in a number 
of different pathways, providing an increased understanding of triclosan
tolerance.

DOI: 10.1089/mdr.2013.0063 
PMID: 24138539  [Indexed for MEDLINE]


235. Bioconjug Chem. 2014 Mar 19;25(3):618-27. doi: 10.1021/bc500033d. Epub 2014 Mar
4.

Direct covalent attachment of DNA microarrays by rapid thiol-ene "click"
chemistry.

Escorihuela J(1), Bañuls MJ, Grijalvo S, Eritja R, Puchades R, Maquieira A.

Author information: 
(1)Centro de Reconocimiento Molecular y Desarrollo Tecnológico, Departamento de
Química, Universitat Politècnica de València , Camino de Vera s/n, 46022
Valencia, Spain.

A rapid strategy for the covalent immobilization of DNA onto silicon-based
materials using the UV-initiated radical thiol-ene reaction is presented in this 
study. Following this approach, thiol- and alkene-modified oligonucleotide probes
were covalently attached in microarray format, reaching immobilization densities 
around 6 pmol·cm(-2). The developed methodology presents the advantages of
spatially controlled probe anchoring (using a photomask), direct attachment
without using cross-linkers (one-pot fashion), and short irradiation times (20
min). Using the described strategy, hybridization efficiencies up to 65% with
full complementary strands were reached. The approach was evaluated by scoring
single-base pair mismatches with discrimination ratios around 15. Moreover, the
efficacy of the proposed DNA detection scheme is further demonstrated through the
assay on a genomic target of bacterial Escherichia coli.

DOI: 10.1021/bc500033d 
PMID: 24559310  [Indexed for MEDLINE]


236. PLoS One. 2014 Mar 12;9(3):e90781. doi: 10.1371/journal.pone.0090781. eCollection
2014.

CaSPIAN: a causal compressive sensing algorithm for discovering directed
interactions in gene networks.

Emad A(1), Milenkovic O(1).

Author information: 
(1)Department of Electrical and Computer Engineering, University of Illinois at
Urbana-Champaign, Urbana, Illinois, United States of America.

We introduce a novel algorithm for inference of causal gene interactions, termed 
CaSPIAN (Causal Subspace Pursuit for Inference and Analysis of Networks), which
is based on coupling compressive sensing and Granger causality techniques. The
core of the approach is to discover sparse linear dependencies between shifted
time series of gene expressions using a sequential list-version of the subspace
pursuit reconstruction algorithm and to estimate the direction of gene
interactions via Granger-type elimination. The method is conceptually simple and 
computationally efficient, and it allows for dealing with noisy measurements. Its
performance as a stand-alone platform without biological side-information was
tested on simulated networks, on the synthetic IRMA network in Saccharomyces
cerevisiae, and on data pertaining to the human HeLa cell network and the SOS
network in E. coli. The results produced by CaSPIAN are compared to the results
of several related algorithms, demonstrating significant improvements in
inference accuracy of documented interactions. These findings highlight the
importance of Granger causality techniques for reducing the number of
false-positives, as well as the influence of noise and sampling period on the
accuracy of the estimates. In addition, the performance of the method was tested 
in conjunction with biological side information of the form of sparse "scaffold
networks", to which new edges were added using available RNA-seq or microarray
data. These biological priors aid in increasing the sensitivity and precision of 
the algorithm in the small sample regime.

DOI: 10.1371/journal.pone.0090781 
PMCID: PMC3951243
PMID: 24622336  [Indexed for MEDLINE]


237. BMC Genomics. 2014 Mar 7;15:181. doi: 10.1186/1471-2164-15-181.

Transcriptome microRNA profiling of bovine mammary epithelial cells challenged
with Escherichia coli or Staphylococcus aureus bacteria reveals pathogen directed
microRNA expression profiles.

Jin W, Ibeagha-Awemu EM, Liang G, Beaudoin F, Zhao X(1), Guan le L.

Author information: 
(1)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB T6G2P5, Canada. xin.zhao@mcgill.ca.

BACKGROUND: MicroRNAs (miRNAs) can post-transcriptionally regulate gene
expression and have been shown to be critical regulators to the fine-tuning of
epithelial immune responses. However, the role of miRNAs in bovine responses to
E. coli and S. aureus, two mastitis causing pathogens, is not well understood.
RESULTS: The global expression of miRNAs in bovine mammary epithelial cells
(MAC-T cells) challenged with and without heat-inactivated Staphylococcus aureus 
(S. aureus) or Escherichia coli (E. coli) bacteria at 0, 6, 12, 24, and 48 hr was
profiled using RNA-Seq. A total of 231 known bovine miRNAs were identified with
more than 10 counts per million in at least one of 13 libraries and 5 miRNAs
including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more 
than 50% of the abundance. One hundred and thirteen novel miRNAs were also
identified and more than one third of them belong to the bta-miR-2284 family.
Seventeen miRNAs were significantly (P < 0.05) differentially regulated by the
presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6
miRNAs differentially regulated within the first 6 hrs post challenge as compared
to 1 miRNA for S. aureus) while S. aureus presented a delayed response. Five
differentially expressed miRNAs (bta-miR-184, miR-24-3p, miR-148, miR-486 and
let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and 
miR-99b) were unique to S. aureus. In addition, our study revealed a temporal
differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, 
miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen
differentially expressed miRNAs indicate a significant enrichment in gene
ontology functional categories in development/cellular processes, biological
regulation as well as cell growth and death. Furthermore, target genes were
significantly enriched in several KEGG pathways including immune system, signal
transduction, cellular process, nervous system, development and human diseases.
CONCLUSION: Using next-generation sequencing, our study identified a pathogen
directed differential regulation of miRNAs in MAC-T cells with roles in immunity 
and development. Our study provides a further confirmation of the involvement of 
mammary epithelia cells in contributing to the immune response to infecting
pathogens and suggests the potential of miRNAs to serve as biomarkers for
diagnosis and development of control measures.

DOI: 10.1186/1471-2164-15-181 
PMCID: PMC4029070
PMID: 24606609  [Indexed for MEDLINE]


238. Science. 2014 Mar 7;343(6175):1246980. doi: 10.1126/science.1246980.

Common genetic variants modulate pathogen-sensing responses in human dendritic
cells.

Lee MN(1), Ye C, Villani AC, Raj T, Li W, Eisenhaure TM, Imboywa SH, Chipendo PI,
Ran FA, Slowikowski K, Ward LD, Raddassi K, McCabe C, Lee MH, Frohlich IY, Hafler
DA, Kellis M, Raychaudhuri S, Zhang F, Stranger BE, Benoist CO, De Jager PL,
Regev A, Hacohen N.

Author information: 
(1)Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard,
Cambridge, MA 02142, USA.

Comment in
    Science. 2014 Mar 7;343(6175):1087-8.
    Nat Rev Immunol. 2014 Apr;14(4):212.

Little is known about how human genetic variation affects the responses to
environmental stimuli in the context of complex diseases. Experimental and
computational approaches were applied to determine the effects of genetic
variation on the induction of pathogen-responsive genes in human dendritic cells.
We identified 121 common genetic variants associated in cis with variation in
expression responses to Escherichia coli lipopolysaccharide, influenza, or
interferon-β (IFN-β). We localized and validated causal variants to binding sites
of pathogen-activated STAT (signal transducer and activator of transcription) and
IRF (IFN-regulatory factor) transcription factors. We also identified a common
variant in IRF7 that is associated in trans with type I IFN induction in response
to influenza infection. Our results reveal common alleles that explain
interindividual variation in pathogen sensing and provide functional annotation
for genetic variants that alter susceptibility to inflammatory diseases.

DOI: 10.1126/science.1246980 
PMCID: PMC4124741
PMID: 24604203  [Indexed for MEDLINE]


239. PLoS One. 2014 Mar 6;9(3):e90447. doi: 10.1371/journal.pone.0090447. eCollection 
2014.

The whole set of constitutive promoters recognized by RNA polymerase RpoD
holoenzyme of Escherichia coli.

Shimada T(1), Yamazaki Y(2), Tanaka K(3), Ishihama A(4).

Author information: 
(1)Department of Frontier Biosience, Hosei University, Koganai, Tokyo, Japan;
Micro-Nano Technology Research Center, Hosei University, Koganai, Tokyo, Japan;
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda,
Yokohama, Japan.
(2)Genetics Strains Research Institute, National Institute of Genetics, Mishima, 
Shizuoka, Japan.
(3)Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda,
Yokohama, Japan.
(4)Department of Frontier Biosience, Hosei University, Koganai, Tokyo, Japan;
Micro-Nano Technology Research Center, Hosei University, Koganai, Tokyo, Japan.

Erratum in
    PLoS One. 2014;9(6):e100908.

The promoter selectivity of Escherichia coli RNA polymerase is determined by the 
sigma subunit with promoter recognition activity. The model prokaryote
Escherichia coli contains seven species of the sigma subunit, each recognizing a 
specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD,
plays a major role in transcription of growth-related genes. Concomitant with the
increase in detection of promoters functioning in vivo under various stressful
conditions, the variation is expanding in the consensus sequence of RpoD
promoters. In order to identify the canonical sequence of "constitutive
promoters" that are recognized by the RNA polymerase holoenzyme containing RpoD
sigma in the absence of supporting transcription factors, an in vitro mixed
transcription assay was carried out using a whole set of variant promoters, each 
harboring one base replacement, within the model promoter with the conserved -35 
and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and
TATAAT(-10), were identified to be ideal for the maximum level of open complex
formation and the highest rate of promoter opening, respectively. For
identification of the full range of constitutive promoters on the E. coli genome,
a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX
screening, and using the reconfirmed consensus promoter sequence, a total of
maximum 669 constitutive promoters were identified, implying that the majority of
hitherto identified promoters represents the TF-dependent "inducible promoters". 
One unique feature of the constitutive promoters is the high level of promoter
sequence conservation, about 85% carrying five-out-of-six agreements with -35 or 
-10 consensus sequence. The list of constitutive promoters provides the community
resource toward estimation of the inducible promoters that operate under various 
stressful conditions in nature.

DOI: 10.1371/journal.pone.0090447 
PMCID: PMC3946193
PMID: 24603758  [Indexed for MEDLINE]


240. PLoS One. 2014 Mar 3;9(3):e90422. doi: 10.1371/journal.pone.0090422. eCollection 
2014.

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes
in growth kinetics induced by an abrupt downshift in water activity.

Kocharunchitt C(1), King T(2), Gobius K(3), Bowman JP(1), Ross T(1).

Author information: 
(1)Food Safety Centre, Tasmanian Institute of Agriculture, University of
Tasmania, Hobart, Tasmania, Australia.
(2)Commonwealth Scientific and Industrial Research Organisation Animal, Food and 
Health Sciences, North Ryde, New South Wales, Australia.
(3)Commonwealth Scientific and Industrial Research Organisation Animal, Food and 
Health Sciences, Werribee, Victoria, Australia.

The present study was undertaken to investigate growth kinetics and
time-dependent change in global expression of Escherichia coli O157∶H7 Sakai upon
an abrupt downshift in water activity (aw). Based on viable count data, shifting 
E. coli from aw 0.993 to aw 0.985 or less caused an apparent loss, then recovery,
of culturability. Exponential growth then resumed at a rate characteristic for
the aw imposed. To understand the responses of this pathogen to abrupt osmotic
stress, we employed an integrated genomic and proteomic approach to characterize 
its cellular response during exposure to a rapid downshift but still within the
growth range from aw 0.993 to aw 0.967. Of particular interest, genes and
proteins with cell envelope-related functions were induced during the initial
loss and subsequent recovery of culturability. This implies that cells undergo
remodeling of their envelope composition, enabling them to adapt to osmotic
stress. Growth at low aw, however, involved up-regulating additional genes and
proteins, which are involved in the biosynthesis of specific amino acids, and
carbohydrate catabolism and energy generation. This suggests their important role
in facilitating growth under such stress. Finally, we highlighted the ability of 
E. coli to activate multiple stress responses by transiently inducing the RpoE
and RpoH regulons to control protein misfolding, while simultaneously activating 
the master stress regulator RpoS to mediate long-term adaptation to
hyperosmolality. This investigation extends our understanding of the potential
mechanisms used by pathogenic E. coli to adapt, survive and grow under osmotic
stress, which could potentially be exploited to aid the selection and/or
development of novel strategies to inactivate this pathogen.

DOI: 10.1371/journal.pone.0090422 
PMCID: PMC3940904
PMID: 24594867  [Indexed for MEDLINE]


241. J Bacteriol. 2014 Mar;196(5):1102-12. doi: 10.1128/JB.01448-13. Epub 2013 Dec 27.

Evidence for moonlighting functions of the θ subunit of Escherichia coli DNA
polymerase III.

Dietrich M(1), Pedró L, García J, Pons M, Hüttener M, Paytubi S, Madrid C, Juárez
A.

Author information: 
(1)Department of Microbiology, Faculty of Biology, University of Barcelona,
Barcelona, Spain.

The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no 
detectable homology to other ORFs in a database). It encodes the θ subunit of the
DNA polymerase III core complex. The precise function of the θ subunit within
this complex is not well established, and loss of holE does not result in a
noticeable phenotype. Paralogs of holE are also present on many conjugative
plasmids and on phage P1 (hot gene). In this study, we provide evidence
indicating that θ (HolE) exhibits structural and functional similarities to a
family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins
that are also encoded by enterobacterial ORFan genes. Microarray studies
comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT
mutants revealed highly similar expression patterns for strains harboring holE
and ydgT alleles. Among the genes differentially regulated in both mutants were
genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed
leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments
with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE 
and YdgT downregulate expression of the tna operon by possibly increasing the
level of Rho-dependent transcription termination at the tna operon's leader
region. Thus, for the first time, a regulatory function can be attributed to
HolE, in addition to its role as structural component of the DNA polymerase III
complex.

DOI: 10.1128/JB.01448-13 
PMCID: PMC3957705
PMID: 24375106  [Indexed for MEDLINE]


242. PLoS Genet. 2014 Feb 20;10(2):e1004120. doi: 10.1371/journal.pgen.1004120.
eCollection 2014 Feb.

Quantitative genome-wide genetic interaction screens reveal global epistatic
relationships of protein complexes in Escherichia coli.

Babu M(1), Arnold R(2), Bundalovic-Torma C(3), Gagarinova A(4), Wong KS(5), Kumar
A(6), Stewart G(7), Samanfar B(8), Aoki H(6), Wagih O(2), Vlasblom J(6), Phanse
S(1), Lad K(6), Yeou Hsiung Yu A(5), Graham C(6), Jin K(1), Brown E(7), Golshani 
A(8), Kim P(2), Moreno-Hagelsieb G(9), Greenblatt J(4), Houry WA(5), Parkinson
J(10), Emili A(4).

Author information: 
(1)Banting and Best Department of Medical Research, Donnelly Centre, University
of Toronto, Toronto, Ontario, Canada ; Department of Biochemistry, Research and
Innovation Centre, University of Regina, Regina, Saskatchewan, Canada.
(2)Banting and Best Department of Medical Research, Donnelly Centre, University
of Toronto, Toronto, Ontario, Canada.
(3)Hospital for Sick Children, Toronto, Ontario, Canada ; Department of
Biochemistry, University of Toronto, Toronto, Ontario, Canada.
(4)Banting and Best Department of Medical Research, Donnelly Centre, University
of Toronto, Toronto, Ontario, Canada ; Department of Molecular Genetics,
University of Toronto, Toronto, Ontario, Canada.
(5)Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
(6)Department of Biochemistry, Research and Innovation Centre, University of
Regina, Regina, Saskatchewan, Canada.
(7)Department of Biochemistry and Biomedical Sciences, McMaster University,
Hamilton, Ontario, Canada.
(8)Department of Biology and Ottawa Institute of Systems Biology, Carleton
University, Ottawa, Ontario, Canada.
(9)Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
(10)Hospital for Sick Children, Toronto, Ontario, Canada ; Department of
Biochemistry, University of Toronto, Toronto, Ontario, Canada ; Department of
Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Large-scale proteomic analyses in Escherichia coli have documented the
composition and physical relationships of multiprotein complexes, but not their
functional organization into biological pathways and processes. Conversely,
genetic interaction (GI) screens can provide insights into the biological role(s)
of individual gene and higher order associations. Combining the information from 
both approaches should elucidate how complexes and pathways intersect
functionally at a systems level. However, such integrative analysis has been
hindered due to the lack of relevant GI data. Here we present a systematic,
unbiased, and quantitative synthetic genetic array screen in E. coli describing
the genetic dependencies and functional cross-talk among over 600,000 digenic
mutant combinations. Combining this epistasis information with putative
functional modules derived from previous proteomic data and genomic context-based
methods revealed unexpected associations, including new components required for
the biogenesis of iron-sulphur and ribosome integrity, and the interplay between 
molecular chaperones and proteases. We find that functionally-linked genes
co-conserved among γ-proteobacteria are far more likely to have correlated GI
profiles than genes with divergent patterns of evolution. Overall, examining
bacterial GIs in the context of protein complexes provides avenues for a deeper
mechanistic understanding of core microbial systems.

DOI: 10.1371/journal.pgen.1004120 
PMCID: PMC3930520
PMID: 24586182  [Indexed for MEDLINE]


243. Sensors (Basel). 2014 Feb 19;14(2):3308-22. doi: 10.3390/s140203308.

Characterization of antimicrobial resistance patterns and detection of virulence 
genes in Campylobacter isolates in Italy.

Di Giannatale E(1), Di Serafino G(2), Zilli K(3), Alessiani A(4), Sacchini L(5), 
Garofolo G(6), Aprea G(7), Marotta F(8).

Author information: 
(1)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
e.digiannatale@izs.it.
(2)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
g.diserafino@izs.it.
(3)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
k.zilli@izs.it.
(4)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
a.alessiani@izs.it.
(5)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
lo.sacchini@izs.it.
(6)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
g.garofolo@izs.it.
(7)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
g.aprea@izs.it.
(8)LNR Campylobacter, IZSAM G.Caporale, via Campo Boario 64100 Teramo, Italy.
f.marotta@izs.it.

Campylobacter has developed resistance to several antimicrobial agents over the
years, including macrolides, quinolones and fluoroquinolones, becoming a
significant public health hazard. A total of 145 strains derived from raw milk,
chicken faeces, chicken carcasses, cattle faeces and human faeces collected from 
various Italian regions, were screened for antimicrobial susceptibility,
molecular characterization (SmaI pulsed-field gel electrophoresis) and detection 
of virulence genes (sequencing and DNA microarray analysis). The prevalence of C.
jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial
susceptibility revealed a high level of resistance for ciprofloxacin (62.76%),
tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter
isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene
profiles were determined using a new microbial diagnostic microarray composed of 
70-mer oligonucleotide probes targeting genes implicated in Campylobacter
pathogenicity. Correspondence between PFGE and microarray clusters was observed. 
Comparisons of PFGE and virulence profiles reflected the high genetic diversity
of the strains examined, leading us to speculate different degrees of
pathogenicity inside Campylobacter populations.

DOI: 10.3390/s140203308 
PMCID: PMC3958300
PMID: 24556669  [Indexed for MEDLINE]


244. PLoS One. 2014 Feb 14;9(2):e87970. doi: 10.1371/journal.pone.0087970. eCollection
2014.

Response of Medicago truncatula seedlings to colonization by Salmonella enterica 
and Escherichia coli O157:H7.

Jayaraman D(1), Valdés-López O(1), Kaspar CW(2), Ané JM(1).

Author information: 
(1)Department of Agronomy, University of Wisconsin-Madison, Madison Madison,
Wisconsin, United States of America.
(2)Department of Bacteriology, University of Wisconsin-Madison, Madison,
Wisconsin, United States of America.

Disease outbreaks due to the consumption of legume seedlings contaminated with
human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella
enterica are reported every year. Besides contaminations occurring during food
processing, pathogens present on the surface or interior of plant tissues are
also responsible for such outbreaks. In the present study, surface and internal
colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella
enterica and Escherichia coli O157:H7 were observed even with inoculum levels as 
low as two bacteria per plant. Furthermore, expression analyses revealed that
approximately 30% of Medicago truncatula genes were commonly regulated in
response to both of these enteric pathogens. This study highlights that very low 
inoculum doses trigger responses from the host plant and that both of these human
enteric pathogens may in part use similar mechanisms to colonize legume
seedlings.

DOI: 10.1371/journal.pone.0087970 
PMCID: PMC3925098
PMID: 24551073  [Indexed for MEDLINE]


245. PLoS One. 2014 Feb 3;9(2):e87038. doi: 10.1371/journal.pone.0087038. eCollection 
2014.

Expression of signaling components in embryonic eyelid epithelium.

Meng Q(1), Jin C(1), Chen Y(1), Chen J(1), Medvedovic M(1), Xia Y(1).

Author information: 
(1)Department of Environmental Health, College of Medicine, University of
Cincinnati, Cincinnati, Ohio, United States of America.

Erratum in
    PLoS One. 2014;9(8):e106783.

Closure of an epithelium opening is a critical morphogenetic event for
development. An excellent example for this process is the transient closure of
embryonic eyelid. Eyelid closure requires shape change and migration of
epithelial cells at the tip of the developing eyelids, and is dictated by
numerous signaling pathways. Here we evaluated gene expression in epithelial
cells isolated from the tip (leading edge, LE) and inner surface epithelium (IE) 
of the eyelid from E15.5 mouse fetuses by laser capture microdissection (LCM). We
showed that the LE and IE cells are different at E15.5, such that IE had higher
expression of muscle specific genes, while LE acquired epithelium identities.
Despite their distinct destinies, these cells were overall similar in expression 
of signaling components for the "eyelid closure pathways". However, while the LE 
cells had more abundant expression of Fgfr2, Erbb2, Shh, Ptch1 and 2, Smo and
Gli2, and Jag1 and Notch1, the IE cells had more abundant expression of Bmp5 and 
Bmpr1a. In addition, the LE cells had more abundant expression of adenomatosis
polyposis coli down-regulated 1 (Apcdd1), but the IE cells had high expression of
Dkk2. Our results suggest that the functionally distinct LE and IE cells have
also differential expression of signaling molecules that may contribute to the
cell-specific responses to morphogenetic signals. The expression pattern suggests
that the EGF, Shh and NOTCH pathways are preferentially active in LE cells, the
BMP pathways are effective in IE cells, and the Wnt pathway may be repressed in
LE and IE cells via different mechanisms.

DOI: 10.1371/journal.pone.0087038 
PMCID: PMC3911929
PMID: 24498290  [Indexed for MEDLINE]


246. Microbiol Immunol. 2014 Feb;58(2):77-86. doi: 10.1111/1348-0421.12120.

Rapid microarray-based DNA genoserotyping of Escherichia coli.

Geue L(1), Monecke S, Engelmann I, Braun S, Slickers P, Ehricht R.

Author information: 
(1)Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health,
Institute of Epidemiology, Wusterhausen.

In this study, an improvement in the oligonucleotide-based DNA microarray for the
genoserotyping of Escherichia coli is presented. Primer and probes for additional
70 O antigen groups were developed. The microarray was transferred to a new
platform, the ArrayStrip format, which allows high through-put tests in 96-well
formats and fully automated microarray analysis. Thus, starting from a single
colony, it is possible to determine within a few hours and a single experiment,
94 of the over 180 known O antigen groups as well as 47 of the 53 different H
antigens. The microarray was initially validated with a set of defined reference 
strains that had previously been serotyped by conventional agglutination in
various reference centers. For further validation of the microarray, 180 clinical
E. coli isolates of human origin (from urine samples, blood cultures, bronchial
secretions, and wound swabs) and 53 E. coli isolates from cattle, pigs, and
poultry were used. A high degree of concordance between the results of classical 
antibody-based serotyping and DNA-based genoserotyping was demonstrated during
validation of the new 70 O antigen groups as well as for the field strains of
human and animal origin. Therefore, this oligonucleotide array is a diagnostic
tool that is user-friendly and more efficient than classical serotyping by
agglutination. Furthermore, the tests can be performed in almost every routine
lab and are easily expanded and standardized.

© 2013 The Societies and Wiley Publishing Asia Pty Ltd.

DOI: 10.1111/1348-0421.12120 
PMID: 24298918  [Indexed for MEDLINE]


247. PLoS One. 2014 Jan 14;9(1):e80160. doi: 10.1371/journal.pone.0080160. eCollection
2014.

Microarray analysis of the Ler regulon in enteropathogenic and enterohaemorrhagic
Escherichia coli strains.

Bingle LE(1), Constantinidou C(1), Shaw RK(1), Islam MS(1), Patel M(1), Snyder
LA(1), Lee DJ(1), Penn CW(1), Busby SJ(1), Pallen MJ(1).

Author information: 
(1)School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United
Kingdom.

The type III protein secretion system is an important pathogenicity factor of
enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes
encoding this apparatus are located on a pathogenicity island (the locus of
enterocyte effacement) and are transcriptionally activated by the master
regulator Ler. In each pathotype Ler is also known to regulate genes located
elsewhere on the chromosome, but the full extent of the Ler regulon is unclear,
especially for enteropathogenic E. coli. The Ler regulon was defined for two
strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic)
in mid and late log phases of growth by DNA microarray analysis of the
transcriptomes of wild-type and ler mutant versions of each strain. In both
strains the Ler regulon is focused on the locus of enterocyte effacement - all
major transcriptional units of which are activated by Ler, with the sole
exception of the LEE1 operon during mid-log phase growth in E2348/69. However,
the Ler regulon does extend more widely and also includes unlinked pathogenicity 
genes: in E2348/69 more than 50 genes outside of this locus were regulated,
including a number of known or potential pathogenicity determinants; in EDL933
only 4 extra-LEE genes, again including known pathogenicity factors, were
activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, 
a number of genes including the plasmid-encoded regulator operon perABC, were
found to be negatively regulated by Ler. Negative regulation by Ler of PerC,
itself a positive regulator of the ler promoter, suggests a negative feedback
loop involving these proteins.

DOI: 10.1371/journal.pone.0080160 
PMCID: PMC3891560
PMID: 24454682  [Indexed for MEDLINE]


248. J Nucleic Acids. 2014;2014:214929. doi: 10.1155/2014/214929. Epub 2014 Apr 15.

Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers
against Shiga Toxins.

Challa S(1), Tzipori S(2), Sheoran A(2).

Author information: 
(1)Department of Infectious Disease and Global Health, Tufts Cummings School of
Veterinary Medicine, Tufts University, 200 Westboro Road, Building 20, North
Grafton, MA 01536, USA ; AstraZeneca, 35 Gatehouse Drive, Waltham, MA 02451, USA.
(2)Department of Infectious Disease and Global Health, Tufts Cummings School of
Veterinary Medicine, Tufts University, 200 Westboro Road, Building 20, North
Grafton, MA 01536, USA.

Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening
hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in
children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more
firmly linked with the development of HUS. In the present study, selective
evolution of ligands by exponential enrichment (SELEX) was used in an attempt to 
identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection,
significant enrichment of aptamer pool was obtained against Stx2, but not against
Stx1, using a RNA aptamer library containing 56 random nucleotides (N56).
Characterization of individual aptamer sequences revealed that six unique RNA
aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding
assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but 
not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa
cells in a flow cytometry assay. However, none of the aptamers neutralized
Stx2-mediated cytotoxicity and death of HeLa cells.

DOI: 10.1155/2014/214929 
PMCID: PMC4009280
PMID: 24839553 


249. Methods Mol Biol. 2014;1103:1-10. doi: 10.1007/978-1-62703-730-3_1.

Identification of regulatory RNA in bacterial genomes by genome-scale mapping of 
transcription start sites.

Singh N(1), Wade JT.

Author information: 
(1)Wadsworth Center, New York State Department of Health, Albany, NY, USA.

The ability to map transcription start sites is critical for studies of gene
regulation and for identification of novel RNAs. Conventional RNA-seq is often
insufficient for identification of transcription start sites due to low coverage 
and/or RNA processing events. We have developed a highly sensitive, genome-scale 
method for detection of transcription start sites in bacteria. This method uses
deep sequencing of cDNA libraries to identify transcription start sites with
strand specificity at nucleotide resolution. Here, we describe the application of
this method for transcription start site identification in Escherichia coli.

DOI: 10.1007/978-1-62703-730-3_1 
PMID: 24318882  [Indexed for MEDLINE]


250. Nat Protoc. 2014 Jan;9(1):146-55. doi: 10.1038/nprot.2014.001. Epub 2013 Dec 19.

Using Spinach-based sensors for fluorescence imaging of intracellular metabolites
and proteins in living bacteria.

Strack RL(1), Song W(1), Jaffrey SR(1).

Author information: 
(1)Department of Pharmacology, Weill Medical College, Cornell University, New
York, New York, USA.

Genetically encoded fluorescent sensors can be valuable tools for studying the
abundance and flux of molecules in living cells. We recently developed a novel
class of sensors composed of RNAs that can be used to detect diverse small
molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic
of GFP, and they have successfully been used to image several metabolites and
proteins in living bacteria. Here we discuss the generation and optimization of
these Spinach-based sensors, which, unlike most currently available genetically
encoded reporters, can be readily generated to any target of interest. We also
provide a detailed protocol for imaging ADP dynamics in living Escherichia coli
after a change from glucose-containing medium to other carbon sources. The entire
procedure typically takes ∼4 d including bacteria transformation and image
analysis. The majority of this protocol is applicable to sensing other
metabolites and proteins in living bacteria.

DOI: 10.1038/nprot.2014.001 
PMCID: PMC4028027
PMID: 24356773  [Indexed for MEDLINE]


251. World J Microbiol Biotechnol. 2013 Dec;29(12):2281-91. doi:
10.1007/s11274-013-1394-1. Epub 2013 Jun 11.

Development of a DNA macroarray for simultaneous detection of multiple foodborne 
pathogenic bacteria in fresh chicken meat.

Kupradit C(1), Rodtong S, Ketudat-Cairns M.

Author information: 
(1)School of Biotechnology, Institute of Agricultural Technology, Suranaree
University of Technology, Nakhon Ratchasima, 30000, Thailand.

A DNA macroarray was developed to provide the ability to detect multiple
foodborne pathogens in fresh chicken meat. Probes targeted to the 16S rRNA and
genus- and species-specific genes, including fimY, ipaH, prfA, and uspA, were
selected for the specific detection of Salmonella spp., Shigella spp., Listeria
monocytogenes, and Escherichia coli, respectively. The combination of target gene
amplification by PCR and a DNA macroarray in our system was able to distinguish
all target bacteria from pure cultures with a detection sensitivity of 10⁵ c.f.u.
ml⁻¹. The DNA macroarray was also applied to 10 fresh chicken meat samples. The
assay validation demonstrated that by combining the enrichment steps for the
target bacteria and the DNA macroarray, all 4 target bacteria could be detected
simultaneously from the fresh chicken samples. The sensitivity of L.
monocytogenes and Shigella boydii detection in the fresh chicken samples was at
least 10 and 3 c.f.u. of the initial contamination in 25 g samples, respectively.
The advantages of our developed protocol are high accuracy and time reduction
when compared to conventional culture. The macroarray developed in our
investigation was cost effective compared to modern oligonucleotide microarray
techniques because there was no expensive equipment required for the detection of
multiple foodborne pathogens.

DOI: 10.1007/s11274-013-1394-1 
PMID: 23754709  [Indexed for MEDLINE]


252. BMC Genomics. 2013 Nov 22;14:822. doi: 10.1186/1471-2164-14-822.

RNA-seq reveals the RNA binding proteins, Hfq and RsmA, play various roles in
virulence, antibiotic production and genomic flux in Serratia sp. ATCC 39006.

Wilf NM, Reid AJ, Ramsay JP, Williamson NR, Croucher NJ, Gatto L, Hester SS,
Goulding D, Barquist L, Lilley KS, Kingsley RA, Dougan G, Salmond GP(1).

Author information: 
(1)Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1QW, UK. gpcs2@cam.ac.uk.

BACKGROUND: Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium
that is virulent in plant and animal models. It produces a red-pigmented
trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic
(Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary
metabolite production in this strain is controlled by a complex regulatory
network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and
major post-transcriptional regulators of gene expression) play opposing roles in 
the regulation of several key phenotypes within S39006. Prodigiosin and
carbapenem production was abolished, and virulence attenuated, in an S39006 ∆hfq 
mutant, while the converse was observed in an S39006 rsmA transposon insertion
mutant.
RESULTS: In order to define the complete regulon of Hfq and RsmA, deep sequencing
of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006
∆hfq and rsmA::Tn mutants. Moreover, we investigated global changes in the
proteome using an LC-MS/MS approach. Analysis of differential gene expression
showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4%
and 19% of the genome, respectively, with some correlation between RNA and
protein expression. Pathways affected include those involved in antibiotic
regulation, virulence, flagella synthesis, and surfactant production. Although
Hfq and RsmA are reported to activate flagellum production in E. coli and an
adherent-invasive E. coli hfq mutant was shown to have no flagella by electron
microscopy, we found that flagellar production was increased in the S39006 rsmA
and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux 
with increased activity of two mobile genetic elements. This was confirmed by
qPCR and analysis of rsmA culture supernatant revealed the presence of prophage
DNA and phage particles. Finally, expression of a hypothetical protein containing
DUF364 increased prodigiosin production and was controlled by a putative 5'
cis-acting regulatory RNA element.
CONCLUSION: Using a combination of transcriptomics and proteomics this study
provides a systems-level understanding of Hfq and RsmA regulation and identifies 
similarities and differences in the regulons of two major regulators.
Additionally our study indicates that RsmA regulates both core and variable
genome regions and contributes to genome stability.

DOI: 10.1186/1471-2164-14-822 
PMCID: PMC4046660
PMID: 24267595  [Indexed for MEDLINE]


253. J Biol Chem. 2013 Nov 15;288(46):32952-62. doi: 10.1074/jbc.M113.507772. Epub
2013 Oct 4.

Search for proteins required for accurate gene expression under oxidative stress:
roles of guanylate kinase and RNA polymerase.

Inokuchi H(1), Ito R, Sekiguchi T, Sekiguchi M.

Author information: 
(1)From the Frontier Research Center and Department of Biochemistry, Fukuoka
Dental College, Fukuoka 814-0193 and.

In aerobically growing cells, in which reactive oxygen species are produced, the 
guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine
as well as cytosine. This mispairing causes alterations in gene expression, and
cells possess mechanisms to prevent such outcomes. In Escherichia coli,
8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced
by mutations in genes related to the prevention of abnormal protein synthesis
under oxidative stress. A genome-wide search for the genes responsible, followed 
by DNA sequence determination, revealed that specific amino acid changes in
guanylate kinase and in the β and β' subunits of RNA polymerase cause elevated
levels of phenotypic suppression, specifically under aerobic conditions. The
involvement of the DnaB, DnaN, and MsbA proteins, which are involved in DNA
replication and in preserving the membrane structure, was also noted.
Interactions of these proteins with each other and also with other molecules may 
be important for preventing errors in gene expression.

DOI: 10.1074/jbc.M113.507772 
PMCID: PMC3829146
PMID: 24097971  [Indexed for MEDLINE]


254. J Microbiol Methods. 2013 Nov;95(2):162-6. doi: 10.1016/j.mimet.2013.08.005. Epub
2013 Aug 24.

Identification of Salmonella Typhimurium-specific DNA aptamers developed using
whole-cell SELEX and FACS analysis.

Moon J(1), Kim G, Lee S, Park S.

Author information: 
(1)Department of Agricultural Engineering, National Academy of Agricultural
Sciences, Suwon 441-707, South Korea.

Conventional methods for detection of infective organisms, such as Salmonella,
are complicated and require multiple steps, and the need for rapid detection has 
increased. Biosensors show great potential for rapid detection of pathogens. In
turn, aptamers have great potential for biosensor assay development, given their 
small size, ease of synthesis and labeling, lack of immunogenicity, a lower cost 
of production than antibodies, and high target specificity. In this study, ssDNA 
aptamers specific to Salmonella Typhimurium were obtained by a whole
bacterium-based systematic evolution of ligands by exponential enrichment (SELEX)
procedure and applied to probing S. Typhimurium. After 10 rounds of selection
with S. Typhimurium as the target and Salmonella Enteritidis, Escherichia coli
and Staphylococcus aureus as counter targets, the highly enriched oligonucleic
acid pool was sorted using flow cytometry. In total, 12 aptamer candidates from
different families were sequenced and grouped. Fluorescent analysis demonstrated 
that aptamer C4 had particularly high binding affinity and selectivity; this
aptamer was then further characterized.

© 2013 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.mimet.2013.08.005 
PMID: 23978634  [Indexed for MEDLINE]


255. Planta. 2013 Nov;238(5):983-9. doi: 10.1007/s00425-013-1937-6. Epub 2013 Aug 6.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular
trichomes.

Sarker LS(1), Demissie ZA, Mahmoud SS.

Author information: 
(1)University of British Columbia, Kelowna, BC, Canada.

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also
contain small amounts of sesquiterpenes, depending on species and environmental
conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 %
of the EO in a few species, including those commercially propagated for EO
production. Here, we report the cloning and functional characterization of
9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x 
intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not
encode a transit peptide, was expressed in Escherichia coli and the recombinant
protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa
recombinant protein specifically converted farnesyl diphosphate to
9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the
monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene
synthases--was not able to derive detectable amounts of any products from the cis
isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing
L. x intermedia flowers and were highly enriched in glandular trichomes, but were
not detected in leaves suggesting that the transcriptional expression of this
gene is spatially and developmentally regulated.

DOI: 10.1007/s00425-013-1937-6 
PMID: 23918183  [Indexed for MEDLINE]


256. J Mol Biol. 2013 Oct 9;425(19):3662-77. doi: 10.1016/j.jmb.2012.12.010. Epub 2012
Dec 28.

Dual posttranscriptional regulation via a cofactor-responsive mRNA leader.

Patterson-Fortin LM(1), Vakulskas CA, Yakhnin H, Babitzke P, Romeo T.

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida,
Gainesville, FL 32611-0700, USA.

Riboswitches are cis-acting mRNA elements that regulate gene expression in
response to ligand binding. Recently, a class of riboswitches was proposed to
respond to the molybdenum cofactor (Moco), which serves as a redox center for
metabolic enzymes. The 5' leader of the Escherichia coli moaABCDE transcript
exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco
biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq
analyses showed that moaA mRNA copurified with the RNA binding protein CsrA
(carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among
its global regulatory roles, CsrA represses stationary phase metabolism and
activates central carbon metabolism. Here, we used gel mobility shift analysis to
determine that CsrA binds specifically and with high affinity to the moaA 5' mRNA
leader. Northern blotting and studies with a series of chromosomal lacZ reporter 
fusions showed that CsrA posttranscriptionally activates moaA expression without 
altering moaA mRNA levels, indicative of translation control. Deletion analyses, 
nucleotide replacement studies and footprinting with CsrA-FeBABE identified two
sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes
changes in moaA RNA structure. We propose that the moaA mRNA leader forms an
aptamer, which serves as a target of posttranscriptional regulation by at least
two different factors, Moco and the protein CsrA. While we are not aware of
similar dual posttranscriptional regulatory mechanisms, additional examples are
likely to emerge.

Copyright © 2012 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.jmb.2012.12.010 
PMCID: PMC3710303
PMID: 23274138  [Indexed for MEDLINE]


257. Acta Med Indones. 2013 Oct;45(4):275-83.

Adenomatous Polyposis Coli, mismatch repair, and microsatellite instability in
colorectal cancer based on different locations.

Effendi-Y S R(1), Zain LH, Siregar GA, Lubis HR, Damanik HA, Laksmi LI,
Chrestella J.

Author information: 
(1)Department of Internal Medicine, Faculty of Medicine, University of Sumatera
Utara, Adam Malik Hospital, Pirngadi Hospital, Medan, Indonesia.

AIM: to examine the protein expression negative (PEN) of Adenomatous Polyposis
Coli (APC), Mismatch Repair (MMR), and Microsatellite Instability (MSI) status of
colorectal cancer (CRC), and establish a comparison of those molecular
characteristics in CRC location among Indonesian patients in Adam Malik Hospital,
Pirngadi Hospital, and other hospitals within the network of Faculty of Medicine 
University of Sumatera Utara Medan Indonesia.
METHODS: this prospective study was conducted from April to December 2012. Fresh 
tissues were obtained from colorectal tumor patients. The APC-PEN, MMR (MLH1,
MSH2, PMS2, MSH6)-PEN, were assessed by immunohistochemistry, and MSI by PCR
using 5 microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250), as
independent variables. The tumour locations as dependent variables were divided
into proximal colon (caecum, ascending colon, transverse colon); distal colon
(splenic flexure, descending colon, sigmoid) and rectum. The comparative study
were done by bivariate and multivariate analysis.
RESULTS: there were 77 cases of colorectal adenocarsinoma. MMR-PEN was found in
54 of 77 (70.1%). MLH1-PEN was different between distal colon and rectal cancer
(p=0.008); MSH6-PEN was different between proximal colon and rectal cancer (p=
0.020). Multivariate analysis showed: MLH1-PEN was related to cancer location
(p=0.006) with OR 0.12 (95% CI 0.026-0.547). It had 0.12 times probability to be 
found in distal than rectum. MLH1-PEN had 10 times higher probability to be found
in proximal than in distal (p=0.037). MSH6-PEN was related to the location
(p=0.026) with OR 0.165 (95% CI 0.034-0.803), and had 0.165 times probability to 
be found in proximal than rectum; and 11 times higher probability in distal than 
proximal colon (p=0.043). APC-PEN was related to the location (p=0.020), with OR 
6.897 (95% CI 1.359-34.995), and 6.89 times higher probability in distal than in 
rectum, with other variables controlled. MSI-H was found in 29 of 77 (37.7%) and 
MSI-L/MSS in 48 (62.3%). The proportion of MSI-H displayed a tendency to occur in
proximal rather than in distal colon or rectal cancer.
CONCLUSION: the underlying carcinogenic pathway or molecular background differs
according to the cancer locations of CRC patients in this region. MLH1-PEN was
prominent in proximal colon cancer, MSH6-PEN in distal colon and rectal cancer,
and APC-PEN in distal colon respectively.


PMID: 24448331  [Indexed for MEDLINE]


258. Biomed Microdevices. 2013 Oct;15(5):821-30. doi: 10.1007/s10544-013-9769-5.

On-chip parallel detection of foodborne pathogens using loop-mediated isothermal 
amplification.

Duarte C(1), Salm E, Dorvel B, Reddy B Jr, Bashir R.

Author information: 
(1)Department of Electrical and Computer Engineering, William L. Everitt
Laboratory, University of Illinois at Urbana-Champaign, 1406 W. Green St.,
Urbana, IL 61801, USA.

According to estimates issued by the Center for Disease Control and Prevention,
one out of six Americans will get sick during this year due to consumption of
contaminated products and there will be 50,000 related hospitalizations. To
control and treat the responsible foodborne diseases, rapid and accurate
detection of pathogens is extremely important. A portable device capable of
performing nucleic acid amplification will enable the effective detection of
infectious agents in multiple settings, leading to better enforcement of food
safety regulations. This work demonstrates the multiplexed detection of food
pathogens through loop-mediated isothermal amplification on a silicon chip.
Silane passivation is used to prevent the adsorption of the polymerase on silicon
oxide, which can severely inhibit nucleic acid amplification. We demonstrate the 
multiplexed screening of virulence genes of Listeria monocytogenes, Escherichia
coli, and Salmonella by dehydrating the corresponding primers in oxidized silicon
wells. Droplets of 30 nL with reagents for nucleic acid amplification and lysate 
of suspected pathogens are arrayed on micro-machined wells with an automated
microinjection system. We show that dehydrated primers re-suspend when other
reagents are microinjected, and the resulting mix can be used to specifically
amplify the targeted gene. Results of characterization experiments demonstrate
sensitivity down to a few templates per reaction, specificity that enables
multiplexed screening, and robustness that allows amplification without DNA
extraction.

DOI: 10.1007/s10544-013-9769-5 
PMID: 23620454  [Indexed for MEDLINE]


259. Biotechnol Bioeng. 2013 Oct;110(10):2573-80. doi: 10.1002/bit.24922. Epub 2013
Apr 22.

In silico maturation of binding-specificity of DNA aptamers against Proteus
mirabilis.

Savory N(1), Lednor D, Tsukakoshi K, Abe K, Yoshida W, Ferri S, Jones BV,
Ikebukuro K.

Author information: 
(1)Department of Biotechnology and Life Science, Tokyo University of Agriculture 
and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

Proteus mirabilis is a prominent cause of catheter-associated urinary tract
infections (CAUTIs) among patients undergoing long-term bladder catheterization. 
There are currently no effective means of preventing P. mirabilis infections, and
strategies for prophylaxis and rapid early diagnosis are urgently required.
Aptamers offer significant potential for development of countermeasures against
P. mirabilis CAUTI and are an ideal class of molecules for the development of
diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX
to identify DNA aptamers that show high affinity for P. mirabilis. While the
aptamers identified displayed high affinity for P. mirabilis cells in dot
blotting assays, they also bound to other uropathogenic bacteria. To improve
aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was 
employed. Two cycles of ISM allowed the identification of an aptamer showing 36% 
higher specificity, evaluated as a ratio of binding signal for P. mirabilis to
that for Escherichia coli (also a cause of CAUTI and the most common urinary
tract pathogen). Aptamers that specifically recognize P. mirabilis would have
diagnostic and therapeutic values and constitute useful tools for studying
membrane-associated proteins in this organism.

Copyright © 2013 Wiley Periodicals, Inc.

DOI: 10.1002/bit.24922 
PMID: 23568752  [Indexed for MEDLINE]


260. IEEE Trans Biomed Circuits Syst. 2013 Oct;7(5):643-54. doi:
10.1109/TBCAS.2012.2230172.

CMOS spectrally-multiplexed FRET-on-a-chip for DNA analysis.

Ho D, Noor MO, Krull UJ, Gulak G, Genov R.

A spectral-multiplexed fluorescence contact imaging microsystem for DNA analysis 
is presented. The microsystem integrates a filterless CMOS Color PhotoGate (CPG) 
sensor that exploits the polysilicon gate as an optical filter, and therefore
does not require an external color filter. The CPG is applied to
fluorescence-based transduction in a spectrally multiplexed format by
differentiating among multiple emission bands, hence replacing the functionality 
of a bank of emission filters. A microsystem has been quantitatively modeled and 
prototyped based on the CPG fabricated in a standard 0.35 μm CMOS technology. The
multi-color imaging capability of the microsystem in analyzing DNA targets has
been validated in the detection of marker gene sequences for spinal muscular
atropy disease and Escherichia coli (E. coli). Spectral-multiplexing enables the 
two DNA targets to be simultaneously detected with a measured detection limits of
240 nM and 210 nM for the two target concentrations at a sample volume of 10 μL
for the green and red transduction channels, respectively.

DOI: 10.1109/TBCAS.2012.2230172 
PMID: 24232625  [Indexed for MEDLINE]


261. J Bacteriol. 2013 Oct;195(19):4496-505. doi: 10.1128/JB.00304-13. Epub 2013 Aug
2.

Identification of the set of genes, including nonannotated morA, under the direct
control of ModE in Escherichia coli.

Kurata T(1), Katayama A, Hiramatsu M, Kiguchi Y, Takeuchi M, Watanabe T,
Ogasawara H, Ishihama A, Yamamoto K.

Author information: 
(1)Department of Frontier Bioscience.

ModE is the molybdate-sensing transcription regulator that controls the
expression of genes related to molybdate homeostasis in Escherichia coli. ModE is
activated by binding molybdate and acts as both an activator and a repressor. By 
genomic systematic evolution of ligands by exponential enrichment (SELEX)
screening and promoter reporter assays, we have identified a total of nine
operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of
which six were activated by ModE and three were repressed. In addition, two
promoters were newly identified and direct transcription of novel genes, referred
to as morA and morB, located on antisense strands of yghW and torY, respectively.
The morA gene encodes a short peptide, MorA, with an unusual initiation codon.
Surprisingly, overexpression of the morA 5' untranslated region exhibited an
inhibitory influence on colony formation of E. coli K-12.

DOI: 10.1128/JB.00304-13 
PMCID: PMC3807462
PMID: 23913318  [Indexed for MEDLINE]


262. Nucleic Acids Res. 2013 Oct;41(19):9090-104. doi: 10.1093/nar/gkt698. Epub 2013
Aug 7.

Direct assessment of transcription fidelity by high-resolution RNA sequencing.

Imashimizu M(1), Oshima T, Lubkowska L, Kashlev M.

Author information: 
(1)Gene Regulation and Chromosome Biology Laboratory, Frederick National
Laboratory for Cancer Research, National Cancer Institute, National Institutes of
Health, Frederick, MD 21702, USA and Graduate School of Biological Sciences, Nara
Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192,
Japan.

Cancerous and aging cells have long been thought to be impacted by transcription 
errors that cause genetic and epigenetic changes. Until now, a lack of
methodology for directly assessing such errors hindered evaluation of their
impact to the cells. We report a high-resolution Illumina RNA-seq method that can
assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies 
in vitro and in vivo. Statistically reliable detection of changes in
transcription fidelity through ∼10(3) nt DNA sites assures that the RNA-seq can
analyze the fidelity in a large number of the sites where errors occur. A
combination of the RNA-seq and biochemical analyses of the positions for the
errors revealed two sequence-specific mechanisms that increase transcription
fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of
nucleotide misincorporation that improves selectivity for the cognate substrate, 
and (ii) increased backtracking of the RNA polymerase that decreases a chance of 
error propagation to the full-length transcript after misincorporation and
provides an opportunity to proofread the error. This method is adoptable to a
genome-wide assessment of transcription fidelity.

DOI: 10.1093/nar/gkt698 
PMCID: PMC3799451
PMID: 23925128  [Indexed for MEDLINE]


263. PLoS One. 2013 Sep 27;8(9):e73299. doi: 10.1371/journal.pone.0073299. eCollection
2013.

Genome-wide analysis of selective constraints on high stability regions of mRNA
reveals multiple compensatory mutations in Escherichia coli.

Mao Y(1), Li Q, Zhang Y, Zhang J, Wei G, Tao S.

Author information: 
(1)College of Life Sciences and State Key Laboratory of Crop Stress Biology in
Arid Areas, Northwest A&F University, Yangling, Shaanxi, China ; Bioinformatics
Center, Northwest A&F University, Yangling, Shaanxi, China.

Message RNA (mRNA) carries a large number of local secondary structures, with
structural stability to participate in the regulations of gene expression. A
worthy question is how the local structural stability is maintained under the
constraint that multiple selective pressures are imposed on mRNA local regions.
Here, we performed the first genome-wide study of natural selection operating on 
high structural stability regions (HSRs) of mRNAs in Escherichia coli. We found
that HSR tends to adjust the folded conformation to reduce the harm of mutations,
showing a high level of mutational robustness. Moreover, guanine preference in
HSR was observed, supporting the hypothesis that the selective constraint for
high structural stability may partly account for the high percentage of G content
in Escherichia coli genome. Notably, we found a substantially reduced synonymous 
substitution rate in HSRs compared with that in their adjacent regions.
Surprisingly and interestingly, the non-key sites in HSRs, which have slight
effect on structural stability, have synonymous substitution rate equivalent to
background regions. To explain this result, we identified compensatory mutations 
in HSRs based on structural stability, and found that a considerable number of
synonymous mutations occur to restore the structural stability decreased heavily 
by the mutations on key sites. Overall, these results suggest a significant role 
of local structural stability as a selective force operating on mRNA, which
furthers our understanding of the constraints imposed on protein-coding RNAs.

DOI: 10.1371/journal.pone.0073299 
PMCID: PMC3785496
PMID: 24086278  [Indexed for MEDLINE]


264. Methods. 2013 Sep 15;63(2):144-59. doi: 10.1016/j.ymeth.2013.04.023. Epub 2013
May 23.

Identifying and characterizing Hfq-RNA interactions.

Faner MA(1), Feig AL.

Author information: 
(1)Department of Chemistry, Wayne State University, 5101 Cass Ave., Detroit, MI, 
United States.

To regulate stress responses and virulence, bacteria use small regulatory RNAs
(sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by 
influencing ribosomal access and RNA decay. A large class of these sRNAs, called 
trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base
pairing between the regulatory RNA and its target mRNA. The resulting network of 
regulation is best characterized in Escherichia coli and Salmonella typhimurium, 
but the importance of Hfq dependent sRNA regulation is recognized in a diverse
population of bacteria. In this review we present the approaches and methods used
to discover Hfq binding RNAs, characterize their interactions and elucidate their
functions.

Copyright © 2013 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ymeth.2013.04.023 
PMCID: PMC3787079
PMID: 23707622  [Indexed for MEDLINE]


265. Water Res. 2013 Sep 15;47(14):4978-85. doi: 10.1016/j.watres.2013.05.041. Epub
2013 May 31.

A dynamic and complex monochloramine stress response in Escherichia coli revealed
by transcriptome analysis.

Holder D(1), Berry D, Dai D, Raskin L, Xi C.

Author information: 
(1)Department of Environmental Health Sciences, University of Michigan, Ann
Arbor, MI, USA.

Despite the widespread use of monochloramine in drinking water treatment, there
is surprisingly little information about its mode of action. In this study, DNA
microarrays were used to investigate the global gene expression of Escherichia
coli cells exposed to sub-lethal concentrations of monochloramine, with a focus
on temporal dynamics. Genes induced by monochloramine were associated with
several stress response functions, including oxidative stress, DNA repair,
multidrug efflux, biofilm formation, antibiotic resistance, and cell wall repair.
The diversity of functional associations supports a model of monochloramine
action involving multiple cellular targets including cell membranes, nucleic
acids, and proteins. These data suggest that E. coli responds to monochloramine
exposure by activating diverse defense responses rather than a single antioxidant
system and the exposure may also induce biofilm formation. The induction of
multidrug efflux pumps and specific antibiotic resistance genes further suggests 
that exposure to monochloramine may contribute to reduced susceptibility to some 
antibiotics.

Copyright © 2013 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.watres.2013.05.041 
PMID: 23866139  [Indexed for MEDLINE]


266. PLoS One. 2013 Sep 11;8(9):e73995. doi: 10.1371/journal.pone.0073995. eCollection
2013.

Genes required for growth at high hydrostatic pressure in Escherichia coli K-12
identified by genome-wide screening.

Black SL(1), Dawson A, Ward FB, Allen RJ.

Author information: 
(1)SUPA School of Physics and Astronomy, University of Edinburgh, Edinburgh,
Midlothian, United Kingdom ; Institute of Cell Biology, School of Biological
Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom.

Despite the fact that much of the global microbial biosphere is believed to exist
in high pressure environments, the effects of hydrostatic pressure on microbial
physiology remain poorly understood. We use a genome-wide screening approach,
combined with a novel high-throughput high-pressure cell culture method, to
investigate the effects of hydrostatic pressure on microbial physiology in vivo. 
The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was 
screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to
the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose
products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and
iscS, whose products were required for growth at 40 MPa. Our results support the 
view that the effects of pressure on cell physiology are pleiotropic, with DNA
replication, cell division, the cytoskeleton and cell envelope physiology all
being potential failure points for cell physiology during growth at elevated
pressure.

DOI: 10.1371/journal.pone.0073995 
PMCID: PMC3770679
PMID: 24040140  [Indexed for MEDLINE]


267. J Biotechnol. 2013 Sep 10;167(3):326-33. doi: 10.1016/j.jbiotec.2013.07.011. Epub
2013 Jul 19.

Enhanced expression of codon optimized interferon gamma in CHO cells.

Chung BK(1), Yusufi FN, Mariati, Yang Y, Lee DY.

Author information: 
(1)Bioprocessing Technology Institute, Agency for Science, Technology and
Research-A*STAR, 20 Biopolis Way #06-01, Singapore 138668, Singapore.

The human interferon-gamma (IFN-γ) is a potential drug candidate for treating
various diseases due to its immunomodulatory properties. The efficient production
of this protein can be achieved through a popular industrial host, Chinese
hamster ovary (CHO) cells. However, recombinant expression of foreign proteins is
typically suboptimal possibly due to the usage of non-native codon patterns
within the coding sequence. Therefore, we demonstrated the application of a
recently developed codon optimization approach to design synthetic IFN-γ coding
sequences for enhanced heterologous expression in CHO cells. For codon
optimization, earlier studies suggested to establish the target usage
distribution pattern in terms of selected design parameters such as individual
codon usage (ICU) and codon context (CC), mainly based on the host's highly
expressed genes. However, our RNA-Seq based transcriptome profiling indicated
that the ICU and CC distribution patterns of different gene expression classes in
CHO cell are relatively similar, unlike other microbial expression hosts,
Escherichia coli and Saccharomyces cerevisiae. This finding was further
corroborated through the in vivo expression of various ICU and CC optimized IFN-γ
in CHO cells. Interestingly, the CC-optimized genes exhibited at least 13-fold
increase in expression level compared to the wild-type IFN-γ while a maximum of
10-fold increase was observed for the ICU-optimized genes. Although design
criteria based on individual codons, such as ICU, have been widely used for gene 
optimization, our experimental results suggested that codon context is relatively
more effective parameter for improving recombinant IFN-γ expression in CHO cells.

Copyright © 2013 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jbiotec.2013.07.011 
PMID: 23876479  [Indexed for MEDLINE]


268. PLoS One. 2013 Sep 10;8(9):e74647. doi: 10.1371/journal.pone.0074647. eCollection
2013.

Bile salts affect expression of Escherichia coli O157:H7 genes for virulence and 
iron acquisition, and promote growth under iron limiting conditions.

Hamner S(1), McInnerney K, Williamson K, Franklin MJ, Ford TE.

Author information: 
(1)Department of Microbiology, Montana State University, Bozeman, Montana, United
States of America.

Bile salts exhibit potent antibacterial properties, acting as detergents to
disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting
the intestinal tract are able to resist bile's antimicrobial effects, relatively 
little is known about how bile influences virulence of enteric pathogens.
Escherichia coli O157:H7 is an important pathogen of humans, capable of causing
severe diarrhea and more serious sequelae. In this study, the transcriptome
response of E. coli O157:H7 to bile was determined. Bile exposure induced
significant changes in mRNA levels of genes related to virulence potential,
including a reduction of mRNA for the 41 genes making up the locus of enterocyte 
effacement (LEE) pathogenicity island. Bile treatment had an unusual effect on
mRNA levels for the entire flagella-chemotaxis regulon, resulting in two- to
four-fold increases in mRNA levels for genes associated with the flagella
hook-basal body structure, but a two-fold decrease for "late" flagella genes
associated with the flagella filament, stator motor, and chemotaxis. Bile salts
also caused increased mRNA levels for seventeen genes associated with iron
scavenging and metabolism, and counteracted the inhibitory effect of the iron
chelating agent 2,2'-dipyridyl on growth of E. coli O157:H7. These findings
suggest that E. coli O157:H7 may use bile as an environmental signal to adapt to 
changing conditions associated with the small intestine, including adaptation to 
an iron-scarce environment.

DOI: 10.1371/journal.pone.0074647 
PMCID: PMC3769235
PMID: 24058617  [Indexed for MEDLINE]


269. Methods. 2013 Sep 1;63(1):60-5. doi: 10.1016/j.ymeth.2013.06.003. Epub 2013 Jun
25.

Detection of non-coding RNA in bacteria and archaea using the DETR'PROK Galaxy
pipeline.

Toffano-Nioche C(1), Luo Y, Kuchly C, Wallon C, Steinbach D, Zytnicki M, Jacq A, 
Gautheret D.

Author information: 
(1)Université Paris-Sud, Institut de Génétique et Microbiologie, CNRS UMR 8621,
Orsay F-91405, France.

RNA-seq experiments are now routinely used for the large scale sequencing of
transcripts. In bacteria or archaea, such deep sequencing experiments typically
produce 10-50 million fragments that cover most of the genome, including
intergenic regions. In this context, the precise delineation of the non-coding
elements is challenging. Non-coding elements include untranslated regions (UTRs) 
of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the
antisense strand of genes (asRNA). Here we present a computational pipeline
(DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework
that takes as input a mapping of deep sequencing reads and performs successive
steps of clustering, comparison with existing annotation and identification of
transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and
asRNAs. We provide a step-by-step description of the protocol using real-life
example data sets from Vibrio splendidus and Escherichia coli.

Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ymeth.2013.06.003 
PMID: 23806640  [Indexed for MEDLINE]


270. Phytother Res. 2013 Sep;27(9):1398-406. doi: 10.1002/ptr.4859. Epub 2012 Dec 17.

The multi-targeted effects of Chrysanthemum herb extract against Escherichia coli
O157:H7.

Kim KS(1), Lim DJ, Yang HJ, Choi EK, Shin MH, Ahn KS, Jung SH, Um JY, Jung HJ,
Lee JH, Lee SG, Jung SK, Jang HJ.

Author information: 
(1)College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee
University, Heogi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea.

The Chrysanthemum lavandulifolium extract, which includes chrysoeriol,
sudachitin, and acacetin, has excellent antibiotic effects on Escherichia coli
O157:H7 (E. coli O157). A notable point is that the antibiotic targets of the
herb extract are similar to the targets of commonly used antibiotic drugs,
including bacterial cell wall biosynthesis, bacterial protein synthesis, and
bacterial DNA replication and repair. In addition, the herbal antibiotic inhibits
the etiological factors that contribute to the pathogenic property. The herbal
sample was extracted and fractionated and then inoculated through a disk
diffusion method to confirm its antibiotic effect against E. coli O157. Total RNA
was isolated from the affected bacterial cells, and its expression level was
analyzed through a microarray analysis. To confirm the accuracy of the microarray
data, a real-time PCR was performed. Three active compounds, chrysoeriol,
sudachitin, and acacetin, were identified with a high-performance liquid
chromatography-electrospray ionization/mass spectrometry chromatogram, and the
disk diffusion study confirmed that chrysoeriol and sudachitin contribute to the 
antibiotic properties of the herb extract. The results demonstrate that the
multi-target efficacy of the herbal sample may indicate the potential for the
development of more effective and safer drugs that will act as substitutes for
existing antibiotics.

Copyright © 2012 John Wiley & Sons, Ltd.

DOI: 10.1002/ptr.4859 
PMID: 23255247  [Indexed for MEDLINE]


271. Planta. 2013 Sep;238(3):487-97. doi: 10.1007/s00425-013-1905-1. Epub 2013 Jun 14.

Regulation of phenylalanine ammonia-lyase (PAL) gene family in wood forming
tissue of Populus trichocarpa.

Shi R(1), Shuford CM, Wang JP, Sun YH, Yang Z, Chen HC, Tunlaya-Anukit S, Li Q,
Liu J, Muddiman DC, Sederoff RR, Chiang VL.

Author information: 
(1)Forest Biotechnology Group, Department of Forestry and Environmental
Resources, North Carolina State University, Raleigh, NC 27695, USA. rshi@ncsu.edu

Phenylalanine ammonia-lyase (PAL) catalyzes the initial step of phenylpropanoid
biosynthesis in plants. Five PAL genes (PtrPAL1 to 5) have been identified in
Populus trichocarpa. These genes are classified into two subgroups according to
their transcript sequence similarity and tissue specificity. However, the
regulation of these genes and their protein functions are not well understood. In
this study, enzymatic properties of each PtrPALs were characterized based on
their recombinant proteins expressed in E.coli. Subcellular localizations of each
PtrPALs in stem wood forming tissue were investigated and individual PtrPAL
protein abundances in cytosol and membrane protein fractions were measured using 
protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Protein/mRNA
ratios of PtrPALs were further verified using RNA-Seq and gel-enhanced liquid
chromatography mass spectrometry (GeLC-MS). All PtrPALs have similar catalytic
properties for the deamination of L-phenylalanine, their major substrate. All
PtrPALs have similar subcellular locations in stem wood forming tissue, with
major amount in the cytosol (93-96 %) and less in the membrane (4-7 %). However, 
the protein/mRNA ratios of subgroup A (PtrPAL2, 4 and 5) are about five times
that of subgroup B (PtrPAL1 and 3) in stem wood forming tissue, while all PtrPALs
have similar transcript abundances. These results indicate a greater functional
significance of subgroup A PtrPALs for stem wood formation, and highlight the
role of gene post-transcriptional regulation.

DOI: 10.1007/s00425-013-1905-1 
PMID: 23765265  [Indexed for MEDLINE]


272. Mar Drugs. 2013 Aug 13;11(8):2894-916. doi: 10.3390/md11082894.

Development of Synechocystis sp. PCC 6803 as a phototrophic cell factory.

Yu Y(1), You L, Liu D, Hollinshead W, Tang YJ, Zhang F.

Author information: 
(1)Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of
Education), School of Pharmaceutical Sciences, Wuhan University, 185 East Lake
Road, Wuhan 430071, China. yuyi@seas.wustl.edu

Cyanobacteria (blue-green algae) play profound roles in ecology and
biogeochemistry. One model cyanobacterial species is the unicellular
cyanobacterium Synechocystis sp. PCC 6803. This species is highly amenable to
genetic modification. Its genome has been sequenced and many systems biology and 
molecular biology tools are available to study this bacterium. Recently,
researchers have put significant efforts into understanding and engineering this 
bacterium to produce chemicals and biofuels from sunlight and CO2. To demonstrate
our perspective on the application of this cyanobacterium as a
photosynthesis-based chassis, we summarize the recent research on Synechocystis
6803 by focusing on five topics: rate-limiting factors for cell cultivation;
molecular tools for genetic modifications; high-throughput system biology for
genome wide analysis; metabolic modeling for physiological prediction and
rational metabolic engineering; and applications in producing diverse chemicals. 
We also discuss the particular challenges for systems analysis and engineering
applications of this microorganism, including precise characterization of
versatile cell metabolism, improvement of product rates and titers, bioprocess
scale-up, and product recovery. Although much progress has been achieved in the
development of Synechocystis 6803 as a phototrophic cell factory, the
biotechnology for "Compounds from Synechocystis" is still significantly lagging
behind those for heterotrophic microbes (e.g., Escherichia coli).

DOI: 10.3390/md11082894 
PMCID: PMC3766872
PMID: 23945601  [Indexed for MEDLINE]


273. Appl Environ Microbiol. 2013 Aug;79(16):4940-50. doi: 10.1128/AEM.00958-13. Epub 
2013 Jun 14.

Whole-genome transcriptional analysis of Escherichia coli during heat
inactivation processes related to industrial cooking.

Guernec A(1), Robichaud-Rincon P, Saucier L.

Author information: 
(1)Department of Animal Science, Faculty of Agriculture and Food Sciences,
Université Laval, Quebec City, Québec, Canada.

Escherichia coli K-12 was grown to the stationary phase, for maximum
physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were
then heated at 58°C or 60°C to reach a process lethality value
\[\mathbf{\left(}{{\mathit{F}}^{\mathit{o}}}_{\mathbf{70}}^{\mathbf{10}}\mathbf{\
right)} \] of 2 or 3 or to a core temperature of 71°C (control industrial cooking
temperature). Growth recovery and cell membrane integrity were evaluated
immediately after heating, and a global transcription analysis was performed
using gene expression microarrays. Only cells heated at 58°C with F(o) = 2 were
still able to grow on liquid or solid BHI broth after heat treatment. However,
their transcriptome did not differ from that of bacteria heated at 58°C with F(o)
= 3 (P value for the false discovery rate [P-FDR] > 0.01), where no growth
recovery was observed posttreatment. Genome-wide transcriptomic data obtained at 
71°C were distinct from those of the other treatments without growth recovery.
Quantification of heat shock gene expression by real-time PCR revealed that dnaK 
and groEL mRNA levels decreased significantly above 60°C to reach levels similar 
to those of control cells at 37°C (P < 0.0001). Furthermore, despite similar
levels of cell inactivation measured by growth on BHI media after heating, 132
and 8 genes were differentially expressed at 71°C compared to 58°C and 60°C at
F(o) = 3, respectively (P-FDR < 0.01). Among them, genes such as aroA, citE,
glyS, oppB, and asd, whose expression was upregulated at 71°C, may be worth
investigating as good biomarkers for accurately determining the efficiency of
heat treatments, especially when cells are too injured to be enumerated using
growth media.

DOI: 10.1128/AEM.00958-13 
PMCID: PMC3754711
PMID: 23770902  [Indexed for MEDLINE]


274. Nucleic Acids Res. 2013 Aug;41(14):e140. doi: 10.1093/nar/gkt444. Epub 2013 May
28.

Computational analysis of bacterial RNA-Seq data.

McClure R(1), Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA,
Vanderpool CK, Tjaden B.

Author information: 
(1)Department of Microbiology, Boston University School of Medicine, Boston, MA
02118, USA.

Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled
tremendous leaps forward in our understanding of bacterial transcriptomes.
However, computational methods for analysis of bacterial transcriptome data have 
not kept pace with the large and growing data sets generated by RNA-seq
technology. Here, we present new algorithms, specific to bacterial gene
structures and transcriptomes, for analysis of RNA-seq data. The algorithms are
implemented in an open source software system called Rockhopper that supports
various stages of bacterial RNA-seq data analysis, including aligning sequencing 
reads to a genome, constructing transcriptome maps, quantifying transcript
abundance, testing for differential gene expression, determining operon
structures and visualizing results. We demonstrate the performance of Rockhopper 
using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with
Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus
pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps
generated by our algorithms are highly accurate when compared with focused
experimental data from E. coli and N. gonorrhoeae, and we validate our system's
ability to identify novel small RNAs, operons and transcription start sites. Our 
results suggest that Rockhopper can be used for efficient and accurate analysis
of bacterial RNA-seq data, and that it can aid with elucidation of bacterial
transcriptomes.

DOI: 10.1093/nar/gkt444 
PMCID: PMC3737546
PMID: 23716638  [Indexed for MEDLINE]


275. BMC Genomics. 2013 Jul 30;14:520. doi: 10.1186/1471-2164-14-520.

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in 
E. coli K12 through accurate full-length transcripts assembling.

Li S(1), Dong X, Su Z.

Author information: 
(1)Department of Bioinformatics and Genomics, College of Computing and
Informatics, The University of North Carolina at Charlotte, 9201 University City 
Blvd, Charlotte, NC 28223, USA.

BACKGROUND: Although prokaryotic gene transcription has been studied over
decades, many aspects of the process remain poorly understood. Particularly,
recent studies have revealed that transcriptomes in many prokaryotes are far more
complex than previously thought. Genes in an operon are often alternatively and
dynamically transcribed under different conditions, and a large portion of genes 
and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA)
transcripts, respectively. Ironically, similar studies have not been conducted in
the model bacterium E coli K12, thus it is unknown whether or not the bacterium
possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes
the major method for analyzing the complexity of prokaryotic transcriptome, it is
still a challenging task to accurately assemble full length transcripts using
short RNA-seq reads.
RESULTS: To fill these gaps, we have profiled the transcriptomes of E. coli K12
under different culture conditions and growth phases using a highly specific
directional RNA-seq technique that can capture various types of transcripts in
the bacterial cells, combined with a highly accurate and robust algorithm and
tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full
length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized
in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA
transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts 
under different culture conditions and growth phases.
CONCLUSIONS: As has been demonstrated in many other prokaryotes, E. coli K12 also
has a highly complex and dynamic transcriptomes under different culture
conditions and growth phases. Such complex and dynamic transcriptomes might play 
important roles in the physiology of the bacterium. TruHMM is a highly accurate
and robust algorithm for assembling full-length transcripts in prokaryotes using 
directional RNA-seq short reads.

DOI: 10.1186/1471-2164-14-520 
PMCID: PMC3734233
PMID: 23899370  [Indexed for MEDLINE]


276. PLoS One. 2013 Jul 25;8(7):e69507. doi: 10.1371/journal.pone.0069507. Print 2013.

Molecular characterization of multidrug resistant hospital isolates using the
antimicrobial resistance determinant microarray.

Leski TA(1), Vora GJ, Barrows BR, Pimentel G, House BL, Nicklasson M, Wasfy M,
Abdel-Maksoud M, Taitt CR.

Author information: 
(1)Center for Bio/Molecular Science and Engineering, US Naval Research
Laboratory, Washington, DC, United States of America.

Molecular methods that enable the detection of antimicrobial resistance
determinants are critical surveillance tools that are necessary to aid in curbing
the spread of antibiotic resistance. In this study, we describe the use of the
Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique
genes that confer resistance to 12 classes of antimicrobial compounds, quaternary
amines and streptothricin for the determination of multidrug resistance (MDR)
gene profiles. Fourteen reference MDR strains, which either were genome,
sequenced or possessed well characterized drug resistance profiles were used to
optimize detection algorithms and threshold criteria to ensure the microarray's
effectiveness for unbiased characterization of antimicrobial resistance
determinants in MDR strains. The subsequent testing of Acinetobacter baumannii,
Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the
presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA 
and CTX-M classes (and OXA and CTX-M subfamilies) of β-lactamases] and their
assemblages which were confirmed by PCR and DNA sequence analysis. When combined 
with results from the reference strains, ~25% of the ARDM content was confirmed
as effective for representing allelic content from both Gram-positive and
-negative species. Taken together, the ARDM identified MDR assemblages containing
six to 18 unique resistance genes in each strain tested, demonstrating its
utility as a powerful tool for molecular epidemiological investigations of
antimicrobial resistance in clinically relevant bacterial pathogens.

DOI: 10.1371/journal.pone.0069507 
PMCID: PMC3723915
PMID: 23936031  [Indexed for MEDLINE]


277. APMIS. 2013 Jul;121(7):634-42. doi: 10.1111/apm.12081. Epub 2013 Jun 12.

A DNA microarray for the versatile diagnosis of infectious diarrhea.

Donatin E(1), Buffet S, Leroy Q, Raoult D, Drancourt M.

Author information: 
(1)Aix Marseille Université, URMITE, UMR63 CNRS 7278, IRD 198, Inserm 1095,
Marseille, France.

Several bacteria, viruses, and parasites cause diarrhea as coinfecting pathogens.
We designed a DNA microarray comprising 60-bp probes spotted 194 times for the
multiplex detection of 33 enteropathogenic bacteria and seven enteropathogenic
viruses, and the archaeon Methanobrevibacter smithii was used as an internal
positive control. Nine pathogen-free stool specimens were used as negative
controls. One of these control specimens was further spiked with Salmonella
enterica as a positive control. The microarray was then tested with 40
pathological stool specimens, comprising S. enterica (n = 30), Campylobacter
jejuni (n = 4), pathogenic Escherichia coli (n = 2), and adenovirus (n = 4). M.
smithii was detected in 47/49 (95.9%) specimens, no pathogen was detected in
negative controls and S. enterica was identified in the S. enterica-spiked
positive control. The overall specificity was 100% and the overall sensitivity
was 97.5% because one S. enterica sample was missed by the microarray. The
multiplexed detection of C. jejuni spiked into an adenovirus-positive stool
sample gave positive results, with fluorescence values of 14.3 and 9.1,
respectively. These data indicate that using the protocol developed in this
article, the DNA array allows for the multiplexed detection of some
enteropathogens in stool samples.

© 2013 APMIS. Published by John Wiley & Sons Ltd.

DOI: 10.1111/apm.12081 
PMID: 23758523  [Indexed for MEDLINE]


278. FEMS Microbiol Lett. 2013 Jul;344(2):159-65. doi: 10.1111/1574-6968.12172. Epub
2013 Jun 3.

Involvement of the ribose operon repressor RbsR in regulation of purine
nucleotide synthesis in Escherichia coli.

Shimada T(1), Kori A, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes
for the transport and initial-step metabolism of d-ribose form a single rbsDACBK 
operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD 
and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In
the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type
transcription factor RbsR, which is encoded by a gene located downstream of this 
ribose operon. At present, the rbs operon is believed to be the only target of
regulation by RbsR. After Genomic SELEX screening, however, we have identified
that RbsR binds not only to the rbs promoter but also to the promoters of a set
of genes involved in purine nucleotide metabolism. Northern blotting analysis
indicated that RbsR represses the purHD operon for de novo synthesis of purine
nucleotide but activates the add and udk genes involved in the salvage pathway of
purine nucleotide synthesis. Taken together, we propose that RbsR is a global
regulator for switch control between the de novo synthesis of purine nucleotides 
and its salvage pathway.

© 2013 Federation of European Microbiological Societies. Published by John Wiley 
& Sons Ltd. All rights reserved.

DOI: 10.1111/1574-6968.12172 
PMID: 23651393  [Indexed for MEDLINE]


279. J Biomed Nanotechnol. 2013 Jul;9(7):1254-60.

Development of a magnetic nanoparticles microarray for simultaneous and simple
detection of foodborne pathogens.

Li S(1), Liu H, Deng Y, Lin L, He N.

Author information: 
(1)Hunan Key Laboratory of Green Packaging and Application of Biological
Nanotechnology, Hunan University of Technology, Zhuzhou 412007, PR China.
solisong@163.com

Foodborne diseases are a widespread and growing public health problem affecting
both developed and developing countries, microbiologically contaminated food and 
water are the major causes of diarrhoeal diseases. Methods based on polymerase
chain reaction (PCR) and microarrays are rapid and sensitive enough to detect
very small quantities of microorganisms, however, the requirement for expensive
equipments limits their application. In the present paper, we describe a method
based on multiplex PCR and magnetic nanoparticles labelling for simultaneous
detection of four major foodborne pathogens, including Escherichia coli O157:H7, 
Salmonella enterica, Vibrio cholera and Campylobacter jejuni. The process
utilizes an oligonucleotide array onto which 5' biotinylated single strand PCR
products were hybridized and visualized with streptavidin-coated magnetic
nanoparticles (SA-MNPs), the signal from which could be detected by the naked
eye, microscope or CCD camera. By employing SA-MNPs as visible labels, the
microarray unambiguously distinguished all 4 pathogens with detection sensitivity
up to 316 CFU/mL. Due to its high sensitivity, specificity and simple detection
procedure, the magnetic bead assay provides a powerful tool for the detection and
identification of foodborne pathogens in a modestly equipped laboratory.


PMID: 23909141  [Indexed for MEDLINE]


280. Metab Eng. 2013 Jul;18:44-52. doi: 10.1016/j.ymben.2013.03.004. Epub 2013 Apr 4.

Genome-wide analysis of redox reactions reveals metabolic engineering targets for
D-lactate overproduction in Escherichia coli.

Kim HJ(1), Hou BK, Lee SG, Kim JS, Lee DW, Lee SJ.

Author information: 
(1)Systems & Synthetic Biology Research Center, Korea Research Institute of
Bioscience and Biotechnology-KRIBB, Daejeon 305-806, Republic of Korea.

Most current metabolic engineering applications rely on the inactivation of
unwanted reactions and the amplification of product-oriented reactions. All of
the biochemical reactions involved with cellular metabolism are tightly
coordinated with the electron flow, which depends on the cellular energy status. 
Thus, the cellular metabolic flux can be controlled either by modulation of the
electron flow or the regulation of redox reactions. This study analyzed the
genome-wide anaerobic fermentation products of 472 Escherichia coli single gene
knockouts, which comprised mainly of dehydrogenases, oxidoreductases, and
redox-related proteins. Many metabolic pathways that were located far from
anaerobic mixed-acid fermentation significantly affected the profiles of lactic
acid, succinic acid, acetic acid, formic acid, and ethanol. Unexpectedly,
D-lactate overproduction was determined by a single gene deletion in
dehydrogenases (e.g., guaB, pyrD, and serA) involved with nucleotide and amino
acid metabolism. Furthermore, the combined knockouts of guaB, pyrD, serA, fnr,
arcA, or arcB genes, which are involved with anaerobic transcription regulation, 
enhanced D-lactate overproduction. These results suggest that the anaerobic
fermentation profiles of E. coli can be tuned via the disruption of peripheral
dehydrogenases in anaerobic conditions.

Copyright © 2013 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ymben.2013.03.004 
PMID: 23563322  [Indexed for MEDLINE]


281. Plasmid. 2013 Jul;70(1):52-60. doi: 10.1016/j.plasmid.2013.01.006. Epub 2013 Jan 
31.

Role of plasmid- and chromosomally encoded Hha proteins in modulation of gene
expression in E. coli O157:H7.

Paytubi S(1), Dietrich M, Queiroz MH, Juárez A.

Author information: 
(1)Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, 
Avda. Diagonal 643, 08028 Barcelona, Spain. spaytubi@ub.edu

H-NS and Hha belong to the nucleoid-associated family of proteins and modulate
gene expression in response to environmental stimuli. Genes coding for these
proteins can be either chromosomally or plasmid-encoded. In this work, we analyse
the regulatory role of the Hha protein encoded in the virulence plasmid of the
enterohemorrhagic Escherichia coli O157:H7 (Hha(pO157)). This plasmid is present 
in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both,
Hha(pO157) and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant
degree of similarity. The hha gene from plasmid pO157 is transcribed from its own
putative promoter and is overexpressed in a chromosomal hha mutant. As its
chromosomal counterpart, Hha(pO157) is able to interact with H-NS. Remarkably,
Hha(pO157) targets only a subset of the genes modulated by Hhachr. This has been 
evidenced by both assaying the ability of Hha(pO157) to complement expression of 
a specific operon (i.e., the haemolysin operon) and by comparing the global
transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives.
Hha(pO157) and Hhachr share some common regulatory features, however they also
display specific targeting of some genes and even a different modulatory role in 
some others.

Copyright © 2013 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.plasmid.2013.01.006 
PMID: 23376462  [Indexed for MEDLINE]


282. J Microbiol Biotechnol. 2013 Jun 28;23(6):878-84.

In vitro selection of RNA aptamer specific to Salmonella typhimurium.

Han SR(1), Lee SW.

Author information: 
(1)Department of Molecular Biology, Institute of Nanosensor and Biotechnology,
Dankook University, Yongin 448-701, Korea.

Salmonella is a major foodborne pathogen that causes a variety of human diseases.
Development of ligands directly and specifically binding to the Salmonella will
be crucial for the rapid detection of, and thus for efficient protection from,
the virulent bacteria. In this study, we identified a RNA aptamer-based ligand
that can specifically recognize Salmonella Typhimurium through SELEX technology. 
To this end, we isolated and characterized an RNase-resistant RNA aptamer that
bound to the OmpC protein of Salmonella Typhimurium with high specificity and
affinity (Kd ~ 20 nM). Of note, the selected aptamer was found to specifically
bind to Salmonella Typhimurium, but neither to Gram-positive bacteria
(Staphylococcus aureus) nor to other Gram-negative bacteria (Escherichia coli
O157:H7). This was evinced by aptamer-immobilized ELISA and aptamer-linked
precipitation experiments. This Salmonella species-specific aptamer could be
useful as a diagnostic ligand against pathogen-caused foodborne sickness.


PMID: 23676911  [Indexed for MEDLINE]


283. PLoS One. 2013 Jun 28;8(6):e66986. doi: 10.1371/journal.pone.0066986. Print 2013.

Bacterial diversity in meconium of preterm neonates and evolution of their fecal 
microbiota during the first month of life.

Moles L(1), Gómez M, Heilig H, Bustos G, Fuentes S, de Vos W, Fernández L,
Rodríguez JM, Jiménez E.

Author information: 
(1)Departamento de Nutrición, Bromatología y Tecnología de los Alimentos,
Universidad Complutense de Madrid, Madrid, Spain.

The establishment and succession of bacterial communities in infants may have a
profound impact in their health, but information about the composition of
meconium microbiota and its evolution in hospitalized preterm infants is scarce. 
In this context, the objective of this work was to characterize the microbiota of
meconium and fecal samples obtained during the first 3 weeks of life from 14
donors using culture and molecular techniques, including DGGE and the Human
Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture
techniques offer a quantification of cultivable bacteria and allow further study 
of the isolate, while molecular techniques provide deeper information on
bacterial diversity. Culture and HITChip results were very similar but the former
showed lower sensitivity. Inter-individual differences were detected in the
microbiota profiles although the meconium microbiota was peculiar and distinct
from that of fecal samples. Bacilli and other Firmicutes were the main bacteria
groups detected in meconium while Proteobacteria dominated in the fecal samples. 
Culture technique showed that Staphylococcus predominated in meconium and that
Enterococcus, together with Gram-negative bacteria such as Escherichia coli,
Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more
abundant in fecal samples. In addition, HITChip results showed the prevalence of 
bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium
samples whereas those related to Enterococcus, Escherichia coli, Klebsiella
pneumoniae and Yersinia predominated in the 3(rd) week feces. This study
highlights that spontaneously-released meconium of preterm neonates contains a
specific microbiota that differs from that of feces obtained after the first week
of life. Our findings indicate that the presence of Serratia was strongly
associated with a higher degree of immaturity and other hospital-related
parameters, including antibiotherapy and mechanical ventilation.

DOI: 10.1371/journal.pone.0066986 
PMCID: PMC3695978
PMID: 23840569  [Indexed for MEDLINE]


284. Dev Biol. 2013 Jun 15;378(2):122-40. doi: 10.1016/j.ydbio.2013.03.020. Epub 2013 
Apr 6.

APC loss-induced intestinal tumorigenesis in Drosophila: Roles of Ras in Wnt
signaling activation and tumor progression.

Wang C(1), Zhao R, Huang P, Yang F, Quan Z, Xu N, Xi R.

Author information: 
(1)National Institute of Biological Sciences, No. 7 Science Park Road,
Zhongguancun Life Science Park, Beijing 102206, China.

Adenomatous polyposis coli (APC) and K-ras are the two most frequently mutated
genes found in human colorectal cancers. In human colorectal cancers, Wnt
signaling activation after the loss of APC is hypothesized to be the key event
for adenoma initiation, whereas additional mutations such as Ras activation are
required for the progression from adenoma to carcinoma. However, accumulating
data have led to conflicting views regarding the precise role of Ras in APC
loss-induced tumorigenesis. Here, using Drosophila midgut as a model system, we
show that in the absence of Ras, APC mutant epithelial cells cannot initiate
hyperplasia, suggesting that Ras plays an essential role in tumor initiation.
Conversely, activating Ras by expressing oncogenic Ras or Raf in APC-deficient
cells led to a blockage of cell differentiation and to preinvasive tumor
outgrowth, characteristics that are shared by advanced colorectal carcinoma in
humans. Mechanistically, we find that Ras is not required for Wnt signaling
activation after APC loss, although Ras hyperactivation is able to potentiate Wnt
signaling by increasing the cytoplasmic and nuclear accumulation of
Armadillo/β-catenin via mechanisms independent of JNK/Rac1 or PI3K-Akt signaling,
partly owing to the downregulation of DE-cadherin. Together with the data from
gene expression analyses, our results indicate that both parallel and cooperative
mechanisms of Wnt and Ras signaling are responsible for the initiation and
progression of intestinal tumorigenesis after APC loss.

Copyright © 2013 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ydbio.2013.03.020 
PMID: 23570874  [Indexed for MEDLINE]


285. Vet Res. 2013 Jun 11;44:40. doi: 10.1186/1297-9716-44-40.

Differential response of bovine mammary epithelial cells to Staphylococcus aureus
or Escherichia coli agonists of the innate immune system.

Gilbert FB(1), Cunha P, Jensen K, Glass EJ, Foucras G, Robert-Granié C, Rupp R,
Rainard P.

Author information: 
(1)INRA, UMR1282, ISP, F 37380, Nouzilly, France. pascal.rainard@tours.inra.fr.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major
pathology of dairy cows. To better understand the differential response of the
mammary gland to these two pathogens, we stimulated bovine mammary epithelial
cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus
culture supernatant (SaS) to compare the transcriptomic profiles of the initial
bMEC response. By using HEK 293 reporter cells for pattern recognition receptors,
the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or
Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that
lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC
were found to be responsive to each of these molecules. Transcriptome profiling
revealed a core innate immune response partly shared by LPS and SaS. However, LPS
induced expression of a significant higher number of genes and the fold changes
were of greater magnitude than those induced by SaS. Microarray data analysis
suggests that the activation pathways and the early chemokine and cytokine
production preceded the defense and stress responses. A major differential
response was the activation of the type I IFN pathway by LPS but not by SaS. The 
higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target
mononuclear leucocytes by LPS than by SaS is likely to be related to the
differential activation of the type I IFN pathway, and could induce a different
profile of the initial recruitment of leucocytes. The MEC responses to the two
stimuli were different, as LPS was associated with NF-κB and Fas signaling
pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It 
is noteworthy that at the protein level secretion of TNF-α and IL-1β was not
induced by either stimulus. These results suggest that the response of MEC to
diffusible stimuli from E. coli and S. aureus contributes to the onset of the
response with differential leucocyte recruitment and distinct inflammatory and
innate immune reactions of the mammary gland to infection.

DOI: 10.1186/1297-9716-44-40 
PMCID: PMC3686618
PMID: 23758654  [Indexed for MEDLINE]


286. Appl Environ Microbiol. 2013 Jun;79(11):3392-9. doi: 10.1128/AEM.00225-13. Epub
2013 Mar 22.

Development of a DNA microarray for molecular identification of all 46 Salmonella
O serogroups.

Guo D(1), Liu B, Liu F, Cao B, Chen M, Hao X, Feng L, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA,
Tianjin, People's Republic of China.

Salmonella is a major cause of food-borne disease in many countries. Serotype
determination of Salmonella is important for disease assessment, infection
control, and epidemiological surveillance. In this study, a microarray system
that targets the O antigen-specific genes was developed for simultaneously
detecting and identifying all 46 Salmonella O serogroups. Of these, 40 serogroups
can be confidently identified, and the remaining 6, in three pairs (serogroups
O67 and B, E1 and E4, and A and D1), need to be further distinguished from each
other using PCR methods or conventional serotyping methods. The microarray was
shown to be highly specific when evaluated against 293 Salmonella strains, 186
Shigella strains, representative Escherichia coli strains, and 10 strains of
other bacterial species. The assay correctly identified 288 (98%) of the
Salmonella strains. The detection sensitivity was determined to be 50 ng genomic 
DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU
per gram inoculated could be detected after enrichment. This newly developed
microarray assay is the first molecular protocol that can be used for the
comprehensive detection and identification of all 46 Salmonella O serogroups.
Compared to the traditional serogrouping method, the microarray provides a
reliable, high-throughput, and sensitive approach that can be used for rapid
identification of multiple Salmonella O serogroups simultaneously.

DOI: 10.1128/AEM.00225-13 
PMCID: PMC3648052
PMID: 23524674  [Indexed for MEDLINE]


287. J Bacteriol. 2013 Jun;195(11):2499-508. doi: 10.1128/JB.02252-12. Epub 2013 Mar
22.

The interacting Cra and KdpE regulators are involved in the expression of
multiple virulence factors in enterohemorrhagic Escherichia coli.

Njoroge JW(1), Gruber C, Sperandio V.

Author information: 
(1)Department of Microbiology, University of Texas Southwestern Medical Center,
Dallas, Texas, USA.

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 codes for
two interacting DNA binding proteins, Cra and KdpE, that coregulate expression of
the locus of enterocyte effacement (LEE) genes in a metabolite-dependent manner. 
Cra is a transcription factor that uses fluctuations in the concentration of
carbon metabolism intermediates to positively regulate virulence of EHEC. KdpE is
a response regulator that activates the transcription of homeostasis genes in
response to salt-induced osmolarity and virulence genes in response to changes in
metabolite concentrations. Here, we probed the transcriptional profiles of the
Δcra, ΔkdpE, and Δcra ΔkdpE mutant strains and show that Cra and KdpE share
several targets besides the LEE, but both Cra and KdpE also have independent
targets. Several genes within O-islands (genomic islands present in EHEC but
absent from E. coli K-12), such as Z0639, Z0640, Z3388, Z4267, and espFu
(encoding an effector necessary for formation of attaching and effacing lesions
on epithelial cells), were directly regulated by both Cra and KdpE, while Z2077
was only regulated by Cra. These studies identified and confirmed new direct
targets for Cra and KdpE that included putative virulence factors as well as
characterized virulence factors, such as EspFu and EspG. These results map out
the role of the two interacting regulators, Cra and KdpE, in EHEC pathogenesis
and global gene regulation.

DOI: 10.1128/JB.02252-12 
PMCID: PMC3676075
PMID: 23524613  [Indexed for MEDLINE]


288. PLoS One. 2013 May 30;8(5):e65466. doi: 10.1371/journal.pone.0065466. Print 2013.

Global transcriptional and phenotypic analyses of Escherichia coli O157:H7 strain
Xuzhou21 and its pO157_Sal cured mutant.

Zhao H(1), Chen C, Xiong Y, Xu X, Lan R, Wang H, Yao X, Bai X, Liu X, Meng Q,
Zhang X, Sun H, Zhao A, Bai X, Cheng Y, Chen Q, Ye C, Xu J.

Author information: 
(1)State Key Laboratory for Infectious Disease Prevention and Control,
Collaborative Innovation Center for Diagnosis and Treatment of Infectious
Disease, National Institute for Communicable Disease Control and Prevention,
Changping, Beijing, China.

Escherichia coli O157:H7 is an important food-borne pathogen that can cause
hemorrhagic colitis and hemolytic-uremic syndrome in humans. pO157_Sal, a novel
conjugative plasmid is present in a Chinese O157:H7 outbreak strain Xuzhou21.
Here we investigated the phenotypic and transcriptional differences between the
wild type strain Xuzhou21 and the pO157_Sal cured mutant strain Xuzhou21m.
RNA-Seq analysis found that all 52 ORFs encoded on pO157_Sal were transcribed.
One hundred and sixty eight chromosomal and pO157 genes were differentially
expressed (≥2 fold difference) between Xuzhou21 and Xuzhou21m. Sixty-seven and
101 genes were up-regulated and down-regulated respectively by pO157_Sal
including genes related to stress response, adaption and virulence. The
plasmid-cured mutant Xuzhou21m grew slower than wild type Xuzhou21 and pO157_Sal 
plasmid complemented strain Xuzhou21c in M9 medium under the condition of high
NaCl or presence of sodium deoxycholate (NaDC), corroborating with the RNA-Seq
data. Seven differentially expressed genes are associated with NaDC resistance,
including the adenine-specific DNA-methyltransferase gene (dam), multidrug efflux
system subunit gene mdtA, hyperosmotically inducible periplasmic protein gene
osmY and oxidation-reduction related genes while two differentially expressed
genes (osmY and pspD) are likely to be related to resistance to osmotic pressure.
A number of differentially expressed genes were virulence associated including
four genes encoding T3SS effectors from the chromosome and ehxD from pO157.
Through complementation of Xuzhou21m with a plasmid construct carrying the
pO157_Sal hha homolog we further showed that the pO157_Sal hha represses the
expression of T3SS effectors. These findings demonstrated that the plasmid
pO157_Sal affects the transcription of the chromosomal and pO157 plasmid genes
and contributes to the enhanced ability to resist stress. We conclude that
pO157_Sal plays an important role in regulating global gene expression and
affects the virulence and adaptation of E. coli O157:H7.

DOI: 10.1371/journal.pone.0065466 
PMCID: PMC3667801
PMID: 23738017  [Indexed for MEDLINE]


289. PLoS One. 2013 May 22;8(5):e62601. doi: 10.1371/journal.pone.0062601. Print 2013.

Promoters of Escherichia coli versus promoter islands: function and structure
comparison.

Panyukov VV(1), Ozoline ON.

Author information: 
(1)Department of Bioinformatics, Institute of Mathematical Problems of Biology,
Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

Expression of bacterial genes takes place under the control of RNA polymerase
with exchangeable σ-subunits and multiple transcription factors. A typical
promoter region contains one or several overlapping promoters. In the latter case
promoters have the same or different σ-specificity and are often subjected to
different regulatory stimuli. Genes, transcribed from multiple promoters, have on
average higher expression levels. However, recently in the genome of Escherichia 
coli we found 78 regions with an extremely large number of potential
transcription start points (promoter islands, PIs). It was shown that all PIs
interact with RNA polymerase in vivo and are able to form transcriptionally
competent open complexes both in vitro and in vivo but their transcriptional
activity measured by oligonucleotide microarrays was very low, if any. Here we
confirmed transcriptional defectiveness of PIs by analyzing the 5'-end specific
RNA-seq data, but showed their ability to produce short oligos (9-14 bases). This
combination of functional properties indicated a deliberate suppression of
transcriptional activity within PIs. According to our data this suppression may
be due to a specific conformation of the DNA double helix, which provides an
ideal platform for interaction with both RNA polymerase and the histone-like
nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen
sites optimized by evolution for staying in a heterochromatin-like state. Since
almost all promoter islands are associated with horizontally acquired genes, we
offer them as specific components of bacterial evolution involved in acquisition 
of foreign genetic material by turning off the expression of toxic or useless
aliens or by providing optimal promoter for beneficial genes. The putative
molecular mechanism underlying the appearance of promoter islands within
recipient genomes is discussed.

DOI: 10.1371/journal.pone.0062601 
PMCID: PMC3661553
PMID: 23717391  [Indexed for MEDLINE]


290. Anal Biochem. 2013 May 1;436(1):22-8. doi: 10.1016/j.ab.2013.01.014. Epub 2013
Jan 26.

Isolation and characterization of DNA aptamers against Escherichia coli using a
bacterial cell-systematic evolution of ligands by exponential enrichment
approach.

Kim YS(1), Song MY, Jurng J, Kim BC.

Author information: 
(1)Center for Environment, Health and Welfare Research, Korea Institute of
Science and Technology, Seoul 136-701, Republic of Korea.

Aptamers are powerful capturing probes against various targets such as proteins, 
small organic compounds, metal ions, and even cells. In this study, we isolated
and characterized single-stranded DNA (ssDNA) aptamers against Escherichia coli. 
A total of 28 ssDNAs were isolated after 10 rounds of selection using a bacterial
cell-SELEX (systematic evolution of ligands by exponential enrichment) process.
Other bacterial species (Klebsiella pneumoniae, Citrobacter freundii,
Enterobacter aerogenes, and Staphylococcus epidermidis) were used for counter
selection to enhance the selectivity of ssDNA aptamers against E. coli. Finally, 
four ssDNA aptamers showed high affinity and selectivity to E. coli, The
dissociation constants (K(d)) of these four ssDNA aptamers to E. coli were
estimated to range from 12.4 to 25.2 nM. These aptamers did not bind to other
bacterial species, including four counter cells, but they showed affinity to
different E. coli strains. The binding of these four aptamers to E. coli was
observed directly by fluorescence microscopy.

Copyright © 2013 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ab.2013.01.014 
PMID: 23357235  [Indexed for MEDLINE]


291. Appl Environ Microbiol. 2013 May;79(9):2891-8. doi: 10.1128/AEM.00205-13. Epub
2013 Feb 22.

Simultaneous quantification of multiple food- and waterborne pathogens by use of 
microfluidic quantitative PCR.

Ishii S(1), Segawa T, Okabe S.

Author information: 
(1)Division of Environmental Engineering, Faculty of Engineering, Hokkaido
University, Sapporo, Hokkaido, Japan. s.ishii@eng.hokudai.ac.jp

The direct quantification of multiple pathogens has been desired for diagnostic
and public health purposes for a long time. In this study, we applied
microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and
quantification of multiple food- and waterborne pathogens. In this system,
multiple singleplex qPCR assays were run under identical detection conditions in 
nanoliter-volume chambers that are present in high densities on a chip. First, we
developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by
using prevalidated TaqMan probes. Specific and sensitive quantification was
achieved by using these qPCR assays. With the addition of two previously
validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric
pathogens, a fecal indicator bacterium (general Escherichia coli), and a process 
control strain in the microfluidic qPCR system. We preamplified the template DNA 
to increase the sensitivity of the qPCR assays. Our results suggested that
preamplification was effective for quantifying small amounts of the template DNA 
without any major impact on the sensitivity, efficiency, and quantitative
performance of qPCR. This microfluidic qPCR system allowed us to detect and
quantify multiple pathogens from fecal samples and environmental water samples
spiked with pathogens at levels as low as 100 cells/liter. These results suggest 
that the routine monitoring of multiple pathogens in food and water samples is
now technically feasible. This method may provide more reliable information for
risk assessment than the current fecal contamination indicator approach.

DOI: 10.1128/AEM.00205-13 
PMCID: PMC3623133
PMID: 23435884  [Indexed for MEDLINE]


292. PLoS One. 2013 Apr 17;8(4):e61975. doi: 10.1371/journal.pone.0061975. Print 2013.

Genome-wide analysis of alternative splicing during dendritic cell response to a 
bacterial challenge.

Rodrigues R(1), Grosso AR, Moita L.

Author information: 
(1)Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa, Lisboa, Portugal.

The immune system relies on the plasticity of its components to produce
appropriate responses to frequent environmental challenges. Dendritic cells (DCs)
are critical initiators of innate immunity and orchestrate the later and more
specific adaptive immunity. The generation of diversity in transcriptional
programs is central for effective immune responses. Alternative splicing is
widely considered a key generator of transcriptional and proteomic complexity,
but its role has been rarely addressed systematically in immune cells. Here we
used splicing-sensitive arrays to assess genome-wide gene- and exon-level
expression profiles in human DCs in response to a bacterial challenge. We find
widespread alternative splicing events and splicing factor transcriptional
signatures induced by an E. coli challenge to human DCs. Alternative splicing
acts in concert with transcriptional modulation, but these two mechanisms of gene
regulation affect primarily distinct functional gene groups. Alternative splicing
is likely to have an important role in DC immunobiology because it affects genes 
known to be involved in DC development, endocytosis, antigen presentation and
cell cycle arrest.

DOI: 10.1371/journal.pone.0061975 
PMCID: PMC3629138
PMID: 23613991  [Indexed for MEDLINE]


293. PLoS One. 2013 Apr 16;8(4):e61850. doi: 10.1371/journal.pone.0061850. Print 2013.

A global transcriptional switch between the attack and growth forms of
Bdellovibrio bacteriovorus.

Karunker I(1), Rotem O, Dori-Bachash M, Jurkevitch E, Sorek R.

Author information: 
(1)Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel.

Bdellovibrio bacteriovorus is an obligate predator of bacteria ubiquitously found
in the environment. Its life cycle is composed of two essential phases: a
free-living, non-replicative, fast swimming attack phase (AP) wherein the
predator searches for prey; and a non-motile, actively dividing growth phase (GP)
in which it consumes the prey. The molecular regulatory mechanisms governing the 
switch between AP and GP are largely unknown. We used RNA-seq to generate a
single-base-resolution map of the Bdellovibrio transcriptome in AP and GP,
revealing a specific "AP" transcriptional program, which is largely mutually
exclusive of the GP program. Based on the expression map, most genes in the
Bdellovibrio genome are classified as "AP only" or "GP only". We experimentally
generated a genome-wide map of 140 AP promoters, controlling the majority of
AP-specific genes. This revealed a common sigma-like DNA binding site highly
similar to the E. coli flagellar genes regulator sigma28 (FliA). Further analyses
suggest that FliA has evolved to become a global AP regulator in Bdellovibrio.
Our results also reveal a non-coding RNA that is massively expressed in AP. This 
ncRNA contains a c-di-GMP riboswitch. We suggest it functions as an intracellular
reservoir for c-di-GMP, playing a role in the rapid switch from AP to GP.

DOI: 10.1371/journal.pone.0061850 
PMCID: PMC3627812
PMID: 23613952  [Indexed for MEDLINE]


294. Anal Chim Acta. 2013 Apr 15;772:87-92. doi: 10.1016/j.aca.2013.02.029. Epub 2013 
Feb 28.

Quantitative detection of well-based DNA array using switchable lanthanide
luminescence.

Karhunen U(1), Soikkeli M, Lahdenperä S, Soukka T.

Author information: 
(1)Division of Biotechnology, University of Turku, Turku, Finland. umskar@utu.fi

In this report a novel wash-free method for multiplexed DNA detection is
demonstrated employing target specific probe pairs and switchable lanthanide
luminescence technology on a solid-phase array. Four oligonucleotide capture
probes, conjugated at 3' to non-luminescent lanthanide ion carrier chelate, were 
immobilized as a small array on the bottom of a microtiter plate well onto which 
a mix of corresponding detection probes, conjugated at 5' to a light absorbing
antenna ligand, were added. In the presence of complementary target nucleic acid 
both the spotted capture probe and the liquid-phase detection probe hybridize
adjacently on the target. Consequently the two non-luminescent label molecules
self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide
luminescence is thereafter measured without a wash step from the spots by
scanning in time-resolved mode. The homogeneous solid-phase array-based method
resulted in quantitative detection of synthetic target oligonucleotides with 0.32
nM and 0.60 nM detection limits in a single target and multiplexed assay,
respectively, corresponding to 3× SD of the background. Also qualitative
detection of PCR-amplified target from Escherichia coli is described.

Copyright © 2013 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.aca.2013.02.029 
PMID: 23540252  [Indexed for MEDLINE]


295. Appl Environ Microbiol. 2013 Apr;79(7):2435-45. doi: 10.1128/AEM.03501-12. Epub
2013 Feb 1.

Evaluating the pathogenic potential of environmental Escherichia coli by using
the Caenorhabditis elegans infection model.

Merkx-Jacques A(1), Coors A, Brousseau R, Masson L, Mazza A, Tien YC, Topp E.

Author information: 
(1)Agriculture and Agri-Food Canada, London, Ontario, Canada.

The detection and abundance of Escherichia coli in water is used to monitor and
mandate the quality of drinking and recreational water. Distinguishing commensal 
waterborne E. coli isolates from those that cause diarrhea or extraintestinal
disease in humans is important for quantifying human health risk. A DNA
microarray was used to evaluate the distribution of virulence genes in 148 E.
coli environmental isolates from a watershed in eastern Ontario, Canada, and in
eight clinical isolates. Their pathogenic potential was evaluated with
Caenorhabditis elegans, and the concordance between the bioassay result and the
pathotype deduced by genotyping was explored. Isolates identified as potentially 
pathogenic on the basis of their complement of virulence genes were significantly
more likely to be pathogenic to C. elegans than those determined to be
potentially nonpathogenic. A number of isolates that were identified as
nonpathogenic on the basis of genotyping were pathogenic in the infection assay, 
suggesting that genotyping did not capture all potentially pathogenic types. The 
detection of the adhesin-encoding genes sfaD, focA, and focG, which encode
adhesins; of iroN2, which encodes a siderophore receptor; of pic, which encodes
an autotransporter protein; and of b1432, which encodes a putative transposase,
was significantly associated with pathogenicity in the infection assay. Overall, 
E. coli isolates predicted to be pathogenic on the basis of genotyping were
indeed so in the C. elegans infection assay. Furthermore, the detection of C.
elegans-infective environmental isolates predicted to be nonpathogenic on the
basis of genotyping suggests that there are hitherto-unrecognized virulence
factors or combinations thereof that are important in the establishment of
infection.

DOI: 10.1128/AEM.03501-12 
PMCID: PMC3623224
PMID: 23377948  [Indexed for MEDLINE]


296. Appl Microbiol Biotechnol. 2013 Apr;97(8):3677-86. doi:
10.1007/s00253-013-4766-4. Epub 2013 Mar 14.

Selection of DNA aptamers for capture and detection of Salmonella Typhimurium
using a whole-cell SELEX approach in conjunction with cell sorting.

Dwivedi HP(1), Smiley RD, Jaykus LA.

Author information: 
(1)Population Health and Pathobiology, College of Veterinary Medicine, North
Carolina State University, Raleigh, NC, USA. hari-prakash.dwivedi@biomerieux.com

Alternative ligands such as nucleic acid aptamers can be used for pathogen
capture and detection and offer advantages over antibodies, including reduced
cost, ease of production and modification, and improved stability. DNA aptamers
demonstrating binding specificity to Salmonella enterica serovar Typhimurium were
identified by whole-cell-systematic evolution of ligands by exponential
enrichment (SELEX) beginning with a combinatorial library of biotin-labeled
single stranded DNA molecules. Aptamer specificity was achieved using whole-cell 
counter-SELEX against select non-Salmonella genera. Aptamers binding to
Salmonella were sorted, cloned, sequenced, and characterized for binding
efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively
high binding affinity with an apparent dissociation constant (K d value) of
1.73 ± 0.54 μM and was selected for further characterization. Binding exclusivity
analysis of S8-7 showed low apparent cross-reactivity with other foodborne
bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate
cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a
ligand for magnetic capture of serially diluted Salmonella Typhimurium cells,
followed by downstream detection using qPCR. The lower limit of detection of the 
aptamer magnetic capture-qPCR assay was 10(2)-10(3) CFU equivalents of Salmonella
Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to
12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole
cells; (b) the application of flow cytometry for aptamer pool selection, thereby 
favoring purification of ligands with both high binding affinity and targeting
abundant cell surface moieties; and (c) the use of pre-labeled primers that
circumvented the need for post-selection ligand labeling. Taken together, this
study provides proof-of-concept that biotinylated aptamers selected by whole-cell
SELEX can be used in a qPCR-based capture-detection platform for Salmonella
Typhimurium.

DOI: 10.1007/s00253-013-4766-4 
PMID: 23494620  [Indexed for MEDLINE]


297. Indian J Med Microbiol. 2013 Apr-Jun;31(2):142-7. doi: 10.4103/0255-0857.115230.

Detection of the common resistance genes in Gram-negative bacteria using gene
chip technology.

Ting C(1), Jun A, Shun Z.

Author information: 
(1)Clinical Research Center of Ningbo No. 2 Hospital, Ningbo, Zhejiang, 315010,
China.

OBJECTIVE: To design a resistance gene detection chip that could, in parallel,
detect common clinical drug resistance genes of Gram-negative bacteria.
MATERIALS AND METHODS: Seventy clinically significant Gram-negative bacilli
(Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas
aeruginosa, Acinetobacter baumannii) were collected. According to the known
resistance gene sequences, we designed and synthesized primers and probes, which 
were used to prepare resistance gene detection chips, and finally we hybridized
and scanned the gene detection chips.
RESULTS: The results between the gene chip and polymerase chain reaction (PCR)
were compared. The rate was consistently 100% in the eight kinds of resistance
genes tested (TEM, SHV, CTX-M, DHA, CIT, VIM, KPC, OXA-23). One strain of
Pseudomonas aeruginosa had the IMP, but it was not found by gene chip.
CONCLUSION: The design of Gram-negative bacteria-resistant gene detection chip
had better application value.

DOI: 10.4103/0255-0857.115230 
PMID: 23867670  [Indexed for MEDLINE]


298. Vet Immunol Immunopathol. 2013 Mar 15;152(1-2):87-92. doi:
10.1016/j.vetimm.2012.09.027. Epub 2012 Sep 26.

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: a
gene expression study.

Schroyen M(1), Goddeeris BM, Stinckens A, Verhelst R, Janssens S, Cox E, Georges 
M, Niewold T, Buys N.

Author information: 
(1)Department Biosystems, K.U. Leuven, Kasteelpark Arenberg 30, 3001 Heverlee,
Belgium.

Diarrhoea in neonatal and early-weaned piglets due to enterotoxigenic Escherichia
coli-F4 (ETEC-F4) is an important problem in the pig farming industry. There is
substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not 
all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13 has 
been found to be in close linkage to the susceptibility of piglets for
ETEC-F4ab,ac. Potential candidate genes on SSC13 have been examined and although 
some polymorphisms were found to be in linkage disequilibrium with the phenotype,
the causative mutation has not yet been found. In this study we are looking at
the expression of porcine genes in relation to ETEC-F4ab,ac. With the aid of the 
Affymetrix GeneChip Porcine Genome Array we were able to find differentially
expressed genes between ETEC-F4ab,ac receptor positive (Fab,acR(+)) piglets
without diarrhoea and F4ab,acR(+) piglets with diarrhoea or F4ab,acR(-) animals. 
Since the susceptibility to ETEC-F4ab,ac was described as a Mendelian trait, it
is not so surprisingly that only two differentially expressed genes, transferrin 
receptor (TFRC) and trefoil factor 1 (TFF1), came out of the analysis. Although
both genes could pass for functional candidate genes only TFRC also mapped to the
region on SSC13 associated with susceptibility for ETEC-F4, which makes TFRC a
positional functional candidate gene. Validation by qRT-PCR confirmed the
differential expression of TFRC and TFF1. In piglets without diarrhoea, the
expression of both genes was higher in F4ab,acR(+) than in F4ab,acR(-) piglets.
Similarly, TFRC and TFF1 expression in F4ab,acR(+) piglets without diarrhoea was 
also higher than in F4ab,acR(+) piglets with diarrhoea. Consequently, although
both genes might not play a role as receptor for F4 fimbriae, they could be of
great importance during an ETEC-F4 outbreak. An upregulation of TFRC can be a
consequence of the piglets ability to raise an effective immune response. An
elevation of TFF1, a protein involved in mucin formation, may also affect the
piglet's capability to cope with ETEC bacteria, rather than being a receptor for 
its fimbriae.

Copyright © 2012 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.vetimm.2012.09.027 
PMID: 23078902  [Indexed for MEDLINE]


299. Mol Biosyst. 2013 Mar;9(3):522-30. doi: 10.1039/c2mb25374g. Epub 2012 Dec 18.

Genome-wide transcriptional responses of Escherichia coli to glyphosate, a potent
inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate
synthase.

Lu W(1), Li L, Chen M, Zhou Z, Zhang W, Ping S, Yan Y, Wang J, Lin M.

Author information: 
(1)Biotechnology Research Institute, Chinese Academy of Agricultural Sciences/Key
Laboratory of Crop Biotechnology, Ministry of Agriculture, Beijing 100081, China.

The shikimate pathway enzymes offer attractive targets for the development of
antimetabolites. Glyphosate is an effective antimetabolite that inhibits
5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway,
thereby resulting in a shortage of the chorismate-derived essential aromatic
amino acids. However, little is known about the genome-wide transcriptional
responses of bacteria to glyphosate shock. In the current study, a transcriptome 
analysis of Escherichia coli (E. coli) exposed to glyphosate identified the
differential expression of 1040 genes, which represent 23.2% of the genome. The
differentially expressed genes are primarily involved in amino acid metabolism,
cell motility, and central carbon metabolism, indicating that the impact of
glyphosate on the shikimate pathway also extends to other metabolic pathways.
Expectedly, almost all genes encoding the proteins for the shikimate and specific
aromatic amino acid pathways were downregulated after the addition of glyphosate.
Furthermore, the expression of many energy- and metabolism-related genes was
repressed. In contrast, glyphosate treatment induced the coordinated upregulation
of at least 50 genes related to cell motility and chemotaxis. The reverse
transcription-quantitative real-time PCR (RT-qPCR) data showed that the
expression profiles of selected genes from the referred pathways were found to be
consistent with the microarray data. The results suggest that the presence of
glyphosate during growth induces metabolic starvation, an energy drain and other 
non-target effects.

DOI: 10.1039/c2mb25374g 
PMID: 23247721  [Indexed for MEDLINE]


300. J Biol Chem. 2013 Feb 22;288(8):5407-16. doi: 10.1074/jbc.M112.408245. Epub 2013 
Jan 10.

Enhanced membrane-tethered mucin 3 (MUC3) expression by a tetrameric branched
peptide with a conserved TFLK motif inhibits bacteria adherence.

Pan Q(1), Tian Y, Li X, Ye J, Liu Y, Song L, Yang Y, Zhu R, He Y, Chen L, Chen W,
Mao X, Peng Z, Wang R.

Author information: 
(1)Department of Gastroenterology, Southwest Hospital, Chongqing, 400038 China.

We investigated whether a synthetic tetrameric branched peptide based on the
conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more
efficient than the monomeric peptide at up-regulating MUC3 expression and
examined the possible mechanism(s) and biological significance of this process.
We used standard solid-phase methods to synthesize a tetrameric branched peptide 
(sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing
10-mer BP. The aberrant expression of transcription factors was analyzed using a 
transcription factor protein/DNA array. MUC3 and relevant transcription factors
were detected using real-time PCR and/or Western blots. The luciferase assay,
EMSA, and ChIP assays were used to analyze the activity of the human MUC3
promoter. The bacterial adherence assay was used to evaluate the in vitro
inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli
serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with
the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP 
induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal
10-mer monomeric peptide, and MUC3 expression was activated through
transcriptional mechanisms, including the induction of multiple transcription
factors and further binding with their cis-elements between nucleotides -242 and 
-62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP
dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the
HT-29-Gal cells compared with the controls. This finding suggests a potential
therapeutic use for this peptide to prevent gastrointestinal infection.

DOI: 10.1074/jbc.M112.408245 
PMCID: PMC3581376
PMID: 23316049  [Indexed for MEDLINE]


301. J Biol Chem. 2013 Feb 22;288(8):5426-42. doi: 10.1074/jbc.M112.431569. Epub 2013 
Jan 1.

The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that
also affects molybdenum cofactor biosynthesis.

Dahl JU(1), Radon C, Bühning M, Nimtz M, Leichert LI, Denis Y, Jourlin-Castelli
C, Iobbi-Nivol C, Méjean V, Leimkühler S.

Author information: 
(1)Institute of Biochemistry and Biology, Department of Molecular Enzymology,
University of Potsdam, 14476 Potsdam, Germany.

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine
for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters,
molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The
sulfur transfer from IscS to various biomolecules is mediated by different
interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS
cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which
bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA 
strain showed that the expression of genes of the moaABCDE operon coding for
proteins involved in molybdenum cofactor biosynthesis is increased under aerobic 
and anaerobic conditions. Additionally, under anaerobic conditions the expression
of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate
reductase were also increased. On the contrary, the activity of all molydoenzymes
analyzed was significantly reduced in the ΔtusA mutant. Characterization of the
ΔtusA strain under aerobic conditions showed an overall low molybdopterin content
and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic
conditions the activity of nitrate reductase was reduced by only 50%, showing
that TusA is not essential for molybdenum cofactor biosynthesis. We present a
model in which we propose that the direction of sulfur transfer for each
sulfur-containing biomolecule is regulated by the availability of the interaction
partner of IscS. We propose that in the absence of TusA, more IscS is available
for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to
a modified expression of several genes.

DOI: 10.1074/jbc.M112.431569 
PMCID: PMC3581435
PMID: 23281480  [Indexed for MEDLINE]


302. Gastroenterology. 2013 Feb;144(2):381-91. doi: 10.1053/j.gastro.2012.10.048. Epub
2012 Nov 7.

Troy, a tumor necrosis factor receptor family member, interacts with lgr5 to
inhibit wnt signaling in intestinal stem cells.

Fafilek B(1), Krausova M, Vojtechova M, Pospichalova V, Tumova L, Sloncova E,
Huranova M, Stancikova J, Hlavata A, Svec J, Sedlacek R, Luksan O, Oliverius M,
Voska L, Jirsa M, Paces J, Kolar M, Krivjanska M, Klimesova K,
Tlaskalova-Hogenova H, Korinek V.

Author information: 
(1)Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
Prague, Czech Republic.

BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the
intestinal epithelia; blocking this pathway reduces the proliferative capacity of
the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal
cancer. We investigated the roles of the Wnt pathway in homeostasis of the
intestinal epithelium and during malignant transformation in human cells and
mice.
METHODS: We performed chromatin immunoprecipitation (ChIP) with DNA microarray
analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human
colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal
tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate.
Intestinal tissues from these mice, as well as Apc(+/Min) and
Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry
and in situ hybridization.
RESULTS: We identified promoter regions of 960 genes that interacted with the Wnt
pathway nuclear effector T-cell factor 4 in 4 different human colorectal
cancer-derived cell lines; 18 of these promoters were present in all chromatin
precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor
receptor superfamily called TROY. Levels of TROY messenger RNA were increased in 
human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in
cells stimulated with the Wnt3a ligand. Expression of Troy was significantly
up-regulated in neoplastic tissues from mice during intestinal tumorigenesis.
Lineage tracing experiments revealed that Troy is produced specifically by
fast-cycling intestinal stem cells. TROY associated with a unique marker of these
cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In
organoids established from the intestinal crypts, Troy suppressed signaling
mediated by R-spondin, a Wnt agonist.
CONCLUSIONS: TROY is up-regulated in human colorectal cancer cell lines and in
intestinal tumors in mice. It functions as a negative modulator of the Wnt
pathway in LGR5-positive stem cells.

Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

DOI: 10.1053/j.gastro.2012.10.048 
PMID: 23142137  [Indexed for MEDLINE]


303. Genes Cells. 2013 Feb;18(2):123-34. doi: 10.1111/gtc.12026. Epub 2013 Jan 10.

Novel regulator PgrR for switch control of peptidoglycan recycling in Escherichia
coli.

Shimada T(1), Yamazaki K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

Peptidoglycan (PG), also designated as murein, forms a skeletal mesh within the
periplasm of bacterial membrane. PG is a metabolically stable cell architecture
in Escherichia coli, but under as yet ill-defined conditions, a portion of PG is 
degraded, of which both amino sugar and peptide moieties are either recycled or
used as self-generated nutrients for cell growth. At present, the control of PG
degradation remains uncharacterized. Using the Genomic SELEX screening system, we
identified an uncharacterized transcription factor YcjZ is a repressor of the
expression of the initial step enzymes for PG peptide degradation. Under nutrient
starvation, the genes encoding the enzymes for PG peptide degradation are
derepressed so as to generate amino acids but are tightly repressed at high
osmotic conditions so as to maintain the rigid membrane for withstanding the
turgor. Taken together, we propose to rename YcjZ as PgrR (regulator of peptide
glycan recycling).

© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of
Japan and Wiley Publishing Asia Pty Ltd.

DOI: 10.1111/gtc.12026 
PMID: 23301696  [Indexed for MEDLINE]


304. Mol Biotechnol. 2013 Feb;53(2):182-8. doi: 10.1007/s12033-012-9512-0.

Detection and genotyping of Campylobacter jejuni and Campylobacter coli by use of
DNA oligonucleotide arrays.

Marotta F(1), Zilli K, Tonelli A, Sacchini L, Alessiani A, Migliorati G, Di
Giannatale E.

Author information: 
(1)Istituto G. Caporale, 64100 Teramo, Italy. f.marotta@izs.it

Campylobacter have emerged as the most common bacterial food-borne illness in the
developed world. The ability to reduce Campylobacter infections in humans is
linked to the full comprehension of the principal key aspects of its infection
cycle. A microbial diagnostic microarray detecting Campylobacter housekeeping,
structural, and virulence associated genes was designed and validated using
genomic DNA from reference and field strains of Campylobacter jejuni and coli
isolated from human, chicken, and raw milk. This microarray was confirmed to be a
powerful diagnostic tool for monitoring emerging Campylobacter pathotypes as well
as for epidemiological, environmental, and phylogenetic studies including the
evaluation of genome plasticity.

DOI: 10.1007/s12033-012-9512-0 
PMID: 22354794  [Indexed for MEDLINE]


305. Database (Oxford). 2013 Jan 17;2013:bas059. doi: 10.1093/database/bas059. Print
2013.

Evidence classification of high-throughput protocols and confidence integration
in RegulonDB.

Weiss V(1), Medina-Rivera A, Huerta AM, Santos-Zavaleta A, Salgado H, Morett E,
Collado-Vides J.

Author information: 
(1)Programa de Genómica Computacional, Centro de Ciencias Genómicas, Universidad 
Nacional Autónoma de México, AP 565-A, Cuernavaca, Morelos 62100, Mexico.
vweiss@ccg.unam.mx

RegulonDB provides curated information on the transcriptional regulatory network 
of Escherichia coli and contains both experimental data and computationally
predicted objects. To account for the heterogeneity of these data, we introduced 
in version 6.0, a two-tier rating system for the strength of evidence,
classifying evidence as either 'weak' or 'strong' (Gama-Castro,S.,
Jimenez-Jacinto,V., Peralta-Gil,M. et al. RegulonDB (Version 6.0): gene
regulation model of Escherichia Coli K-12 beyond transcription, active
(experimental) annotated promoters and textpresso navigation. Nucleic Acids Res.,
2008;36:D120-D124.). We now add to our classification scheme the classification
of high-throughput evidence, including chromatin immunoprecipitation (ChIP) and
RNA-seq technologies. To integrate these data into RegulonDB, we present two
strategies for the evaluation of confidence, statistical validation and
independent cross-validation. Statistical validation involves verification of
ChIP data for transcription factor-binding sites, using tools for motif discovery
and quality assessment of the discovered matrices. Independent cross-validation
combines independent evidence with the intention to mutually exclude false
positives. Both statistical validation and cross-validation allow to upgrade
subsets of data that are supported by weak evidence to a higher confidence level.
Likewise, cross-validation of strong confidence data extends our two-tier rating 
system to a three-tier system by introducing a third confidence score
'confirmed'. Database URL: http://regulondb.ccg.unam.mx/

DOI: 10.1093/database/bas059 
PMCID: PMC3548332
PMID: 23327937  [Indexed for MEDLINE]


306. BMC Genomics. 2013 Jan 16;14:36. doi: 10.1186/1471-2164-14-36.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially
modulate transcriptional responses in neighbouring uninfected bovine mammary
gland quarters.

Jensen K(1), Günther J, Talbot R, Petzl W, Zerbe H, Schuberth HJ, Seyfert HM,
Glass EJ.

Author information: 
(1)Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School
of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG,
UK. kirsty.jensen@roslin.ed.ac.uk

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the
infection of the mammary gland by various micro-organisms. Although the
transcriptional response of bovine mammary gland cells to in vitro infection has 
been studied, the interplay and consequences of these responses in the in vivo
environment of the mammary gland are less clear. Previously mammary gland
quarters were considered to be unaffected by events occurring in neighbouring
quarters. More recently infection of individual quarters with mastitis causing
pathogens, especially Escherichia coli, has been shown to influence the
physiology of neighbouring uninfected quarters. Therefore, the transcriptional
responses of uninfected mammary gland quarters adjacent to quarters infected with
two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were
compared.
RESULTS: The bacteriologically sterile, within-animal control quarters exhibited 
a transcriptional response to the infection of neighbouring quarters. The
greatest response was associated with E. coli infection, while a weaker, yet
significant, response occurred during S. aureus infection. The transcriptional
responses of these uninfected quarters included the enhanced expression of many
genes previously associated with mammary gland infections. Comparison of the
transcriptional response of uninfected quarters to S. aureus and E. coli
infection identified 187 differentially expressed genes, which were particularly 
associated with cellular responses, e.g. response to stress. The most affected
network identified by Ingenuity Pathway analysis has the immunosuppressor
transforming growth factor beta 1 (TGFB1) at its hub and largely consists of
genes more highly expressed in control quarters from S. aureus infected cows.
CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of
neighbouring quarters and the responses were dependent on pathogen type.
Therefore, bovine udder quarters exhibit interdependence and should not be
considered as separate functional entities. This suggests that mastitis pathogens
not only interact directly with host mammary cells, but also influence discrete
sites some distance away, which will affect their response to the subsequent
spread of the infection. Understanding the underlying mechanisms may provide
further clues for ways to control mammary gland infections. These results also
have implications for the design of experimental studies investigating immune
regulatory mechanisms in the bovine mammary gland.

DOI: 10.1186/1471-2164-14-36 
PMCID: PMC3598231
PMID: 23324411  [Indexed for MEDLINE]


307. Ann Agric Environ Med. 2013;20(2):273-5.

Virulence and antibiotic resistance of Escherichia coli isolated from rooks.

Kmet V(1), Drugdova Z, Kmetova M, Stanko M.

Author information: 
(1)Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovakia. 
kmetv@saske.sk

With regard to antibiotic resistance studies in various model animals in the
urban environment, the presented study focused on the rook, many behavioural and 
ecological aspects of which are important from an epidemiological point of view. 
A total of 130 Escherichia coli strains isolated from rook faeces during a
two-year period (2011-2012) were investigated for antibiotic resistance and
virulence. Resistance to ampicillin (60%) and streptomycin (40%) were the most
frequent, followed by resistance to fluoroquinolones (ciprofloxacin-22% and
enrofloxacin-24%), tetracycline (18%), cotrimoxazol (17%) and florfenicol (14%). 
Ceftiofur resistance occured in 10.7% of strains and cefquinom resistance in 1.5%
of strains. Twenty-five E.coli strains with a higher level of MICs of
cephalosporins (over 2mg/L of ceftazidime and ceftriaxon) and fluoroquinolones
were selected for detection of betalactamase genes (CTX-M, CMY), plasmid-mediated
quinolone resistance qnrS, integrase 1, and for APEC (avian pathogenic E.coli)
virulence factors (iutA, cvaC, iss, tsh, ibeA, papC, kpsII). Genes of CTX-M1,
CMY-2, integrase 1, papC, cvaC, iutA were detected in one strain of E.coli, and
qnrS, integrase 1, iss, cvaC, tsh were detected in another E.coli. DNA microarray
revealed the absence of verotoxin and enterotoxin genes and pathogenicity
islands. The results show that rooks can serve as a reservoir of
antibiotic-resistant E. coli with avian pathogenic virulence factors for the
human population, and potentially transmit such E.coli over long distances.


PMID: 23772573  [Indexed for MEDLINE]


308. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2013;30(8):1422-36. 
doi: 10.1080/19440049.2012.743192. Epub 2012 Dec 3.

Genomic paradigms for food-borne enteric pathogen analysis at the USFDA: case
studies highlighting method utility, integration and resolution.

Elkins CA(1), Kotewicz ML, Jackson SA, Lacher DW, Abu-Ali GS, Patel IR.

Author information: 
(1)Division of Molecular Biology, Center for Food Safety and Applied Nutrition
(CFSAN), United States Food and Drug Administration (USFDA), Laurel, MD 20708,
USA. chris.elkins@fda.hhs.gov

Modern risk control and food safety practices involving food-borne bacterial
pathogens are benefiting from new genomic technologies for rapid, yet highly
specific, strain characterisations. Within the United States Food and Drug
Administration (USFDA) Center for Food Safety and Applied Nutrition (CFSAN),
optical genome mapping and DNA microarray genotyping have been used for several
years to quickly assess genomic architecture and gene content, respectively, for 
outbreak strain subtyping and to enhance retrospective trace-back analyses. The
application and relative utility of each method varies with outbreak scenario and
the suspect pathogen, with comparative analytical power enhanced by database
scale and depth. Integration of these two technologies allows high-resolution
scrutiny of the genomic landscapes of enteric food-borne pathogens with notable
examples including Shiga toxin-producing Escherichia coli (STEC) and Salmonella
enterica serovars from a variety of food commodities. Moreover, the recent
application of whole genome sequencing technologies to food-borne pathogen
outbreaks and surveillance has enhanced resolution to the single nucleotide
scale. This new wealth of sequence data will support more refined next-generation
custom microarray designs, targeted re-sequencing and "genomic signature
recognition" approaches involving a combination of genes and single nucleotide
polymorphism detection to distil strain-specific fingerprinting to a minimised
scale. This paper examines the utility of microarrays and optical mapping in
analysing outbreaks, reviews best practices and the limits of these technologies 
for pathogen differentiation, and it considers future integration with whole
genome sequencing efforts.

DOI: 10.1080/19440049.2012.743192 
PMID: 23199033  [Indexed for MEDLINE]


309. Genes Genet Syst. 2013;88(4):233-40.

Development of a system for discovery of genetic interactions for essential genes
in Escherichia coli K-12.

Yong HT(1), Yamamoto N, Takeuchi R, Hsieh YJ, Conrad TM, Datsenko KA, Nakayashiki
T, Wanner BL, Mori H.

Author information: 
(1)Graduate School of Biological Science, Nara Institute of Science and
Technology.

Genetic interaction networks are especially useful for functional assignment of
genes and gaining new insights into the systems-level organization of the cell.
While studying interactions of nonessential genes can be relatively
straight-forward via use of deletion mutants, different approaches must be used
to reveal interactions of essential genes due to their indispensability. One
method shown to be useful for revealing interactions of essential genes requires 
tagging the query protein. However, this approach can be complicated by
mutational effects of potential hypomorphic alleles. Here, we describe a pilot
study for a new scheme of systematically studying the interactions of essential
genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from 
which the essential gene is conditionally expressed. The essential gene is
expressed at lower levels, producing a moderate growth defect in a query host.
Secondary mutations are introduced into the query host by conjugation and the
resultant exconjugants are scored for growth by imaging them over time. We report
results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and
yjgP, showing (on average) interactions with nearly 80 nonessential genes. This
system should prove useful for genome-wide analyses of other essential genes in
E. coli K-12.


PMID: 24463526  [Indexed for MEDLINE]


310. Genomics. 2013 Jan;101(1):1-11. doi: 10.1016/j.ygeno.2012.10.003. Epub 2012 Oct
22.

Structural attributes of nucleotide sequences in promoter regions of
supercoiling-sensitive genes: how to relate microarray expression data with
genomic sequences.

Kravatskaya GI(1), Chechetkin VR, Kravatsky YV, Tumanyan VG.

Author information: 
(1)Engelhardt Institute of Molecular Biology of Russian Academy of Sciences,
Russia. gk@eimb.ru

The level of supercoiling in the chromosome can affect gene expression. To
clarify the basis of supercoiling sensitivity, we analyzed the structural
features of nucleotide sequences in the vicinity of promoters for the genes with 
expression enhanced and decreased in response to loss of chromosomal supercoiling
in Escherichia coli. Fourier analysis of promoter sequences for
supercoiling-sensitive genes reveals the tendency in selection of sequences with 
helical periodicities close to 10nt for relaxation-induced genes and to 11nt for 
relaxation-repressed genes. The helical periodicities in the subsets of promoters
recognized by RNA polymerase with different sigma factors were also studied. A
special procedure was developed for the study of correlations between the
intensities of periodicities in promoter sequences and the expression levels of
corresponding genes. Significant correlations of expression with the AT content
and with AT periodicities about 10, 11, and 50nt indicate their role in
regulation of supercoiling-sensitive genes.

Copyright © 2012 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ygeno.2012.10.003 
PMID: 23085385  [Indexed for MEDLINE]


311. Infect Immun. 2013 Jan;81(1):122-32. doi: 10.1128/IAI.00676-12. Epub 2012 Oct 22.

Characterization of the AggR regulon in enteroaggregative Escherichia coli.

Morin N(1), Santiago AE, Ernst RK, Guillot SJ, Nataro JP.

Author information: 
(1)Department of Microbiology and Immunology, University of Maryland School of
Medicine, Baltimore, Maryland, USA.

AggR is a transcriptional regulator of enteroaggregative Escherichia coli (EAEC) 
and has been proposed as the defining factor for typical EAEC strains. Expression
of multiple putative virulence factors, including the aggregative adherence
fimbriae (AAF), dispersin, the dispersin translocator Aat, and the Aai type VI
secretion system, have been found to be regulated by AggR. Here, we confirm the
existence of at least 44 AggR-regulated genes using DNA microarray and real-time 
quantitative reverse transcription-PCR (qRT-PCR); these genes include chromosomal
and plasmid-borne loci and 19 previously unsuspected genes. Two previously
uncharacterized virulence plasmid-encoded open reading frames (ORFs) (designated 
ORF3 and ORF4) exhibit significant identity with isoprenoid biosynthesis genes of
Bacteria and Archaea. The predicted ORF4 product is closely related to
isopentenyl isomerase (IDI) enzymes, whereas the predicted product of the
adjacent ORF3 exhibits an aspartate-rich region that is common among
trans-isoprenyl phosphate synthases. We show that mutations in these ORFs confer 
changes in bacterial surface properties. AggR coordinately controls expression of
a large number of EAEC genes.

DOI: 10.1128/IAI.00676-12 
PMCID: PMC3536136
PMID: 23090962  [Indexed for MEDLINE]


312. J Biomol Struct Dyn. 2013;31(1):115-24. doi: 10.1080/07391102.2012.691368. Epub
2012 Jul 18.

Identification of self-consistent modulons from bacterial microarray expression
data with the help of structured regulon gene sets.

Permina EA(1), Medvedeva YA, Baeck PM, Hegde SR, Mande SC, Makeev VJ.

Author information: 
(1)Vavilov Institute of General Genetics, 3 Gubkina str. , Moscow, GSP-1, 119991,
Russia. elizabeth.permina@vigg.ru

Identification of bacterial modulons from series of gene expression measurements 
on microarrays is a principal problem, especially relevant for inadequately
studied but practically important species. Usage of a priori information on
regulatory interactions helps to evaluate parameters for regulatory subnetwork
inference. We suggest a procedure for modulon construction where a seed regulon
is iteratively updated with genes having expression patterns similar to those for
regulon member genes. A set of genes essential for a regulon is used to control
modulon updating. Essential genes for a regulon were selected as a subset of
regulon genes highly related by different measures to each other. Using
Escherichia coli as a model, we studied how modulon identification depends on the
data, including the microarray experiments set, the adopted relevance measure and
the regulon itself. We have found that results of modulon identification are
highly dependent on all parameters studied and thus the resulting modulon varies 
substantially depending on the identification procedure. Yet, modulons that were 
identified correctly displayed higher stability during iterations, which allows
developing a procedure for reliable modulon identification in the case of less
studied species where the known regulatory interactions are sparse.

DOI: 10.1080/07391102.2012.691368 
PMID: 22803819  [Indexed for MEDLINE]


313. Methods Mol Biol. 2013;985:223-46. doi: 10.1007/978-1-62703-299-5_12.

Trackable multiplex recombineering for gene-trait mapping in E. coli.

Mansell TJ(1), Warner JR, Gill RT.

Author information: 
(1)Department of Chemical and Biological Engineering, Engineering Center,
University of Colorado Boulder, Boulder, CO, USA.

Recent advances in homologous recombination in Escherichia coli have enabled
improved genome engineering by multiplex recombineering. In this chapter, we
present trackable multiplex recombineering (TRMR), a method for gene-trait
mapping which creates simulated knockdown and overexpression mutants for
virtually all genes in the E. coli genome. The method combines oligonucleotide
synthesis with multiplex recombineering to create two libraries comprising of
over 8,000 E. coli strains in total that can be selected for traits of interest
via high-throughput screening or selection. DNA barcodes included in the
recombineering cassette allow for rapid characterization of a naïve or selected
population via DNA microarray analysis. Important considerations for
oligonucleotide design, DNA library construction, recombineering, strain
characterization, and selection are discussed.

DOI: 10.1007/978-1-62703-299-5_12 
PMID: 23417807  [Indexed for MEDLINE]


314. PLoS One. 2013;8(3):e57860. doi: 10.1371/journal.pone.0057860. Epub 2013 Mar 5.

Effect of simulated microgravity on E. coli K12 MG1655 growth and gene
expression.

Arunasri K(1), Adil M, Venu Charan K, Suvro C, Himabindu Reddy S, Shivaji S.

Author information: 
(1)Centre for Cellular and Molecular Biology, Hyderabad, India.

This study demonstrates the effects of simulated microgravity on E. coli K 12
MG1655 grown on LB medium supplemented with glycerol. Global gene expression
analysis indicated that the expressions of hundred genes were significantly
altered in simulated microgravity conditions compared to that of normal gravity
conditions. Under these conditions genes coding for adaptation to stress are up
regulated (sufE and ssrA) and simultaneously genes coding for membrane
transporters (ompC, exbB, actP, mgtA, cysW and nikB) and carbohydrate catabolic
processes (ldcC, ptsA, rhaD and rhaS) are down regulated. The enhanced growth in 
simulated gravity conditions may be because of the adequate supply of
energy/reducing equivalents and up regulation of genes involved in DNA
replication (srmB) and repression of the genes encoding for nucleoside metabolism
(dfp, pyrD and spoT). In addition, E. coli cultured in LB medium supplemented
with glycerol (so as to protect the cells from freezing temperatures) do not
exhibit multiple stress responses that are normally observed when cells are
exposed to microgravity in LB medium without glycerol.

DOI: 10.1371/journal.pone.0057860 
PMCID: PMC3589462
PMID: 23472115  [Indexed for MEDLINE]


315. PLoS One. 2013;8(2):e55594. doi: 10.1371/journal.pone.0055594. Epub 2013 Feb 8.

Pseudomonas aeruginosa PA1006, which plays a role in molybdenum homeostasis, is
required for nitrate utilization, biofilm formation, and virulence.

Filiatrault MJ(1), Tombline G, Wagner VE, Van Alst N, Rumbaugh K, Sokol P,
Schwingel J, Iglewski BH.

Author information: 
(1)Department of Microbiology and Immunology, University of Rochester School of
Medicine and Dentistry, Rochester, New York, USA.

Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen.
Herein, we demonstrate that the PA1006 protein is critical for all nitrate
reductase activities, growth as a biofilm in a continuous flow system, as well as
virulence in mouse burn and rat lung model systems. Microarray analysis revealed 
that ΔPA1006 cells displayed extensive alterations in gene expression including
nitrate-responsive, quorum sensing (including PQS production), and iron-regulated
genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors,
members of the TCA cycle, and Type VI Secretion System components. Phenotype
Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also
revealed a reduced ability to utilize a number of TCA cycle intermediates as well
as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these 
data indicate that the loss of PA1006 confers extensive changes in Pae
metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data
from the accompanying study, loss of PA1006 persulfuration and/or molybdenum
homeostasis are likely the cause of extensive metabolic alterations that impact
biofilm development and virulence in the ΔPA1006 mutant.

DOI: 10.1371/journal.pone.0055594 
PMCID: PMC3568122
PMID: 23409004  [Indexed for MEDLINE]


316. PLoS One. 2013;8(1):e48979. doi: 10.1371/journal.pone.0048979. Epub 2013 Jan 9.

Gene expression-based classifiers identify Staphylococcus aureus infection in
mice and humans.

Ahn SH(1), Tsalik EL, Cyr DD, Zhang Y, van Velkinburgh JC, Langley RJ, Glickman
SW, Cairns CB, Zaas AK, Rivers EP, Otero RM, Veldman T, Kingsmore SF, Lucas J,
Woods CW, Ginsburg GS, Fowler VG Jr.

Author information: 
(1)Division of Infectious Diseases and International Health, Department of
Medicine, Duke University, Durham, North Carolina, USA.

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and
uncertainty lead to treatment delays and inappropriate antibiotic use. A growing 
literature suggests the host's inflammatory response to the pathogen represents a
potential tool to improve upon current diagnostics. The hypothesis of this study 
is that the host responds differently to S. aureus than to E. coli infection in a
quantifiable way, providing a new diagnostic avenue. This study uses Bayesian
sparse factor modeling and penalized binary regression to define peripheral blood
gene-expression classifiers of murine and human S. aureus infection. The
murine-derived classifier distinguished S. aureus infection from healthy controls
and Escherichia coli-infected mice across a range of conditions (mouse and
bacterial strain, time post infection) and was validated in outbred mice
(AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects
distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC
0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus
infection share common biological pathways, allowing the murine model to classify
S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers
were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). 
The approach described here lends insight into the conserved and disparate
pathways utilized by mice and humans in response to these infections.
Furthermore, this study advances our understanding of S. aureus infection; the
host response to it; and identifies new diagnostic and therapeutic avenues.

DOI: 10.1371/journal.pone.0048979 
PMCID: PMC3541361
PMID: 23326304  [Indexed for MEDLINE]


317. Toxins (Basel). 2012 Dec 14;4(12):1517-34. doi: 10.3390/toxins4121517.

Verotoxin A subunit protects lymphocytes and T cell lines against X4 HIV
infection in vitro.

Shi PL(1), Binnington B, Sakac D, Katsman Y, Ramkumar S, Gariepy J, Kim M, Branch
DR, Lingwood C.

Author information: 
(1)Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.
peilinss@yahoo.ca

Our previous genetic, pharmacological and analogue protection studies identified 
the glycosphingolipid, Gb(3) (globotriaosylceramide, Pk blood group antigen) as a
natural resistance factor for HIV infection. Gb(3) is a B cell marker (CD77), but
a fraction of activated peripheral blood mononuclear cells (PBMCs) can also
express Gb(3). Activated PBMCs predominantly comprise CD4+ T-cells, the primary
HIV infection target. Gb(3) is the sole receptor for Escherichia coli verotoxins 
(VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A)
non-covalently associated with five smaller receptor-binding B subunits. The
effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined.
Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small
Gb(3)+/CD4+ T-cell subset was eliminated but, surprisingly, remaining CD4+ T-cell
HIV-1(IIIB) (and HIV-1(Ba-L)) susceptibility was significantly reduced. The
Gb(3)-Jurkat T-cell line was similarly protected by brief VT exposure prior to
HIV-1(IIIB) infection. The efficacy of the VT1A subunit alone confirmed receptor 
independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect.
Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by
50%. This may relate to the mechanism of action since HIV replication requires
primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated
up regulation of 30 genes. Three of the top four were histone genes, suggesting
HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement
of HIV infection, consistent with a histone-mediated mechanism. We speculate that
VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell
susceptibility.

DOI: 10.3390/toxins4121517 
PMCID: PMC3528260
PMID: 23242319  [Indexed for MEDLINE]


318. Avian Dis. 2012 Dec;56(4):732-6.

Strong concordance between transcriptomic patterns of spleen and peripheral blood
leukocytes in response to avian pathogenic Escherichia coli infection.

Sandford EE(1), Orr M, Li X, Zhou H, Johnson TJ, Kariyawasam S, Liu P, Nolan LK, 
Lamont SJ.

Author information: 
(1)Department of Animal Science, Iowa State University, Ames, IA 50011, USA.

Avian pathogenic Escherichia coli (APEC) causes morbidity in chickens and
exhibits zoonotic potential. Understanding host transcriptional responses to
infection aids the understanding of protective mechanisms and serves to inform
future colibacillosis control strategies. Transcriptomes of spleen and peripheral
blood leukocytes (PBLs) of the same individual birds in response to APEC
infection were compared to identify common response patterns and connecting
pathways. More than 100 genes in three contrasts examining pathology and
infection status were significantly differentially expressed in both tissues and 
similarly regulated. Tissue-specific differences in catalytic activity, however, 
appear between birds with mild and severe pathology responses. Early expression
differences, between birds with severe pathology and uninfected controls, in the 
mitogen-activated protein kinase pathway in PBLs precede spleen responses in the 
p53 and cytokine-cytokine receptor pathways. Tissue bianalysis is useful in
identifying genes and pathways important to the response to APEC, whose role
might otherwise be underestimated in importance.

DOI: 10.1637/10261-060512-Reg.1 
PMID: 23397846  [Indexed for MEDLINE]


319. Biotechniques. 2012 Dec;53(6):373-80. doi: 10.2144/000113937.

cDNA normalization by hydroxyapatite chromatography to enrich transcriptome
diversity in RNA-seq applications.

Vandernoot VA(1), Langevin SA, Solberg OD, Lane PD, Curtis DJ, Bent ZW, Williams 
KP, Patel KD, Schoeniger JS, Branda SS, Lane TW.

Author information: 
(1)Biotechnology and Bioengineering Department, Sandia National Laboratories,
Livermore, CA, USA.

Second-generation sequencing (SGS) has become the preferred method for RNA
transcriptome profiling of organisms and single cells. However, SGS analysis of
transcriptome diversity (including protein-coding transcripts and regulatory
non-coding RNAs) is inefficient unless the sample of interest is first depleted
of nucleic acids derived from ribosomal RNA (rRNA), which typically account for
up to 95% of total intracellular RNA content. Here we describe a novel microscale
hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and
prokaryotic high abundant rRNA species, thereby increasing sequence coverage
depth and transcript diversity across non-rRNA populations. RNA-seq analysis of
Escherichia coli K-12 and human intracellular total RNA showed that HAC-based
normalization enriched for all non-ribosomal RNA species regardless of RNA
transcript abundance or length when compared with untreated controls. Microcolumn
HAC normalization generated rRNA-depleted cDNA libraries comparable to the
well-established duplex specific nuclease (DSN) normalization and Ribo-Zero
rRNA-depletion methods, thus establishing microscale HAC as an effective, cost
saving, and non-destructive alternative normalization technique.

DOI: 10.2144/000113937 
PMID: 23227988  [Indexed for MEDLINE]


320. J Clin Microbiol. 2012 Dec;50(12):3990-8. doi: 10.1128/JCM.00990-12. Epub 2012
Oct 3.

Direct detection and genotyping of Klebsiella pneumoniae carbapenemases from
urine by use of a new DNA microarray test.

Peter H(1), Berggrav K, Thomas P, Pfeifer Y, Witte W, Templeton K, Bachmann TT.

Author information: 
(1)Division of Pathway Medicine, University of Edinburgh, Medical School,
Edinburgh, United Kingdom.

Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to
antibiotic therapy, as they confer resistance to carbapenems, which are used to
treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we
describe the development and evaluation of a DNA microarray for the detection and
genotyping of KPC genes (bla(KPC)) within a 5-h period. To test the whole assay
procedure (DNA extraction plus a DNA microarray assay) directly from clinical
specimens, we compared two commercial DNA extraction kits (the QIAprep Spin
miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the
direct DNA extraction from urine samples (dilution series spiked in human urine).
Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 ×
10(5) CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, 
on average, for K. pneumoniae (Norgen). This study presents, for the first time, 
the combination of a new KPC microarray with commercial sample preparation for
detecting and genotyping microbial pathogens directly from clinical specimens;
this paves the way toward tests providing epidemiological and diagnostic data,
enabling better antimicrobial stewardship.

DOI: 10.1128/JCM.00990-12 
PMCID: PMC3502996
PMID: 23035190  [Indexed for MEDLINE]


321. J Microbiol Methods. 2012 Dec;91(3):341-53. doi: 10.1016/j.mimet.2012.09.016.
Epub 2012 Sep 27.

Development of bacteria identification array to detect lactobacilli in Thai
fermented sausage.

Rungrassamee W(1), Tosukhowong A, Klanchui A, Maibunkaew S, Plengvidhya V,
Karoonuthaisiri N.

Author information: 
(1)Microarray Laboratory, National Center for Genetic Engineering and
Biotechnology (BIOTEC), Khlong Luang, Pathum Thani, Thailand.
wanilada.run@biotec.or.th

To improve the quality and safety of food products, there is a need in the food
industry for a reliable method for simultaneously monitoring multiple bacterial
strains. Microarray technology is a high-throughput screening approach that can
provide an alternative for bacteria detection. A total of 164 bacteria-specific
probes were designed from 16S rRNA gene sequences to target 12 bacteria species, 
including lactic acid bacteria and selected food pathogens. After fabrication
onto aminosilane-coated slides, hybridization conditions of the array were
optimized for high specificity and signal intensities. The array was applied to
detect 12 bacteria individually and was specific to all (Lactobacillus plantarum 
group, L. fermentum, L. brevis, L. delbrueckii, L. casei, L. sakei, Escherichia
coli, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes)
except L. animalis. Multiplex detection using mixed bacteria populations was
evaluated and accurate detection was obtained. The feasibility of using the array
to detect the target bacteria in food was evaluated with Thai fermented sausages 
(Nham). Meat samples were collected on days 2, 3 and 7 after natural
fermentation, L. plantarum-inoculated fermentation and L. brevis-inoculated
fermentation before applying to the array. The naturally-fermented Nham contained
L. sakei, L. delbrueckii, L. plantarum and L. fermentum. The L.
plantarum-inoculated Nham showed a similar lactic acid bacteria population but
the positive signal level for L. plantarum was higher than with natural
fermentation. The L. brevis-inoculated Nham contained L. brevis, L. plantarum, L.
delbrueckii and L. fermentum. The array was used to monitor bacteria population
dynamics during the fermentation process. The naturally-fermented and L.
brevis-inoculated samples showed lower positive signal levels of L. plantarum on 
day 2, but signals gradually increased on days 3 and 7 of the fermentation. In
contrast, the L. plantarum-started fermentation showed a higher positive signal
level on day 2 than the natural and L. brevis-inoculated samples, and the
positive signal level remained high on days 3 and 7. The bacteria identification 
array was proven to be useful as an alternative method to detect and monitor
target bacteria populations during food fermentation.

Copyright © 2012 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.mimet.2012.09.016 
PMID: 23022427  [Indexed for MEDLINE]


322. Microbiologyopen. 2012 Dec;1(4):381-94. doi: 10.1002/mbo3.42. Epub 2012 Oct 8.

A novel regulator RcdA of the csgD gene encoding the master regulator of biofilm 
formation in Escherichia coli.

Shimada T(1), Katayama Y, Kawakita S, Ogasawara H, Nakano M, Yamamoto K, Ishihama
A.

Author information: 
(1)Department of Frontier Bioscience and Research Center for Micro-Nano
Technology, Hosei University, Koganei, Tokyo, 184-8584, Japan.

The FixJ/LuxR family transcription factor CsgD is a master regulator of biofilm
formation in Escherichia coli. Previously, we identified more than 10
transcription factors that participate in regulation of the csgD promoter. After 
genomic SELEX screening of regulation targets, an uncharacterized TetR-type
transcription factor YbjK was found to be involved in regulation of the csgD
promoter. In addition, a number of stress-response genes were found to be under
the direct control of YbjK. Taken together, we propose to rename it to RcdA
(regulator of csgD). One unique feature of RcdA is its mode of DNA binding. Gel
shift, DNase-I footprinting, and atomic force microscopic (AFM) analyses
indicated that RcdA is a DNA-binding protein with a high level of cooperativity, 
with which it covers the entire surface of probe DNA through protein-protein
interaction and moreover it induces the formation of aggregates of DNA-RcdA
complexes.

© 2012 The Authors. Published by Blackwell Publishing Ltd.

DOI: 10.1002/mbo3.42 
PMCID: PMC3535384
PMID: 23233451  [Indexed for MEDLINE]


323. J Biol Chem. 2012 Nov 9;287(46):38812-23. doi: 10.1074/jbc.M112.406173. Epub 2012
Sep 25.

Copper starvation-inducible protein for cytochrome oxidase biogenesis in
Bradyrhizobium japonicum.

Serventi F(1), Youard ZA, Murset V, Huwiler S, Bühler D, Richter M, Luchsinger R,
Fischer HM, Brogioli R, Niederer M, Hennecke H.

Author information: 
(1)Institute of Microbiology, Eidgenössische Technische Hochschule Zürich (ETH
Zürich), Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.

Microarray analysis of Bradyrhizobium japonicum grown under copper limitation
uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as
an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and 
PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic
membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered
in other bacteria, namely PCu(A)C and YcnJ, where they play a role in cytochrome 
oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE
operon led to a pleiotropic phenotype, including defects in the aa(3)-type
cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. 
Complementation analyses revealed that, under our assay conditions, the tested
functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE.
The B. japonicum genome harbors a second pcuC-like gene (blr7088), which,
however, did not functionally replace the mutated pcuC. The PcuC protein was
overexpressed in Escherichia coli, purified to homogeneity, and shown to bind
Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His(79),
Met(90), His(113), and Met(115) by alanine perturbed copper binding. This
corroborates the previously purported role of this protein as a periplasmic
copper chaperone for the formation of the Cu(A) center on the aa(3)-type
cytochrome oxidase. In addition, we provide evidence that PcuC and the copper
chaperone ScoI are important for the symbiotically essential, Cu(A)-free
cbb(3)-type cytochrome oxidase specifically in endosymbiotic bacteroids of
soybean root nodules, which could explain the symbiosis-defective phenotype of
the pcuC and scoI mutants.

DOI: 10.1074/jbc.M112.406173 
PMCID: PMC3493923
PMID: 23012364  [Indexed for MEDLINE]


324. Anal Chem. 2012 Nov 6;84(21):8966-9. doi: 10.1021/ac302902s. Epub 2012 Oct 22.

Aptamer-based viability impedimetric sensor for bacteria.

Labib M, Zamay AS, Kolovskaya OS, Reshetneva IT, Zamay GS, Kibbee RJ, Sattar SA, 
Zamay TN, Berezovski MV.

The development of an aptamer-based viability impedimetric sensor for bacteria
(AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella
typhimurium were selected via the cell-systematic evolution of ligands by
exponential enrichment (SELEX) technique. Twelve rounds of selection were
performed; each comprises a positive selection step against viable S. typhimurium
and a negative selection step against heat killed S. typhimurium and a mixture of
related pathogens, including Salmonella enteritidis, Escherichia coli,
Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii to ensure
the species specificity of the selected aptamers. The DNA sequence showing the
highest binding affinity to the bacteria was further integrated into an
impedimetric sensor via self-assembly onto a gold nanoparticle-modified
screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly
selective and can successfully detect S. typhimurium down to 600 CFU mL(-1)
(equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from
other Salmonella species, including S. enteritidis and S. choleraesuis. This
report is envisaged to open a new venue for the aptamer-based viability sensing
of a variety of microorganisms, particularly viable but nonculturable (VBNC)
bacteria, using a rapid, economic, and label-free electrochemical platform.

DOI: 10.1021/ac302902s 
PMID: 23075417  [Indexed for MEDLINE]


325. J Biol Chem. 2012 Nov 2;287(45):37926-38. doi: 10.1074/jbc.M112.386284. Epub 2012
Sep 17.

Activation of NF-κB protein prevents the transition from juvenile ovary to testis
and promotes ovarian development in zebrafish.

Pradhan A(1), Khalaf H, Ochsner SA, Sreenivasan R, Koskinen J, Karlsson M,
Karlsson J, McKenna NJ, Orbán L, Olsson PE.

Author information: 
(1)Department of Biology, Örebro Life Science Center, School of Science and
Technology, Örebro University, SE-701 82 Örebro, Sweden.

Testis differentiation in zebrafish involves juvenile ovary to testis
transformation initiated by an apoptotic wave. The molecular regulation of this
transformation process is not fully understood. NF-κB is activated at an early
stage of development and has been shown to interact with steroidogenic factor-1
in mammals, leading to the suppression of anti-Müllerian hormone (Amh) gene
expression. Because steroidogenic factor-1 and Amh are important for proper
testis development, NF-κB-mediated induction of anti-apoptotic genes could,
therefore, also play a role in zebrafish gonad differentiation. The aim of this
study was to examine the potential role of NF-κB in zebrafish gonad
differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli
activated the NF-κB pathways and resulted in an increased ratio of females from
30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed
elevated expression of NF-κB-regulated genes. To confirm the involvement of
NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium
deoxycholate, a known inducer of NF-κB or NF-κB activation inhibitor (NAI).
Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and
resulted in an increased proportion of females from 25 to 45%, whereas the
inhibition of NF-κB using NAI resulted in a decrease in females from 45 to 20%.
This study provides proof for an essential role of NF-κB in gonadal
differentiation of zebrafish and represents an important step toward the complete
understanding of the complicated process of sex differentiation in this species
and possibly other cyprinid teleosts as well.

DOI: 10.1074/jbc.M112.386284 
PMCID: PMC3488064
PMID: 22988238  [Indexed for MEDLINE]


326. J Appl Microbiol. 2012 Nov;113(5):1076-86. doi: 10.1111/j.1365-2672.2012.05412.x.
Epub 2012 Aug 20.

Gene expression profiling of Escherichia coli in response to interactions with
the lettuce rhizosphere.

Hou Z(1), Fink RC, Black EP, Sugawara M, Zhang Z, Diez-Gonzalez F, Sadowsky MJ.

Author information: 
(1)Department of Food Science and Nutrition, University of Minnesota, St. Paul,
MN, USA.

AIMS: The objective of this study was to examine transcriptional changes in
Escherichia coli when the bacterium was growing in the lettuce rhizoshpere.
METHODS AND RESULTS: A combination of microarray analyses, colonization assays
and confocal microscopy was used to gain a more complete understanding of
bacterial genes involved in the colonization and growth of E. coli K12 in the
lettuce root rhizosphere using a novel hydroponic assay system. After 3 days of
interaction with lettuce roots, E. coli genes involved in protein synthesis,
stress responses and attachment were up-regulated. Mutants in curli production
(crl, csgA) and flagella synthesis (fliN) had a reduced capacity to attach to
roots as determined by bacterial counts and by confocal laser scanning
microscopy.
CONCLUSIONS: This study indicates that E. coli K12 has the capability to colonize
lettuce roots by using attachment genes and can readily adapt to the rhizosphere 
of lettuce plants.
SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study show curli production
and biofilm modulation genes are important for rhizosphere colonization and may
provide useful targets to disrupt this process. Further studies using pathogenic 
strains will provide additional information about lettuce-E. coli interactions.

© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied
Microbiology.

DOI: 10.1111/j.1365-2672.2012.05412.x 
PMID: 22830299  [Indexed for MEDLINE]


327. Transcription. 2012 Nov-Dec;3(6):300-4. doi: 10.4161/trns.21903. Epub 2012 Nov 1.

A new way to start: nanoRNA-mediated priming of transcription initiation.

Nickels BE(1).

Author information: 
(1)Waksman Institute and Department of Genetics, Rutgers University, Piscataway, 
NJ, USA. bnickels@waksman.rutgers.edu

A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs,
species termed "nanoRNAs," to prime transcription initiation in Escherichia coli.
Priming of transcription initiation with nanoRNAs represents a previously
undocumented component of transcription start site selection and gene expression.

DOI: 10.4161/trns.21903 
PMCID: PMC3630185
PMID: 23117822  [Indexed for MEDLINE]


328. Bioconjug Chem. 2012 Oct 17;23(10):2121-8. doi: 10.1021/bc300333a. Epub 2012 Sep 
28.

Development of oligonucleotide microarrays onto Si-based surfaces via thioether
linkage mediated by UV irradiation.

Escorihuela J(1), Bañuls MJ, Puchades R, Maquieira Á.

Author information: 
(1)Centro de Reconocimiento Molecular y Desarrollo Tecnológico, Departamento de
Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia,
Spain.

Selective covalent immobilization of thiolated oligonucleotides onto an
epoxy-functionalized silicon-substrate can be achieved via light radiation (365
nm). Following this approach, thiol-modified oligonucleotide probes were
covalently attached as microarrays, reaching an immobilization density of 2.5
pmol·cm(-2), with a yield of 53%. The developed method presents the advantages of
spatially controlled probe anchoring (by means of using a photomask), direct
attachment without using cross-linkers, and short irradiation times (10 min).
Hybridization efficiencies up to 65%, with full complementary strands, were
reached. The approach was evaluated by scoring single nucleotide polymorphisms
with a discrimination ratio around 15. Moreover, sensitive and selective
detection of bacterial Escherichia coli was demonstrated.

DOI: 10.1021/bc300333a 
PMID: 22992053  [Indexed for MEDLINE]


329. Chemistry. 2012 Oct 15;18(42):13320-30. doi: 10.1002/chem.201201662. Epub 2012
Sep 20.

Synthesis of deoxynucleoside triphosphates that include proline, urea, or
sulfonamide groups and their polymerase incorporation into DNA.

Hollenstein M(1).

Author information: 
(1)Department of Chemistry & Biochemistry, University of Bern, Freiestrasse 3,
3012 Bern, Switzerland. hollenstein@dcb.unibe.ch

To expand the chemical array available for DNA sequences in the context of in
vitro selection, I present herein the synthesis of five nucleoside triphosphate
analogues containing side chains capable of organocatalysis. The synthesis
involved the coupling of L-proline-containing residues (dU(tP)TP and dU(cP)TP), a
dipeptide (dU(FP)TP), a urea derivative (dU(Bpu)TP), and a sulfamide residue
(dU(Bs)TP) to a suitably protected common intermediate, followed by
triphosphorylation. These modified dNTPs were shown to be excellent substrates
for the Vent (exo(-)) and Pwo DNA polymerases, as well as the Klenow fragment of 
E. coli DNA polymerase I, although they were only acceptable substrates for the
9°N(m) polymerase. All of the modified dNTPs, with the exception of dU(Bpu)TP,
were readily incorporated into DNA by the polymerase chain reaction (PCR).
Modified oligonucleotides efficiently served as templates for PCR for the
regeneration of unmodified DNA. Thermal denaturation experiments showed that
these modifications are tolerated in the major groove. Overall, these heavily
modified dNTPs are excellent candidates for SELEX.

Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI: 10.1002/chem.201201662 
PMID: 22996052  [Indexed for MEDLINE]


330. Anal Chem. 2012 Oct 2;84(19):8114-7. doi: 10.1021/ac302217u. Epub 2012 Sep 14.

Aptamer-based impedimetric sensor for bacterial typing.

Labib M(1), Zamay AS, Kolovskaya OS, Reshetneva IT, Zamay GS, Kibbee RJ, Sattar
SA, Zamay TN, Berezovski MV.

Author information: 
(1)Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario
K1N 6N5, Canada.

The development of an aptamer-based impedimetric sensor for typing of bacteria
(AIST-B) is presented. Highly specific DNA aptamers to Salmonella enteritidis
were selected via Cell-SELEX technique. Twelve rounds of selection were
performed; each comprises a positive selection step against S. enteritidis and a 
negative selection step against a mixture of related pathogens, including
Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, and Citrobacter freundii, to ensure the species-specificity of the
selected aptamers. After sequencing of the pool showing the highest binding
affinity to S. enteritidis, a DNA sequence of high affinity to the bacteria was
integrated into an impedimetric sensor via self-assembly onto a gold
nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably,
this aptasensor is highly selective and can successfully detect S. enteritidis
down to 600 CFU mL(-1) (equivalent to 18 CFU in 30 μL assay volume) in 10 min and
distinguish it from other Salmonella species, including S. typhimurium and S.
choleraesuis. This report is envisaged to open a new venue for the aptamer-based 
typing of a variety of microorganisms using a rapid, economic, and label-free
electrochemical platform.

DOI: 10.1021/ac302217u 
PMID: 22971146  [Indexed for MEDLINE]


331. Anim Genet. 2012 Oct;43(5):525-34. doi: 10.1111/j.1365-2052.2011.02287.x. Epub
2011 Nov 7.

Microarray analysis of differential gene expression in sensitive and resistant
pig to Escherichia coli F18.

Bao WB(1), Ye L, Pan ZY, Zhu J, Du ZD, Zhu GQ, Huang XG, Wu SL.

Author information: 
(1)College of Animal Science and Technology, Yangzhou University, Yangzhou
225009, Jiangsu Province, China.

In this study, Agilent two-colour microarray-based gene expression profiling was 
used to detect differential gene expression in duodenal tissues collected from
eight full-sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity
and resistance to Escherichia coli F18). Using a two-fold change minimum
threshold, we found 18 genes that were differentially expressed (10 up-regulated 
and eight down-regulated) between the sensitive and resistant animal groups. Our 
gene ontology analysis revealed that these differentially expressed genes are
involved in a variety of biological processes, including immune responses,
extracellular modification (e.g. glycosylation), cell adhesion and signal
transduction, all of which are related to the anabolic metabolism of glycolipids,
as well as to inflammation- and immune-related pathways. Based on the genes
identified in the screen and the pathway analysis results, real-time PCR was used
to test the involvement of ST3GAL1 and A genes (of glycolipid-related pathways), 
SLA-1 and SLA-3 genes (of inflammation- and immune-related pathways), as well as 
the differential genes FUT1, TAP1 and SLA-DQA. Subsequently, real-time PCR was
performed to validate seven differentially expressed genes screened out by the
microarray approach, and sufficient consistency was observed between the two
methods. The results support the conclusion that these genes are related to the
E. coli F18 receptor and susceptibility to E. coli F18.

© 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for
Animal Genetics.

DOI: 10.1111/j.1365-2052.2011.02287.x 
PMID: 22497274  [Indexed for MEDLINE]


332. Mol Biosyst. 2012 Oct;8(10):2593-604. doi: 10.1039/c2mb25069a.

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under
nitrate conditions using different levels of omics data.

Toya Y(1), Nakahigashi K, Tomita M, Shimizu K.

Author information: 
(1)Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
ytoya@sfc.keio.ac.jp

It is of practical interest to investigate the effect of nitrates on bacterial
metabolic regulation of both fermentation and energy generation, as compared to
aerobic and anaerobic growth without nitrates. Although gene level regulation has
previously been studied for nitrate assimilation, it is important to understand
this metabolic regulation in terms of global regulators. In the present study,
therefore, we measured gene expression using DNA microarrays, intracellular
metabolite concentrations using CE-TOFMS, and metabolic fluxes using the
(13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory
gene for anoxic response control, ArcA) mutant to compare the metabolic state
under nitrate conditions to that under aerobic and anaerobic conditions without
nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In
wild-type, although the measured metabolite concentrations changed very little
among the three culture conditions, the TCA cycle and the pentose phosphate
pathway fluxes were significantly different under each condition. These results
suggested that the ATP production rate was 29% higher under nitrate conditions
than that under anaerobic conditions, whereas the ATP production rate was 10%
lower than that under aerobic conditions. The flux changes in the TCA cycle were 
caused by changes in control at the gene expression level. In ΔarcA mutant, the
TCA cycle flux was significantly increased (4.4 times higher than that of the
wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio
increased approximately two-fold compared to that of the wild-type strain.

DOI: 10.1039/c2mb25069a 
PMID: 22790675  [Indexed for MEDLINE]


333. BMC Genomics. 2012 Sep 25;13:509. doi: 10.1186/1471-2164-13-509.

Transcriptomic analysis of peritoneal cells in a mouse model of sepsis:
confirmatory and novel results in early and late sepsis.

Bhatty M(1), Fan R, Muir WM, Pruett SB, Nanduri B.

Author information: 
(1)Department of Basic Sciences, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762, USA.

BACKGROUND: The events leading to sepsis start with an invasive infection of a
primary organ of the body followed by an overwhelming systemic response.
Intra-abdominal infections are the second most common cause of sepsis. Peritoneal
fluid is the primary site of infection in these cases. A microarray-based
approach was used to study the temporal changes in cells from the peritoneal
cavity of septic mice and to identify potential biomarkers and therapeutic
targets for this subset of sepsis patients.
RESULTS: We conducted microarray analysis of the peritoneal cells of mice
infected with a non-pathogenic strain of Escherichia coli. Differentially
expressed genes were identified at two early (1 h, 2 h) and one late time point
(18 h). A multiplexed bead array analysis was used to confirm protein expression 
for several cytokines which showed differential expression at different time
points based on the microarray data. Gene Ontology based hypothesis testing
identified a positive bias of differentially expressed genes associated with
cellular development and cell death at 2 h and 18 h respectively. Most
differentially expressed genes common to all 3 time points had an immune response
related function, consistent with the observation that a few bacteria are still
present at 18 h.
CONCLUSIONS: Transcriptional regulators like PLAGL2, EBF1, TCF7, KLF10 and SBNO2,
previously not described in sepsis, are differentially expressed at early and
late time points. Expression pattern for key biomarkers in this study is similar 
to that reported in human sepsis, indicating the suitability of this model for
future studies of sepsis, and the observed differences in gene expression suggest
species differences or differences in the response of blood leukocytes and
peritoneal leukocytes.

DOI: 10.1186/1471-2164-13-509 
PMCID: PMC3621573
PMID: 23009705  [Indexed for MEDLINE]


334. Vet Microbiol. 2012 Sep 14;159(1-2):163-70. doi: 10.1016/j.vetmic.2012.03.033.
Epub 2012 Mar 30.

Phylogroup and lpfA influence epithelial invasion by mastitis associated
Escherichia coli.

Dogan B(1), Rishniw M, Bruant G, Harel J, Schukken YH, Simpson KW.

Author information: 
(1)Department of Clinical Sciences, College of Veterinary Medicine, Cornell
University, Ithaca, NY, USA. be16@cornell.edu

Escherichia coli infection is one of the most common causes of bovine mastitis in
well managed dairies. Although E. coli infections are usually transient, E. coli 
can also cause persistent intramammary infections. We sought to determine whether
E. coli isolates recovered from either transient or persistent intramammary
infections differed both genetically and in their ability to invade mammary
epithelial cells. E. coli isolates from transient (EC(trans), n=16) and
persistent (EC(pers), n=12) mastitis cases were compared for differences in
overall genotype, virulence genes, serotype, phylogroup (A, B1, B2, D), and
invasion of bovine mammary epithelial cells, MAC-T by microarray analysis,
suppressive subtractive hybridization, PCR and gentamicin protection assays.
EC(trans) and EC(pers) were diverse in overall genotype and serotype, and were
predominantly of phylogroups A and B1. Both EC(trans) and EC(pers) contained
genes encoding type II, IV and VI secretion systems, long polar fimbriae (lpfA)
and iron acquisition, and lacked genes associated with virulence in diarrheagenic
E. coli. EC(trans) had fewer virulence genes than EC(pers) (p<0.05), but no
individual virulence genes were unique to either group. In phylogroup A, EC(pers)
were more invasive than EC(trans) (p<0.05), but no difference was observed
between them in phylogroup B1. Enhanced epithelial cell invasion was associated
with lpfA (p<0.05). Our findings indicate that a genetically diverse group of E. 
coli is associated with transient and persistent mastitis. We did not identify a 
set of bacterial genes to account for phenotypic differences. However, we found
that mastitis phenotype, phylogroup and presence of lpfA were associated with the
ability to invade cultured bovine mammary epithelial cells.

Published by Elsevier B.V.

DOI: 10.1016/j.vetmic.2012.03.033 
PMID: 22510704  [Indexed for MEDLINE]


335. J Clin Invest. 2012 Sep;122(9):3248-59. doi: 10.1172/JCI61216. Epub 2012 Aug 6.

Notch1 counteracts WNT/β-catenin signaling through chromatin modification in
colorectal cancer.

Kim HA(1), Koo BK, Cho JH, Kim YY, Seong J, Chang HJ, Oh YM, Stange DE, Park JG, 
Hwang D, Kong YY.

Author information: 
(1)Department of Biological Sciences, College of Natural Sciences, Seoul National
University, Seoul, Republic of Korea.

Crosstalk between the Notch and wingless-type MMTV integration site (WNT)
signaling pathways has been investigated for many developmental processes.
However, this negative correlation between Notch and WNT/β-catenin signaling
activity has been studied primarily in normal developmental and physiological
processes in which negative feedback loops for both signaling pathways are
intact. We found that Notch1 signaling retained the capability of suppressing the
expression of WNT target genes in colorectal cancers even when β-catenin
destruction by the adenomatous polyposis coli (APC) complex was disabled.
Activation of Notch1 converted high-grade adenoma into low-grade adenoma in an
Apcmin mouse colon cancer model and suppressed the expression of WNT target genes
in human colorectal cancer cells through epigenetic modification recruiting
histone methyltransferase SET domain bifurcated 1 (SETDB1). Extensive microarray 
analysis of human colorectal cancers also showed a negative correlation between
the Notch1 target gene, Notch-regulated ankyrin repeat protein 1 (NRARP), and WNT
target genes. Notch is known to be a strong promoter of tumor initiation, but
here we uncovered an unexpected suppressive role of Notch1 on WNT/β-catenin
target genes involved in colorectal cancer.

DOI: 10.1172/JCI61216 
PMCID: PMC3428081
PMID: 22863622  [Indexed for MEDLINE]


336. Nat Biotechnol. 2012 Sep;30(9):868-875. doi: 10.1038/nbt.2316.

Combinatorial discovery of polymers resistant to bacterial attachment.

Hook AL(#)(1), Chang CY(#)(2), Yang J(#)(1), Luckett J(2), Cockayne A(2),
Atkinson S(2), Mei Y(3), Bayston R(4), Irvine DJ(5), Langer R(3)(6)(7), Anderson 
DG(3)(6)(7), Williams P(2), Davies MC(1), Alexander MR(1).

Author information: 
(1)Laboratory of Biophysics and Surface Analysis, University of Nottingham,
Nottingham, UK.
(2)School of Molecular Medical Sciences, University of Nottingham, Nottingham,
UK.
(3)Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts, USA.
(4)School of Clinical Sciences, Queen's Medical Centre, University of Nottingham,
Nottingham, UK.
(5)School of Chemistry, University of Nottingham, Nottingham, UK.
(6)David H. Koch Institute for Integrative Cancer Research, Massachusetts
Institute of Technology, Cambridge, Massachusetts, USA.
(7)Harvard-MIT Division of Health Science Technology, Massachusetts Institute of 
Technology, Cambridge, Massachusetts, USA.
(#)Contributed equally

Erratum in
    Nat Biotechnol. 2014 Jun;32(6):592.

Bacterial attachment and subsequent biofilm formation pose key challenges to the 
optimal performance of medical devices. In this study, we determined the
attachment of selected bacterial species to hundreds of polymeric materials in a 
high-throughput microarray format. Using this method, we identified a group of
structurally related materials comprising ester and cyclic hydrocarbon moieties
that substantially reduced the attachment of pathogenic bacteria (Pseudomonas
aeruginosa, Staphylococcus aureus and Escherichia coli). Coating silicone with
these 'hit' materials achieved up to a 30-fold (96.7%) reduction in the surface
area covered by bacteria compared with a commercial silver hydrogel coating in
vitro, and the same material coatings were effective at reducing bacterial
attachment in vivo in a mouse implant infection model. These polymers represent a
class of materials that reduce the attachment of bacteria that could not have
been predicted to have this property from the current understanding of
bacteria-surface interactions.

DOI: 10.1038/nbt.2316 
PMCID: PMC3796337
PMID: 22885723  [Indexed for MEDLINE]


337. Int J Cancer. 2012 Aug 1;131(3):673-82. doi: 10.1002/ijc.26419. Epub 2011 Oct 23.

Multicenter study identified molecular blood-born protein signatures for Wilms
Tumor.

Schmitt J(1), Heisel S, Keller A, Leidinger P, Ludwig N, Habel N, Furtwängler R, 
Nourkami-Tutdibi N, Wegert J, Grundy P, Gessler M, Graf N, Lenhof HP, Meese E.

Author information: 
(1)Department of Human Genetics, Medical School, Saarland University, 66421
Homburg, Germany. jana.schmitt@uniklinikum-saarland.de

Wilms Tumor (WT) is the most common renal childhood tumor. Recently, we reported 
a cDNA microarray expression pattern that varied between WTs with different risk 
histology. Since the Societé Internationale d'Oncologie Pédiatrique (SIOP) in
Europe initiates treatment without a histological confirmation, it is important
to identify blood-born markers that indicate WT development. In a multicenter
study, we established an autoantibody signature by using an array with 1,827
recombinant E. coli clones. This array was screened with sera of patients with WT
recruited by SIOP or the Children's Oncology Group (COG). We report an extended
number of antigens that are reactive with autoantibodies present in sera from
patients with WT. We established an autoantibody signature that separates
untreated patients with WT recruited in SIOP from non-WT controls with a
specificity of 0.83 and a sensitivity of 0.82 at standard deviations of 0.02 and 
0.04, respectively. Likewise, patients recruited in the COG in the United States 
were separated from the controls with an accuracy of 0.83 at a standard deviation
of 0.02. Proteins that were most significant include zinc finger proteins (e.g., 
ZFP 346), ribosomal proteins and the protein fascin that has been associated with
various types of cancer including renal cell carcinoma. Our study provides first 
evidence for autoantibody signatures for WTs and suggests that these may be most 
informative before chemotherapy. We present the first multicenter study of
autoantibody signatures in patients with WT. We established an autoantibody
signature that separates patients with WT from controls.

Copyright © 2011 UICC.

DOI: 10.1002/ijc.26419 
PMID: 21913182  [Indexed for MEDLINE]


338. Res Vet Sci. 2012 Aug;93(1):23-7. doi: 10.1016/j.rvsc.2011.06.016. Epub 2011 Jul 
12.

Virulence genes in bla(CTX-M) Escherichia coli isolates from chickens and humans.

Randall L(1), Wu G, Phillips N, Coldham N, Mevius D, Teale C.

Author information: 
(1)Veterinary Laboratories Agency (Weybridge), Department of Bacteriology, New
Haw, Addlestone, Surrey KT15 3NB, United Kingdom. l.randall@vla.defra.gsi.gov.uk

The aim of this study was to determine the presence of virulence genes in
isolates of CTX-M Escherichia coli from diseased chickens, from healthy chickens 
and from urinary tract infections in people. Three CTX-M E. coli strains from
three different instances of disease in poultry (two of which were E. coli
related) were tested for bla(CTX-M) sequence type and replicon type.
Additionally, they were tested for the presence of 56 virulence genes (encoding
fimbriae, adhesins, toxins, microcins and iron acquisition genes) using a
micro-array. Results were compared to the virulence genes present in isolates
from 26 healthy chickens and from 10 people with urinary tract infections. All
genes found in isolates from diseased birds, including the astA (heat stable
toxin) and tsh (temperature sensitive haemagglutinin) genes which have previously
been associated with colibacillosis in chickens, were also present in isolates
from healthy birds. However, 6/10 of the virulence genes found were exclusive to 
isolates from humans. Genes exclusive to chicken isolates included ireA
(sidephore receptor), lpfA (long polar fimbriae), mchF (microcin transporter
protein) and tsh whilst genes exclusive to human isolates included ctdB
(cytolethal distending toxin), nfaE (non-fimbrial adhesion), senB (plasmid
encoded enterotoxin) and toxB (toxin B). The results support previous findings
that CTX-M E. coli strains in chickens are generally different from those causing
disease in humans, but genes such as astA and tsh in isolates from diseased birds
with colisepticaemia were also present in isolates from healthy birds.

Crown Copyright © 2011. Published by Elsevier India Pvt Ltd. All rights reserved.

DOI: 10.1016/j.rvsc.2011.06.016 
PMID: 21752412  [Indexed for MEDLINE]


339. J Biomed Sci. 2012 Jul 31;19:69. doi: 10.1186/1423-0127-19-69.

Whole blood-derived microRNA signatures in mice exposed to lipopolysaccharides.

Hsieh CH(1), Rau CS, Jeng JC, Chen YC, Lu TH, Wu CJ, Wu YC, Tzeng SL, Yang JC.

Author information: 
(1)Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung
Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan.

BACKGROUND: Lipopolysaccharide (LPS) is recognized as the most potent microbial
mediator presaging the threat of invasion of Gram-negative bacteria that
implicated in the pathogenesis of sepsis and septic shock. This study was
designed to examine the microRNA (miRNA) expression in whole blood from mice
injected with intraperitoneal LPS.
METHODS: C57BL/6 mice received intraperitoneal injections of varying
concentrations (range, 10-1000 μg) of LPS from different bacteria, including
Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella
enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS
injection. Whole blood samples were obtained and tissues, including lung, brain, 
liver, and spleen, were harvested for miRNA expression analysis using an miRNA
array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in
the whole blood of C57BL/6 and Tlr4(-/-) mice injected with LPS was quantified
using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice 
injected with lipoteichoic acid (LTA) from Staphylococcus aureus.
RESULTS: Following LPS injection, a significant increase of 15 miRNAs was
observed in the whole blood. Among them, only 3 miRNAs showed up-regulated
expression in the lung, but no miRNAs showed a high expression level in the other
examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b,
miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on
real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h
and persisted for at least 6 h. Exposure to LPS from different bacteria did not
induce significantly different expression of these miRNA targets. Additionally,
significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and
miR-107 were observed in whole blood of Tlr4(-/-) mice. In contrast, LTA exposure
induced moderate expression of miR-451 but not of the other 7 miRNA targets.
CONCLUSIONS: We identified a specific whole blood-derived miRNA signature in mice
exposed to LPS, but not to LTA, from different gram-negative bacteria. These
whole blood-derived miRNAs are promising as biomarkers for LPS exposure.

DOI: 10.1186/1423-0127-19-69 
PMCID: PMC3419134
PMID: 22849760  [Indexed for MEDLINE]


340. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12740-5. doi:
10.1073/pnas.1205124109. Epub 2012 Jul 16.

Large mutational target size for rapid emergence of bacterial persistence.

Girgis HS(1), Harris K, Tavazoie S.

Author information: 
(1)Department of Molecular Biology, Princeton University, Princeton, NJ 08544,
USA.

Phenotypic heterogeneity displayed by a clonal bacterial population permits a
small fraction of cells to survive prolonged exposure to antibiotics. Although
first described over 60 y ago, the molecular mechanisms underlying this behavior,
termed persistence, remain largely unknown. To systematically explore the genetic
basis of persistence, we selected a library of transposon-mutagenized Escherichia
coli cells for survival to multiple rounds of lethal ampicillin exposure.
Application of microarray-based genetic footprinting revealed a large number of
loci that drastically elevate persistence frequency through null mutations and
domain disruptions. In one case, the C-terminal disruption of methionyl-tRNA
synthetase (MetG) results in a 10,000-fold higher persistence frequency than wild
type. We discovered a mechanism by which null mutations in transketolase A (tktA)
and glycerol-3-phosphate (G3P) dehydrogenase (glpD) increase persistence through 
metabolic flux alterations that increase intracellular levels of the
growth-inhibitory metabolite methylglyoxal. Systematic double-mutant analyses
revealed the genetic network context in which such persistent mutants function.
Our findings reveal a large mutational target size for increasing persistence
frequency, which has fundamental implications for the emergence of antibiotic
tolerance in the clinical setting.

DOI: 10.1073/pnas.1205124109 
PMCID: PMC3411964
PMID: 22802628  [Indexed for MEDLINE]


341. Nat Methods. 2012 Jul 15;9(8):796-804. doi: 10.1038/nmeth.2016.

Wisdom of crowds for robust gene network inference.

Marbach D(1), Costello JC, Küffner R, Vega NM, Prill RJ, Camacho DM, Allison KR; 
DREAM5 Consortium, Kellis M, Collins JJ, Stolovitzky G.

Collaborators: Aderhold A, Allison KR, Bonneau R, Camacho DM, Chen Y, Collins JJ,
Cordero F, Costello JC, Crane M, Dondelinger F, Drton M, Esposito R, Foygel R, de
la Fuente A, Gertheiss J, Geurts P, Greenfield A, Grzegorczyk M, Haury AC, Holmes
B, Hothorn T, Husmeier D, Huynh-Thu VA, Irrthum A, Kellis M, Karlebach G, Küffner
R, Lèbre S, De Leo V, Madar A, Mani S, Marbach D, Mordelet F, Ostrer H, Ouyang Z,
Pandya R, Petri T, Pinna A, Poultney CS, Prill RJ, Rezny S, Ruskin HJ, Saeys Y,
Shamir R, Sîrbu A, Song M, Soranzo N, Statnikov A, Stolovitzky G, Vega N,
Vera-Licona P, Vert JP, Visconti A, Wang H, Wehenkel L, Windhager L, Zhang Y,
Zimmer R.

Author information: 
(1)Computer Science and Artificial Intelligence Laboratory, Massachusetts
Institute of Technology, Cambridge, Massachusetts, USA.

Reconstructing gene regulatory networks from high-throughput data is a
long-standing challenge. Through the Dialogue on Reverse Engineering Assessment
and Methods (DREAM) project, we performed a comprehensive blind assessment of
over 30 network inference methods on Escherichia coli, Staphylococcus aureus,
Saccharomyces cerevisiae and in silico microarray data. We characterize the
performance, data requirements and inherent biases of different inference
approaches, and we provide guidelines for algorithm application and development. 
We observed that no single inference method performs optimally across all data
sets. In contrast, integration of predictions from multiple inference methods
shows robust and high performance across diverse data sets. We thereby
constructed high-confidence networks for E. coli and S. aureus, each comprising
~1,700 transcriptional interactions at a precision of ~50%. We experimentally
tested 53 previously unobserved regulatory interactions in E. coli, of which 23
(43%) were supported. Our results establish community-based methods as a powerful
and robust tool for the inference of transcriptional gene regulatory networks.

DOI: 10.1038/nmeth.2016 
PMCID: PMC3512113
PMID: 22796662  [Indexed for MEDLINE]


342. J Proteome Res. 2012 Jul 6;11(7):3680-9. doi: 10.1021/pr300147z. Epub 2012 Jun
21.

Tissue proteomics by one-dimensional gel electrophoresis combined with label-free
protein quantification.

Vasilj A(1), Gentzel M, Ueberham E, Gebhardt R, Shevchenko A.

Author information: 
(1)Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.

Label-free methods streamline quantitative proteomics of tissues by alleviating
the need for metabolic labeling of proteins with stable isotopes. Here we detail 
and implement solutions to common problems in label-free data processing geared
toward tissue proteomics by one-dimensional gel electrophoresis followed by
liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification
pipeline showed high levels of performance in terms of duplicate reproducibility,
linear dynamic range, and number of proteins identified and quantified. When
applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we
demonstrated an 8-fold increase in the number of statistically significant
changing proteins compared to alternative approaches, including many more
previously unidentified hydrophobic proteins. Better proteome coverage and
quantification accuracy revealed molecular details of the perturbed energy
metabolism.

DOI: 10.1021/pr300147z 
PMID: 22671763  [Indexed for MEDLINE]


343. J Bacteriol. 2012 Jul;194(13):3437-47. doi: 10.1128/JB.00097-12. Epub 2012 Apr
20.

Mechanism for regulation of the putrescine utilization pathway by the
transcription factor PuuR in Escherichia coli K-12.

Nemoto N(1), Kurihara S, Kitahara Y, Asada K, Kato K, Suzuki H.

Author information: 
(1)Division of Applied Biology, Graduate School of Science and Technology, Kyoto 
Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto, Japan.

In Escherichia coli, putrescine is metabolized to succinate for use as a carbon
and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene
in the puu gene cluster encodes a transcription factor, PuuR, which has a
helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR
mutant, in which three amino acid residues in the helix-turn-helix DNA binding
motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested
that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I 
footprint analyses suggested that PuuR binds to the promoter regions of puuA and 
puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition
sites was diminished with increasing putrescine concentrations in vitro. These
results suggest that PuuR regulates the intracellular putrescine concentration by
the transcriptional regulation of genes in the Puu pathway, including puuR
itself. The puu gene cluster is found in E. coli and closely related
enterobacteria, but this gene cluster is uncommon in other bacterial groups. E.
coli and related enterobacteria may have gained the Puu pathway as an adaptation 
for survival in the mammalian intestine, an environment in which polyamines exist
at relatively high concentrations.

DOI: 10.1128/JB.00097-12 
PMCID: PMC3434745
PMID: 22522900  [Indexed for MEDLINE]


344. J Dairy Sci. 2012 Jul;95(7):3852-64. doi: 10.3168/jds.2011-5251.

Genomic analysis of between-cow variation in dermal fibroblast response to
lipopolysaccharide.

Kandasamy S(1), Kerr DE.

Author information: 
(1)Department of Animal Science, College of Agriculture and Life Sciences,
University of Vermont, Burlington 05405, USA.

The innate immune response plays a major role in defense against mastitis-causing
pathogens. Identification of existing variation in innate immune signaling among 
cows and the underlying molecular causes for the variation may help in design of 
new mastitis control strategies. The dermal fibroblast has been used as a model
cell type to explore between-cow variation in the ability of cells to produce
IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears
related to an animal's ability to respond to in vivo challenge with LPS or
Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of
cows and microarray-based genomic analysis were used to investigate the cause(s) 
for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low
response phenotype (LR(array)) and another with a high response phenotype
(HR(array)), were selected from our collection of fibroblast cultures established
from 88 cows. The LR(array) fibroblast culture produced approximately 5-fold less
IL-8 and IL-6 protein in response to 24-h LPS treatment than the HR(array)
fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2
cultures before and after 8-h LPS treatment revealed a combined LPS-induced
differential expression of 321 transcripts, indicating the robust response
capability of the fibroblast cell. Under basal conditions, the microarray
analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the
LR(array) fibroblasts compared with the HR(array) fibroblasts, and this was
associated with a marked reduction in expression of genes regulated by the
TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20,
MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was
confirmed and extended by quantitative PCR analysis (QPCR) that revealed a
33-fold lower expression of TLR4 in the LR(array) fibroblast culture. After LPS
treatment, the difference in TLR4 expression increased to almost 50-fold and was 
associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA
sequence variations were identified in the proximal 1,300-bp promoter region of
the TLR4 gene, and microarray analysis did not reveal a molecular explanation for
the reduced TLR4 expression under either basal conditions or following exposure
to LPS. The attenuated innate immune response of the LR(array) fibroblast culture
to LPS may be caused by reduced TLR4 receptor expression. Also, the primary
dermal fibroblast cells can be used to examine underlying causes for between-cow 
variations in key immune response pathways.

Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc.
All rights reserved.

DOI: 10.3168/jds.2011-5251 
PMCID: PMC4235160
PMID: 22720940  [Indexed for MEDLINE]


345. J Nanosci Nanotechnol. 2012 Jul;12(7):5138-42.

Rapid detection of food pathogens using RNA aptamers-immobilized slide.

Maeng JS(1), Kim N, Kim CT, Han SR, Lee YJ, Lee SW, Lee MH, Cho YJ.

Author information: 
(1)Bio-Nanotechnology Research Center Korea Food Research Institute, Songnam
463-746, Korea.

The purpose of this study was to develop a simple and rapid detection system for 
foodborne bacteria, which consisted of an optical microscope and its slide chip
with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA
aptamers were built by the method of SELEX (systematic evolution of ligands by
exponential enrichment) for components of cell wall, LPS (lipopolysaccharide)
from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell
membrane protein of OmpC from Salmonella typhimurium, respectively. These
aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to
be immobilized on silver surface which was, in advance, fabricated on glass
slide, using a spin-coating method. To confirm that each aptamers retained its
specific binding activities to their antigenic live bacteria, microscopic view of
bound cells immobilized on silver film were observed. Furthermore, we observed
the fluorescence-emitting bacteria-aptamer complex immobilized on silver film
after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16
dT-linker molecules. As a result, the RNA aptamers-immobilized slide system
developed in this study was a useful new tool to rapidly monitor individual food 
pathogens.


PMID: 22966534  [Indexed for MEDLINE]


346. Mol Biotechnol. 2012 Jul;51(3):283-8. doi: 10.1007/s12033-011-9466-7.

Genetic characterization of Escherichia coli O157:H7 strains isolated from the
one-humped camel (Camelus dromedarius) by using microarray DNA technology.

Salehi TZ(1), Tonelli A, Mazza A, Staji H, Badagliacca P, Tamai IA, Jamshidi R,
Harel J, Lelli R, Masson L.

Author information: 
(1)Department of Microbiology, Faculty of Veterinary Medicine, University of
Tehran, Gharib Avenue, Azadi Street 14155-6453, Tehran, Iran.

From the Camelidae family members, several serotypes of Escherichia coli (E.
coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal
samples. To date Shiga toxin-producing E. coli (STEC) strains have never been
typed in one-humped camel (Camelus dromedarius). In the present study, two E.
coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence 
gene profiles were determined using a custom E. coli virulence DNA microarray,
composed of 70-mer oligonucleotide probes targeting 264 virulence or related
genes of known E. coli pathotypes. Both strains displayed positive hybridization 
signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA,
espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen
specific probe, various virulence plasmid (pO157) probes like katP in addition to
other accessory virulence genes characterized in STEC.

DOI: 10.1007/s12033-011-9466-7 
PMID: 22081366  [Indexed for MEDLINE]


347. J Immunol. 2012 Jun 15;188(12):6300-8. doi: 10.4049/jimmunol.1200256. Epub 2012
May 11.

Leukemia inhibitory factor signaling is required for lung protection during
pneumonia.

Quinton LJ(1), Mizgerd JP, Hilliard KL, Jones MR, Kwon CY, Allen E.

Author information: 
(1)Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA.

Lung infections represent a tremendous disease burden and a leading cause of
acute lung injury. STAT3 signaling is essential for controlling lung injury
during pneumonia. We previously identified LIF as a prominent STAT3-activating
cytokine expressed in the airspaces of pneumonic lungs, but its physiological
significance in this setting has never been explored. To do so, Escherichia coli 
was intratracheally instilled into C57BL/6 mice in the presence of neutralizing
anti-LIF IgG or control IgG. Anti-LIF completely eliminated lung LIF detection
and markedly exacerbated lung injury compared with control mice as evidenced by
airspace albumin content, lung liquid accumulation, and histological analysis.
Although lung bacteriology was equivalent between groups, bacteremia was more
prevalent with anti-LIF treatment, suggestive of compromised barrier function
rather than impaired antibacterial defense as the cause of dissemination.
Inflammatory cytokine expression was also exaggerated in anti-LIF-treated lungs, 
albeit after injury had ensued. Interestingly, alveolar neutrophil recruitment
was modestly but significantly reduced compared with control mice despite
elevated cytokine levels, indicating that inflammatory injury was not a
consequence of excessive neutrophilic alveolitis. Lastly, the lung transcriptome 
was dramatically remodeled during pneumonia, but far more so following LIF
neutralization, with gene changes implicating cell death and epithelial
homeostasis among other processes relevant to tissue injury. From these findings,
we conclude that endogenous LIF facilitates tissue protection during pneumonia.
The LIF-STAT3 axis is identified in this study as a critical determinant of lung 
injury with clinical implications for pneumonia patients.

DOI: 10.4049/jimmunol.1200256 
PMCID: PMC3370070
PMID: 22581855  [Indexed for MEDLINE]


348. Br J Nutr. 2012 Jun;107(11):1591-602. doi: 10.1017/S0007114511004910. Epub 2011
Sep 29.

Stool-fermented Plantago ovata husk induces apoptosis in colorectal cancer cells 
independently of molecular phenotype.

Sohn VR(1), Giros A, Xicola RM, Fluvià L, Grzybowski M, Anguera A, Llor X.

Author information: 
(1)Digestive Diseases and Nutrition Section, Department of Medicine and Cancer
Center, University of Illinois at Chicago, 840 South Wood Street (M/C 716),
Chicago, IL 60612, USA.

Several studies have suggested that the partially fermentable fibre Plantago
ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We
studied the potentially pro-apoptotic effect of PO and the implicated mechanisms 
in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29,
SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the 
human colon. The fermentation products of PO induced apoptosis in all primary
tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli,
β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both
intrinsic and extrinsic pathways were implicated. The intrinsic pathway was
activated through a shift in the balance towards a pro-apoptotic environment with
an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) 
and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and 
LoVo cells. This resulted in mitochondrial membrane depolarisation, increased
expression of caspase activators second mitochondria-derived activator of
caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome
member apoptotic protease activating factor 1 and down-regulation of inhibitors
of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The
extrinsic pathway was activated presumably through the up-regulation of death
receptor (DR5). Some important differences were seen between primary tumour and
metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable
up-regulation of TNF-α ligand along with death-inducing signalling complex
components receptor interacting protein and TNF-α receptor 1-associated death
domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), 
an inhibitor of both spontaneous death ligand-independent and death
receptor-mediated apoptosis, was significantly down-regulated after PO treatment 
in all primary tumour cells, but not in metastatic LoVo. These findings suggest
that PO could potentially be a useful chemotherapy adjuvant.

DOI: 10.1017/S0007114511004910 
PMID: 22018732  [Indexed for MEDLINE]


349. Int J Gynecol Cancer. 2012 Jun;22(5):732-41. doi: 10.1097/IGC.0b013e3182510496.

Not all fat is equal: differential gene expression and potential therapeutic
targets in subcutaneous adipose, visceral adipose, and endometrium of obese women
with and without endometrial cancer.

Modesitt SC(1), Hsu JY, Chowbina SR, Lawrence RT, Hoehn KL.

Author information: 
(1)Division of Gynecologic Oncology, Obstetrics and Gynecology Department,
University of Virginia Health System, Charlottesville, VA 22908-0712, USA.
scm6h@virginia.edu

OBJECTIVE: To identify obesity-related cancer genes in endometrial and adipose
tissue depots of body mass index-matched morbidly obese women with and without
endometrial cancer.
METHODS: Eight women undergoing hysterectomy (4 women with and 4 women without
endometrial cancer) were matched by age (52.6 years) and body mass index (44.5
kg/m). Endometrium, visceral adipose tissue, and subcutaneous adipose tissue were
collected and subjected to microarray analysis using Affymetrix Human Genome U133
Plus 2.0 Arrays. Gene set enrichment analysis used to extract biological
information from the gene expression data and t test metric ranked and compared
genes in the expression data set. Protein expression was measured in the
endometrial samples, and serum was collected for hormone/metabolite assays.
RESULTS: No significant differences were detected in hormone/metabolite levels
between groups. Gene set enrichment analysis comparisons demonstrated that
endometrial, visceral adipose and subcutaneous adipose tissues displayed 40, 47, 
and 38 alternatively regulated gene set pathways when comparing patients with and
without cancer. Nineteen gene sets were alternately regulated in both visceral
and subcutaneous adipose tissues; however, eighteen of these were regulated in
the opposite direction. Five pathways were significantly and alternately
regulated in all 3 tissue types and included glycolysis/gluconeogenesis,
ribosome, peroxisome proliferator activated receptor signaling, pathogenic
Escherichia coli infection, and natural killer-mediated cytotoxicity. In the
malignant endometrium, liver kinase B1 underexpression was observed in all
patients with cancer. Liver kinase B1 underexpression decreased adenosine
monophosphate-activated protein kinase activity toward acetyl-CoA carboxylase and
implied enhanced lipid biosynthesis in obesity-induced endometrial cancer.
CONCLUSIONS: Subcutaneous and visceral adipose tissue depots have opposite
patterns of gene expression in obese patients with and without endometrial
cancer. The altered de novo lipogenesis and individual gene targets identified
provide new potential targets for cancer treatment and prevention for at-risk
obese women.

DOI: 10.1097/IGC.0b013e3182510496 
PMID: 22635025  [Indexed for MEDLINE]


350. J Inorg Biochem. 2012 Jun;111:164-72. doi: 10.1016/j.jinorgbio.2011.11.022. Epub 
2011 Dec 2.

Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.

Hensley MP(1), Gunasekera TS, Easton JA, Sigdel TK, Sugarbaker SA, Klingbeil L,
Breece RM, Tierney DL, Crowder MW.

Author information: 
(1)Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University,
Oxford, OH 45056, United States.

RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E.
coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes
were significantly up-regulated and 146 genes were significantly down-regulated
in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, 
which encodes for a periplasmic protein known to be involved with Zn(II) import, 
(2) yodA, which encodes for a periplasmic protein with unknown function, and (3) 
ykgM, which encodes for a ribosomal protein that is thought to be a paralog of
ribosomal protein L31. YodA was over-expressed and purified as a maltose binding 
protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II).
Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand,
MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a 
ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers.
Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in
MBP-L31 and that the other three cysteines do not bind metal. These results are
discussed in light of Zn(II) starvation model that has been postulated for B.
subtilis.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.jinorgbio.2011.11.022 
PMCID: PMC3325339
PMID: 22196016  [Indexed for MEDLINE]


351. Microbiology. 2012 Jun;158(Pt 6):1482-92. doi: 10.1099/mic.0.057745-0. Epub 2012 
Mar 22.

Novel regulation targets of the metal-response BasS-BasR two-component system of 
Escherichia coli.

Ogasawara H(1), Shinohara S, Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

The BasS-BasR two-component system is known as an iron- and zinc-sensing
transcription regulator in Escherichia coli, but so far only a few genes have
been identified to be under the direct control of phosphorylated BasR. Using
Genomic SELEX (systematic evolution of ligands by exponential enrichment)
screening, we have identified a total of at least 38 binding sites of
phosphorylated BasR on the E. coli genome, and based on the BasR-binding sites,
have predicted more than 20 novel targets of regulation. By DNase I footprint
analysis for high-affinity BasR-binding sites, a direct repeat of a TTAAnnTT
sequence was identified as the BasR box. Transcription regulation in vivo of the 
target genes was confirmed after Northern blot analysis of target gene mRNAs from
both wild-type E. coli and an otherwise isogenic basR deletion mutant. The BasR
regulon can be classified into three groups of genes: group 1 includes the genes 
for the formation and modification of membrane structure; group 2 includes genes 
for modulation of membrane functions; and group 3 includes genes for
stress-response cell functions, including csgD, the master regulator of biofilm
formation.

DOI: 10.1099/mic.0.057745-0 
PMID: 22442305  [Indexed for MEDLINE]


352. J Vis Exp. 2012 May 28;(63). pii: 3961. doi: 10.3791/3961.

Detection of bacteria using fluorogenic DNAzymes.

Aguirre SD(1), Ali MM, Kanda P, Li Y.

Author information: 
(1)Department of Biochemistry and Biomedical Sciences, McMaster University,
Canada.

Outbreaks linked to food-borne and hospital-acquired pathogens account for
millions of deaths and hospitalizations as well as colossal economic losses each 
and every year. Prevention of such outbreaks and minimization of the impact of an
ongoing epidemic place an ever-increasing demand for analytical methods that can 
accurately identify culprit pathogens at the earliest stage. Although there is a 
large array of effective methods for pathogen detection, none of them can satisfy
all the following five premier requirements embodied for an ideal detection
method: high specificity (detecting only the bacterium of interest), high
sensitivity (capable of detecting as low as a single live bacterial cell), short 
time-to-results (minutes to hours), great operational simplicity (no need for
lengthy sampling procedures and the use of specialized equipment), and cost
effectiveness. For example, classical microbiological methods are highly specific
but require a long time (days to weeks) to acquire a definitive result.(1) PCR-
and antibody-based techniques offer shorter waiting times (hours to days), but
they require the use of expensive reagents and/or sophisticated equipment.(2-4)
Consequently, there is still a great demand for scientific research towards
developing innovative bacterial detection methods that offer improved
characteristics in one or more of the aforementioned requirements. Our laboratory
is interested in examining the potential of DNAzymes as a novel class of
molecular probes for biosensing applications including bacterial detection.(5)
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made
single-stranded DNA molecules with the capability of catalyzing chemical
reactions.(6-8) These molecules can be isolated from a vast random-sequence DNA
pool (which contains as many as 10(16) individual sequences) by a process known
as "in vitro selection" or "SELEX" (systematic evolution of ligands by
exponential enrichment).(9-16) These special DNA molecules have been widely
examined in recent years as molecular tools for biosensing applications.(6-8) Our
laboratory has established in vitro selection procedures for isolating
RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs
as analytical tools.(17-29) RFDs catalyze the cleavage of a DNA-RNA chimeric
substrate at a single ribonucleotide junction (R) that is flanked by a
fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the
uncleaved substrate minimal fluorescence. However, the cleavage event leads to
the separation of F and Q, which is accompanied by significant increase of
fluorescence intensity. More recently, we developed a method of isolating RFDs
for bacterial detection.(5) These special RFDs were isolated to "light up" in the
presence of the crude extracellular mixture (CEM) left behind by a specific type 
of bacteria in their environment or in the media they are cultured (Fig. 1). The 
use of crude mixture circumvents the tedious process of purifying and identifying
a suitable target from the microbe of interest for biosensor development (which
could take months or years to complete). The use of extracellular targets means
the assaying procedure is simple because there is no need for steps to obtain
intracellular targets. Using the above approach, we derived an RFD that cleaves
its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli 
(CEM-EC).(5) This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be
strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other
bacteria (Fig. 3). Here we present the key experimental procedures for setting up
E. coli detection assays using RFD-EC1 and representative results.

DOI: 10.3791/3961 
PMCID: PMC3466938
PMID: 22688431  [Indexed for MEDLINE]


353. Front Cell Infect Microbiol. 2012 May 11;2:61. doi: 10.3389/fcimb.2012.00061.
eCollection 2012.

O-antigen and virulence profiling of shiga toxin-producing Escherichia coli by a 
rapid and cost-effective DNA microarray colorimetric method.

Quiñones B(1), Swimley MS, Narm KE, Patel RN, Cooley MB, Mandrell RE.

Author information: 
(1)Produce Safety and Microbiology Research Unit, Western Regional Research
Center, U.S. Department of Agriculture/Agricultural Research Service Albany, CA, 
USA. beatriz.quinones@ars.usda.gov

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne
illness worldwide. The present study developed the use of DNA microarrays with
the ampliPHOX colorimetric method to rapidly detect and genotype STEC strains. A 
low-density 30-mer oligonucleotide DNA microarray was designed to target
O-antigen gene clusters of 11 E. coli serogroups (O26, O45, O91, O103, O104,
O111, O113, O121, O128, O145, and O157) that have been associated with the
majority of STEC infections. In addition, the DNA microarray targeted 11
virulence genes, encoding adhesins, cytotoxins, proteases, and receptor proteins,
which have been implicated in conferring increased ability to cause disease for
STEC. Results from the validation experiments demonstrated that this
microarray-based colorimetric method allowed for a rapid and accurate genotyping 
of STEC reference strains from environmental and clinical sources and from
distinct geographical locations. Positive hybridization signals were detected
only for probes targeting serotype and virulence genes known to be present in the
STEC reference strains. Quantification analysis indicated that the mean pixel
intensities of the signal for probes targeting O-antigen or virulence genes were 
at least three times higher when compared to the background. Furthermore, this
microarray-based colorimetric method was then employed to genotype a group of E. 
coli isolates from watershed sediment and animal fecal samples that were
collected from an important region for leafy-vegetable production in the central 
coast of California. The results indicated an accurate identification of O-type
and virulence genes in the tested isolates and confirmed that the ampliPHOX
colorimetric method with low-density DNA microarrays enabled a fast assessment of
the virulence potential of STEC using low-cost reagents and instrumentation.

DOI: 10.3389/fcimb.2012.00061 
PMCID: PMC3417394
PMID: 22919652  [Indexed for MEDLINE]


354. Appl Environ Microbiol. 2012 May;78(9):3379-86. doi: 10.1128/AEM.07199-11. Epub
2012 Feb 17.

Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C
plasmid.

Lang KS(1), Danzeisen JL, Xu W, Johnson TJ.

Author information: 
(1)Department of Veterinary and Biomedical Sciences, College of Veterinary
Medicine, University of Minnesota, St. Paul, Minnesota, USA.

The multidrug resistance-encoding plasmids belonging to the IncA/C
incompatibility group have recently emerged among Escherichia coli and Salmonella
enterica strains in the United States. These plasmids have a unique genetic
structure compared to other enterobacterial plasmid types, a broad host range,
and a propensity to acquire large numbers of antimicrobial resistance genes via
their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C
plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C
plasmids under laboratory growth and in the face of selective pressure. The
effects of ampicillin, florfenicol, or streptomycin exposure were compared to
those on cells left untreated at logarithmic phase using Illumina platform-based 
RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking
antibiotics, much of the backbone of pAR060302 was transcriptionally inactive,
including its putative transfer regions. A few plasmid backbone genes of interest
were highly transcribed, including genes of a putative toxin-antitoxin system and
an H-NS-like transcriptional regulator. In contrast, numerous genes within the
accessory regions of pAR060302 were highly transcribed, including the resistance 
genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin
resulted in no genes being differentially expressed compared to controls lacking 
antibiotics, suggesting that many of the resistance-associated genes are not
differentially expressed due to exposure to these antibiotics. In contrast,
florfenicol treatment resulted in the upregulation of floR and numerous
chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that 
it mitigates the fitness costs of carrying resistance-associated genes through
global regulation with its transcriptional regulators.

DOI: 10.1128/AEM.07199-11 
PMCID: PMC3346456
PMID: 22344651  [Indexed for MEDLINE]


355. Clin Infect Dis. 2012 May;54(9):1314-21. doi: 10.1093/cid/cis036.

"Silent" dissemination of Klebsiella pneumoniae isolates bearing K. pneumoniae
carbapenemase in a long-term care facility for children and young adults in
Northeast Ohio.

Viau RA(1), Hujer AM, Marshall SH, Perez F, Hujer KM, Briceño DF, Dul M, Jacobs
MR, Grossberg R, Toltzis P, Bonomo RA.

Author information: 
(1)Department of Medicine, Jacobi Medical Center, Albert Einstein College of
Medicine, Bronx, New York, USA. robert.bonomo@va.gov

BACKGROUND: Klebsiella pneumoniae isolates harboring the K. pneumoniae
carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in
both acute and long-term care facilities (LTCFs). As part of a study conducted in
2004 to determine the risk of stool colonization with extended-spectrum
cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that
exhibited nonsusceptibility to extended-spectrum cephalosporins were detected.
All were gastrointestinal carriage isolates that were not associated with
infection.
METHODS: Reassessment of the carbapenem minimum inhibitory concentrations using
revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered
carbapenem resistance. To further investigate, a DNA microarray assay,
PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR
(rep-PCR) and multilocus sequence typing (MLST) were performed.
RESULTS: The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) 
was identified by PCR amplification and sequencing of the amplicon. In addition, 
a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed
"low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated 
that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98%
similar). According to MLST, all isolates belonged to sequence type 36. This
sequence type has not been previously linked with bla(KPC) carriage. Plasmids
from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia 
coli J-53 recipients.
CONCLUSIONS: Our findings reveal the "silent" dissemination of bla(KPC-3) as part
of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and
establish the first report, to our knowledge, of K. pneumoniae containing
bla(KPC-3) in an LTCF caring for neurologically impaired children and young
adults.

DOI: 10.1093/cid/cis036 
PMCID: PMC3404693
PMID: 22492318  [Indexed for MEDLINE]


356. J Clin Microbiol. 2012 May;50(5):1632-9. doi: 10.1128/JCM.06115-11. Epub 2012 Feb
8.

Using nucleic acid microarrays to perform molecular epidemiology and detect novel
β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world.

Lascols C(1), Hackel M, Hujer AM, Marshall SH, Bouchillon SK, Hoban DJ, Hawser
SP, Badal RE, Bonomo RA.

Author information: 
(1)International Health Management Associates, Schaumburg, Illinois, USA.
clascols@ihmainc.com

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and
carbapenemase-producing Enterobacteriaceae is a major concern in both hospital
and community settings. Rapid identification of these resistant pathogens and the
genetic determinants they possess is needed to assist in clinical practice and
epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae,
Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically
ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in
2008-2009 from a longitudinal surveillance study (SMART) was examined using an in
vitro nucleic acid-based microarray. This approach was used to detect and
identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9,
and 8/25) and bla(KPC) genes and was combined with selective PCR amplification
and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC)
genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified 
as possessing at least one bla(ESBL) gene (95.2% concordance), and 59
phenotypically ESBL-negative isolates, used as negative controls, were negative. 
Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n =
19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129),
and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common
bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12).
Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL)
genes in a current global population; we report that CTX-M-15 is still the
dominant ESBL and provide the first report of the new β-lactamase variants
bla(SHV-129) and bla(KPC-11).

DOI: 10.1128/JCM.06115-11 
PMCID: PMC3347121
PMID: 22322349  [Indexed for MEDLINE]


357. Lab Invest. 2012 May;92(5):735-43. doi: 10.1038/labinvest.2012.46. Epub 2012 Mar 
12.

FOSL1 as a candidate target gene for 11q12 rearrangements in desmoplastic
fibroblastoma.

Macchia G(1), Trombetta D, Möller E, Mertens F, Storlazzi CT, Debiec-Rychter M,
Sciot R, Nord KH.

Author information: 
(1)Department of Clinical Genetics, University and Regional Laboratories, Skåne
University Hospital, Lund University, Lund, Sweden.
gemmamacchia@biologia.uniba.it

Desmoplastic fibroblastoma (DF) is a benign fibroblastic/myofibroblastic tumor.
Cytogenetic analyses have revealed consistent rearrangement of chromosome band
11q12, strongly suggesting that this region harbors a gene of pathogenetic
importance. To identify the target gene of the 11q12 rearrangements, we analyzed 
six cases diagnosed as DF using chromosome banding, fluorescence in situ
hybridization (FISH), single-nucleotide polymorphism array and gene expression
approaches. Different structural rearrangements involving 11q12 were found in
five of the six cases. Metaphase FISH analyses in two of them mapped the 11q12
breakpoints to an ~20-kb region, harboring FOSL1. Global gene expression
profiling followed by quantitative real-time PCR showed that FOSL1 was expressed 
at higher levels in DF with 11q12 rearrangements than in desmoid-type
fibromatoses. Furthermore, FOSL1 was not upregulated in the single case of DF
that did not show cytogenetic involvement of 11q12; instead this tumor was found 
to display a hemizygous loss on 5q, including the APC (adenomatous polyposis
coli) locus, raising the possibility that it actually was a misdiagnosed Gardner 
fibroma. 5'RACE-PCR in two 11q12-positive DF did not identify any fusion
transcripts. Thus, in agreement with the finding at chromosome banding analysis
that varying translocation partners are involved in the 11q12 rearrangement, the 
molecular data suggest that the functional outcome of the 11q12 rearrangements is
deregulated expression of FOSL1.

DOI: 10.1038/labinvest.2012.46 
PMID: 22411068  [Indexed for MEDLINE]


358. Plant J. 2012 May;70(4):650-65. doi: 10.1111/j.1365-313X.2012.04920.x. Epub 2012 
Mar 8.

Inducible NAD overproduction in Arabidopsis alters metabolic pools and gene
expression correlated with increased salicylate content and resistance to
Pst-AvrRpm1.

Pétriacq P(1), de Bont L, Hager J, Didierlaurent L, Mauve C, Guérard F, Noctor G,
Pelletier S, Renou JP, Tcherkez G, Gakière B.

Author information: 
(1)Institut de Biologie des Plantes, CNRS UMR 8618, Bâtiment 630, Université
Paris-Sud 11, Orsay Cedex, France. pierre.petriacq@u-psud.fr

Plant development and function are underpinned by redox reactions that depend on 
co-factors such as nicotinamide adenine dinucleotide (NAD). NAD has recently been
shown to be involved in several signalling pathways that are associated with
stress tolerance or defence responses. However, the mechanisms by which NAD
influences plant gene regulation, metabolism and physiology still remain unclear.
Here, we took advantage of Arabidopsis thaliana lines that overexpressed the nadC
gene from E. coli, which encodes the NAD biosynthesis enzyme quinolinate
phosphoribosyltransferase (QPT). Upon incubation with quinolinate, these lines
accumulated NAD and were thus used as inducible systems to determine the
consequences of an increased NAD content in leaves. Metabolic profiling showed
clear changes in several metabolites such as aspartate-derived amino acids and
NAD-derived nicotinic acid. Large-scale transcriptomic analyses indicated that
NAD promoted the induction of various pathogen-related genes such as the
salicylic acid (SA)-responsive defence marker PR1. Extensive comparison with
transcriptomic databases further showed that gene expression under high NAD
content was similar to that obtained under biotic stress, eliciting conditions or
SA treatment. Upon inoculation with the avirulent strain of Pseudomonas syringae 
pv. tomato Pst-AvrRpm1, the nadC lines showed enhanced resistance to bacteria
infection and exhibited an ICS1-dependent build-up of both conjugated and free SA
pools. We therefore concluded that higher NAD contents are beneficial for plant
immunity by stimulating SA-dependent signalling and pathogen resistance.

Published 2012. This article is a US Government work and is in the public domain 
in the USA.

DOI: 10.1111/j.1365-313X.2012.04920.x 
PMID: 22268572  [Indexed for MEDLINE]


359. Biochem Biophys Res Commun. 2012 Apr 27;421(1):119-23. doi:
10.1016/j.bbrc.2012.03.127. Epub 2012 Apr 3.

Genome-wide screening of Escherichia coli genes involved in execution and
promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is
essential for plasmid acceptance in recipient cells.

Kurono N(1), Matsuda A, Etchuya R, Sobue R, Sasaki Y, Ito M, Ando T, Maeda S.

Author information: 
(1)Graduate School of Humanities and Sciences, Nara Women's University,
Kitauoya-nishimachi, Nara 630-8506, Japan.

Acquisition of new genetic traits by horizontal gene transfer is a bacterial
strategy for adaptation to the environment. We previously showed that Escherichia
coli can transmit non-conjugative plasmids laterally in a co-culture containing
strains with and without the plasmid. In this study, using the Keio collection, a
comprehensive library of E. coli knock-out mutants for non-essential genes, we
screened for genes responsible for the execution and promotion of cell-to-cell
plasmid transfer in recipient cells. By stepwise screening of 'transfer-down'
mutants, two essential genes and six promoting genes were obtained. One of the
essential genes was priA, which is involved in DNA replication. This priA mutant 
was also unable to be transformed by artificial transformation methods, probably 
due to the deficiency of the plasmid maintenance function. The other essential
gene was rodZ (yfgA), a gene involved in the regulation of rod-shaped structure
of E. coli cells. This rodZ mutant was transformable by all three methods of
artificial transformation tested, suggesting that this gene is essential for
cell-to-cell plasmid transfer but not for artificial transformation. These are
the first data that suggest that rodZ plays an essential role in DNA acquisition.

Copyright © 2012 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.bbrc.2012.03.127 
PMID: 22497891  [Indexed for MEDLINE]


360. Appl Environ Microbiol. 2012 Apr;78(7):2452-5. doi: 10.1128/AEM.07783-11. Epub
2012 Jan 20.

Increased furan tolerance in Escherichia coli due to a cryptic ucpA gene.

Wang X(1), Miller EN, Yomano LP, Shanmugam KT, Ingram LO.

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida, USA.

Expression arrays were used to identify 4 putative oxidoreductases that were
upregulated (>3-fold) by furfural (15 mM, 15 min). Plasmid expression of one
(ucpA) increased furan tolerance in ethanologenic strain LY180 and wild-type
strain W. Deleting ucpA decreased furfural tolerance. Although the mechanism
remains unknown, the cryptic ucpA gene is now associated with a phenotype: furan 
resistance.

DOI: 10.1128/AEM.07783-11 
PMCID: PMC3302629
PMID: 22267665  [Indexed for MEDLINE]


361. Nucleic Acids Res. 2012 Apr;40(7):2846-61. doi: 10.1093/nar/gkr1141. Epub 2011
Dec 1.

An in silico model for identification of small RNAs in whole bacterial genomes:
characterization of antisense RNAs in pathogenic Escherichia coli and
Streptococcus agalactiae strains.

Pichon C(1), du Merle L, Caliot ME, Trieu-Cuot P, Le Bouguénec C.

Author information: 
(1)Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram Positif,
25-28 Rue du Docteur Roux, F-75724 Paris, France.

Characterization of small non-coding ribonucleic acids (sRNA) among the large
volume of data generated by high-throughput RNA-seq or tiling microarray analyses
remains a challenge. Thus, there is still a need for accurate in silico
prediction methods to identify sRNAs within a given bacterial species. After
years of effort, dedicated software were developed based on comparative genomic
analyses or mathematical/statistical models. Although these genomic analyses
enabled sRNAs in intergenic regions to be efficiently identified, they all failed
to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand
complementary to that which encodes the protein. The statistical models enabled
any genomic region to be analyzed theorically but not efficiently. We present a
new model for in silico identification of sRNA and asRNA candidates within an
entire bacterial genome. This model was successfully used to analyze the
Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In
both bacteria, numerous asRNAs are transcribed from the complementary strand of
genes located in pathogenicity islands, strongly suggesting that these asRNAs are
regulators of the virulence expression. In particular, we characterized an asRNA 
that acted as an enhancer-like regulator of the type 1 fimbriae production
involved in the virulence of extra-intestinal pathogenic E. coli.

DOI: 10.1093/nar/gkr1141 
PMCID: PMC3326304
PMID: 22139924  [Indexed for MEDLINE]


362. RNA Biol. 2012 Apr;9(4):458-68. doi: 10.4161/rna.19065. Epub 2012 Feb 16.

The small RNA RybA regulates key-genes in the biosynthesis of aromatic amino
acids under peroxide stress in E. coli.

Gerstle K(1), Klätschke K, Hahn U, Piganeau N.

Author information: 
(1)Department of Chemistry, Institute for Biochemistry and Molecular Biology,
Hamburg University, Hamburg, Germany.

In bacteria, adaptive response to external stimuli is often regulated by small
RNAs (sRNAs). In Escherichia coli, the organism in which sRNAs have been best
characterized so far, no function could be attributed to 40 out of 79 sRNAs. Here
we decipher the function of RybA, one of these orphan sRNAs. RybA was discovered 
in 2001 by Wassarman et al. using comparative genomics. This sRNA is conserved
between E. coli, Salmonella typhimurium and Klebsiella pneumoniae. We determined 
the expression pattern of RybA under different growth conditions and identified
its exact 5' and 3' ends. Using microarray and Northern analysis we show that,
under peroxide stress, the absence of RybA leads to an upregulation of key genes 
of the TyrR regulon involved in the metabolism of aromatic compounds including
the aromatic amino acids. Although containing an open reading frame, which might 
have an independent function, RybA does not require translation for this activity
and therefore acts at the RNA level. Furthermore we demonstrate that regulation
requires the transcription regulator TyrR. The mechanism of activation of TyrR,
probably the primary target of RybA, remains to be elucidated. The downregulation
of aromatic amino acid biosynthesis might regulate the cellular concentration of 
chorismate and its availability for other downstream products like ubiquinone or 
enterobactin. While ubiquinone participates in the defense against oxidative
stress in the cytoplasmic membrane, enterobactin is involved in iron import and
is therefore detrimental under oxidative stress.

DOI: 10.4161/rna.19065 
PMID: 22336764  [Indexed for MEDLINE]


363. RNA Biol. 2012 Apr;9(4):469-88. doi: 10.4161/rna.19317. Epub 2012 Feb 16.

Dynamics of Salmonella small RNA expression in non-growing bacteria located
inside eukaryotic cells.

Ortega AD(1), Gonzalo-Asensio J, García-del Portillo F.

Author information: 
(1)Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

Small non-coding regulatory RNAs (sRNAs) have been studied in many bacterial
pathogens during infection. However, few studies have focused on how
intracellular pathogens modulate sRNA expression inside eukaryotic cells. Here,
we monitored expression of all known sRNAs of Salmonella enterica serovar
Typhimurium (S. Typhimurium) in bacteria located inside fibroblasts, a host cell 
type in which this pathogen restrains growth. sRNA sequences known in S.
Typhimurium and Escherichia coli were searched in the genome of S. Typhimurium
virulent strain SL1344, the subject of this study. Expression of 84 distinct
sRNAs was compared in extra- and intracellular bacteria. Non-proliferating
intracellular bacteria upregulated six sRNAs, including IsrA, IsrG, IstR-2,
RyhB-1, RyhB-2 and RseX while repressed the expression of the sRNAs DsrA, GlmZ,
IsrH-1, IsrI, SraL, SroC, SsrS(6S) and RydC. Interestingly, IsrH-1 was previously
reported as an sRNA induced by S. Typhimurium inside macrophages. Kinetic
analyses unraveled changing expression patterns for some sRNAs along the
infection. InvR and T44 expression dropped after an initial induction phase while
IstR-2 was induced exclusively at late infection times (> 6 h). Studies focused
on the Salmonella-specific sRNA RyhB-2 revealed that intracellular bacteria use
this sRNA to regulate negatively YeaQ, a cis-encoded protein of unknown function.
RyhB-2, together with RyhB-1, contributes to attenuate intracellular bacterial
growth. To our knowledge, these data represent the first comprehensive study of
S. Typhimurium sRNA expression in intracellular bacteria and provide the first
insights into sRNAs that may direct pathogen adaptation to a non-proliferative
state inside the host cell.

DOI: 10.4161/rna.19317 
PMID: 22336761  [Indexed for MEDLINE]


364. J Vis Exp. 2012 Mar 2;(61):e3890. doi: 10.3791/3890.

On-chip isotachophoresis for separation of ions and purification of nucleic
acids.

Garcia-Schwarz G(1), Rogacs A, Bahga SS, Santiago JG.

Author information: 
(1)Mechanical Engineering, Stanford University, USA.

Electrokinetic techniques are a staple of microscale applications because of
their unique ability to perform a variety of fluidic and electrophoretic
processes in simple, compact systems with no moving parts. Isotachophoresis (ITP)
is a simple and very robust electrokinetic technique that can achieve
million-fold preconcentration and efficient separation and extraction based on
ionic mobility. For example, we have demonstrated the application of ITP to
separation and sensitive detection of unlabeled ionic molecules (e.g. toxins,
DNA, rRNA, miRNA) with little or no sample preparation and to extraction and
purification of nucleic acids from complex matrices including cell culture,
urine, and blood. ITP achieves focusing and separation using an applied electric 
field and two buffers within a fluidic channel system. For anionic analytes, the 
leading electrolyte (LE) buffer is chosen such that its anions have higher
effective electrophoretic mobility than the anions of the trailing electrolyte
(TE) buffer (Effective mobility describes the observable drift velocity of an ion
and takes into account the ionization state of the ion, as described in detail by
Persat et al.). After establishing an interface between the TE and LE, an
electric field is applied such that LE ions move away from the region occupied by
TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but
cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter
called the "ITP interface"). Further, the TE and LE form regions of respectively 
low and high conductivity, which establish a steep electric field gradient at the
ITP interface. This field gradient preconcentrates sample species as they focus. 
Proper choice of TE and LE results in focusing and purification of target species
from other non-focused species and, eventually, separation and segregation of
sample species. We here review the physical principles underlying ITP and discuss
two standard modes of operation: "peak" and "plateau" modes. In peak mode,
relatively dilute sample ions focus together within overlapping narrow peaks at
the ITP interface. In plateau mode, more abundant sample ions reach a
steady-state concentration and segregate into adjoining plateau-like zones
ordered by their effective mobility. Peak and plateau modes arise out of the same
underlying physics, but represent distinct regimes differentiated by the initial 
analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a
peak mode assay for the extraction of nucleic acids from E. coli cell culture. We
conclude by presenting a plateau mode assay, where we use a non-focusing tracer
(NFT) species to visualize the separation and perform quantitation of amino
acids.

DOI: 10.3791/3890 
PMCID: PMC3399465
PMID: 22415002  [Indexed for MEDLINE]


365. Gastroenterology. 2012 Mar;142(3):562-571.e2. doi: 10.1053/j.gastro.2011.11.026. 
Epub 2011 Nov 22.

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression
of β-catenin.

Freeman TJ(1), Smith JJ, Chen X, Washington MK, Roland JT, Means AL, Eschrich SA,
Yeatman TJ, Deane NG, Beauchamp RD.

Author information: 
(1)Department of Surgery, Vanderbilt University Medical Center, Nashville,
Tennessee 37232-2730, USA.

BACKGROUND & AIMS: Mutational inactivation of adenomatous polyposis coli (APC) is
an early event in colorectal cancer (CRC) progression that affects the stability 
and increases the activity of β-catenin, a mediator of Wnt signaling. Progression
of CRC also involves inactivation of signaling via transforming growth factor β
and bone morphogenetic protein (BMP), which are tumor suppressors. However, the
interactions between these pathways are not clear. We investigated the effects of
loss of the transcription factor Smad4 on levels of β-catenin messenger RNA
(mRNA) and Wnt signaling.
METHODS: We used microarray analysis to associate levels of Smad4 and β-catenin
mRNA in colorectal tumor samples from 250 patients. We performed
oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T)
and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480
colon cancer cells. We analyzed adenomas from (APC(Δ1638/+)) and (APC(Δ1638/+)) ×
(K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection.
RESULTS: In human CRC samples, reduced levels of Smad4 correlated with increased 
levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA 
and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T
cells increased binding of RNA polymerase II to the β-catenin gene. Expression of
Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice 
with heterozygous disruption of Apc(APC(Δ1638/+)), Smad4-deficient intestinal
adenomas had increased levels of β-catenin mRNA and expression of Wnt target
genes compared with adenomas from APC(Δ1638/+) mice that expressed Smad4.
CONCLUSIONS: Transcription of β-catenin is inhibited by BMP signaling to Smad4.
These findings provide important information about the interaction among
transforming growth factor β, BMP, and Wnt signaling pathways in progression of
CRC.

Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

DOI: 10.1053/j.gastro.2011.11.026 
PMCID: PMC3343368
PMID: 22115830  [Indexed for MEDLINE]


366. Infect Immun. 2012 Mar;80(3):1232-42. doi: 10.1128/IAI.06138-11. Epub 2012 Jan 3.

Analysis of global transcriptional profiles of enterotoxigenic Escherichia coli
isolate E24377A.

Sahl JW(1), Rasko DA.

Author information: 
(1)Department of Microbiology and Immunology and Institute for Genome Sciences,
University of Maryland School of Medicine, Baltimore, Maryland, USA.

Enterotoxigenic Escherichia coli (ETEC) is an important pathogenic variant
(pathovar) of E. coli in developing countries from a human health perspective,
causing significant morbidity and mortality. Previous studies have examined
specific regulatory networks in ETEC, although little is known about the global
effects of inter- and intrakingdom signaling on the expression of virulence and
colonization factors in ETEC. In this study, an E. coli/Shigella pan-genome
microarray, combined with quantitative reverse transcriptase PCR (qRT-PCR) and
RNA sequencing (RNA-seq), was used to quantify the expression of ETEC virulence
and colonization factors. Biologically relevant chemical signals were combined
with ETEC isolate E24377A during growth in either Luria broth (LB) or Dulbecco's 
modified Eagle medium (DMEM), and transcription was examined during different
phases of the growth cycle; chemical signals examined included glucose, bile
salts, and preconditioned media from E. coli/Shigella isolates. The results
demonstrate that the presence of bile salts, which are found in the intestine and
thought to be bactericidal, upregulates the expression of many ETEC virulence
factors, including heat-stable (estA) and heat-labile (eltA) enterotoxin genes.
In contrast, the ETEC colonization factors CS1 and CS3 were downregulated in the 
presence of bile, consistent with findings in studies of other enteric pathogens.
RNA-seq analysis demonstrated that one of the most differentially expressed genes
in the presence of bile is a unique plasmid-encoded AraC-like transcriptional
regulator (peaR); other previously unknown genetic elements were found as well.
These results provide transcriptional targets and putative mechanisms that should
help improve understanding of the global regulatory networks and virulence
expression in this important human pathogen.

DOI: 10.1128/IAI.06138-11 
PMCID: PMC3294641
PMID: 22215741  [Indexed for MEDLINE]


367. J Basic Microbiol. 2012 Feb;52(1):27-34. doi: 10.1002/jobm.201000458. Epub 2011
Jun 9.

Simultaneous analysis of foodborne pathogenic bacteria by an oligonucleotide
microarray assay.

Hu Y(1), Liu J, Xia D, Chen S.

Author information: 
(1)The Center for Disease Control and Prevention of Guangzhou, Guangzhou, China. 
huyushan1976@gmail.com

A rapid and accurate method for simultaneous identification of foodborne
infectious pathogens was developed based on oligonucleotide microarray
technology. The proposed identification method is based on PCR amplification of
the target region of the groEL genes with degenerate primers, followed by the PCR
products hybridization with oligonucleotide probes specific for species. The
groEL gene amplification products of seventeen species of pathogenic bacteria
were hybridized to the oligonucleotide array. Hybridization results were analyzed
with digoxigenin-linked enzyme reaction. Results indicated that fifteen species
of pathogenic bacteria showed high sensitivity and specificity for the
oligonucleotide array, while two other species gave cross-reaction with the E.
coli. Our results suggested that microarray analysis of foodborne infectious
pathogens might be very useful for simultaneous identification of bacterial
pathogens. The oligonucleotide array can also be applied to samples collected in 
clinical settings of foodborne infections. The superiority of oligonucleotide
array over other tests lies on its rapidity, accuracy and efficiency in the
diagnosis, treatment and control of foodborne infections.

Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI: 10.1002/jobm.201000458 
PMID: 21656816  [Indexed for MEDLINE]


368. J Clin Invest. 2012 Feb;122(2):759-76. doi: 10.1172/JCI57313. Epub 2012 Jan 9.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga
toxin-associated hemolytic uremic syndrome in humans and mice.

Petruzziello-Pellegrini TN(1), Yuen DA, Page AV, Patel S, Soltyk AM, Matouk CC,
Wong DK, Turgeon PJ, Fish JE, Ho JJ, Steer BM, Khajoee V, Tigdi J, Lee WL, Motto 
DG, Advani A, Gilbert RE, Karumanchi SA, Robinson LA, Tarr PI, Liles WC, Brunton 
JL, Marsden PA.

Author information: 
(1)Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario, Canada.

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It
often occurs after gastrointestinal infection with E. coli O157:H7, which
produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and
renal injury. Stx-mediated changes in endothelial phenotype have been linked to
the pathogenesis of HUS. Here we report our studies investigating Stx-induced
changes in gene expression and their contribution to the pathogenesis of HUS. Stx
function by inactivating host ribosomes but can also alter gene expression at
concentrations that minimally affect global protein synthesis. Gene expression
profiling of human microvascular endothelium treated with Stx implicated a role
for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand
(stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The
changes in gene expression required a catalytically active Stx A subunit and were
mediated by enhanced transcription and mRNA stability. Stx also enhanced the
association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of
Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4,
CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1
interaction decreased endothelial activation and organ injury and improved animal
survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels
were elevated in individuals who progressed to HUS. Collectively, these data
implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest 
novel therapeutic strategies for prevention and/or treatment of complications
associated with E. coli O157:H7 infection.

DOI: 10.1172/JCI57313 
PMCID: PMC3266777
PMID: 22232208  [Indexed for MEDLINE]


369. Nucleic Acids Res. 2012 Feb;40(4):1856-67. doi: 10.1093/nar/gkr892. Epub 2011 Nov
3.

Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA
aptamer: insight into RNA-binding properties of bacterial Hfq.

Someya T(1), Baba S, Fujimoto M, Kawai G, Kumasaka T, Nakamura K.

Author information: 
(1)Graduate School of Life and Environmental Sciences, University of Tsukuba,
1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305-8572, Japan. tsomeya@biol.tsukuba.ac.jp

Bacterial Hfq is a protein that plays an important role in the regulation of
genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more
sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However,
functional and structural information about Bacillus subtilis Hfq (BsHfq)
including the RNA sequences that specifically bind to it remain unknown. Here, we
describe RNA aptamers including fragment (AG)(3)A that are recognized by BsHfq
and crystal structures of the BsHfq-(AG)(3)A complex at 2.2 Å resolution.
Mutational and structural studies revealed that the RNA fragment binds to the
distal site, one of the two binding sites on Hfq, and identified amino acid
residues that are critical for sequence-specific interactions between BsHfq and
(AG)(3)A. In particular, R32 appears to interact with G bases in (AG)(3)A.
Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and
protein-RNA interaction patterns engaged in the R32 residues of BsHfq-(AG)(3)A
differ from those of EcHfq-poly(A). These findings provide novel insight into how
the Hfq homologue recognizes RNA.

DOI: 10.1093/nar/gkr892 
PMCID: PMC3287200
PMID: 22053080  [Indexed for MEDLINE]


370. BMC Genomics. 2012 Jan 24;13:37. doi: 10.1186/1471-2164-13-37.

Expression profiling reveals Spot 42 small RNA as a key regulator in the central 
metabolism of Aliivibrio salmonicida.

Hansen GA(1), Ahmad R, Hjerde E, Fenton CG, Willassen NP, Haugen P.

Author information: 
(1)Department of chemistry, Faculty of science and technology, University of
Tromsø, N-9037, Tromsø, Norway.

BACKGROUND: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an 
abundant, small and unstable RNA. Its biological role has remained obscure until 
recently, and is today implicated in having broader roles in the central and
secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous 
in the Vibrionaceae family of gamma-proteobacteria. One member of this family,
Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon.
Its genome encodes Spot 42 with 84% identity to E. coli Spot 42.
RESULTS: We generated a A. salmonicida spf deletion mutant. We then used
microarray and Northern blot analyses to monitor global effects on the
transcriptome in order to provide insights into the biological roles of Spot 42
in this bacterium. In the presence of glucose, we found a surprisingly large
number of ≥ 2X differentially expressed genes, and several major cellular
processes were affected. A gene encoding a pirin-like protein showed an on/off
expression pattern in the presence/absence of Spot 42, which suggests that Spot
42 plays a key regulatory role in the central metabolism by regulating the switch
between fermentation and respiration. Interestingly, we discovered an sRNA named 
VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an
expression pattern opposite to that of Spot 42, and its expression is repressed
by glucose.
CONCLUSIONS: We hypothesize that Spot 42 plays a key role in the central
metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via
pirin.

DOI: 10.1186/1471-2164-13-37 
PMCID: PMC3295665
PMID: 22272603  [Indexed for MEDLINE]


371. Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1269-74. doi:
10.1073/pnas.1113246109. Epub 2012 Jan 9.

Gut inflammation can boost horizontal gene transfer between pathogenic and
commensal Enterobacteriaceae.

Stecher B(1), Denzler R, Maier L, Bernet F, Sanders MJ, Pickard DJ, Barthel M,
Westendorf AM, Krogfelt KA, Walker AW, Ackermann M, Dobrindt U, Thomson NR, Hardt
WD.

Author information: 
(1)Institute of Microbiology, ETH Zürich, 8093 Zürich, Switzerland.

The mammalian gut harbors a dense microbial community interacting in multiple
ways, including horizontal gene transfer (HGT). Pangenome analyses established
particularly high levels of genetic flux between Gram-negative
Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT
are incompletely understood. Using a mouse colitis model, we found that
Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of
resident commensal Escherichia coli. These blooms boosted conjugative HGT of the 
colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli.
Transconjugation efficiencies of ~100% in vivo were attributable to high
intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little.
Under normal conditions, HGT was blocked by the commensal microbiota inhibiting
contact-dependent conjugation between Enterobacteriaceae. Our data show that
pathogen-driven inflammatory responses in the gut can generate transient
enterobacterial blooms in which conjugative transfer occurs at unprecedented
rates. These blooms may favor reassortment of plasmid-encoded genes between
pathogens and commensals fostering the spread of fitness-, virulence-, and
antibiotic-resistance determinants.

DOI: 10.1073/pnas.1113246109 
PMCID: PMC3268327
PMID: 22232693  [Indexed for MEDLINE]


372. Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1347-52. doi:
10.1073/pnas.1118018109. Epub 2012 Jan 9.

Digital RNA sequencing minimizes sequence-dependent bias and amplification noise 
with optimized single-molecule barcodes.

Shiroguchi K(1), Jia TZ, Sims PA, Xie XS.

Author information: 
(1)Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
MA 02138, USA.

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is
hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic 
to exponential PCR amplification. We developed a simple strategy for mitigating
these complications, allowing truly digital RNA-Seq. Following reverse
transcription, a large set of barcode sequences is added in excess, and nearly
every cDNA molecule is uniquely labeled by random attachment of barcode sequences
to both ends. After PCR, we applied paired-end deep sequencing to read the two
barcodes and cDNA sequences. Rather than counting the number of reads, RNA
abundance is measured based on the number of unique barcode sequences observed
for a given cDNA sequence. We optimized the barcodes to be unambiguously
identifiable, even in the presence of multiple sequencing errors. This method
allows counting with single-copy resolution despite sequence-dependent bias and
PCR-amplification noise, and is analogous to digital PCR but amendable to
quantifying a whole transcriptome. We demonstrated transcriptome profiling of
Escherichia coli with more accurate and reproducible quantification than
conventional RNA-Seq.

DOI: 10.1073/pnas.1118018109 
PMCID: PMC3268301
PMID: 22232676  [Indexed for MEDLINE]


373. Biosens Bioelectron. 2012 Jan 15;31(1):330-6. doi: 10.1016/j.bios.2011.10.043.
Epub 2011 Oct 31.

Sensitive label-free oligonucleotide-based microfluidic detection of mercury (II)
ion by using exonuclease I.

Yuan M(1), Zhu Y, Lou X, Chen C, Wei G, Lan M, Zhao J.

Author information: 
(1)State Key Lab of Transducer Technology, Shanghai Institute of Microsystem and 
Information Technology, Chinese Academy of Sciences, 865 Changning Rd, Shanghai
200050, China.

Mercury is a highly toxic metal that can cause significant harm to humans and
aquatic ecosystems. This paper describes a novel approach for mercury (Hg(2+))
ion detection by using label-free oligonucleotide probes and Escherichia coli
exonuclease I (Exo I) in a microfluidic electrophoretic separated platform. Two
single-stranded DNAs (ssDNA) TT-21 and TT-44 with 7 Thymine-Thymine mispairs are 
employed to capture mercury ions. Due to the coordination structure of
T-Hg(2+)-T, these ssDNAs are folded into hairpin-like double-stranded DNAs
(dsDNA) which are more difficult to be digested by Exo I, as confirmed by
polyacrylamide gel electrophoresis (PAGE) analysis. A series of microfluidic
capillary electrophoretic separation studies are carried out to investigate the
effect of Exo I and mercury ion concentrations on the detected fluorescence
intensity. This method has demonstrated a high sensitivity of mercury ion
detection with the limit of detection around 15 nM or 3 ppb. An excellent
selectivity of the probe for mercury ions over five interference ions Fe(3+),
Cd(2+), Pb(2+), Cu(2+) and Ca(2+) is also revealed. This method could potentially
be used for mercury ion detection with high sensitivity and reliability.

Copyright © 2011 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.bios.2011.10.043 
PMID: 22172497  [Indexed for MEDLINE]


374. Biochem Biophys Res Commun. 2012 Jan 6;417(1):414-20. doi:
10.1016/j.bbrc.2011.11.130. Epub 2011 Dec 7.

In vitro selection of Escherichia coli O157:H7-specific RNA aptamer.

Lee YJ(1), Han SR, Maeng JS, Cho YJ, Lee SW.

Author information: 
(1)Department of Molecular Biology, Institute of Nanosensor and Biotechnology,
Dankook University, Yongin, Republic of Korea.

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes
life-threatening symptoms in humans worldwide. To rapidly and properly identify
the pathogen and avoid its toxic effects, ligands which can directly and
specifically bind to the virulent E. coli O157:H7 serotype should be identified. 
In this study, a RNA aptamer-based ligand which can specifically distinguish the 
pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX
method. To this end, an RNA library was first incubated with the E. coli K12
strain, and the RNAs binding to the strain were discarded. The precluded RNAs
were then used for the selection of O157:H7-specific aptamers. After 6 rounds of 
the subtractive cell-SELEX process, the selected aptamer was found to
specifically bind to the O157:H7 serotype, but not to the K12 strain. This was
evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an
aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer
that distinguishes between the virulent serotype and the nonpathogenic strain
specifically bound to an O157:H7-specific lipopolysaccharide which includes the O
antigen. This novel O157:H7-specific aptamer could be of potential application as
a diagnostic ligand against the pathogen-related food borne illness.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.bbrc.2011.11.130 
PMID: 22166202  [Indexed for MEDLINE]


375. Stat Appl Genet Mol Biol. 2012 Jan 6;11(1):Article 2. doi:
10.2202/1544-6115.1695.

Transcriptional network inference from functional similarity and expression data:
a global supervised approach.

Ambroise J(1), Robert A, Macq B, Gala JL.

Author information: 
(1)Université Catholique de Louvain.

An important challenge in system biology is the inference of biological networks 
from postgenomic data. Among these biological networks, a gene transcriptional
regulatory network focuses on interactions existing between transcription factors
(TFs) and and their corresponding target genes. A large number of reverse
engineering algorithms were proposed to infer such networks from gene expression 
profiles, but most current methods have relatively low predictive performances.
In this paper, we introduce the novel TNIFSED method (Transcriptional Network
Inference from Functional Similarity and Expression Data), that infers a
transcriptional network from the integration of correlations and partial
correlations of gene expression profiles and gene functional similarities through
a supervised classifier. In the current work, TNIFSED was applied to predict the 
transcriptional network in Escherichia coli and in Saccharomyces cerevisiae,
using datasets of 445 and 170 affymetrix arrays, respectively. Using the area
under the curve of the receiver operating characteristics and the F-measure as
indicators, we showed the predictive performance of TNIFSED to be better than
unsupervised state-of-the-art methods. TNIFSED performed slightly worse than the 
supervised SIRENE algorithm for the target genes identification of the TF having 
a wide range of yet identified target genes but better for TF having only few
identified target genes. Our results indicate that TNIFSED is complementary to
the SIRENE algorithm, and particularly suitable to discover target genes of
"orphan" TFs.

DOI: 10.2202/1544-6115.1695 
PMID: 22499684  [Indexed for MEDLINE]


376. Antimicrob Agents Chemother. 2012 Jan;56(1):518-25. doi: 10.1128/AAC.00736-11.
Epub 2011 Nov 7.

Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field
isolates is associated with antimony resistance.

Ashutosh(1), Garg M, Sundar S, Duncan R, Nakhasi HL, Goyal N.

Author information: 
(1)Division of Biochemistry, Central Drug Research Institute, Council of
Scientific and Industrial Research, Chattar Manzil Palace, Lucknow, India.

Emergence of resistance to pentavalent antimonials has become a severe obstacle
in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The
mechanisms operating in laboratory-generated strains are somewhat known, but the 
determinants of clinical antimony resistance are not well understood. By
utilizing a DNA microarray expression profiling approach, we identified a gene
encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan
Leishmania donovani (LdMAPK1) that was consistently downregulated in
antimony-resistant field isolates. The expression level of the gene was validated
by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed
at the protein level in resistant isolates. Primary structure analysis of LdMAPK1
revealed the presence of all of the characteristic features of MAPK1. When
expressed in Escherichia coli, the recombinant enzyme showed kinase activity with
myelin basic protein as the substrate and was inhibited by staurosporine.
Interestingly, overexpression of this gene in a drug-sensitive laboratory strain 
and a resistant field isolate resulted in increased the sensitivity of the
transfectants to potassium antimony tartrate, suggesting that it has a role in
antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may
be in part correlated with antimony drug resistance in Indian VL isolates.

DOI: 10.1128/AAC.00736-11 
PMCID: PMC3256019
PMID: 22064540  [Indexed for MEDLINE]


377. Appl Environ Microbiol. 2012 Jan;78(1):70-80. doi: 10.1128/AEM.06785-11. Epub
2011 Oct 28.

Engineering of bacterial methyl ketone synthesis for biofuels.

Goh EB(1), Baidoo EE, Keasling JD, Beller HR.

Author information: 
(1)Joint BioEnergy Institute, Emeryville, California, USA.

We have engineered Escherichia coli to overproduce saturated and monounsaturated 
aliphatic methyl ketones in the C₁₁ to C₁₅ (diesel) range; this group of methyl
ketones includes 2-undecanone and 2-tridecanone, which are of importance to the
flavor and fragrance industry and also have favorable cetane numbers (as we
report here). We describe specific improvements that resulted in a 700-fold
enhancement in methyl ketone titer relative to that of a fatty acid-overproducing
E. coli strain, including the following: (i) overproduction of β-ketoacyl
coenzyme A (CoA) thioesters achieved by modification of the β-oxidation pathway
(specifically, overexpression of a heterologous acyl-CoA oxidase and native FadB 
and chromosomal deletion of fadA) and (ii) overexpression of a native
thioesterase (FadM). FadM was previously associated with oleic acid degradation, 
not methyl ketone synthesis, but outperformed a recently identified methyl ketone
synthase (Solanum habrochaites MKS2 [ShMKS2], a thioesterase from wild tomato) in
β-ketoacyl-CoA-overproducing strains tested. Whole-genome transcriptional
(microarray) studies led to the discovery that FadM is a valuable catalyst for
enhancing methyl ketone production. The use of a two-phase system with decane
enhanced methyl ketone production by 4- to 7-fold in addition to increases from
genetic modifications.

DOI: 10.1128/AEM.06785-11 
PMCID: PMC3255637
PMID: 22038610  [Indexed for MEDLINE]


378. Comput Math Methods Med. 2012;2012:953086. doi: 10.1155/2012/953086. Epub 2012
Mar 5.

A Bayesian approach for decision making on the identification of genes with
different expression levels: an application to Escherichia coli bacterium data.

Saraiva EF(1), Louzada F, Milan LA, Meira S, Cobre J.

Author information: 
(1)FACET, Universidade Federal da Grande Dourados, Brazil.

A common interest in gene expression data analysis is to identify from a large
pool of candidate genes the genes that present significant changes in expression 
levels between a treatment and a control biological condition. Usually, it is
done using a statistic value and a cutoff value that are used to separate the
genes differentially and nondifferentially expressed. In this paper, we propose a
Bayesian approach to identify genes differentially expressed calculating
sequentially credibility intervals from predictive densities which are
constructed using the sampled mean treatment effect from all genes in study
excluding the treatment effect of genes previously identified with statistical
evidence for difference. We compare our Bayesian approach with the standard ones 
based on the use of the t-test and modified t-tests via a simulation study, using
small sample sizes which are common in gene expression data analysis. Results
obtained report evidence that the proposed approach performs better than standard
ones, especially for cases with mean differences and increases in treatment
variance in relation to control variance. We also apply the methodologies to a
well-known publicly available data set on Escherichia coli bacterium.

DOI: 10.1155/2012/953086 
PMCID: PMC3306789
PMID: 22474543  [Indexed for MEDLINE]


379. In Vivo. 2012 Jan-Feb;26(1):39-45.

Gene expression changes induced by HIPEC in a murine model of gastric cancer.

Graziosi L(1), Mencarelli A, Renga B, Santorelli C, Cantarella F, Bugiantella W, 
Cavazzoni E, Donini A, Fiorucci S.

Author information: 
(1)Department of Surgery, School of Medicine, University of Perugia, Santa Maria 
della Misericordia Hospital, Perugia, Italy. luiginagraziosi@yahoo.it

BACKGROUND: Peritoneal carcinogenesis (PC) is the most frequent pattern of
metastasis in patients with locally advanced gastric cancer. Despite this, there 
is a consensus on the use of cytoreductive surgery and hyperthermic
intraperitoneal chemotherapy (HIPEC) for the treatment of PC from gastric cancer.
The molecular mechanisms involved in beneficial effects of HIPEC remain
unexplored.
MATERIALS AND METHODS: Human gastric cancer MKN45 cells were injected into the
peritoneal cavity of immune-deficient NOD-SCID mice. After induction of PC, the
animals were randomized into five groups: HIPEC with mitomycin and cisplatin;
normothermic intraperitoneal chemotherapy (NIPEC); normothermic intraperitoneal
saline; hyperthermic intraperitoneal saline alone; no treatment. After 10 days of
treatment, the mice were sacrificed and the extent of PC was assessed.
RESULTS: Compared with the other groups of treatment, HIPEC reduced the extent
and severity of peritoneal dissemination as measured by assessing the total
number of peritoneal and mesenteric nodules (p<0,05) and the HIPEC procedure
increased median survival significantly. By gene array analysis, HIPEC was found 
to effectively modulate the expression of a subset of genes involved in formation
of peritoneal metastasis, including adenomatous polyposis coli; beta (3) subunit 
of the integrin gene; chemokine stromal cell-derived factor-1 receptor; spleen
tyrosine kinase; vascular endothelial growth factor receptor 3; collagen, type
IV, alpha 2 and Carbossi-terminal binding proteins 1.
CONCLUSION: In the present study we have provided evidence that HIPEC protects
against peritoneal dissemination in a mouse model of peritoneal gastric
carcinogenesis and brings about specific changes in gene expression wich may be
related to this protection.


PMID: 22210714  [Indexed for MEDLINE]


380. J Microbiol Methods. 2012 Jan;88(1):110-6. doi: 10.1016/j.mimet.2011.10.021. Epub
2011 Nov 12.

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria
monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter
sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and
Pseudomonas fluorescens in milk and meat samples.

Chiang YC(1), Tsen HY, Chen HY, Chang YH, Lin CK, Chen CY, Pai WY.

Author information: 
(1)Department of Food Science and Technology, Hung Kuang University, No. 34,
Chung Chie Rd., Shalu, Taichung 433, Taiwan, ROC. honda224@sunrise.hk.edu.tw

Food products, such as milk and meat products including cheese, milk powder,
fermented milk, sausage, etc. are susceptible to the contamination by pathogenic 
and deteriorative bacteria. These bacteria include Listeria monocytogens,
Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7,
Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and
Pseudomonas fluorescens, etc. Traditional methods for the detection of these
microorganisms are laborious and time consuming. Therefore, rapid and accurate
diagnostic methods are needed. In this study, we designed the DNA probes and PCR 
primers for the detection of aforementioned microorganisms. By using two sets of 
multiplex PCR, followed by a chromogenic macroarray system, these organisms in
milk or other food products could be simultaneously detected. When the system was
used for the inspection of milk or meat homogenate containing 10(0) target cells 
per milliliter or gram of the sample, all these bacterial species could be
identified after an 8h pre-enrichment step. The system consisting of a multiplex 
PCR step followed by macroarray allowed us to detect multiple target bacterial
species simultaneously without the use of agarose gel electrophoresis. Compared
to the commonly used multiplex PCR method, this approach has the additional
advantage of detecting more bacterial strains because some bacterial strains
generate PCR products with the same molecular sizes which can be differentiated
by macroarray but not by electrophoresis.

Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

DOI: 10.1016/j.mimet.2011.10.021 
PMID: 22101309  [Indexed for MEDLINE]


381. J Pathol. 2012 Jan;226(1):73-83. doi: 10.1002/path.2972. Epub 2011 Oct 18.

The C-terminus of Apc does not influence intestinal adenoma development or
progression.

Lewis A(1), Davis H, Deheragoda M, Pollard P, Nye E, Jeffery R, Segditsas S, East
P, Poulsom R, Stamp G, Wright N, Tomlinson I.

Author information: 
(1)Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human 
Genetics, University of Oxford, Oxford, UK. lewisa@well.ox.ac.uk

Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours.
These mutations are almost always protein-truncating, deleting both central
domains that regulate Wnt signalling and C-terminal domains that interact with
the cytoskeleton. The importance of Wnt dysregulation for colorectal
tumourigenesis is well characterized. It is, however, unclear whether loss of
C-terminal functions contributes to tumourigenesis, although this protein region 
has been implicated in cellular processes--including polarity, migration,
mitosis, and chromosomal instability (CIN)—that have been postulated as critical 
for the development and progression of intestinal tumours. Since almost all APC
mutations in human patients disrupt both central and C-terminal regions, we
created a mouse model to test the role of the C-terminus of APC in intestinal
tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc
that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but 
leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T)
animals. The latter allele represented the most commonly found human APC mutation
and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus.
Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in
number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas
were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the
Wnt signalling pathway was observed in tumours from both mice, no evidence of
differential C-terminus functions (such as cell migration, CIN, or localization
of APC and EB1) was seen. We conclude that the C-terminus of APC does not
influence intestinal adenoma development or progression.

Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by 
John Wiley & Sons, Ltd.

DOI: 10.1002/path.2972 
PMCID: PMC4610063
PMID: 22009253  [Indexed for MEDLINE]


382. PLoS Comput Biol. 2012;8(2):e1002391. doi: 10.1371/journal.pcbi.1002391. Epub
2012 Feb 23.

Robust detection of hierarchical communities from Escherichia coli gene
expression data.

Treviño S 3rd(1), Sun Y, Cooper TF, Bassler KE.

Author information: 
(1)Department of Physics, University of Houston, Houston, Texas, United States of
America.

Determining the functional structure of biological networks is a central goal of 
systems biology. One approach is to analyze gene expression data to infer a
network of gene interactions on the basis of their correlated responses to
environmental and genetic perturbations. The inferred network can then be
analyzed to identify functional communities. However, commonly used algorithms
can yield unreliable results due to experimental noise, algorithmic
stochasticity, and the influence of arbitrarily chosen parameter values.
Furthermore, the results obtained typically provide only a simplistic view of the
network partitioned into disjoint communities and provide no information of the
relationship between communities. Here, we present methods to robustly detect
co-regulated and functionally enriched gene communities and demonstrate their
application and validity for Escherichia coli gene expression data. Applying a
recently developed community detection algorithm to the network of interactions
identified with the context likelihood of relatedness (CLR) method, we show that 
a hierarchy of network communities can be identified. These communities
significantly enrich for gene ontology (GO) terms, consistent with them
representing biologically meaningful groups. Further, analysis of the most
significantly enriched communities identified several candidate new regulatory
interactions. The robustness of our methods is demonstrated by showing that a
core set of functional communities is reliably found when artificial noise,
modeling experimental noise, is added to the data. We find that noise mainly acts
conservatively, increasing the relatedness required for a network link to be
reliably assigned and decreasing the size of the core communities, rather than
causing association of genes into new communities.

DOI: 10.1371/journal.pcbi.1002391 
PMCID: PMC3285575
PMID: 22383870  [Indexed for MEDLINE]


383. PLoS Genet. 2012 Jan;8(1):e1002443. doi: 10.1371/journal.pgen.1002443. Epub 2012 
Jan 19.

A genome-wide analysis of promoter-mediated phenotypic noise in Escherichia coli.

Silander OK(1), Nikolic N, Zaslaver A, Bren A, Kikoin I, Alon U, Ackermann M.

Author information: 
(1)Computational and Systems Biology, Biozentrum, University of Basel, Basel,
Switzerland. olinsilander@gmail.com

Gene expression is subject to random perturbations that lead to fluctuations in
the rate of protein production. As a consequence, for any given protein,
genetically identical organisms living in a constant environment will contain
different amounts of that particular protein, resulting in different phenotypes. 
This phenomenon is known as "phenotypic noise." In bacterial systems, previous
studies have shown that, for specific genes, both transcriptional and
translational processes affect phenotypic noise. Here, we focus on how the
promoter regions of genes affect noise and ask whether levels of
promoter-mediated noise are correlated with genes' functional attributes, using
data for over 60% of all promoters in Escherichia coli. We find that essential
genes and genes with a high degree of evolutionary conservation have promoters
that confer low levels of noise. We also find that the level of noise cannot be
attributed to the evolutionary time that different genes have spent in the genome
of E. coli. In contrast to previous results in eukaryotes, we find no association
between promoter-mediated noise and gene expression plasticity. These results are
consistent with the hypothesis that, in bacteria, natural selection can act to
reduce gene expression noise and that some of this noise is controlled through
the sequence of the promoter region alone.

DOI: 10.1371/journal.pgen.1002443 
PMCID: PMC3261926
PMID: 22275871  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no competing
interests exist.


384. PLoS One. 2012;7(12):e51179. doi: 10.1371/journal.pone.0051179. Epub 2012 Dec 14.

Enhancing E. coli tolerance towards oxidative stress via engineering its global
regulator cAMP receptor protein (CRP).

Basak S(1), Jiang R.

Author information: 
(1)School of Chemical & Biomedical Engineering, Nanyang Technological University,
Singapore, Singapore.

Oxidative damage to microbial hosts often occurs under stressful conditions
during bioprocessing. Classical strain engineering approaches are usually both
time-consuming and labor intensive. Here, we aim to improve E. coli performance
under oxidative stress via engineering its global regulator cAMP receptor protein
(CRP), which can directly or indirectly regulate redox-sensing regulators SoxR
and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed
to introduce modifications to CRP, and three mutants (OM1~OM3) were identified
with improved tolerance via H(2)O(2) enrichment selection. The best mutant OM3
could grow in 12 mM H(2)O(2) with the growth rate of 0.6 h(-1), whereas the
growth of wild type was completely inhibited at this H(2)O(2) concentration. OM3 
also elicited enhanced thermotolerance at 48°C as well as resistance against
cumene hydroperoxide. The investigation about intracellular reactive oxygen
species (ROS), which determines cell viability, indicated that the accumulation
of ROS in OM3 was always lower than in WT with or without H(2)O(2) treatment.
Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have
demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS-
and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay
suggested that catalase (katE) could be a major antioxidant enzyme in OM3 instead
of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this 
is the first work on improving E. coli oxidative stress resistance by reframing
its transcription machinery through its native global regulator. The positive
outcome of this approach may suggest that engineering CRP can be successfully
implemented as an efficient strain engineering alternative for E. coli.

DOI: 10.1371/journal.pone.0051179 
PMCID: PMC3522674
PMID: 23251448  [Indexed for MEDLINE]


385. PLoS One. 2012;7(10):e47005. doi: 10.1371/journal.pone.0047005. Epub 2012 Oct 8.

Genomic diversity of Escherichia isolates from diverse habitats.

Oh S(1), Buddenborg S, Yoder-Himes DR, Tiedje JM, Konstantinidis KT.

Author information: 
(1)School of Civil and Environmental Engineering, Georgia Institute of
Technology, Atlanta, Georgia, United States of America.

Our understanding of the Escherichia genus is heavily biased toward pathogenic or
commensal isolates from human or animal hosts. Recent studies have recovered
Escherichia isolates that persist, and even grow, outside these hosts. Although
the environmental isolates are typically phylogenetically distinct, they are
highly related to and phenotypically indistinguishable from their human
counterparts, including for the coliform test. To gain insights into the genomic 
diversity of Escherichia isolates from diverse habitats, including freshwater,
soil, animal, and human sources, we carried out comparative DNA-DNA
hybridizations using a multi-genome E. coli DNA microarray. The microarray was
validated based on hybridizations with selected strains whose genome sequences
were available and used to assess the frequency of microarray false positive and 
negative signals. Our results showed that human fecal isolates share two sets of 
genes (n>90) that are rarely found among environmental isolates, including genes 
presumably important for evading host immune mechanisms (e.g., a multi-drug
transporter for acids and antimicrobials) and adhering to epithelial cells (e.g.,
hemolysin E and fimbrial-like adhesin protein). These results imply that
environmental isolates are characterized by decreased ability to colonize host
cells relative to human isolates. Our study also provides gene markers that can
distinguish human isolates from those of warm-blooded animal and environmental
origins, and thus can be used to more reliably assess fecal contamination in
natural ecosystems.

DOI: 10.1371/journal.pone.0047005 
PMCID: PMC3466228
PMID: 23056556  [Indexed for MEDLINE]


386. PLoS One. 2012;7(8):e43862. doi: 10.1371/journal.pone.0043862. Epub 2012 Aug 27.

Modeling formamide denaturation of probe-target hybrids for improved microarray
probe design in microbial diagnostics.

Yilmaz LS(1), Loy A, Wright ES, Wagner M, Noguera DR.

Author information: 
(1)Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, Massachusetts, United States of America.
lutfu.yilmaz@umassmed.edu

Application of high-density microarrays to the diagnostic analysis of microbial
communities is challenged by the optimization of oligonucleotide probe
sensitivity and specificity, as it is generally unfeasible to experimentally test
thousands of probes. This study investigated the adjustment of hybridization
stringency using formamide with the idea that sensitivity and specificity can be 
optimized during probe design if the hybridization efficiency of oligonucleotides
with target and non-target molecules can be predicted as a function of formamide 
concentration. Sigmoidal denaturation profiles were obtained using fluorescently 
labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target
with increasing concentrations of formamide in the hybridization buffer. A linear
free energy model (LFEM) was developed and microarray-specific nearest neighbor
rules were derived. The model simulated formamide melting with a denaturant
m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per
percent of formamide added (v/v). Using the LFEM and specific probe sets, free
energy rules were systematically established to predict the stability of single
and double mismatches, including bulged and tandem mismatches. The absolute error
in predicting the position of experimental denaturation profiles was less than 5%
formamide for more than 90 percent of probes, enabling a practical level of
accuracy in probe design. The potential of the modeling approach for probe design
and optimization is demonstrated using a dataset including the 16S rRNA gene of
Rhodobacter sphaeroides as an additional target molecule. The LFEM and
thermodynamic databases were incorporated into a computational tool (ProbeMelt)
that is freely available at http://DECIPHER.cee.wisc.edu.

DOI: 10.1371/journal.pone.0043862 
PMCID: PMC3428302
PMID: 22952791  [Indexed for MEDLINE]


387. PLoS One. 2012;7(8):e43455. doi: 10.1371/journal.pone.0043455. Epub 2012 Aug 17.

The timing of IFNβ production affects early innate responses to Listeria
monocytogenes and determines the overall outcome of lethal infection.

Pontiroli F(1), Dussurget O, Zanoni I, Urbano M, Beretta O, Granucci F,
Ricciardi-Castagnoli P, Cossart P, Foti M.

Author information: 
(1)Department of Biotechnology and Bioscience, University of Milano-Bicocca,
Milan, Italy.

Dendritic cells (DCs) and natural killer (NK) cells are essential components of
the innate immunity and play a crucial role in the first phase of host defense
against infections and tumors. Listeria monocytogenes (Lm) is an intracellular
pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have
shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection.
We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the 
molecular mechanisms underlying DCs interaction with Lm. Using a functional
genomics approach, we found that Lm infection induced a cluster of late response 
genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I
INFs were produced at the maximal level only at 24 h post infection indicating
that the regulation of IFNs in the context of Lm infection is delayed compared to
the rapid response observed with viral pathogens. We showed that during Lm
infection, IFNγ production and cytotoxic activity were severely impaired in NK
cells compared to E. coli infection. These defects were restored by providing an 
exogenous source of IFNβ during the initial phase of bacterial challenge.
Moreover, when treated with IFNβ during early infection, NK cells were able to
reduce bacterial titer in the spleen and significantly improve survival of
infected mice. These findings show that the timing of IFNβ production is
fundamental to the efficient control of the bacterium during the early innate
phase of Lm infection.

DOI: 10.1371/journal.pone.0043455 
PMCID: PMC3422257
PMID: 22912878  [Indexed for MEDLINE]


388. PLoS One. 2012;7(7):e41041. doi: 10.1371/journal.pone.0041041. Epub 2012 Jul 17.

Role of heat-stable enterotoxins in the induction of early immune responses in
piglets after infection with enterotoxigenic Escherichia coli.

Loos M(1), Geens M, Schauvliege S, Gasthuys F, van der Meulen J, Dubreuil JD,
Goddeeris BM, Niewold T, Cox E.

Author information: 
(1)Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Ghent
University, Merelbeke, Belgium. michaela.loos@ugent.be

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST)
and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in
piglets. However, the relative importance of the different enterotoxins in host
immune responses against ETEC infection has been poorly defined. In the present
study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+)
ETEC strain were constructed that lack the expression of LT in combination with
one or both types of ST enterotoxins (STa and/or STb). The small intestinal
segment perfusion (SISP) technique and microarray analysis were used to study
host early immune responses induced by these mutant strains 4 h after infection
in comparison to the wild type strain and a PBS control. Simultaneously, net
fluid absorption of pig small intestinal mucosa was measured 4 h after infection,
allowing us to correlate enterotoxin secretion with gene regulation. Microarray
analysis showed on the one hand a non-toxin related general antibacterial
response comprising genes such as PAP, MMP1 and IL8. On the other hand, results
suggest a dominant role for STb in small intestinal secretion early after
post-weaning infection, as well as in the induced innate immune response through 
differential regulation of immune mediators like interleukin 1 and interleukin
17.

DOI: 10.1371/journal.pone.0041041 
PMCID: PMC3398878
PMID: 22815904  [Indexed for MEDLINE]


389. PLoS One. 2012;7(7):e40360. doi: 10.1371/journal.pone.0040360. Epub 2012 Jul 11.

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated
during prospective surveillance of a newly opened hospital in Iraq.

Huang XZ(1), Frye JG, Chahine MA, Glenn LM, Ake JA, Su W, Nikolich MP, Lesho EP.

Author information: 
(1)Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver
Spring, Maryland, United States of America. xiaozhe.huang1.ctr@us.army.mil

BACKGROUND: Gram-negative multidrug-resistant (MDR) bacteria are major causes of 
nosocomial infections, and antibiotic resistance in these organisms is often
plasmid mediated. Data are scarce pertaining to molecular mechanisms of
antibiotic resistance in resource constrained areas such as Iraq.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n =
38) and randomly selected non-MDR counterparts (n = 41) isolated from patients,
healthcare workers and environmental surfaces in a newly opened hospital in Iraq 
were investigated to characterize plasmids found in these isolates and determine 
their contribution to antibiotic resistance. Our results demonstrated that MDR E.
coli and K. pneumoniae isolates harbored significantly more (≥ 3) plasmids
compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01).
Various large plasmids (~52 to 100 kb) from representative isolates were
confirmed to contain multiple resistance genes by DNA microarray analysis.
Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla(TEM1),
bla(AMPC), bla(CTX-M-15), bla(OXA-1), bla(VIM-2) and bla(SHV)),
sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol
(cat) resistance genes were detected on these plasmids. Additionally, multiple
plasmids carrying multiple antibiotic resistance genes were found in the same
host strain. Genetic transfer-associated genes were identified on the plasmids
from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, 
B/O, K, I1 and N) were detected in the isolates, while globally disseminated
IncA/C and IncHI1 plasmids were not detected in these isolates.
CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the 
plasmids found in Enterobacteriaceae isolated following the opening of a new
hospital in Iraq. The information provided here furthers our understanding of the
mechanisms of drug resistance in this specific region and their evolutionary
relationship with other parts of world. The large plasmids, carrying resistance
genes and transfer-associated genes, may be potential factors for regional
dissemination of antibiotic resistance.

DOI: 10.1371/journal.pone.0040360 
PMCID: PMC3394789
PMID: 22808141  [Indexed for MEDLINE]


390. PLoS One. 2012;7(5):e38133. doi: 10.1371/journal.pone.0038133. Epub 2012 May 30.

Role of operon aaoSo-mutT in antioxidant defense in Streptococcus
oligofermentans.

Zhou P(1), Liu L, Tong H, Dong X.

Author information: 
(1)State Key Laboratory of Microbial Resources, Institute of Microbiology,
Chinese Academy of Sciences, Beijing, China.

Previously, we have found that an insertional inactivation of aao(So), a gene
encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of
Streptococcus oligofermentans. Here, we found that aao(So) and mutT, a homolog of
pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of
either gene did not impair the growth of S. oligofermentans, but double deletion 
of both aao(So) and mutT was lethal. Quantitative PCR showed that the transcript 
abundance of mutT was reduced for 13-fold in the aao(So) insertional mutant,
indicating that gene polarity derived from the inactivation of aao(So) attenuated
the expression of mutT. Enzymatic assays were conducted to determine the
biochemical functions of LAAO and MutT of S. oligofermentans. The results
indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H(2)O(2)
(min · mg protein)(-1)]; and MutT showed the pyrophosphohydrolase activity, which
removed mutagens such as 8-oxo-dGTP. Like paraquat, aao(So) mutations increased
the expression of SOD, and addition of aminoacetone (final concentration, 5 mM)
decreased the mutant's growth by 11%, indicating that the aao(So) mutants are
under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in 
both the deletion and insertional gene mutants of aao(So). Electron spin
resonance spectroscopy showed increased hydroxyl radicals in both types of
aao(So) mutant. This demonstrated that inactivation of aao(So) caused the
elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our 
study indicates that the presence of both LAAO and MutT can prevent endogenous
metabolites-generated ROS and mutagens. In this way, we were able to determine
the role of the aao(So)-mutT operon in antioxidant defense in S. oligofermentans.

DOI: 10.1371/journal.pone.0038133 
PMCID: PMC3364214
PMID: 22666463  [Indexed for MEDLINE]


391. PLoS One. 2012;7(5):e36720. doi: 10.1371/journal.pone.0036720. Epub 2012 May 16.

BC4707 is a major facilitator superfamily multidrug resistance transport protein 
from Bacillus cereus implicated in fluoroquinolone tolerance.

Simm R(1), Vörös A, Ekman JV, Sødring M, Nes I, Kroeger JK, Saidijam M, Bettaney 
KE, Henderson PJ, Salkinoja-Salonen M, Kolstø AB.

Author information: 
(1)Laboratory for Microbial Dynamics, Department of Pharmaceutical Biosciences,
School of Pharmacy, University of Oslo, Oslo, Norway.

Transcriptional profiling highlighted a subset of genes encoding putative
multidrug transporters in the pathogen Bacillus cereus that were up-regulated
during stress produced by bile salts. One of these multidrug transporters
(BC4707) was selected for investigation. Functional characterization of the
BC4707 protein in Escherichia coli revealed a role in the energized efflux of
xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in
Bacillus cereus ATCC14579 suggested a more specific, but modest role in the
efflux of norfloxacin. In addition to this, transcriptional analyses showed that 
BC4707 is also expressed during growth of B. cereus under non-stressful
conditions where it may have a role in the normal physiology of the bacteria.
Altogether, the results indicate that bc4707, which is part of the core genome of
the B. cereus group of bacteria, encodes a multidrug resistance efflux protein
that is likely involved in maintaining intracellular homeostasis during growth of
the bacteria.

DOI: 10.1371/journal.pone.0036720 
PMCID: PMC3353944
PMID: 22615800  [Indexed for MEDLINE]


392. PLoS One. 2012;7(5):e36697. doi: 10.1371/journal.pone.0036697. Epub 2012 May 14.

Stable gene targeting in human cells using single-strand oligonucleotides with
modified bases.

Rios X(1), Briggs AW, Christodoulou D, Gorham JM, Seidman JG, Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, Boston, Massachusetts, United 
States of America.

Recent advances allow multiplexed genome engineering in E. coli, employing easily
designed oligonucleotides to edit multiple loci simultaneously. A similar
technology in human cells would greatly expedite functional genomics, both by
enhancing our ability to test how individual variants such as single nucleotide
polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing
simultaneous mutation of multiple loci. However, oligo-mediated targeting of
human cells is currently limited by low targeting efficiencies and low survival
of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that
use of modified base analogs can increase targeting efficiency, in part by
avoiding the mismatch repair machinery. We investigate the effects of
oligonucleotide toxicity and find a strong correlation between the number of
phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated
at a frequency of ~0.05% with an optimized oligonucleotide design combining
modified bases and reduced number of phosphorothioate bonds. We provide evidence 
from comparative RNA-seq analysis suggesting cellular immunity induced by the
oligonucleotides might contribute to the low viability of oligo-corrected cells. 
Further optimization of this method should allow rapid and scalable genome
engineering in human cells.

DOI: 10.1371/journal.pone.0036697 
PMCID: PMC3351460
PMID: 22615794  [Indexed for MEDLINE]


393. PLoS One. 2012;7(3):e32127. doi: 10.1371/journal.pone.0032127. Epub 2012 Mar 23.

A genome-wide association study identifies two novel promising candidate genes
affecting Escherichia coli F4ab/F4ac susceptibility in swine.

Fu WX(1), Liu Y, Lu X, Niu XY, Ding XD, Liu JF, Zhang Q.

Author information: 
(1)Key Laboratory of Animal Genetics Breeding and Reproduction, Ministry of
Agriculture, College of Animal Science and Technology, China Agricultural
University, Beijing, China.

Enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbria is the major
pathogenic bacteria causing diarrhoea in neonatal and post-weaning piglets.
Previous studies have revealed that the susceptibility to ETEC F4ab/F4ac is an
autosomal Mendelian dominant trait and the loci controlling the F4ab/F4ac
receptor are located on SSC13q41, between markers SW207 and S0283. To pinpoint
these loci and further validate previous findings, we performed a genome-wide
association study (GWAS) using a two generation family-based population,
consisting of 301 piglets with phenotypes of susceptibility to ETEC F4ab/F4ac by 
the vitro adhesion test. The DNA of all piglets and their parents was genotyped
using the Illumina PorcineSNP60 BeadChip, and 50,972 and 50,483 SNPs were
available for F4ab and F4ac susceptibility, respectively, in the association
analysis after quality control. In summary, 28 and 18 significant SNPs (p<0.05)
were detected associated with F4ab and F4ac susceptibility respectively at
genome-wide significance level. From these significant findings, two novel
candidate genes, HEG1 and ITGB5, were firstly identified as the most promising
genes underlying F4ab/F4ac susceptibility in swine according to their functions
and positions. Our findings herein provide a novel evidence for unravelling
genetic mechanism of diarrhoea risk in piglets.

DOI: 10.1371/journal.pone.0032127 
PMCID: PMC3311625
PMID: 22457712  [Indexed for MEDLINE]


394. PLoS One. 2012;7(3):e33256. doi: 10.1371/journal.pone.0033256. Epub 2012 Mar 15.

Bleomycin sensitivity in Escherichia coli is medium-dependent.

Xu T(1), Brown W, Marinus MG.

Author information: 
(1)Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets
primarily the furanose rings of DNA and in the presence of ferrous ions produces 
oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth
medium and exposed to low concentrations of BLM contain double-strand breaks and 
require homologous recombination to survive. To a lesser extent, the cells also
require the abasic (AP) endonucleases associated with base excision repair,
presumably to repair oxidative damage. As expected, there is strong induction of 
the SOS system in treated cells. In contrast, E. coli cells growing in glucose or
glycerol minimal medium are resistant to the lethal action of BLM and do not
require either homologous recombination functions or AP-endonucleases for
survival. DNA ligase activity, however, is needed for cells growing in minimal
medium to resist the lethal effects of BLM. There is weak SOS induction in such
treated cells.

DOI: 10.1371/journal.pone.0033256 
PMCID: PMC3305319
PMID: 22438905  [Indexed for MEDLINE]


395. PLoS One. 2012;7(2):e31969. doi: 10.1371/journal.pone.0031969. Epub 2012 Feb 29.

Interpreting patterns of gene expression: signatures of coregulation, the data
processing inequality, and triplet motifs.

Ku WL(1), Duggal G, Li Y, Girvan M, Ott E.

Author information: 
(1)Department of Physics and the Institute for Physical Science and Technology,
University of Maryland, College Park, Maryland, United States of America.
wlku@umd.edu

Various methods of reconstructing transcriptional regulatory networks infer
transcriptional regulatory interactions (TRIs) between strongly coexpressed gene 
pairs (as determined from microarray experiments measuring mRNA levels).
Alternatively, however, the coexpression of two genes might imply that they are
coregulated by one or more transcription factors (TFs), and do not necessarily
share a direct regulatory interaction. We explore whether and under what
circumstances gene pairs with a high degree of coexpression are more likely to
indicate TRIs, coregulation or both. Here we use established TRIs in combination 
with microarray expression data from both Escherichia coli (a prokaryote) and
Saccharomyces cerevisiae (a eukaryote) to assess the accuracy of predictions of
coregulated gene pairs and TRIs from coexpressed gene pairs. We find that
coexpressed gene pairs are more likely to indicate coregulation than TRIs for
Saccharomyces cerevisiae, but the incidence of TRIs in highly coexpressed gene
pairs is higher for Escherichia coli. The data processing inequality (DPI) has
previously been applied for the inference of TRIs. We consider the case where a
transcription factor gene is known to regulate two genes (one of which is a
transcription factor gene) that are known not to regulate one another. According 
to the DPI, the non-interacting gene pairs should have the smallest mutual
information among all pairs in the triplets. While this is sometimes the case for
Escherichia coli, we find that it is almost always not the case for Saccharomyces
cerevisiae. This brings into question the usefulness of the DPI sometimes
employed to infer TRIs from expression data. Finally, we observe that when a TF
gene is known to regulate two other genes, it is rarely the case that one
regulatory interaction is positively correlated and the other interaction is
negatively correlated. Typically both are either positively or negatively
correlated.

DOI: 10.1371/journal.pone.0031969 
PMCID: PMC3290541
PMID: 22393375  [Indexed for MEDLINE]


396. PLoS Pathog. 2012;8(8):e1002864. doi: 10.1371/journal.ppat.1002864. Epub 2012 Aug
9.

Genome-wide analysis of germline signaling genes regulating longevity and innate 
immunity in the nematode Pristionchus pacificus.

Rae R(1), Sinha A, Sommer RJ.

Author information: 
(1)Department of Evolutionary Biology, Max Planck Institute for Developmental
Biology, Tübingen, Germany.

Removal of the reproductive system of many animals including fish, flies,
nematodes, mice and humans can increase lifespan through mechanisms largely
unknown. The abrogation of the germline in Caenorhabditis elegans increases
longevity by 60% due to a signal emitted from the somatic gonad. Apart from
increased longevity, germline-less C. elegans is also resistant to other
environmental stressors such as feeding on bacterial pathogens. However, the
evolutionary conservation of this pathogen resistance, its genetic basis and an
understanding of genes involved in producing this extraordinary survival
phenotype are currently unknown. To study these evolutionary aspects we used the 
necromenic nematode Pristionchus pacificus, which is a genetic model system used 
in comparison to C. elegans. By ablation of germline precursor cells and
subsequent feeding on the pathogen Serratia marcescens we discovered that P.
pacificus shows remarkable resistance to bacterial pathogens and that this
response is evolutionarily conserved across the Genus Pristionchus. To gain a
mechanistic understanding of the increased resistance to bacterial pathogens and 
longevity in germline-ablated P. pacificus we used whole genome microarrays to
profile the transcriptional response comparing germline ablated versus un-ablated
animals when fed S. marcescens. We show that lipid metabolism, maintenance of the
proteasome, insulin signaling and nuclear pore complexes are essential for
germline deficient phenotypes with more than 3,300 genes being differentially
expressed. In contrast, gene expression of germline-less P. pacificus on E. coli 
(longevity) and S. marcescens (immunity) is very similar with only 244 genes
differentially expressed indicating that longevity is due to abundant gene
expression also involved in immunity. By testing existing mutants of
Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved
function of both genes in resistance to bacterial pathogens and longevity. This
is the first study to show that the influence of the reproductive system on
extending lifespan and innate immunity is conserved in evolution.

DOI: 10.1371/journal.ppat.1002864 
PMCID: PMC3415453
PMID: 22912581  [Indexed for MEDLINE]


397. Pol J Microbiol. 2012;61(2):105-10.

Virulence genes profiles and phylogenetic origin of Escherichia coli from acute
and chronic intestinal diseases revealed by comparative genomic hybridization
microarray.

Sobieszczańska B(1), Kasprzykowska U, Turniak M, Maciejewski H, Franiczek R,
Duda-Madej A.

Author information: 
(1)University of Medicine, Department of Microbiology, Wrocław, Poland.
bmsobie@gmail.com

The association between Escherichia coli virulence factors and chronic intestinal
disorders is mostly unknown. The presented study compared the distribution of
virulence genes and phylogroups among E. coli isolated from chronic intestinal
disorders such as Crohn's disease and irritable bowel syndrome (IBS) with strains
isolated from patients with acute diarrhea as a control group. The presence of
159 virulence genes corresponding to known E. coli pathotypes was determined
among 78 E. coli archive strains isolated from IBS, acute diarrhea and Crohn's
disease using CGH microarray. E. coli isolated from IBS demonstrated a mosaic of 
virulence genes specific to enteropathogenic, enterotoxigenic, enterohemorrhagic 
E. coli strains and Shigella species. In contrast, virulence factors and
phylogroups distribution among E. coli isolated from children with acute diarrhea
was similar to extraintestinal E. coli strains that probably acquired some
virulence genes. The acquisition of virulence genes might have an impact on
diarrheagenic potential of these strains. On the other hand, E. coli isolated
from children with Crohn's disease seem to be similar to adherent-invasive E.
coli strains (AIEC), as it lack most known virulence genes. The presented study
showed that these analyzed groups of E. coli strains differed from each other
with the respect to the distribution of virulence genes. The differences in gene 
content support the idea that the participation of E. coli in chronic intestinal 
diseases is mostly related to virulence potential of these strains.


PMID: 23163209  [Indexed for MEDLINE]


398. Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(9):485-508.

Prokaryotic genome regulation: a revolutionary paradigm.

Ishihama A(1).

Author information: 
(1)Department of Frontier Bioscience and Micro-Nano Technology Research Center,
Hosei University, Koganei, Tokyo 184-8584, Japan. aishiham@hosei.ac.jp

After determination of the whole genome sequence, the research frontier of
bacterial molecular genetics has shifted to reveal the genome regulation under
stressful conditions in nature. The gene selectivity of RNA polymerase is
modulated after interaction with two groups of regulatory proteins, 7 sigma
factors and 300 transcription factors. For identification of regulation targets
of transcription factors in Escherichia coli, we have developed Genomic SELEX
system and subjected to screening the binding sites of these factors on the
genome. The number of regulation targets by a single transcription factor was
more than those hitherto recognized, ranging up to hundreds of promoters. The
number of transcription factors involved in regulation of a single promoter also 
increased to as many as 30 regulators. The multi-target transcription factors and
the multi-factor promoters were assembled into complex networks of transcription 
regulation. The most complex network was identified in the regulation cascades of
transcription of two master regulators for planktonic growth and biofilm
formation.


PMCID: PMC3511978
PMID: 23138451  [Indexed for MEDLINE]


399. J Biol Chem. 2011 Dec 30;286(52):44726-38. doi: 10.1074/jbc.M111.290684. Epub
2011 Oct 28.

Determination of target sequence bound by PapX, repressor of bacterial motility, 
in flhD promoter using systematic evolution of ligands by exponential enrichment 
(SELEX) and high throughput sequencing.

Reiss DJ(1), Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, Michigan 48104, USA.

Most uncomplicated urinary tract infections (UTIs) are caused by uropathogenic
Escherichia coli (UPEC). Both motility and adherence are integral to UTI
pathogenesis, yet they represent opposing forces. Therefore, it is logical to
reciprocally regulate these functions. In UPEC strain CFT073, PapX, a
non-structural protein encoded by one of the two pap operons encoding P fimbria
adherence factor, represses flagella-mediated motility and is a putative member
of the winged helix transcription factor family. The mechanism of this
repression, however, is not understood. papX is found preferentially in more
virulent UPEC isolates, being significantly more prevalent in pyelonephritis
strains (53% of isolates) than in asymptomatic bacteriuria (32%) or
fecal/commensal (12.5%) strains. To examine PapX structure-function, we generated
papX linker insertion and site-directed mutants, which identified two key
residues for PapX function (Lys(54) and Arg(127)) within domains predicted by
modeling with I-TASSER software to be important for dimerization and DNA binding,
respectively. To determine the PapX binding site in the CFT073 genome, systematic
evolution of ligands by exponential enrichment (SELEX) in conjunction with high
throughput sequencing was utilized for the first time to determine a novel
binding site for a bacterial transcription factor. This method identified a 29-bp
binding site within the flhDC promoter (TTACGGTGAGTTATTTTAACTGTGCGCAA), centered 
410 bp upstream of the flhD translational start site. Gel shift experiments
demonstrated that PapX binds directly to this site to repress transcription of
flagellar genes.

DOI: 10.1074/jbc.M111.290684 
PMCID: PMC3247938
PMID: 22039053  [Indexed for MEDLINE]


400. Antimicrob Agents Chemother. 2011 Dec;55(12):5850-60. doi: 10.1128/AAC.00498-11. 
Epub 2011 Sep 19.

Fluoroquinolone efflux in Streptococcus suis is mediated by SatAB and not by
SmrA.

Escudero JA(1), San Millan A, Gutierrez B, Hidalgo L, La Ragione RM, AbuOun M,
Galimand M, Ferrándiz MJ, Domínguez L, de la Campa AG, Gonzalez-Zorn B.

Author information: 
(1)Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad
Complutense de Madrid, Madrid, Spain.

Streptococcus suis is an emerging zoonotic pathogen. With the lack of an
effective vaccine, antibiotics remain the main tool to fight infections caused by
this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone 
(FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging 
to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS),
respectively, have been analyzed in the fluoroquinolone-resistant clinical
isolate BB1013. Genes encoding these pumps were overexpressed either
constitutively or in the presence of ciprofloxacin in this strain. These genes
could not be cloned in plasmids in Escherichia coli despite strong expression
repression. Finally, site-directed insertion of smrA and satAB in the amy locus
of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the
functional expression and study of both pumps. Results showed that SatAB is a
narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin),
susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone
resistance. Chromosomal integration in Bacillus is a novel method for studying
efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the
possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

DOI: 10.1128/AAC.00498-11 
PMCID: PMC3232752
PMID: 21930876  [Indexed for MEDLINE]


401. Environ Microbiol. 2011 Dec;13(12):3149-62. doi:
10.1111/j.1462-2920.2011.02607.x. Epub 2011 Oct 9.

Enterobactin is required for biofilm development in reduced-genome Escherichia
coli.

May T(1), Okabe S.

Author information: 
(1)Division of Environmental Engineering, Graduate School of Engineering,
Hokkaido University, Kita-13, Nishi-8, Kita-Ku, Sapporo, Hokkaido 060-8628,
Japan.

A variety of bacterial cell surface structures and quorum signalling molecules
play a role in biofilm development in Escherichia coli. However, here we show
that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental
E. coli genome, including the genes involved in the synthesis of various cell
surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers
and the autoinducer-2 signalling molecule, is able to develop mature biofilms.
Using temporal gene expression profiling, we investigated phenotypic changes in
reduced-genome biofilms in relation with the genes encoding the synthesis of
different amino acids that were differentially expressed during biofilm
formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes,
as involved in biofilm formation by the reduced-genome E. coli. Of these, for a
first time, we demonstrated that overproduction of entB and yahK, which encode an
enterobactin for iron transport and a hypothetical oxidoreductase protein,
respectively, promoted biofilm development and maturation. Our results indicate
that specific types of genes contribute to phenotypic changes in reduced-genome
E. coli biofilms. In addition, this work demonstrates that the functions of
biofilm-specific genes could be analysed through experiments using the
reduced-genome E. coli.

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

DOI: 10.1111/j.1462-2920.2011.02607.x 
PMID: 21980953  [Indexed for MEDLINE]


402. J Hosp Infect. 2011 Dec;79(4):349-53. doi: 10.1016/j.jhin.2011.08.005. Epub 2011 
Oct 1.

Distribution of extended-spectrum beta-lactamase genes using a commercial DNA
micro-array system.

Nijhuis RH(1), van Zwet AA, Savelkoul PH, Roovers EA, Bosboom RW, Postma B, van
Griethuysen AJ.

Author information: 
(1)Department of Medical Microbiology and Medical Immunology, Rijnstate, Velp,
The Netherlands. rnijhuis@rijnstate.nl

Extended-spectrum beta-lactamase (ESBL) genes are distributed worldwide and their
epidemiology is complex. Using the Check-ESBL assay, the distribution of class A 
ESBL genes in clinical isolates of aerobic Gram-negative bacilli from three
laboratories in the East of The Netherlands was determined. Four patient
categories were distinguished: (i) patients admitted to an intensive care unit
(ICU); (ii) non-ICU inpatients; (iii) outpatients admitted less than a year
before collection of the isolate, (<1); (iv) outpatients admitted more than
one-year prior to isolate collection or who had never been hospitalized (>1).
From February 2009 until March 2010, out of 491 putative ESBL-positive isolates
detected by the Vitek2 or Phoenix automated sensitivity testing systems, ESBL
genes were detected in 247 (50.3%) by the Check-ESBL assay. Of these, 116 were
from hospitalized patients (35 ICU, 81 non-ICU) and 131 were from outpatients (43
<1, 88 >1). In all, 274 ESBL genes were identified in these 247 isolates: 153
CTX-M-1 group (predominantly in E. coli and K. pneumoniae, 70.4% and 51.6%
respectively), 67 CTX-M-9 group (predominantly in E. cloacae, 57.9%), 32 SHV, 14 
TEM and 8 CTX-M-2 group. ESBL-producing E. cloacae were significantly more common
in hospitalized patients than in outpatients, 20.7% and 3.8% respectively
(P=0.001). CTX-M-9 group ESBLs were significantly more prevalent in ICU patients 
(P=0.003), whereas SHV ESBLs were more common in hospitalized patients than in
outpatients (P<0.001). There was no significant difference in distribution of
ESBL genes between the two outpatient groups.

Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All
rights reserved.

DOI: 10.1016/j.jhin.2011.08.005 
PMID: 21962834  [Indexed for MEDLINE]


403. Mol Cells. 2011 Dec;32(6):527-33. doi: 10.1007/s10059-011-0156-x. Epub 2011 Nov
3.

DNA aptamers against the receptor binding region of hemagglutinin prevent avian
influenza viral infection.

Choi SK(1), Lee C, Lee KS, Choe SY, Mo IP, Seong RH, Hong S, Jeon SH.

Author information: 
(1)Department of Life Science, Hallym University, Chuncheon 200-702, Korea.

The entrance of influenza virus into host cells is facilitated by the attachment 
of the globular region of viral hemagglutinin to the sialic acid receptors on
host cell surfaces. In this study, we have cloned the cDNA fragment encoding the 
entire globular region (residues 101-257) of hemagglutinin of the H9N2 type avian
influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9
peptide), which was expressed and purified in E. coli, was used for the
immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific
DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 
aptamers were newly isolated. A random mixture of these aptamers showed an
increased level of binding affinity to the H9 peptide. The sequence alignment
analysis of these aptamers revealed that 6 aptamers have highly conserved
consensus sequences. Among these, aptamer C7 showed the highest similarity to the
consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new
modified aptamer designated as C7-35M. This new aptamer showed strong binding
capability to the viral particles. Furthermore, it could prevent MDCK cells from 
viral infection by strong binding to the viral particles. These results suggest
that our aptamers can recognize the hemagglutinin protein of avian influenza
virus and inhibit the binding of the virus to target receptors required for the
penetration of host cells.

DOI: 10.1007/s10059-011-0156-x 
PMCID: PMC3887679
PMID: 22058017  [Indexed for MEDLINE]


404. BMC Genomics. 2011 Nov 28;12:583. doi: 10.1186/1471-2164-12-583.

The association of DNA damage response and nucleotide level modulation with the
antibacterial mechanism of the anti-folate drug trimethoprim.

Sangurdekar DP(1), Zhang Z, Khodursky AB.

Author information: 
(1)Lewis-Sigler Institute for Integrative Genomics, Princeton University, 132
Carl C. Icahn Laboratory, Princeton University, Washington Road, Princeton NJ
08540, USA.

BACKGROUND: Trimethoprim is a widely prescribed antibiotic for a variety of
bacterial infections. It belongs to a class of anti-metabolites - antifolates -
which includes drugs used against malarial parasites and in cancer therapy.
However, spread of bacterial resistance to the drug has severely hampered its
clinical use and has necessitated further investigations into its mechanism of
action and treatment regimen. Trimethoprim selectively starves bacterial cells
for tetrahydrofolate, a vital cofactor necessary for the synthesis of several
metabolites. The outcome (bacteriostatic or bactericidal) of such starvation,
however, depends on the availability of folate-dependent metabolites in the
growth medium. To characterize this dependency, we investigated in detail the
regulatory and structural components of Escherichia coli cellular response to
trimethoprim in controlled growth and supplementation conditions.
RESULTS: We surveyed transcriptional responses to trimethoprim treatment during
bacteriostatic and bactericidal conditions and analyzed associated gene
sets/pathways. Concurrent starvation of all folate dependent metabolites caused
growth arrest, and this was accompanied by induction of general stress and
stringent responses. Three gene sets were significantly associated with the
bactericidal effect of TMP in different media including LB: genes of the SOS
regulon, genes of the pyrimidine nucleotide biosynthetic pathway and members of
the multiple antibiotic resistance (mar) regulon controlled by the MarR
repressor. However, the SOS response was identified as the only universal
transcriptional signature associated with the loss of viability by direct thymine
starvation or by folate stress. We also used genome-wide gene knock-out screen to
uncover means of sensitization of bacteria to the drug. We observed that among a 
number of candidate genes and pathways, the effect of knock-outs in the
deoxyribose nucleotide salvage pathway, encoded by the deoCABD operon and under
the control of the DeoR repressor, was most informative.
CONCLUSION: Transcriptional induction of DNA damage response is an essential
feature of the bactericidal effect of trimethoprim. Either the observation of the
transcriptional response or DNA damage itself, or both, is made possible by
thymine starvation when other folate-dependent metabolites are not limited. The
effect of DNA damage by the drug takes place prior to its bactericidal effect, at
the beginning of the lag stage of the treatment. Mutations in the deoxyribose
nucleotide salvage pathway can affect duration of the lag as well as the rate of 
killing. This information can be used to postulate certain mechanistic
differences between direct thymine starvation in thymidylate synthase deficient
mutants and thymine starvation by anti-folate inhibitors.

© 2011 Sangurdekar et al; licensee BioMed Central Ltd.

DOI: 10.1186/1471-2164-12-583 
PMCID: PMC3258297
PMID: 22122981  [Indexed for MEDLINE]


405. Anal Chem. 2011 Nov 15;83(22):8531-6. doi: 10.1021/ac201823v. Epub 2011 Oct 17.

Carbon nanoparticles as detection labels in antibody microarrays. Detection of
genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

Noguera PS(1), Posthuma-Trumpie GA, van Tuil M, van der Wal FJ, de Boer A, Moers 
AP, van Amerongen A.

Author information: 
(1)Biomolecular Sensing & Diagnostics, Wageningen UR Food & Biobased Research,
Wageningen, The Netherlands.

The present study demonstrates that carbon nanoparticles (CNPs) can be used as
labels in microarrays. CNPs were used in nucleic acid microarray immunoassays
(NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli
(STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) 
and the gene for E. coli 16S (hui). Optimization was performed using a
Box-Behnken design, and the limit of detection for each virulence factor was
established. Finally, this NAMIA using CNPs was tested with DNA from 48 field
strains originating from cattle feces, and its performance was evaluated by
comparing results with those achieved by the reference method q-PCR. All factors 
tested gave sensitivity and specificity values higher than 0.80 and efficiency
values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k
> 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a 
perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the
developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is 
indeed fit for purpose, i.e., it is valuable for fast screening of amplified
genetic material such as E. coli virulence factors. This also proves the
applicability of CNPs in microarrays.

DOI: 10.1021/ac201823v 
PMID: 21936575  [Indexed for MEDLINE]


406. Antimicrob Agents Chemother. 2011 Nov;55(11):5043-53. doi: 10.1128/AAC.05009-11. 
Epub 2011 Aug 15.

Antibacterial mechanism of action of arylamide foldamers.

Mensa B(1), Kim YH, Choi S, Scott R, Caputo GA, DeGrado WF.

Author information: 
(1)Department of Biochemistry and Biophysics, University of Pennsylvania, 1010
Stellar Chance Laboratories, 422 Curie Blvd, Philadelphia, Pennsylvania
19104-4860, USA.

Small arylamide foldamers designed to mimic the amphiphilic nature of
antimicrobial peptides (AMPs) have shown potent bactericidal activity against
both Gram-negative and Gram-positive strains without many of the drawbacks of
natural AMPs. These foldamers were shown to cause large changes in the
permeability of the outer membrane of Escherichia coli. They cause more limited
permeabilization of the inner membrane which reaches critical levels
corresponding with the time required to bring about bacterial cell death.
Transcriptional profiling of E. coli treated with sublethal concentrations of the
arylamides showed induction of genes related to membrane and oxidative stresses, 
with some overlap with the effects observed for polymyxin B. Protein secretion
into the periplasm and the outer membrane is also compromised, possibly
contributing to the lethality of the arylamide compounds. The induction of
membrane stress response regulons such as rcs coupled with morphological changes 
at the membrane observed by electron microscopy suggests that the activity of the
arylamides at the membrane represents a significant contribution to their
mechanism of action.

DOI: 10.1128/AAC.05009-11 
PMCID: PMC3195038
PMID: 21844313  [Indexed for MEDLINE]


407. Mol Cell Biol. 2011 Nov;31(22):4593-608. doi: 10.1128/MCB.05798-11. Epub 2011 Sep
19.

Distinct functions of Sox2 control self-renewal and differentiation in the
osteoblast lineage.

Seo E(1), Basu-Roy U, Zavadil J, Basilico C, Mansukhani A.

Author information: 
(1)Department of Microbiology, NYU School of Medicine, New York, NY 10016, USA.

The transcription factor Sox2 is a key player in the maintenance of pluripotency 
and "stemness." We have previously shown that Sox2 maintains self-renewal in the 
osteoblast lineage while inhibiting differentiation (U. Basu-Roy et al., Cell
Death Differ. 17:1345-1353, 2010; A. Mansukhani, D. Ambrosetti, G. Holmes, L.
Cornivelli, and C. Basilico, J. Cell Biol. 168:1065-1076, 2005). Sox2 also
interferes with Wnt signaling by binding β-catenin, a central mediator of the Wnt
pathway. Here we show that these multiple functions of Sox2 are encoded in
distinct domains. The self-renewal function of Sox2 is dependent on its
transcriptional activity and requires both its DNA-binding and C-terminal
activation regions, while only the third C-terminal transactivation (TA) region
is required for binding β-catenin and interfering with Wnt-induced transcription.
The results of gene expression analysis upon Sox2 deletion strongly support the
notion that Sox2 maintains stemness. We show also that Sox2 suppresses
differentiation by attenuating Wnt signaling by posttranscriptional and
transcriptional mechanisms and that adenomatous polyposis coli (APC) and GSK3β,
which are negative regulators of the Wnt pathway, are direct Sox2 targets in
osteoblasts. Several genes, such as the FoxP1 and BMI-1 genes, that are
associated with stemness are downregulated upon Sox2 inactivation. Constitutive
expression of the Polycomb complex member BMI-1 can bypass the Sox2 requirement
for self-renewal but does not affect differentiation. Our results establish a
connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1 
as a key mediator of Sox2 function.

DOI: 10.1128/MCB.05798-11 
PMCID: PMC3209254
PMID: 21930787  [Indexed for MEDLINE]


408. Nucleic Acids Res. 2011 Nov 1;39(20):e140. doi: 10.1093/nar/gkr617. Epub 2011 Aug
31.

Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq.

Yi H(1), Cho YJ, Won S, Lee JE, Jin Yu H, Kim S, Schroth GP, Luo S, Chun J.

Author information: 
(1)Institute of Molecular Biology and Genetics, School of Biological Sciences &
Institute of Bioinformatics (BIOMAX), Seoul National University, Seoul, Republic 
of Korea.

Next-generation sequencing has great potential for application in bacterial
transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to 
select mRNAs over rRNAs; therefore, rRNA removal is a critical step in
sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme
that, at high temperatures, degrades duplex DNA in preference to single-stranded 
DNA. DSN treatment has been successfully used to normalize the relative
transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms.
In this study, we demonstrate the utility of this method to remove rRNA from
prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by
comparing it with the conventional subtractive hybridization (Hyb) method.
Illumina deep sequencing was performed to obtain transcriptomes from Escherichia 
coli grown under four growth conditions. The results clearly showed that our DSN 
treatment was more efficient at removing rRNA than the Hyb method was, while
preserving the original relative abundance of mRNA species in bacterial cells.
Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment
should be preferred to Hyb-based methods.

DOI: 10.1093/nar/gkr617 
PMCID: PMC3203590
PMID: 21880599  [Indexed for MEDLINE]


409. PLoS Genet. 2011 Nov;7(11):e1002377. doi: 10.1371/journal.pgen.1002377. Epub 2011
Nov 17.

Genetic interaction maps in Escherichia coli reveal functional crosstalk among
cell envelope biogenesis pathways.

Babu M(1), Díaz-Mejía JJ, Vlasblom J, Gagarinova A, Phanse S, Graham C, Yousif F,
Ding H, Xiong X, Nazarians-Armavil A, Alamgir M, Ali M, Pogoutse O, Pe'er A,
Arnold R, Michaut M, Parkinson J, Golshani A, Whitfield C, Wodak SJ,
Moreno-Hagelsieb G, Greenblatt JF, Emili A.

Author information: 
(1)Banting and Best Department of Medical Research, Donnelly Centre, University
of Toronto, Toronto, Canada. mohan.babu@utoronto.ca

As the interface between a microbe and its environment, the bacterial cell
envelope has broad biological and clinical significance. While numerous
biosynthesis genes and pathways have been identified and studied in isolation,
how these intersect functionally to ensure envelope integrity during adaptive
responses to environmental challenge remains unclear. To this end, we performed
high-density synthetic genetic screens to generate quantitative functional
association maps encompassing virtually the entire cell envelope biosynthetic
machinery of Escherichia coli under both auxotrophic (rich medium) and
prototrophic (minimal medium) culture conditions. The differential patterns of
genetic interactions detected among > 235,000 digenic mutant combinations tested 
reveal unexpected condition-specific functional crosstalk and genetic backup
mechanisms that ensure stress-resistant envelope assembly and maintenance. These 
networks also provide insights into the global systems connectivity and dynamic
functional reorganization of a universal bacterial structure that is both broadly
conserved among eubacteria (including pathogens) and an important target.

DOI: 10.1371/journal.pgen.1002377 
PMCID: PMC3219608
PMID: 22125496  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no competing
interests exist.


410. J Mol Biol. 2011 Oct 14;413(1):51-65. doi: 10.1016/j.jmb.2011.07.063. Epub 2011
Aug 3.

Degenerate RNA packaging signals in the genome of Satellite Tobacco Necrosis
Virus: implications for the assembly of a T=1 capsid.

Bunka DH(1), Lane SW, Lane CL, Dykeman EC, Ford RJ, Barker AM, Twarock R,
Phillips SE, Stockley PG.

Author information: 
(1)Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds
LS2 9JT, UK.

Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle
expressed in Escherichia coli, we have established conditions for in vitro
disassembly and reassembly of the viral capsid. In vivo assembly is dependent on 
the presence of the coat protein (CP) N-terminal region, and in vitro assembly
requires RNA. Using immobilised CP monomers under reassembly conditions with
"free" CP subunits, we have prepared a range of partially assembled CP species
for RNA aptamer selection. SELEX directed against the RNA-binding face of the
STNV CP resulted in the isolation of several clones, one of which (B3) matches
the STNV-1 genome in 16 out of 25 nucleotide positions, including across a
statistically significant 10/10 stretch. This 10-base region folds into a
stem-loop displaying the motif ACAA and has been shown to bind to STNV CP.
Analysis of the other aptamer sequences reveals that the majority can be folded
into stem-loops displaying versions of this motif. Using a sequence and secondary
structure search motif to analyse the genomic sequence of STNV-1, we identified
30 stem-loops displaying the sequence motif AxxA. The implication is that there
are many stem-loops in the genome carrying essential recognition features for
binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold
showed that only 8 out of 30 of these stem-loops would be formed in the
lowest-energy structure. These results are consistent with an assembly mechanism 
based on kinetically driven folding of the RNA.

Copyright © 2011 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.jmb.2011.07.063 
PMID: 21839093  [Indexed for MEDLINE]


411. Arch Microbiol. 2011 Oct;193(10):711-22. doi: 10.1007/s00203-011-0708-7. Epub
2011 May 11.

Biofilm formation in Escherichia coli cra mutants is impaired due to
down-regulation of curli biosynthesis.

Reshamwala SM(1), Noronha SB.

Author information: 
(1)Department of Biosciences and Bioengineering, Indian Institute of Technology
Bombay, Powai, Mumbai, Maharashtra, 400076, India.

Cra is a pleiotropic regulatory protein that controls carbon and energy flux in
enteric bacteria. Recent studies have shown that Cra also regulates other cell
processes and influences biofilm formation. The purpose of the present study was 
to investigate the role of Cra in biofilm formation in Escherichia coli. Congo
red-binding studies suggested that curli biosynthesis is impaired in cra mutants.
Microarray analysis of wild-type and mutant E. coli cultivated in conditions
promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC
and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of
genes required for curli production is regulated by Cra. Four putative
Cra-binding sites were identified in the curli intergenic region, which were
experimentally validated by performing electromobility shift assays.
Site-directed mutagenesis of three Cra-binding sites in the promoter region of
the csgDEFG operon suggests that Cra activates transcription of this operon upon 
binding to operator regions both downstream and upstream of the transcription
start site. Based on the Cra-binding sites identified in this and other studies, 
the Cra consensus sequence is refined.

DOI: 10.1007/s00203-011-0708-7 
PMID: 21559929  [Indexed for MEDLINE]


412. J Immunol. 2011 Oct 1;187(7):3683-93. doi: 10.4049/jimmunol.1101397. Epub 2011
Aug 24.

Genome-wide expression profiling and mutagenesis studies reveal that
lipopolysaccharide responsiveness appears to be absolutely dependent on TLR4 and 
MD-2 expression and is dependent upon intermolecular ionic interactions.

Meng J(1), Gong M, Björkbacka H, Golenbock DT.

Author information: 
(1)Division of Infectious Diseases and Immunology, University of Massachusetts
Medical School, Worcester, MA 01605, USA. jianmin.meng@umassmed.edu

Lipid A (a hexaacylated 1,4' bisphosphate) is a potent immune stimulant for
TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates
signal transduction. Historically, studies also suggested the existence of
TLR4/MD-2-independent LPS signaling. In this article, we define the role of TLR4 
and MD-2 in LPS signaling by using genome-wide expression profiling in TLR4- and 
MD-2-deficient macrophages after stimulation with peptidoglycan-free LPS and
synthetic Escherichia coli lipid A. Of the 1396 genes significantly induced or
repressed by any one of the treatments in the wild-type macrophages, none was
present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2
complex is the only receptor for endotoxin and that both are required for
responses to LPS. Using a molecular genetics approach, we investigated the
mechanism of TLR4/MD-2 activation by combining the known crystal structure of
TLR4/MD-2 with computer modeling. According to our murine TLR4/MD-2-activation
model, the two phosphates on lipid A were predicted to interact extensively with 
the two positively charged patches on mouse TLR4. When either positive patch was 
abolished by mutagenesis into Ala, the responses to LPS and lipid A were nearly
abrogated. However, the MyD88-dependent and -independent pathways were impaired
to the same extent, indicating that the adjuvant activity of monophosphorylated
lipid A most likely arises from its decreased potential to induce an active
receptor complex and not more downstream signaling events. Hence, we concluded
that ionic interactions between lipid A and TLR4 are essential for optimal LPS
receptor activation.

DOI: 10.4049/jimmunol.1101397 
PMCID: PMC3178671
PMID: 21865549  [Indexed for MEDLINE]


413. J Microbiol Methods. 2011 Oct;87(1):105-10. doi: 10.1016/j.mimet.2011.07.019.
Epub 2011 Jul 30.

Rapid O serogroup identification of the ten most clinically relevant STECs by
Luminex microbead-based suspension array.

Lin A(1), Nguyen L, Lee T, Clotilde LM, Kase JA, Son I, Carter JM, Lauzon CR.

Author information: 
(1)U.S. Food and Drug Administration San Francisco District Laboratory, Alameda, 
CA 94502, USA. andrew.lin@fda.hhs.gov

Identification and serotyping of Shiga toxin-producing Escherichia coli during
foodborne outbreaks can aid in matching clinical, food, and environmental
isolates when trying to identify the source of illness and ultimately food
contamination. Herein we describe a Luminex microbead-based suspension array to
identify the O serogroup of the ten most clinically relevant STECs: O26, O45,
O91, O103, O111, O113, O121, O128, O145, and O157. The use of PCR followed by
Luminex xMAP® technology enables the detection of multiple analytes in a single
multiplex reaction with high throughput capabilities. One hundred and fourteen
STEC isolates were correctly identified with no false positives among forty-six
other organisms using this assay. Assay performance was tested in multiple
laboratories using a panel of eleven different STEC serogroups on the Bio-Plex
200 and MAGPIX instruments. The STEC microbead-based suspension array can be
performed in a 96-well plate format for high throughput screening in less than
4h. Furthermore, it is expandable, allowing for the addition of O serogroups
should the need arise.

Published by Elsevier B.V.

DOI: 10.1016/j.mimet.2011.07.019 
PMID: 21835211  [Indexed for MEDLINE]


414. J Nutr. 2011 Oct;141(10):1805-12. doi: 10.3945/jn.111.140178. Epub 2011 Aug 31.

Whole blueberry powder modulates the growth and metastasis of MDA-MB-231 triple
negative breast tumors in nude mice.

Adams LS(1), Kanaya N, Phung S, Liu Z, Chen S.

Author information: 
(1)Division of Tumor Cell Biology, Beckman Research Institute of the City of
Hope, Duarte, CA, USA.

Previous studies in our laboratory demonstrated that blueberry (BB) extract
exhibited antitumor activity against MDA-MB-231 triple negative breast cancer
(TNBC) cells and decreased metastatic potential in vitro. The current study
tested 2 doses of whole BB powder, 5 and 10% (wt:wt) in the diet, against
MDA-MB-231 tumor growth in female nude mice. In this study, tumor volume was 75% 
lower in mice fed the 5% BB diet and 60% lower in mice fed the 10% BB diet than
in control mice (P ≤ 0.05). Tumor cell proliferation (Ki-67) was lower in the 5
and 10% BB-fed mice and cell death (Caspase 3) was greater in the 10% BB-fed mice
compared to control mice (P ≤ 0.05). Gene analysis of tumor tissues from the 5%
BB-fed mice revealed significantly altered expression of genes important to
inflammation, cancer, and metastasis, specifically, Wnt signaling,
thrombospondin-2, IL-13, and IFNγ. To confirm effects on Wnt signaling, analysis 
of tumor tissues from 5% BB-fed mice revealed lower β-catenin expression and
glycogen synthase kinase-3β phosphorylation with greater expression of the
β-catenin inhibitory protein adenomatous polyposis coli compared to controls. A
second study tested the ability of the 5% BB diet to inhibit
MDA-MB-231-luc-D3H2LN metastasis in vivo. In this study, 5% BB-fed mice developed
70% fewer liver metastases (P = 0.04) and 25% fewer lymph node metastases (P =
0.09) compared to control mice. This study demonstrates the oral antitumor and
metastasis activity of whole BB powder against TNBC in mice.

DOI: 10.3945/jn.111.140178 
PMCID: PMC3174855
PMID: 21880954  [Indexed for MEDLINE]


415. Mol Microbiol. 2011 Oct;82(2):378-97. doi: 10.1111/j.1365-2958.2011.07818.x. Epub
2011 Sep 14.

Novel roles of LeuO in transcription regulation of E. coli genome: antagonistic
interplay with the universal silencer H-NS.

Shimada T(1), Bridier A, Briandet R, Ishihama A.

Author information: 
(1)Hosei University, Department of Frontier Bioscience, Koganei, Tokyo 184-8540, 
Japan INRA UMR1319 MICALIS, 78352 Jouy-en-Josas, France AgroParisTech UMR
MICALIS, 91300 Massy, France.

LeuO, the regulator of leucine biosynthesis operon of Escherichia coli, is
involved in the regulation of as yet unspecified genes affecting the stress
response and pathogenesis expression. To get insights into the regulatory role(s)
of LeuO, Genomic SELEX screening has been performed to identify the whole set of 
its regulation targets. A total of 140 LeuO-binding sites were identified on the 
E. coli genome, of which as many as 133 (95%) were found to contain the binding
sites of H-NS, the universal silencer of stress-response genes, supporting the
concept that LeuO plays an antagonistic role with anti-silencing activity.
Western blot analysis indicated that H-NS predominates in growing phase; however,
after prolonged culture for 1 week, H-NS decreased instead LeuO increased,
supporting the anti-silencing role of LeuO. In concert with this model, a set of 
stress-response genes including cryptic chaperone/usher-type fimbriae operons are
under the control of antagonistic interplay between LeuO and H-NS. Confocal laser
scanning microscopic observation in flow-chambers showed that the mutants lacking
leuO and some fimbriae genes are defective in biofilm formation or form altered
biofilm architecture. Taken together we propose that LeuO is a major player in
antagonistic interplay against the universal silencer H-NS.

© 2011 Blackwell Publishing Ltd.

DOI: 10.1111/j.1365-2958.2011.07818.x 
PMID: 21883529  [Indexed for MEDLINE]


416. Zhonghua Wai Ke Za Zhi. 2011 Oct 1;49(10):927-33.

[Genomics study of steroid induced femoral head necrosis of rats].

[Article in Chinese]

Wu CL(1), Mao Q, Liu H, Tong PJ, Jin HT, Xiao LW.

Author information: 
(1)Institute of Orthopedics and Traumatology, Zhejiang Chinese Medical
University, Hangzhou 310053, China.

OBJECTIVES: To identify significantly differentially expression genes of
steroid-induced femoral head necrosis (SINFH) of rats by gene chip, and to find
out the potential factors and molecular mechanisms that oxidative stress
originate or strengthen the SINFH.
METHODS: Twenty Wistar rats were divided into experimental group and control
group randomly. E. coli endotoxin was given to all rats at a dose of 20 µg/kg
body weight by daily i.p. for two times. Then methylprednisolone (40 mg/kg) or
saline was daily injected into the left gluteus muscle of the rats in
experimental group and control group respectively. Six weeks later, the mRNA was 
extracted from the femoral head of rats in every group, and the cDNA were
obtained by inverse transcript, then carried out microarray detection. The
quantitative RT-PCR was used to confirm the result of microarray, and the
differentially expressed genes were analyzed for the functional annotation by
gene ontology (GO).
RESULTS: Compared to the control group, 190 genes in the experimental group were 
differentially expressed, with 52 up-regulated and 138 down-regulated. Of these
genes, 102 are known (have deposited in GeneBank), while 88 of them are unknown. 
The known genes can be divided into several families according to their
biological functions, such as: oxidative stress, apoptosis, signal transduction, 
angiogenesis, extracellular matrix, lipid metabolism, and gene transcription
related genes. The results of quantitative RT-PCR are consistent with gene-chip
results.
CONCLUSIONS: The occurrence of SINFH is a complicated process affected by
multiple factors and signaling pathways. Our findings indicate that many genes
which are involved in different signaling pathways were differentially expressed 
between SINFH rats and normal rats.


PMID: 22321685  [Indexed for MEDLINE]


417. BMC Genomics. 2011 Sep 27;12:469. doi: 10.1186/1471-2164-12-469.

Spleen transcriptome response to infection with avian pathogenic Escherichia coli
in broiler chickens.

Sandford EE(1), Orr M, Balfanz E, Bowerman N, Li X, Zhou H, Johnson TJ,
Kariyawasam S, Liu P, Nolan LK, Lamont SJ.

Author information: 
(1)Department of Animal Science, Iowa State University, Ames, IA 50011, USA.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is detrimental to poultry
health and its zoonotic potential is a food safety concern. Regulation of
antimicrobials in food-production animals has put greater focus on enhancing host
resistance to bacterial infections through genetics. To better define effective
mechanism of host resistance, global gene expression in the spleen of chickens,
harvested at two times post-infection (PI) with APEC, was measured using
microarray technology, in a design that will enable investigation of effects of
vaccination, challenge, and pathology level.
RESULTS: There were 1,101 genes significantly differentially expressed between
severely infected and non-infected groups on day 1 PI and 1,723 on day 5 PI. Very
little difference was seen between mildly infected and non-infected groups on
either time point. Between birds exhibiting mild and severe pathology, there were
2 significantly differentially expressed genes on day 1 PI and 799 on day 5 PI.
Groups with greater pathology had more genes with increased expression than
decreased expression levels. Several predominate immune pathways, Toll-like
receptor, Jak-STAT, and cytokine signaling, were represented between challenged
and non-challenged groups. Vaccination had, surprisingly, no detectible effect on
gene expression, although it significantly protected the birds from observable
gross lesions. Functional characterization of significantly expressed genes
revealed unique gene ontology classifications during each time point, with many
unique to a particular treatment or class contrast.
CONCLUSIONS: More severe pathology caused by APEC infection was associated with a
high level of gene expression differences and increase in gene expression levels.
Many of the significantly differentially expressed genes were unique to a
particular treatment, pathology level or time point. The present study not only
investigates the transcriptomic regulations of APEC infection, but also the
degree of pathology associated with that infection. This study will allow for
greater discovery into host mechanisms for disease resistance, providing targets 
for marker assisted selection and advanced drug development.

DOI: 10.1186/1471-2164-12-469 
PMCID: PMC3190404
PMID: 21951686  [Indexed for MEDLINE]


418. J Biosci. 2011 Sep;36(4):587-601.

Analysis of phage Mu DNA transposition by whole-genome Escherichia coli tiling
arrays reveals a complex relationship to distribution of target selection protein
B, transcription and chromosome architectural elements.

Ge J(1), Lou Z, Cui H, Shang L, Harshey RM.

Author information: 
(1)Section of Molecular Genetics and Microbiology and Institute of Cellular and
Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.

Of all known transposable elements, phage Mu exhibits the highest transposition
efficiency and the lowest target specificity. In vitro, MuB protein is
responsible for target choice. In this work, we provide a comprehensive
assessment of the genome-wide distribution of MuB and its relationship to Mu
target selection using high-resolution Escherichia coli tiling DNA arrays. We
have also assessed how MuB binding and Mu transposition are influenced by
chromosome-organizing elements such as AT-rich DNA signatures, or the binding of 
the nucleoid-associated protein Fis, or processes such as transcription. The
results confirm and extend previous biochemical and lower resolution in vivo
data. Despite the generally random nature of Mu transposition and MuB binding,
there were hot and cold insertion sites and MuB binding sites in the genome, and 
differences between the hottest and coldest sites were large. The new data also
suggest that MuB distribution and subsequent Mu integration is responsive to DNA 
sequences that contribute to the structural organization of the chromosome.


PMCID: PMC3712764
PMID: 21857106  [Indexed for MEDLINE]


419. Mol Immunol. 2011 Sep;48(15-16):2113-21. doi: 10.1016/j.molimm.2011.07.002. Epub 
2011 Jul 30.

Inflammatory response of porcine epithelial IPEC J2 cells to enterotoxigenic E.
coli infection is modulated by zinc supplementation.

Sargeant HR(1), Miller HM, Shaw MA.

Author information: 
(1)Faculty of Biological Sciences, University of Leeds, Clarendon Road, Leeds LS2
9JT, United Kingdom.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in pigs and
humans. The duration and severity of diarrhoea can be controlled using zinc
supplementation, typically pharmacological levels of zinc oxide in pigs. In this 
study, IPEC J2 cells were used as an in vitro model of intestinal ETEC infection,
with separate and simultaneous zinc treatment. Genomic analysis identified
increased expression of a variety of innate immune response genes (NF-κB targets)
in response to ETEC exposure, and several stress response genes in response to
zinc exposure, provided as ZnO. Expression of genes involved in the innate immune
response was reduced when cells were simultaneously exposed to ZnO, and it is
suggested that ZnO treatment inhibits the induction of NF-κB in response to
pathogens, possibly through up-regulated heat shock proteins. A similar response 
in vivo with consequent down-regulation in the inflammatory response would reduce
further pathogen invasion, maintain normal gut function and maintain growth.

Copyright © 2011 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.molimm.2011.07.002 
PMID: 21803424  [Indexed for MEDLINE]


420. Appl Environ Microbiol. 2011 Aug 15;77(16):5671-81. doi: 10.1128/AEM.05321-11.
Epub 2011 Jul 8.

Biochemical characterization and relative expression levels of multiple
carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola
23 grown on an ester-enriched substrate.

Kabel MA(1), Yeoman CJ, Han Y, Dodd D, Abbas CA, de Bont JA, Morrison M, Cann IK,
Mackie RI.

Author information: 
(1)Royal Nedalco, Bergen op Zoom, The Netherlands.

We measured expression and used biochemical characterization of multiple
carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola
23 grown on an ester-enriched substrate to gain insight into the carbohydrate
esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola
23 genome contains 16 genes predicted to encode carbohydrate esterase activity,
and based on microarray data, four of these were upregulated >2-fold at the
transcriptional level during growth on an ester-enriched oligosaccharide
(XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn
oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, 
and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and
Axe1-6A), were heterologously expressed in Escherichia coli, purified, and
biochemically characterized. All four enzymes showed the highest activity at
physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. 
ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme)
released ferulic acid from methylferulate, wheat bran, corn fiber, and
XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid 
esters, but exhibited negligible activity on sugar acetates. As expected, the P. 
ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase
family domains (CE1 and CE6), released ferulic acid from the same substrates as
Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various
acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not
assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that
had activity toward a broad range of mostly nonpolymeric acetylated substrates
along with AcXOS. All enzymes were inhibited by the proximal location of other
side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The
unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it
the ability to hydrolyze substituents on the xylan backbone and enhances its
capacity to efficiently degrade hemicellulose.

DOI: 10.1128/AEM.05321-11 
PMCID: PMC3165261
PMID: 21742923  [Indexed for MEDLINE]


421. J Vis Exp. 2011 Aug 11;(54). pii: 2864. doi: 10.3791/2864.

Competitive genomic screens of barcoded yeast libraries.

Smith AM(1), Durbic T, Oh J, Urbanus M, Proctor M, Heisler LE, Giaever G, Nislow 
C.

Author information: 
(1)Banting and Best Department of Medical Research and Department of Molecular
Genetics, University of Toronto, Canada.

By virtue of advances in next generation sequencing technologies, we have access 
to new genome sequences almost daily. The tempo of these advances is
accelerating, promising greater depth and breadth. In light of these
extraordinary advances, the need for fast, parallel methods to define gene
function becomes ever more important. Collections of genome-wide deletion mutants
in yeasts and E. coli have served as workhorses for functional characterization
of gene function, but this approach is not scalable, current gene-deletion
approaches require each of the thousands of genes that comprise a genome to be
deleted and verified. Only after this work is complete can we pursue
high-throughput phenotyping. Over the past decade, our laboratory has refined a
portfolio of competitive, miniaturized, high-throughput genome-wide assays that
can be performed in parallel. This parallelization is possible because of the
inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode
serving as a proxy for the mutation and one can measure the barcode abundance to 
assess mutant fitness. In this study, we seek to fill the gap between DNA
sequence and barcoded mutant collections. To accomplish this we introduce a
combined transposon disruption-barcoding approach that opens up parallel barcode 
assays to newly sequenced, but poorly characterized microbes. To illustrate this 
approach we present a new Candida albicans barcoded disruption collection and
describe how both microarray-based and next generation sequencing-based platforms
can be used to collect 10,000-1,000,000 gene-gene and drug-gene interactions in a
single experiment.

DOI: 10.3791/2864 
PMCID: PMC3211125
PMID: 21860376  [Indexed for MEDLINE]


422. Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12875-80. doi:
10.1073/pnas.1109379108. Epub 2011 Jul 18.

Small RNAs endow a transcriptional activator with essential repressor functions
for single-tier control of a global stress regulon.

Gogol EB(1), Rhodius VA, Papenfort K, Vogel J, Gross CA.

Author information: 
(1)Department of Microbiology and Immunology, and Department of Cell and Tissue
Biology, University of California, San Francisco, CA 94158, USA.

The Escherichia coli σ(E) envelope stress response monitors and repairs the outer
membrane, a function central to the life of Gram-negative bacteria. The σ(E)
stress response was characterized as a single-tier activation network comprised
of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly
expressed sRNAs were thought to carry out the specialized function of halting de 
novo synthesis of several abundant porins when envelope homeostasis was
perturbed. Using a systematic target profiling and validation approach we
discovered that MicA and RybB are each global mRNA repressors of both distinct
and shared targets, and that the two sRNAs constitute a posttranscriptional
repression arm whose regulatory scope rivals that of the protein-based σ(E)
activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and
new nonporin targets predict roles for MicA and RybB in crosstalk with other
regulatory responses. This work also provides an example of evolutionarily
unrelated sRNAs that are coinduced and bind the same targets, but at different
sites. Our finding that expression of either MicA or RybB sRNA protects the cell 
from the loss of viability experienced when σ(E) activity is inadequate
illustrates the importance of the posttranscriptional repression arm of the
response. σ(E) is a paradigm of a single-tier stress response with a clear
division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a
transcriptional factor intrinsically restricted to gene activation (σ(E)) with
the opposite repressor function.

DOI: 10.1073/pnas.1109379108 
PMCID: PMC3150882
PMID: 21768388  [Indexed for MEDLINE]


423. J Biol Chem. 2011 Jul 29;286(30):26576-84. doi: 10.1074/jbc.M111.243261. Epub
2011 Jun 3.

The multidrug efflux pump MdtEF protects against nitrosative damage during the
anaerobic respiration in Escherichia coli.

Zhang Y(1), Xiao M, Horiyama T, Zhang Y, Li X, Nishino K, Yan A.

Author information: 
(1)School of Biological Sciences, University of Hong Kong, Hong Kong Special
Administrative Region, Hong Kong, China.

Drug efflux represents an important protection mechanism in bacteria to withstand
antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of 
all transporters in bacterial genomes, yet the expression and functions of only a
handful of them have been studied. Among the 20 efflux genes encoded in the
Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively
expressed. The expression, activities, and physiological functions of the
remaining efflux genes are poorly understood. In this study we identified a
dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic
growth condition of E. coli, which is independent of antibiotic exposure. We
found that expression of MdtEF is up-regulated more than 20-fold under anaerobic 
conditions by the global transcription factor ArcA, resulting in increased efflux
activity and enhanced drug tolerance in anaerobically grown E. coli. Cells
lacking mdtEF display a significantly decreased survival rate under the condition
of anaerobic respiration of nitrate. Deletion of the genes responsible for the
biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal
electron acceptor during the anaerobic respiration restores the decreased
survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole
nitrosative derivatives, a class of toxic byproducts formed and accumulated
within E. coli when the bacterium respires nitrate under anaerobic conditions.
Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated 
during the anaerobic physiology of E. coli to protect the bacterium from
nitrosative damage through expelling the nitrosyl indole derivatives out of the
cells.

DOI: 10.1074/jbc.M111.243261 
PMCID: PMC3143622
PMID: 21642439  [Indexed for MEDLINE]


424. Bioinformatics. 2011 Jul 15;27(14):1948-56. doi: 10.1093/bioinformatics/btr307.
Epub 2011 May 18.

An ensemble biclustering approach for querying gene expression compendia with
experimental lists.

De Smet R(1), Marchal K.

Author information: 
(1)Department of Plant Systems Biology, VIB, Ghent University, Technologiepark
927, Ghent, Belgium.

MOTIVATION: Query-based biclustering techniques allow interrogating a gene
expression compendium with a given gene or gene list. They do so by searching for
genes in the compendium that have a profile close to the average expression
profile of the genes in this query-list. As it can often not be guaranteed that
the genes in a long query-list will all be mutually coexpressed, it is advisable 
to use each gene separately as a query. This approach, however, leaves the user
with a tedious post-processing of partially redundant biclustering results. The
fact that for each query-gene multiple parameter settings need to be tested in
order to detect the 'most optimal bicluster size' adds to the redundancy problem.
RESULTS: To aid with this post-processing, we developed an ensemble approach to
be used in combination with query-based biclustering. The method relies on a
specifically designed consensus matrix in which the biclustering outcomes for
multiple query-genes and for different possible parameter settings are merged in 
a statistically robust way. Clustering of this matrix results in distinct,
non-redundant consensus biclusters that maximally reflect the information
contained within the original query-based biclustering results. The usefulness of
the developed approach is illustrated on a biological case study in Escherichia
coli.
AVAILABILITY AND IMPLEMENTATION: Compiled Matlab code is available from
http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Information_DeSmet_2011/.

DOI: 10.1093/bioinformatics/btr307 
PMID: 21593133  [Indexed for MEDLINE]


425. BMC Genomics. 2011 Jul 6;12:349. doi: 10.1186/1471-2164-12-349.

Investigating the global genomic diversity of Escherichia coli using a
multi-genome DNA microarray platform with novel gene prediction strategies.

Jackson SA(1), Patel IR, Barnaba T, LeClerc JE, Cebula TA.

Author information: 
(1)Division of Molecular Biology, Office of Applied Research and Safety
Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug
Administration, Laurel, Maryland 20708, USA. scott.jackson@fda.hhs.gov

BACKGROUND: The gene content of a diverse group of 183 unique Escherichia coli
and Shigella isolates was determined using the Affymetrix GeneChip® E. coli
Genome 2.0 Array, originally designed for transcriptome analysis, as a genotyping
tool. The probe set design utilized by this array provided the opportunity to
determine the gene content of each strain very accurately and reliably. This
array constitutes 10,112 independent genes representing four individual E. coli
genomes, therefore providing the ability to survey genes of several different
pathogen types. The entire ECOR collection, 80 EHEC-like isolates, and a diverse 
set of isolates from our FDA strain repository were included in our analysis.
RESULTS: From this study we were able to define sets of genes that correspond to,
and therefore define, the EHEC pathogen type. Furthermore, our sampling of 63
unique strains of O157:H7 showed the ability of this array to discriminate
between closely related strains. We found that individual strains of O157:H7
differed, on average, by 197 probe sets. Finally, we describe an analysis method 
that utilizes the power of the probe sets to determine accurately the
presence/absence of each gene represented on this array.
CONCLUSIONS: These elements provide insights into understanding the microbial
diversity that exists within extant E. coli populations. Moreover, these data
demonstrate that this novel microarray-based analysis is a powerful tool in the
field of molecular epidemiology and the newly emerging field of microbial
forensics.

DOI: 10.1186/1471-2164-12-349 
PMCID: PMC3146454
PMID: 21733163  [Indexed for MEDLINE]


426. Curr Opin Gastroenterol. 2011 Jul;27(4):328-34. doi:
10.1097/MOG.0b013e3283463b45.

Defective innate immunity in inflammatory bowel disease: a Crohn's disease
exclusivity?

Marks DJ(1).

Author information: 
(1)Centre for Molecular Medicine, University College London, London, UK.
d.marks@ucl.ac.uk

PURPOSE OF REVIEW: This review summarizes the recent developments in support of
the immunodeficiency model of Crohn's disease.
RECENT FINDINGS: The demonstration of impaired acute inflammation in Crohn's
disease provides a novel mechanism for its pathogenesis, with diminished
macrophage cytokine production and neutrophil recruitment leading to reduced
bacterial clearance. The innate immune response may be further overwhelmed by
other factors. The mucosal barrier in Crohn's patients is disrupted, with
abnormal ultrastructure as well as antibacterial defensin deficiency. Specific
bacterial agents may contribute and one promising candidate, adherent-invasive
Escherichia coli, has recently been described. An interaction between Nod2 and
the autophagy system has been elucidated, with direct consequences for bacterial 
clearance, and the most recent genome-wide association study meta-analysis has
extended the number of Crohn's disease susceptibility loci to 71. The spectrum of
congenital immunodeficiency disorders recognized to develop Crohn's-like
inflammatory bowel disease is also expanding. Conversely, no specific
immunodeficiency has so far been observed in ulcerative colitis, in which the
defect appears to be failure of inflammation termination and resolution.
SUMMARY: Recent advances continue to highlight defects in innate immunity in
Crohn's patients. Similar abnormalities may extend to other granulomatous
disorders, but not diseases such as ulcerative colitis.

DOI: 10.1097/MOG.0b013e3283463b45 
PMID: 21483259  [Indexed for MEDLINE]


427. Epidemiol Infect. 2011 Jul;139(7):1088-96. doi: 10.1017/S095026881000213X. Epub
2010 Sep 15.

Microarray-based detection of virulence genes in verotoxigenic Escherichia coli
O157:H7 strains from Swedish cattle.

Söderlund R(1), Aspán A(1), La Ragione RM(2), Eriksson E(1), Boqvist S(3).

Author information: 
(1)National Veterinary Institute, Uppsala, Sweden.
(2)Department for Food and Environmental Safety, Veterinary Laboratories Agency, 
Addlestone, Surrey, UK.
(3)Department of Biomedical Sciences and Veterinary Public Health, Swedish
University of Agricultural Sciences, Uppsala, Sweden.

Verotoxigenic Escherichia coli (VTEC) serotype O157:H7 strains from a Swedish
cattle prevalence study (n=32), and livestock-derived strains linked to human
disease (n=13), were characterized by microarray and PCR detection of virulence
genes. The overall aim of the study was to investigate the distribution of known 
virulence determinants and determine which genes are linked to increased
pathogenicity in humans. A core set of 18 genes or gene variants were found in
all strains, while seven genes were variably present. This suggests that the
majority of VTEC O157:H7 found in Swedish cattle carry a broad repertoire of
virulence genes and should be considered potentially harmful to humans. A single 
virulence gene type was significantly associated with strains linked to human
disease cases (P=0.012), but no genetic trait to explain the increased virulence 
of this genotype could be found.

DOI: 10.1017/S095026881000213X 
PMID: 20843386  [Indexed for MEDLINE]


428. Infect Immun. 2011 Jul;79(7):2619-31. doi: 10.1128/IAI.05152-11. Epub 2011 Apr
19.

Transcriptome of Proteus mirabilis in the murine urinary tract: virulence and
nitrogen assimilation gene expression.

Pearson MM(1), Yep A, Smith SN, Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, Michigan 48109, USA.

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary 
tract infections. In this study, microarrays were used to analyze P. mirabilis
gene expression in vivo from experimentally infected mice. Urine was collected at
1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine
for transcriptional analysis. Across nine microarrays, 471 genes were upregulated
and 82 were downregulated in vivo compared to in vitro broth culture. Genes
upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae,
urease, iron uptake systems, amino acid and peptide transporters, pyruvate
metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. 
Flagella were downregulated. Ammonia assimilation gene glnA (glutamine
synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was
upregulated in vivo. Contrary to our expectations, ammonia availability due to
urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant
was growth deficient in minimal medium with citrate as the sole carbon source,
and loss of gdhA resulted in a significant fitness defect in the mouse model of
urinary tract infection. Unlike Escherichia coli, which represses gdhA and
upregulates glnA in vivo and cannot utilize citrate, the data suggest that P.
mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and
this ability contributes to the pathogenic potential of P. mirabilis in the
urinary tract.

DOI: 10.1128/IAI.05152-11 
PMCID: PMC3191972
PMID: 21505083  [Indexed for MEDLINE]


429. Nucleic Acids Res. 2011 Jul;39(13):5338-55. doi: 10.1093/nar/gkr129. Epub 2011
Mar 11.

In vitro transcription profiling of the σS subunit of bacterial RNA polymerase:
re-definition of the σS regulon and identification of σS-specific promoter
sequence elements.

Maciag A(1), Peano C, Pietrelli A, Egli T, De Bellis G, Landini P.

Author information: 
(1)Department of Biomolecular Sciences and Biotechnology, Università degli Studi 
di Milano, Milan, Italy.

Specific promoter recognition by bacterial RNA polymerase is mediated by σ
subunits, which assemble with RNA polymerase core enzyme (E) during transcription
initiation. However, σ(70) (the housekeeping σ subunit) and σ(S) (an alternative 
σ subunit mostly active during slow growth) recognize almost identical promoter
sequences, thus raising the question of how promoter selectivity is achieved in
the bacterial cell. To identify novel sequence determinants for selective
promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA
polymerase saturated either with σ(70) (Eσ(70)) or with σ(S) (Eσ(S)) using the
whole Escherichia coli genome as DNA template. We found that Eσ(70), in the
absence of any additional transcription factor, preferentially transcribes genes 
associated with fast growth (e.g. ribosomal operons). In contrast, Eσ(S)
efficiently transcribes genes involved in stress responses, secondary metabolism 
as well as RNAs from intergenic regions with yet-unknown function. Promoter
sequence comparison suggests that, in addition to different conservation of the
-35 sequence and of the UP element, selective promoter recognition by either form
of RNA polymerase can be affected by the A/T content in the -10/+1 region.
Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the
-10/+1 region could improve promoter recognition by Eσ(S).

DOI: 10.1093/nar/gkr129 
PMCID: PMC3141248
PMID: 21398637  [Indexed for MEDLINE]


430. Microb Cell Fact. 2011 Jun 30;10:52. doi: 10.1186/1475-2859-10-52.

The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli 
K-12 and E. coli B at high density growth.

Son YJ(1), Phue JN, Trinh LB, Lee SJ, Shiloach J.

Author information: 
(1)Biotechnology Core Laboratory, National Institute of Diabetes and Digestive
and Kidney Diseases/ NIH, Bethesda, MD 20892, USA.

BACKGROUND: E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to
glucose concentration and, therefore, grows faster and produces less acetate than
E. coli K-12, especially when growing to high cell densities at high glucose
concentration. By performing genomic analysis, it was demonstrated that the cause
of this difference in sensitivity to the glucose concentration is the result of
the differences in the central carbon metabolism activity. We hypothesized that
the global transcription regulator Cra (FruR) is constitutively expressed in E.
coli B and may be responsible for the different behaviour of the two strains. To 
investigate this possibility and better understand the function of Cra in the two
strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared
and their growth behaviour and gene expression at high glucose were evaluated
using microarray and real-time PCR.
RESULTS: The deletion of the cra gene in E. coli B (BL21) minimally affected the 
growth and maximal acetate accumulation, while the deletion of the same gene in
E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate
concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA
(gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate 
producing gene) were down-regulated in both strains, while acs (acetate uptake
gene) was down-regulated only in E.coli B (BL21). These transcriptional
differences had little effect on acetate and pyruvate production. Additionally,
it was found that the lower growth of E. coli K-12 (JM109) strain was the result 
of transcription inhibition of the osmoprotectant producing bet operon (betABT).
CONCLUSIONS: The transcriptional changes caused by the deletion of cra gene did
not affect the activity of the central carbon metabolism, suggesting that Cra
does not act alone; rather it interacts with other pleiotropic regulators to
create a network of metabolic effects. An unexpected outcome of this work is the 
finding that cra deletion caused transcription inhibition of the bet operon in E.
coli K-12 (JM109) but did not affect this operon transcription in E. coli B
(BL21). This property, together with the insensitivity to high glucose
concentrations, makes this the E. coli B (BL21) strain more resistant to
environmental changes.

DOI: 10.1186/1475-2859-10-52 
PMCID: PMC3146397
PMID: 21718532  [Indexed for MEDLINE]


431. BMC Vet Res. 2011 Jun 3;7:24. doi: 10.1186/1746-6148-7-24.

Mouldy feed, mycotoxins and Shiga toxin - producing Escherichia coli colonization
associated with Jejunal Hemorrhage Syndrome in beef cattle.

Baines D(1), Erb S, Turkington K, Kuldau G, Juba J, Masson L, Mazza A, Roberts R.

Author information: 
(1)Lethbridge Research Centre, 5403 1 Avenue South, Lethbridge, AB, T1J 4B1,
Canada. danica.baines@agr.gc.ca

BACKGROUND: Both O157 and non-O157 Shiga toxin - producing Escherichia coli
(STECs) cause serious human disease outbreaks through the consumption of
contaminated foods. Cattle are considered the main reservoir but it is unclear
how STECs affect mature animals. Neonatal calves are the susceptible age class
for STEC infections causing severe enteritis. In an earlier study, we determined 
that mycotoxins and STECs were part of the disease complex for dairy cattle with 
Jejunal Hemorrhage Syndrome (JHS). For STECs to play a role in the development of
JHS, we hypothesized that STEC colonization should also be evident in beef cattle
with JHS. Aggressive medical and surgical therapies are effective for JHS, but
rely on early recognition of clinical signs for optimal outcomes suggesting that 
novel approaches must be developed for managing this disease. The main objective 
of this study was to confirm that mouldy feeds, mycotoxins and STEC colonization 
were associated with the development of JHS in beef cattle.
RESULTS: Beef cattle developed JHS after consuming feed containing several types 
of mycotoxigenic fungi including Fusarium poae, F. verticillioides, F.
sporotrichioides, Penicillium roqueforti and Aspergillus fumigatus. Mixtures of
STECs colonized the mucosa in the hemorrhaged tissues of the cattle and no other 
pathogen was identified. The STECs expressed Stx1 and Stx2, but more
significantly, Stxs were also present in the blood collected from the lumen of
the hemorrhaged jejunum. Feed extracts containing mycotoxins were toxic to
enterocytes and 0.1% of a prebiotic, Celmanax Trademark, removed the cytotoxicity
in vitro. The inclusion of a prebiotic in the care program for symptomatic beef
calves was associated with 69% recovery.
CONCLUSIONS: The current study confirmed that STECs and mycotoxins are part of
the disease complex for JHS in beef cattle. Mycotoxigenic fungi are only relevant
in that they produce the mycotoxins deposited in the feed. A prebiotic, Celmanax 
Trademark, acted as a mycotoxin binder in vitro and interfered with the
progression of disease.

DOI: 10.1186/1746-6148-7-24 
PMCID: PMC3135542
PMID: 21639911  [Indexed for MEDLINE]


432. Can J Microbiol. 2011 Jun;57(6):453-9. doi: 10.1139/w11-030. Epub 2011 May 31.

Aptamer selection for the detection of Escherichia coli K88.

Li H(1), Ding X, Peng Z, Deng L, Wang D, Chen H, He Q.

Author information: 
(1)The Co-construction Laboratory of Microbial Molecular Biology of Province
Department and Ministry of Science and Technology, College of Life Sciences,
Hunan Normal University, Changsha, People's Republic of China.

In this study, the first group of single-stranded DNA aptamers that are highly
specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an
enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by
Exponential Enrichment) procedure, during which the K88 fimbriae protein was used
as the target and bovine serum albumin as counter targets. These aptamers were
applied successfully in the detection of ETEC K88. They were then grouped under
different families based on the similarity of their secondary structure and the
homology of their primary sequence. Four sequences from different families were
deliberately chosen for further characterization by fluorescence analysis. Having
the advantage of high sensitivity, fluorescence photometry was selected as
single-stranded DNA quantification method during the SELEX process. Aptamers with
the highest specificity and affinity were analyzed to evaluate binding ability
with E. coli. Since ETEC K88 is the only type of bacterium that expressed
abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein
were able to specifically identify ETEC K88 among other bacteria. This method of 
detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC
K88.

DOI: 10.1139/w11-030 
PMID: 21627466  [Indexed for MEDLINE]


433. Foodborne Pathog Dis. 2011 Jun;8(6):663-79. doi: 10.1089/fpd.2010.0695. Epub 2011
Mar 8.

Related antimicrobial resistance genes detected in different bacterial species
co-isolated from swine fecal samples.

Frye JG(1), Lindsey RL, Meinersmann RJ, Berrang ME, Jackson CR, Englen MD, Turpin
JB, Fedorka-Cray PJ.

Author information: 
(1)Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Richard B. 
Russell Research Center, Agricultural Research Service, U.S. Department of
Agriculture, Athens, Georgia 30605, USA. jonathan.frye@ars.usda.gov

A potential factor leading to the spread of antimicrobial resistance (AR) in
bacteria is the horizontal transfer of resistance genes between bacteria in
animals or their environment. To investigate this, swine fecal samples were
collected on-farm and cultured for Escherichia coli, Salmonella enterica,
Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. 
Forty-nine of the samples from which all four bacteria were recovered were
selected yielding a total of 196 isolates for analysis. Isolates were tested for 
antimicrobial susceptibility followed by hybridization to a DNA microarray
designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the
same fecal sample had the most AR genes in common among the four bacteria. Genes 
detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), 
β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR),
sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and 
tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus
isolated from the same sample frequently had tet(O) and aphA-3 genes detected in 
common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal 
sample shared resistance genes at a significant level (χ², p < 0.0000001). These 
data suggest that there may have been horizontal exchange of AR genes between
these bacteria or there may be a common source of AR genes in the swine
environment for E. coli and Salmonella.

DOI: 10.1089/fpd.2010.0695 
PMID: 21385089  [Indexed for MEDLINE]


434. Foodborne Pathog Dis. 2011 Jun;8(6):705-11. doi: 10.1089/fpd.2010.0753. Epub 2011
Feb 2.

Identification of Escherichia coli O157 by using a novel colorimetric detection
method with DNA microarrays.

Quiñones B(1), Swimley MS, Taylor AW, Dawson ED.

Author information: 
(1)Produce Safety and Microbiology Research Unit, Western Regional Research
Center, Agricultural Research Service, U.S. Department of Agriculture, Albany,
California 94710, USA. beatriz.quinones@ars.usda.gov

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne
illness worldwide. To evaluate better methods to rapidly detect and genotype E.
coli O157 strains, the present study evaluated the use of ampliPHOX, a novel
colorimetric detection method based on photopolymerization, for pathogen
identification with DNA microarrays. A low-density DNA oligonucleotide microarray
was designed to target stx1 and stx2 genes encoding Shiga toxin production, the
eae gene coding for adherence membrane protein, and the per gene encoding the
O157-antigen perosamine synthetase. Results from the validation experiments
demonstrated that the use of ampliPHOX allowed the accurate genotyping of the
tested E. coli strains, and positive hybridization signals were observed for only
probes targeting virulence genes present in the reference strains. Quantification
showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 
76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were
determined for probes where no polymer was formed due to lack of specific
hybridization. Sensitivity tests demonstrated that the sensitivity threshold for 
E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in
combination with photopolymerization allowed the rapid and accurate genotyping of
E. coli O157 strains.

DOI: 10.1089/fpd.2010.0753 
PMCID: PMC3122933
PMID: 21288130  [Indexed for MEDLINE]


435. Infect Immun. 2011 Jun;79(6):2430-9. doi: 10.1128/IAI.01199-10. Epub 2011 Mar 21.

Genotype and phenotypes of an intestine-adapted Escherichia coli K-12 mutant
selected by animal passage for superior colonization.

Fabich AJ(1), Leatham MP, Grissom JE, Wiley G, Lai H, Najar F, Roe BA, Cohen PS, 
Conway T.

Author information: 
(1)Advanced Center for Genome Technology, Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.

We previously isolated a spontaneous mutant of Escherichia coli K-12, strain
MG1655, following passage through the streptomycin-treated mouse intestine, that 
has colonization traits superior to the wild-type parent strain (M. P. Leatham et
al., Infect. Immun. 73:8039-8049, 2005). This intestine-adapted strain (E. coli
MG1655*) grew faster on several different carbon sources than the wild type and
was nonmotile due to deletion of the flhD gene. We now report the results of
several high-throughput genomic analysis approaches to further characterize E.
coli MG1655*. Whole-genome pyrosequencing did not reveal any changes on its
genome, aside from the deletion at the flhDC locus, that could explain the
colonization advantage of E. coli MG1655*. Microarray analysis revealed modest
yet significant induction of catabolic gene systems across the genome in both E. 
coli MG1655* and an isogenic flhD mutant constructed in the laboratory.
Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of
both E. coli MG1655* and the isogenic flhD mutant to oxidize a variety of carbon 
sources. The results show that intestine-adapted E. coli MG1655* is more fit than
the wild type for intestinal colonization, because loss of FlhD results in
elevated expression of genes involved in carbon and energy metabolism, resulting 
in more efficient carbon source utilization and a higher intestinal population.
Hence, mutations that enhance metabolic efficiency confer a colonization
advantage.

DOI: 10.1128/IAI.01199-10 
PMCID: PMC3125843
PMID: 21422176  [Indexed for MEDLINE]


436. PLoS Comput Biol. 2011 Jun;7(6):e1002060. doi: 10.1371/journal.pcbi.1002060. Epub
2011 Jun 30.

Differential producibility analysis (DPA) of transcriptomic data with metabolic
networks: deconstructing the metabolic response of M. tuberculosis.

Bonde BK(1), Beste DJ, Laing E, Kierzek AM, McFadden J.

Author information: 
(1)Microbial Sciences Division, Faculty of Health and Medical Sciences,
University of Surrey, Guildford, United Kingdom.

A general paucity of knowledge about the metabolic state of Mycobacterium
tuberculosis within the host environment is a major factor impeding development
of novel drugs against tuberculosis. Current experimental methods do not allow
direct determination of the global metabolic state of a bacterial pathogen in
vivo, but the transcriptional activity of all encoded genes has been investigated
in numerous microarray studies. We describe a novel algorithm, Differential
Producibility Analysis (DPA) that uses a metabolic network to extract metabolic
signals from transcriptome data. The method utilizes Flux Balance Analysis (FBA) 
to identify the set of genes that affect the ability to produce each metabolite
in the network. Subsequently, Rank Product Analysis is used to identify those
metabolites predicted to be most affected by a transcriptional signal. We first
apply DPA to investigate the metabolic response of E. coli to both anaerobic
growth and inactivation of the FNR global regulator. DPA successfully extracts
metabolic signals that correspond to experimental data and provides novel
metabolic insights. We next apply DPA to investigate the metabolic response of M.
tuberculosis to the macrophage environment, human sputum and a range of in vitro 
environmental perturbations. The analysis revealed a previously unrecognized
feature of the response of M. tuberculosis to the macrophage environment: a
down-regulation of genes influencing metabolites in central metabolism and
concomitant up-regulation of genes that influence synthesis of cell wall
components and virulence factors. DPA suggests that a significant feature of the 
response of the tubercle bacillus to the intracellular environment is a
channeling of resources towards remodeling of its cell envelope, possibly in
preparation for attack by host defenses. DPA may be used to unravel the
mechanisms of virulence and persistence of M. tuberculosis and other pathogens
and may have general application for extracting metabolic signals from other
"-omics" data.

DOI: 10.1371/journal.pcbi.1002060 
PMCID: PMC3127818
PMID: 21738454  [Indexed for MEDLINE]


437. BMC Genomics. 2011 May 13;12(1):238. doi: 10.1186/1471-2164-12-238.

Escherichia coli genome-wide promoter analysis: identification of additional AtoC
binding target elements.

Pilalis E(1), Chatziioannou AA, Grigoroudis AI, Panagiotidis CA, Kolisis FN,
Kyriakidis DA.

Author information: 
(1)Institute of Biological Research and Biotechnology, National Hellenic Research
Foundation, Athens, Greece.

BACKGROUND: Studies on bacterial signal transduction systems have revealed
complex networks of functional interactions, where the response regulators play a
pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB
operon genes, and the subsequent catabolism of short-chain fatty acids, upon
acetoacetate induction. Transcriptome and phenotypic analyses suggested that
atoSC is also involved in several other cellular activities, although we have
recently reported a palindromic repeat within the atoDAEB promoter as the single,
cis-regulatory binding site of the AtoC response regulator. In this work, we used
a computational approach to explore the presence of yet unidentified AtoC binding
sites within other parts of the E. coli genome.
RESULTS: Through the implementation of a computational de novo motif detection
workflow, a set of candidate motifs was generated, representing putative AtoC
binding targets within the E. coli genome. In order to assess the biological
relevance of the motifs and to select for experimental validation of those
sequences related robustly with distinct cellular functions, we implemented a
novel approach that applies Gene Ontology Term Analysis to the motif hits and
selected those that were qualified through this procedure. The computational
results were validated using Chromatin Immunoprecipitation assays to assess the
in vivo binding of AtoC to the predicted sites. This process verified twenty-two 
additional AtoC binding sites, located not only within intergenic regions, but
also within gene-encoding sequences.
CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has 
indicated an AtoC-related cross-regulatory function. This highlights the
significance of computational genome-wide approaches in elucidating complex
patterns of bacterial cell regulation.

DOI: 10.1186/1471-2164-12-238 
PMCID: PMC3118216
PMID: 21569465  [Indexed for MEDLINE]


438. BMC Genomics. 2011 May 11;12(1):225. doi: 10.1186/1471-2164-12-225.

Strengthening insights into host responses to mastitis infection in ruminants by 
combining heterogeneous microarray data sources.

Genini S(1), Badaoui B, Sclep G, Bishop SC, Waddington D, Pinard van der Laan MH,
Klopp C, Cabau C, Seyfert HM, Petzl W, Jensen K, Glass EJ, de Greeff A, Smith HE,
Smits MA, Olsaker I, Boman GM, Pisoni G, Moroni P, Castiglioni B, Cremonesi P,
Del Corvo M, Foulon E, Foucras G, Rupp R, Giuffra E.

Author information: 
(1)Parco Tecnologico Padano - CERSA, Via Einstein, 26900 Lodi, Italy.
geninis@vet.upenn.edu

BACKGROUND: Gene expression profiling studies of mastitis in ruminants have
provided key but fragmented knowledge for the understanding of the disease. A
systematic combination of different expression profiling studies via
meta-analysis techniques has the potential to test the extensibility of
conclusions based on single studies. Using the program Pointillist, we performed 
meta-analysis of transcription-profiling data from six independent studies of
infections with mammary gland pathogens, including samples from cattle challenged
in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in 
vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells
infected in vitro with S. aureus. We combined different time points from those
studies, testing different responses to mastitis infection: overall (common
signature), early stage, late stage, and cattle-specific.
RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four
meta-analysis combinations share biological functions and pathways (e.g. protein 
ubiquitination and polyamine regulation) which are intrinsic to the general
disease response. In the overall response, pathways related to immune response
and inflammation, as well as biological functions related to lipid metabolism
were altered. This latter observation is consistent with the milk fat content
depression commonly observed during mastitis infection. Complementarities between
early and late stage responses were found, with a prominence of metabolic and
stress signals in the early stage and of the immune response related to the lipid
metabolism in the late stage; both mechanisms apparently modulated by few genes, 
including XBP1 and SREBF1.The cattle-specific response was characterized by
alteration of the immune response and by modification of lipid metabolism.
Comparison of E. coli and S. aureus infections in cattle in vivo revealed that
affected genes showing opposite regulation had the same altered biological
functions and provided evidence that E. coli caused a stronger host response.
CONCLUSIONS: This meta-analysis approach reinforces previous findings but also
reveals several novel themes, including the involvement of genes, biological
functions, and pathways that were not identified in individual studies. As such, 
it provides an interesting proof of principle for future studies combining
information from diverse heterogeneous sources.

DOI: 10.1186/1471-2164-12-225 
PMCID: PMC3118214
PMID: 21569310  [Indexed for MEDLINE]


439. Nano Lett. 2011 May 11;11(5):2083-7. doi: 10.1021/nl2005687. Epub 2011 Apr 14.

Functionalized micromachines for selective and rapid isolation of nucleic acid
targets from complex samples.

Kagan D(1), Campuzano S, Balasubramanian S, Kuralay F, Flechsig GU, Wang J.

Author information: 
(1)Department of Nanoengineering, University of California-San Diego, La Jolla,
California 92093, United States.

The transport properties of single-strand DNA probe-modified self-propelling
micromachines are exploited for "on-the-fly" hybridization and selective
single-step isolation of target nucleic acids from "raw" microliter biological
samples (serum, urine, crude E. coli lysate, saliva). The rapid movement of the
guided modified microrockets induces fluid convection, which enhances the
hybridization efficiency, thus enabling the rapid and selective isolation of
nucleic acid targets from untreated samples. The integration of these autonomous 
microrockets into a lab-on-chip device that provides both nucleic acid isolation 
and downstream analysis could thus be attractive for diverse applications.

DOI: 10.1021/nl2005687 
PMID: 21491941  [Indexed for MEDLINE]


440. Infect Immun. 2011 May;79(5):1951-60. doi: 10.1128/IAI.01230-10. Epub 2011 Feb
28.

Transcriptome analysis of avian pathogenic Escherichia coli O1 in chicken serum
reveals adaptive responses to systemic infection.

Li G(1), Tivendale KA, Liu P, Feng Y, Wannemuehler Y, Cai W, Mangiamele P,
Johnson TJ, Constantinidou C, Penn CW, Nolan LK.

Author information: 
(1)Department of Veterinary Microbiology and Preventive Medicine, College of
Veterinary Medicine, Iowa State University, 1802 University Blvd., Ames, IA
50011, USA.

Infections of avian pathogenic Escherichia coli (APEC) result in annual
multimillion-dollar losses to the poultry industry. Despite this, little is known
about the mechanisms by which APEC survives and grows in the bloodstream. Thus,
the aim of this study was to identify molecular mechanisms enabling APEC to
survive and grow in this critical host environment. To do so, we compared the
transcriptome of APEC O1 during growth in Luria-Bertani broth and chicken serum. 
Several categories of genes, predicted to contribute to adaptation and growth in 
the avian host, were identified. These included several known virulence genes and
genes involved in adaptive metabolism, protein transport, biosynthesis pathways, 
stress resistance, and virulence regulation. Several genes with unknown function,
which were localized to pathogenicity islands or APEC O1's large virulence
plasmid, pAPEC-O1-ColBM, were also identified, suggesting that they too
contribute to survival in serum. The significantly upregulated genes dnaK, dnaJ, 
phoP, and ybtA were subsequently subjected to mutational analysis to confirm
their role in conferring a competitive advantage during infection. This
genome-wide analysis provides novel insight into processes that are important to 
the pathogenesis of APEC O1.

DOI: 10.1128/IAI.01230-10 
PMCID: PMC3088125
PMID: 21357721  [Indexed for MEDLINE]


441. J Bacteriol. 2011 May;193(10):2587-97. doi: 10.1128/JB.01468-10. Epub 2011 Mar
18.

Role of the biofilm master regulator CsgD in cross-regulation between biofilm
formation and flagellar synthesis.

Ogasawara H(1), Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan.

CsgD, the master regulator of biofilm formation, activates the synthesis of curli
fimbriae and extracellular polysaccharides in Escherichia coli. To obtain
insights into its regulatory role, we have identified a total of 20 novel
regulation target genes on the E. coli genome by using chromatin
immunoprecipitation (ChIP)-on-chip analysis with a high-density DNA microarray.
By DNase I footprinting, the consensus CsgD-binding sequence predicted from a
total of 18 target sites was found to include AAAAGNG(N(2))AAAWW. After a
promoter-lacZ fusion assay, the CsgD targets were classified into two groups:
group I genes, such as fliE and yhbT, are repressed by CsgD, while group II
genes, including yccT and adrA, are activated by CsgD. The fliE and fliEFGH
operons for flagellum formation are directly repressed by CsgD, while CsgD
activates the adrA gene, which encodes an enzyme for synthesis of cyclic di-GMP, 
a bacterial second messenger, which in turn inhibits flagellum production and
rotation. Taking these findings together, we propose that the cell motility for
planktonic growth is repressed by CsgD, thereby promoting the switch to biofilm
formation.

DOI: 10.1128/JB.01468-10 
PMCID: PMC3133154
PMID: 21421764  [Indexed for MEDLINE]


442. J Bacteriol. 2011 May;193(9):2186-96. doi: 10.1128/JB.00001-11. Epub 2011 Mar 4.

The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide
toxicity by diminishing the amount of intracellular unincorporated iron.

Liu Y(1), Bauer SC, Imlay JA.

Author information: 
(1)Department of Microbiology, University of Illinois at Urbana-Champaign, 601
South Goodwin Ave., Urbana, IL 61801, USA.

Hydrogen peroxide (H(2)O(2)) is commonly formed in microbial habitats by either
chemical oxidation processes or host defense responses. H(2)O(2) can penetrate
membranes and damage key intracellular biomolecules, including DNA and
iron-dependent enzymes. Bacteria defend themselves against this H(2)O(2) by
inducing a regulon that engages multiple defensive strategies. A previous
microarray study suggested that yaaA, an uncharacterized gene found in many
bacteria, was induced by H(2)O(2) in Escherichia coli as part of its OxyR
regulon. Here we confirm that yaaA is a key element of the stress response to
H(2)O(2). In a catalase/peroxidase-deficient (Hpx(-)) background, yaaA deletion
mutants grew poorly, filamented extensively, and lost substantial viability when 
they were cultured in aerobic LB medium. The results from a thyA forward
mutagenesis assay and the growth defect of the yaaA deletion in a
recombination-deficient (recA56) background indicated that yaaA mutants
accumulated high levels of DNA damage. The growth defect of yaaA mutants could be
suppressed by either the addition of iron chelators or mutations that slowed iron
import, indicating that the DNA damage was caused by the Fenton reaction.
Spin-trapping experiments confirmed that Hpx(-) yaaA cells had a higher hydroxyl 
radical (HO(•)) level. Electron paramagnetic resonance spectroscopy analysis
showed that the proximate cause was an unusually high level of intracellular
unincorporated iron. These results demonstrate that during periods of H(2)O(2)
stress the induction of YaaA is a critical device to suppress intracellular iron 
levels; it thereby attenuates the Fenton reaction and the DNA damage that would
otherwise result. The molecular mechanism of YaaA action remains unknown.

DOI: 10.1128/JB.00001-11 
PMCID: PMC3133076
PMID: 21378183  [Indexed for MEDLINE]


443. Mol Biosyst. 2011 May;7(5):1613-20. doi: 10.1039/c0mb00336k. Epub 2011 Mar 7.

Genome-wide transcriptome and proteome analysis of Escherichia coli expressing
IrrE, a global regulator of Deinococcus radiodurans.

Zhou Z(1), Zhang W, Chen M, Pan J, Lu W, Ping S, Yan Y, Hou X, Yuan M, Zhan Y,
Lin M.

Author information: 
(1)Biotechnology Research Institute, Chinese Academy of Agricultural Sciences,
Key Laboratory of Crop Biotechnology, Ministry of Agriculture, Beijing 100081,
China.

Gram-negative bacterium Escherichia coli and the Gram-positive Deinococcus
radiodurans fundamentally differ in their cell structures and gene regulations.
We have previously reported that IrrE, a Deinococcus genus-specific global
regulator, confers significantly enhanced tolerance to various abiotic stresses. 
To better understand the global effects of IrrE on the regulatory networks, we
carried out combined transcriptome and proteome analysis of E. coli expressing
the IrrE protein. Our analysis showed that 216 (4.8%) of all E. coli genes were
induced and 149 (3.3%) genes were repressed, including those for trehalose
biosynthesis, nucleotides biosynthesis, carbon source utilization, amino acid
utilization, acid resistance, a hydrogenase and an oxidase. Also regulated were
the EvgSA two-component system, the GadE, GadX and PurR master regulators, and 10
transcription factors (AppY, GadW, YhiF, AsnC, BetI, CynR, MhpR, PrpR, TdcA and
KdgR). These results demonstrated that IrrE acts as global regulator and
consequently improves abiotic stress tolerances in the heterologous host E. coli.
The implication of our findings is discussed in relation to the evolutionary role
of horizontal gene transfer in bacterial regulatory networks and environmental
adaptation.

DOI: 10.1039/c0mb00336k 
PMID: 21380435  [Indexed for MEDLINE]


444. Nat Biotechnol. 2011 May;29(5):449-52. doi: 10.1038/nbt.1847. Epub 2011 Apr 24.

Parallel on-chip gene synthesis and application to optimization of protein
expression.

Quan J(1), Saaem I, Tang N, Ma S, Negre N, Gong H, White KP, Tian J.

Author information: 
(1)Department of Biomedical Engineering, Duke University, Durham, North Carolina,
USA.

Comment in
    Nat Rev Genet. 2011 Jun;12(6):381.

Low-cost, high-throughput gene synthesis and precise control of protein
expression are of critical importance to synthetic biology and biotechnology.
Here we describe the development of an on-chip gene synthesis technology, which
integrates on a single microchip the synthesis of DNA oligonucleotides using
inkjet printing, isothermal oligonucleotide amplification and parallel gene
assembly. Use of a mismatch-specific endonuclease for error correction results in
an error rate of ~0.19 errors per kb. We applied this approach to synthesize
pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila
protein antigens, which were then screened for expression in Escherichia coli. In
one round of synthesis and screening, we obtained DNA sequences that were
expressed at a wide range of levels, from zero to almost 60% of the total cell
protein mass. This technology may facilitate systematic investigation of the
molecular mechanisms of protein translation and the design, construction and
evolution of macromolecular machines, metabolic networks and synthetic cells.

DOI: 10.1038/nbt.1847 
PMID: 21516083  [Indexed for MEDLINE]


445. PLoS One. 2011 Apr 29;6(4):e19175. doi: 10.1371/journal.pone.0019175.

Comparing de novo genome assembly: the long and short of it.

Narzisi G(1), Mishra B.

Author information: 
(1)Courant Institute of Mathematical Sciences, New York University, New York, New
York, United States of America. narzisi@nyu.edu

Recent advances in DNA sequencing technology and their focal role in Genome Wide 
Association Studies (GWAS) have rekindled a growing interest in the whole-genome 
sequence assembly (WGSA) problem, thereby, inundating the field with a plethora
of new formalizations, algorithms, heuristics and implementations. And yet, scant
attention has been paid to comparative assessments of these assemblers' quality
and accuracy. No commonly accepted and standardized method for comparison exists 
yet. Even worse, widely used metrics to compare the assembled sequences emphasize
only size, poorly capturing the contig quality and accuracy. This paper addresses
these concerns: it highlights common anomalies in assembly accuracy through a
rigorous study of several assemblers, compared under both standard metrics (N50, 
coverage, contig sizes, etc.) as well as a more comprehensive metric
(Feature-Response Curves, FRC) that is introduced here; FRC transparently
captures the trade-offs between contigs' quality against their sizes. For this
purpose, most of the publicly available major sequence assemblers--both for
low-coverage long (Sanger) and high-coverage short (Illumina) reads
technologies--are compared. These assemblers are applied to microbial
(Escherichia coli, Brucella, Wolbachia, Staphylococcus, Helicobacter) and partial
human genome sequences (Chr. Y), using sequence reads of various read-lengths,
coverages, accuracies, and with and without mate-pairs. It is hoped that, based
on these evaluations, computational biologists will identify innovative sequence 
assembly paradigms, bioinformaticists will determine promising approaches for
developing "next-generation" assemblers, and biotechnologists will formulate more
meaningful design desiderata for sequencing technology platforms. A new software 
tool for computing the FRC metric has been developed and is available through the
AMOS open-source consortium.

DOI: 10.1371/journal.pone.0019175 
PMCID: PMC3084767
PMID: 21559467  [Indexed for MEDLINE]


446. J Biol Chem. 2011 Apr 22;286(16):14315-23. doi: 10.1074/jbc.M110.200741. Epub
2011 Feb 14.

Polynucleotide phosphorylase activity may be modulated by metabolites in
Escherichia coli.

Nurmohamed S(1), Vincent HA, Titman CM, Chandran V, Pears MR, Du D, Griffin JL,
Callaghan AJ, Luisi BF.

Author information: 
(1)Department of Biochemistry, University of Cambridge, 80 Tennis Court Road,
Cambridge CB2 1GA, United Kingdom.

RNA turnover is an essential element of cellular homeostasis and response to
environmental change. Whether the ribonucleases that mediate RNA turnover can
respond to cellular metabolic status is an unresolved question. Here we present
evidence that the Krebs cycle metabolite citrate affects the activity of
Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that
cellular metabolism is affected widely by PNPase activity. An E. coli strain that
requires PNPase for viability has suppressed growth in the presence of increased 
citrate concentration. Transcriptome analysis reveals a PNPase-mediated response 
to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro,
citrate directly binds and modulates PNPase activity, as predicted by
crystallographic data. Binding of metal-chelated citrate in the active site at
physiological concentrations appears to inhibit enzyme activity. However,
metal-free citrate is bound at a vestigial active site, where it stimulates
PNPase activity. Mutagenesis data confirmed a potential role of this vestigial
site as an allosteric binding pocket that recognizes metal-free citrate.
Collectively, these findings suggest that RNA degradative pathways communicate
with central metabolism. This communication appears to be part of a feedback
network that may contribute to global regulation of metabolism and cellular
energy efficiency.

DOI: 10.1074/jbc.M110.200741 
PMCID: PMC3077632
PMID: 21324911  [Indexed for MEDLINE]


447. Anal Chem. 2011 Apr 15;83(8):3153-60. doi: 10.1021/ac2002214. Epub 2011 Mar 21.

Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica , and
Campylobacter jejuni by combining stopped polymerase chain reaction with
chemiluminescence flow-through DNA microarray analysis.

Donhauser SC(1), Niessner R, Seidel M.

Author information: 
(1)Chair for Analytical Chemistry and Institute of Hydrochemistry, Technische
Universität München, Marchioninistrasse 17, D-81377 München, Germany.

Rapid analysis of pathogenic bacteria is essential for food and water control to 
preserve the public health. Therefore, we report on a chemiluminescence (CL)
flow-through DNA microarray assay for the rapid and sensitive quantification of
the pathogenic bacteria Escherichia coli O157:H7, Salmonella enterica , and
Campylobacter jejuni in water. Using the stopped polymerase chain reaction (PCR) 
strategy, the amount of amplified target DNA was strongly dependent on the
applied cell concentration. The amplification was stopped at the logarithmic
phase of the PCR to quantify the DNA products on the DNA microarray chip. The
generation of single-stranded DNA sequences is essential for DNA hybridization
assays on microarrays. Therefore, the DNA strands of the PCR products were
separated by streptavidin-conjugated magnetic nanoparticles. This was achieved by
introducing a reverse primer labeled with biotin together with a digoxigenin
labeled forward primer for CL microarray imaging. A conjugate of an
antidigoxigenin antibody and horseradish peroxidase recognized the
digoxigenin-labeled antistrands bound to the probes on the microarray surface and
catalyzed the reaction of luminol and hydrogen peroxide. The generated light
emission was recorded by a sensitive charge-coupled device (CCD) camera. The
quantification was conducted by a flow-through CL microarray readout system. The 
DNA microarrays were based on an NHS-activated poly(ethylene glycol)-modified
glass substrate. The DNA probes which have the same DNA sequence as the reverse
primer were immobilized on this surface. The full assay was characterized by
spiking experiments with heat-inactivated bacteria in water. The total assay time
was 3.5 h, and the detection limits determined on CL microarrays were for E. coli
O157:H7, S. enterica , and C. jejuni 136, 500, and 1 cell/mL, respectively. The
results of the DNA microarray assay were comparable to the SYBR green-based
assays analyzed with a real-time PCR device. The advantage of the new microarray 
analysis method is seen in the ability of a high multiplex degree on DNA
microarrays, the high specificity of DNA hybridization on DNA microarrays, and
the possibility to get quantitative results on an automated CL flow-through
microarray analysis system.

© 2011 American Chemical Society

DOI: 10.1021/ac2002214 
PMID: 21417213  [Indexed for MEDLINE]


448. Appl Environ Microbiol. 2011 Apr;77(8):2734-48. doi: 10.1128/AEM.02321-10. Epub
2011 Mar 4.

Prevalence, distribution, and diversity of Salmonella enterica in a major produce
region of California.

Gorski L(1), Parker CT, Liang A, Cooley MB, Jay-Russell MT, Gordus AG, Atwill ER,
Mandrell RE.

Author information: 
(1)Produce Safety and Microbiology Research Unit, USDA, Agricultural Research
Service, Albany, CA 94710, USA. lisa.gorski@ars.usda.gov

A survey was initiated to determine the prevalence of Salmonella enterica in the 
environment in and around Monterey County, CA, a major agriculture region of the 
United States. Trypticase soy broth enrichment cultures of samples of
soil/sediment (n = 617), water (n = 252), wildlife (n = 476), cattle feces (n =
795), and preharvest lettuce and spinach (n = 261) tested originally for the
presence of pathogenic Escherichia coli were kept in frozen storage and later
used to test for the presence of S. enterica. A multipathogen oligonucleotide
microarray was employed to identify a subset of samples that might contain
Salmonella in order to test various culture methods to survey a larger number of 
samples. Fifty-five of 2,401 (2.3%) samples yielded Salmonella, representing
samples obtained from 20 different locations in Monterey and San Benito Counties.
Water had the highest percentage of positives (7.1%) among sample types. Wildlife
yielded 20 positive samples, the highest number among sample types, with positive
samples from birds (n = 105), coyotes (n = 40), deer (n = 104), elk (n = 39),
wild pig (n = 41), and skunk (n = 13). Only 16 (2.6%) of the soil/sediment
samples tested positive, and none of the produce samples had detectable
Salmonella. Sixteen different serotypes were identified among the isolates,
including S. enterica serotypes Give, Typhimurium, Montevideo, and Infantis.
Fifty-four strains were sensitive to 12 tested antibiotics; one S. Montevideo
strain was resistant to streptomycin and gentamicin. Pulsed-field gel
electrophoresis (PFGE) analysis of the isolates revealed over 40 different
pulsotypes. Several strains were isolated from water, wildlife, or soil over a
period of several months, suggesting that they were persistent in this
environment.

DOI: 10.1128/AEM.02321-10 
PMCID: PMC3126348
PMID: 21378057  [Indexed for MEDLINE]


449. Appl Environ Microbiol. 2011 Apr;77(7):2309-16. doi: 10.1128/AEM.02094-10. Epub
2011 Jan 28.

Prevalence, development, and molecular mechanisms of bacteriocin resistance in
Campylobacter.

Hoang KV(1), Stern NJ, Saxton AM, Xu F, Zeng X, Lin J.

Author information: 
(1)Department of Animal Science, The University of Tennessee, 2640 Morgan Circle 
Drive, Knoxville, TN 37996-4574, USA.

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow
or broad spectra of antimicrobial activity. Recently, several unique
anti-Campylobacter BCNs have been identified from commensal bacteria isolated
from chicken intestines. These BCNs dramatically reduced C. jejuni colonization
in poultry and are being directed toward on-farm control of Campylobacter.
However, no information concerning prevalence, development, and mechanisms of BCN
resistance in Campylobacter exists. In this study, susceptibilities of 137 C.
jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and
E-760 were examined. Only one C. coli strain displayed resistance to the BCNs
(MIC, 64 μg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 
μg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro
selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16
μg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and
interspecies levels among Campylobacter strains by biphasic natural
transformation. Genomic examination of the OR-7-resistant mutants by using DNA
microarray and random transposon mutagenesis revealed that the multidrug efflux
pump CmeABC contributes to both intrinsic resistance and acquired resistance to
the BCNs. Altogether, this study represents the first report of and a major step 
forward in understanding BCN resistance in Campylobacter, which will facilitate
the development of effective BCN-based strategies to reduce the Campylobacter
loads in poultry.

DOI: 10.1128/AEM.02094-10 
PMCID: PMC3067428
PMID: 21278269  [Indexed for MEDLINE]


450. Nucleic Acids Res. 2011 Apr;39(7):e41. doi: 10.1093/nar/gkq1275. Epub 2010 Dec
10.

COMODO: an adaptive coclustering strategy to identify conserved coexpression
modules between organisms.

Zarrineh P(1), Fierro AC, Sánchez-Rodríguez A, De Moor B, Engelen K, Marchal K.

Author information: 
(1)Department of Electrical Engineering, Katholieke Universiteit Leuven,
Kasteelpark Arenberg 20, 3001 Leuven, Belgium.

Increasingly large-scale expression compendia for different species are becoming 
available. By exploiting the modularity of the coexpression network, these
compendia can be used to identify biological processes for which the expression
behavior is conserved over different species. However, comparing module networks 
across species is not trivial. The definition of a biologically meaningful module
is not a fixed one and changing the distance threshold that defines the degree of
coexpression gives rise to different modules. As a result when comparing modules 
across species, many different partially overlapping conserved module pairs
across species exist and deciding which pair is most relevant is hard. Therefore,
we developed a method referred to as conserved modules across organisms (COMODO) 
that uses an objective selection criterium to identify conserved expression
modules between two species. The method uses as input microarray data and a gene 
homology map and provides as output pairs of conserved modules and searches for
the pair of modules for which the number of sharing homologs is statistically
most significant relative to the size of the linked modules. To demonstrate its
principle, we applied COMODO to study coexpression conservation between the two
well-studied bacteria Escherichia coli and Bacillus subtilis. COMODO is available
at:
http://homes.esat.kuleuven.be/∼kmarchal/Supplementary_Information_Zarrineh_2010/c
omodo/index.html.

DOI: 10.1093/nar/gkq1275 
PMCID: PMC3074154
PMID: 21149270  [Indexed for MEDLINE]


451. Nucleic Acids Res. 2011 Apr;39(8):3188-203. doi: 10.1093/nar/gkq1242. Epub 2010
Dec 11.

Analysis of Escherichia coli RNase E and RNase III activity in vivo using tiling 
microarrays.

Stead MB(1), Marshburn S, Mohanty BK, Mitra J, Pena Castillo L, Ray D, van Bakel 
H, Hughes TR, Kushner SR.

Author information: 
(1)Department of Genetics, University of Georgia, Athens, GA 30605, USA.

Tiling microarrays have proven to be a valuable tool for gaining insights into
the transcriptomes of microbial organisms grown under various nutritional or
stress conditions. Here, we describe the use of such an array, constructed at the
level of 20 nt resolution for the Escherichia coli MG1655 genome, to observe
genome-wide changes in the steady-state RNA levels in mutants defective in either
RNase E or RNase III. The array data were validated by comparison to previously
published results for a variety of specific transcripts as well as independent
northern analysis of additional mRNAs and sRNAs. In the absence of RNase E, 60%
of the annotated coding sequences showed either increases or decreases in their
steady-state levels. In contrast, only 12% of the coding sequences were affected 
in the absence of RNase III. Unexpectedly, many coding sequences showed decreased
abundance in the RNase E mutant, while more than half of the annotated sRNAs
showed changes in abundance. Furthermore, the steady-state levels of many
transcripts showed overlapping effects of both ribonucleases. Data are also
presented demonstrating how the arrays were used to identify potential new genes,
RNase III cleavage sites and the direct or indirect control of specific
biological pathways.

DOI: 10.1093/nar/gkq1242 
PMCID: PMC3082872
PMID: 21149258  [Indexed for MEDLINE]


452. Microb Cell Fact. 2011 Mar 25;10:18. doi: 10.1186/1475-2859-10-18.

Evolution combined with genomic study elucidates genetic bases of isobutanol
tolerance in Escherichia coli.

Minty JJ(1), Lesnefsky AA, Lin F, Chen Y, Zaroff TA, Veloso AB, Xie B, McConnell 
CA, Ward RJ, Schwartz DR, Rouillard JM, Gao Y, Gulari E, Lin XN.

Author information: 
(1)Department of Chemical Engineering, University of Michigan, Ann Arbor, MI
48109, USA.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated
high yield microbial production, but the toxicity of this molecule reduces
fermentation volumetric productivity and final titer. Organic solvent tolerance
is a complex, multigenic phenotype that has been recalcitrant to rational
engineering approaches. We apply experimental evolution followed by genome
resequencing and a gene expression study to elucidate genetic bases of adaptation
to exogenous isobutanol stress.
RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic
pleiotropy between minimal and rich medium, and appear to be specific to the
effects of longer chain alcohols. By examining genotypic adaptation in multiple
independent lineages, we find evidence of parallel evolution in marC, hfq, mdh,
acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced
RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the 
complex, multigenic nature of solvent tolerance, we observe adaptations in a
diversity of cellular processes. Many adaptations appear to involve epistasis
between different mutations, implying a rugged fitness landscape for isobutanol
tolerance. We observe a trend of evolution targeting post-transcriptional
regulation and high centrality nodes of biochemical networks. Collectively, the
genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol
stress based on remodeling the cell envelope and surprisingly, stress response
attenuation.
CONCLUSIONS: We have discovered a set of genotypic adaptations that confer
increased tolerance to exogenous isobutanol stress. Our results are immediately
useful to further efforts to engineer more isobutanol tolerant host strains of E.
coli for isobutanol production. We suggest that rpoS and post-transcriptional
regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis 
targets for future global phenotype engineering.

DOI: 10.1186/1475-2859-10-18 
PMCID: PMC3071312
PMID: 21435272  [Indexed for MEDLINE]


453. Vet Microbiol. 2011 Mar 24;148(2-4):434-5. doi: 10.1016/j.vetmic.2010.08.033.
Epub 2010 Sep 8.

Plasmid-mediated quinolone resistance gene detected in Escherichia coli from
cattle.

Kirchner M(1), Wearing H, Teale C.

Author information: 
(1)Veterinary Laboratories Agency, Woodham, Lane, New Haw, Addlestone, Surrey
KT15 3NB, United Kingdom. m.kirchner@vla.defra.gsi.gov.uk

Fluoroquinolones resistance in bacteria can be due to chromosomal and
plasmid-mediated mechanisms. Of growing concern is the acquisition of genes
encoding quinolone resistance in combination with other resistance mechanisms
such as extended-spectrum beta-lactamases. In this study we describe the
identification of an isolate of Escherichia coli from cattle which carried qnrS1 
in combination with a blaCTX-M gene, although they were not co-localised on the
same plasmid. In addition, using a DNA array it was possible to identify several 
other antimicrobial resistance genes in this isolate. This is the first report of
a qnr gene in E. coli from cattle in the UK and highlights the need for
surveillance of these emerging resistance mechanisms.

Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

DOI: 10.1016/j.vetmic.2010.08.033 
PMID: 20884136  [Indexed for MEDLINE]


454. Proc Natl Acad Sci U S A. 2011 Mar 15;108 Suppl 1:4672-9. doi:
10.1073/pnas.1000091107. Epub 2010 Aug 2.

Substrate-driven gene expression in Roseburia inulinivorans: importance of
inducible enzymes in the utilization of inulin and starch.

Scott KP(1), Martin JC, Chassard C, Clerget M, Potrykus J, Campbell G, Mayer CD, 
Young P, Rucklidge G, Ramsay AG, Flint HJ.

Author information: 
(1)Rowett Institute of Nutrition and Health, University of Aberdeen, Bucksburn,
Aberdeen AB21 9SB, United Kingdom. kps@rowett.ac.uk

Roseburia inulinivorans is a recently identified motile representative of the
Firmicutes that contributes to butyrate formation from a variety of dietary
polysaccharide substrates in the human large intestine. Microarray analysis was
used here to investigate substrate-driven gene-expression changes in R.
inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization
genes induced during growth on inulin included one encoding a
β-fructofuranosidase protein that was prominent in the proteome of inulin-grown
cells. This cluster also included a 6-phosphofructokinase and an ABC transport
system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a
fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system
(PTS II transport enzyme). Real-time PCR analysis showed that the
β-fructofuranosidase and adjacent ABC transport protein showed greatest induction
during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked
sugar phosphotransferase transport system were most strongly up-regulated during 
growth on fructose, indicating that these two clusters play distinct roles in the
use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in
Escherichia coli and shown to hydrolyze fructans ranging from inulin down to
sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on
starch included the major extracellular α-amylase and two distinct
α-glucanotransferases together with a gene encoding a flagellin protein. The
latter response may be concerned with improving bacterial access to insoluble
starch particles.

DOI: 10.1073/pnas.1000091107 
PMCID: PMC3063597
PMID: 20679207  [Indexed for MEDLINE]


455. IET Syst Biol. 2011 Mar;5(2):95-102. doi: 10.1049/iet-syb.2010.0041.

Reconstruction of transcriptional network from microarray data using combined
mutual information and network-assisted regression.

Wang XD(1), Qi YX, Jiang ZL.

Author information: 
(1)Shanghai Jiao Tong University, Institute of Mechanobiology and Medical
Engineering, Shanghai, People's Republic of China.

Many methods had been developed on inferring transcriptional network from gene
expression. However, it is still necessary to design new method that discloses
more detailed and exact network information. Using network-assisted regression,
the authors combined the averaged three-way mutual information (AMI3) and
non-linear ordinary differential equation (ODE) model to infer the
transcriptional network, and to obtain both the topological structure and the
regulatory dynamics. Synthetic and experimental data were used to evaluate the
performance of the above approach. In comparison with the previous methods based 
on mutual information, AMI3 obtained higher precision with the same sensitivity. 
To describe the regulatory dynamics between transcription factors and target
genes, network-assisted regression and regression without network, respectively, 
were applied in the steady-state and time series microarray data. The results
revealed that comparing with regression without network, network-assisted
regression increased the precision, but decreased the fitting goodness. Then, the
authors reconstructed the transcriptional network of Escherichia coli and
simulated the regulatory dynamics of genes. Furthermore, the authors' approach
identified potential transcription factors regulating yeast cell cycle. In
conclusion, network-assisted regression, combined AMI3 and ODE model, was a more 
precisely to infer the topological structure and the regulatory dynamics of
transcriptional network from microarray data. [Includes supplementary material].

DOI: 10.1049/iet-syb.2010.0041 
PMID: 21405197  [Indexed for MEDLINE]


456. Infect Immun. 2011 Mar;79(3):1016-24. doi: 10.1128/IAI.00927-10. Epub 2010 Dec
13.

Activation of motility by sensing short-chain fatty acids via two steps in a
flagellar gene regulatory cascade in enterohemorrhagic Escherichia coli.

Tobe T(1), Nakanishi N, Sugimoto N.

Author information: 
(1)Department of Microbiology and Immunology, Graduate School of Medicine, Osaka 
University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
torutobe@bact.med.osaka-u.ac.jp

The regulated expression of virulence genes is critical for successful infection 
by an intestinal pathogen. Bacteria rely on sensing environmental signals to find
preferable niches and reach the infectious state. Orally ingested
enterohemorrhagic Escherichia coli (EHEC) travels through the gastrointestinal
tract and encounters a variety of environmental factors, some of which act as
triggering signals for the induction of virulence genes. Butyrate, one of the
main short-chain fatty acids (SCFAs), is such a signal, enhancing the expression 
of genes for intimate attachment and type III secretion. We further explored the 
role of SCFAs and found a positive effect of SCFAs on flagellar expression.
Although EHEC did not produce flagella when grown in Dulbecco's modified Eagle's 
medium (DMEM), a tissue culture medium that enhances virulence gene expression,
the addition of SCFAs to the medium induced the production of flagella, and the
EHEC bacteria became motile. Among SCFAs, butyrate simultaneously activates both 
virulence and flagellar genes. Flagella did not affect initial adherence, and
they were not expressed in adherent bacteria during microcolony formation. SCFAs 
activated flagellar genes via two regulatory steps. Butyrate activated the flhDC 
regulatory genes through leucine-responsive regulatory protein (Lrp), which is
also a regulator of virulence genes. However, butyrate, acetate, and propionate
also activated downstream genes independently of flhDC activation. Consequently, 
when encountering increased concentrations of SCFAs, which are abundant in
acetate, in the intestine, EHEC first activates flagellar production and
motility, followed by genes involved in adherence and type III secretion, which
leads to efficient adherence in a preferable niche.

DOI: 10.1128/IAI.00927-10 
PMCID: PMC3067497
PMID: 21149585  [Indexed for MEDLINE]


457. J Ind Microbiol Biotechnol. 2011 Mar;38(3):431-9. doi: 10.1007/s10295-010-0787-5.
Epub 2010 Jul 30.

YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA
that are involved in furfural tolerance.

Turner PC(1), Miller EN, Jarboe LR, Baggett CL, Shanmugam KT, Ingram LO.

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida, Box
110700, Gainesville, FL 32611, USA.

Previous results have demonstrated that the silencing of adjacent genes encoding 
NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased
furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ
Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from
the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream 
gene. YqhC shares homology with transcriptional regulators belonging to the
AraC/XylS family and was shown to act as a positive regulator of the adjacent
operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a
chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and
by a direct comparison of furfural resistance and NADPH-dependent furfural
reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA
orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also
present in more distantly related Gram-negative bacteria. Disruption of yqhC
offers a useful approach to increase furfural tolerance in bacteria.

DOI: 10.1007/s10295-010-0787-5 
PMID: 20676725  [Indexed for MEDLINE]


458. BMC Genomics. 2011 Feb 28;12:130. doi: 10.1186/1471-2164-12-130.

In depth analysis of genes and pathways of the mammary gland involved in the
pathogenesis of bovine Escherichia coli-mastitis.

Buitenhuis B(1), Røntved CM, Edwards SM, Ingvartsen KL, Sørensen P.

Author information: 
(1)Department of Genetics and Biotechnology, Faculty of Agricultural Sciences,
Aarhus University, Blichers allé 20, P,O, Box 50, DK-8830 Tjele, Denmark.
bart.buitenhuis@agrsci.dk

BACKGROUND: Bovine mastitis is one of the most costly and prevalent diseases
affecting dairy cows worldwide. In order to develop new strategies to prevent
Escherichia coli-induced mastitis, a detailed understanding of the molecular
mechanisms underlying the host immune response to an E. coli infection is
necessary. To this end, we performed a global gene-expression analysis of mammary
gland tissue collected from dairy cows that had been exposed to a controlled E.
coli infection. Biopsy samples of healthy and infected utter tissue were
collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent
the acute phase response (APR) and chronic stage, respectively. Differentially
expressed (DE) genes for each stage were analyzed and the DE genes detected at T 
= 24 h were also compared to data collected from two previous E. coli mastitis
studies that were carried out on post mortem tissue.
RESULTS: Nine-hundred-eighty-two transcripts were found to be differentially
expressed in infected tissue at T = 24 (P < 0.05). Up-regulated transcripts (699)
were largely associated with immune response functions, while the down-regulated 
transcripts (229) were principally involved in fat metabolism. At T = 192 h, all 
of the up-regulated transcripts were associated with tissue healing processes.
Comparison of T = 24 h DE genes detected in the three E. coli mastitis studies
revealed 248 were common and mainly involved immune response functions. KEGG
pathway analysis indicated that these genes were involved in 12 pathways related 
to the pro-inflammatory response and APR, but also identified significant
representation of two unexpected pathways: natural killer cell-mediated
cytotoxicity pathway (KEGG04650) and the Rig-I-like receptor signalling pathway
(KEGG04622).
CONCLUSIONS: In E. coli-induced mastitis, infected mammary gland tissue was found
to significantly up-regulate expression of genes related to the immune response
and down-regulate genes related to fat metabolism. Up to 25% of the DE immune
response genes common to the three E. coli mastitis studies at T = 24 h were
independent of E. coli strain and dose, cow lactation stage and number, tissue
collection method and gene analysis method used. Hence, these DE genes likely
represent important mediators of the local APR against E. coli in the mammary
gland.

DOI: 10.1186/1471-2164-12-130 
PMCID: PMC3053262
PMID: 21352611  [Indexed for MEDLINE]


459. BMC Microbiol. 2011 Feb 23;11:39. doi: 10.1186/1471-2180-11-39.

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia
pestis.

Gao H(1), Zhang Y, Han Y, Yang L, Liu X, Guo Z, Tan Y, Huang X, Zhou D, Yang R.

Author information: 
(1)State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of
Microbiology and Epidemiology, Beijing 100071, PR China.

BACKGROUND: The osmotic regulator OmpR in Escherichia coli regulates
differentially the expression of major porin proteins OmpF and OmpC. In Yersinia 
enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and
survival within macrophages. However, the phenotypic and regulatory roles of OmpR
in Y. pestis are not yet fully understood.
RESULTS: Y. pestis OmpR is involved in building resistance against phagocytosis
and controls the adaptation to various stressful conditions met in macrophages.
The ompR mutation likely did not affect the virulence of Y. pestis strain 201
that was a human-avirulent enzootic strain. The microarray-based comparative
transcriptome analysis disclosed a set of 224 genes whose expressions were
affected by the ompR mutation, indicating the global regulatory role of OmpR in
Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16
OmpR-dependent genes, for which OmpR consensus-like sequences were found within
their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with
the increase of medium osmolarity, which was mediated by OmpR occupying the
target promoter regions in a tandem manner.
CONCLUSION: OmpR contributes to the resistance against phagocytosis or survival
within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis
OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. 
There is an inducible expressions of the pore-forming proteins OmpF, C, and × at 
high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in
E. coli. The main difference is that ompF expression is not repressed at high
osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal 
OmpR-binding site for ompF.

DOI: 10.1186/1471-2180-11-39 
PMCID: PMC3050692
PMID: 21345178  [Indexed for MEDLINE]


460. BMC Genomics. 2011 Feb 21;12:123. doi: 10.1186/1471-2164-12-123.

Comparative tissue transcriptomics reveal prompt inter-organ communication in
response to local bacterial kidney infection.

Boekel J(1), Källskog O, Rydén-Aulin M, Rhen M, Richter-Dahlfors A.

Author information: 
(1)Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska
Institutet, Retzius väg 8,Stockholm, Sweden.

BACKGROUND: Mucosal infections elicit inflammatory responses via regulated
signaling pathways. Infection outcome depends strongly on early events occurring 
immediately when bacteria start interacting with cells in the mucosal membrane.
Hitherto reported transcription profiles on host-pathogen interactions are
strongly biased towards in vitro studies. To detail the local in vivo genetic
response to infection, we here profiled host gene expression in a recent
experimental model that assures high spatial and temporal control of
uropathogenic Escherichia coli (UPEC) infection within the kidney of a live rat.
RESULTS: Transcriptional profiling of tissue biopsies from UPEC-infected kidney
tissue revealed 59 differentially expressed genes 8 h post-infection. Their
relevance for the infection process was supported by a Gene Ontology (GO)
analysis. Early differential expression at 3 h and 5 h post-infection was of low 
statistical significance, which correlated to the low degree of infection.
Comparative transcriptomics analysis of the 8 h data set and online available
studies of early local infection and inflammation defined a core of 80 genes
constituting a "General tissue response to early local bacterial infections".
Among these, 25% were annotated as interferon-γ (IFN-γ) regulated. Subsequent
experimental analyses confirmed a systemic increase of IFN-γ in rats with an
ongoing local kidney infection, correlating to splenic, rather than renal Ifng
induction and suggested this inter-organ communication to be mediated by
interleukin (IL)-23. The use of comparative transcriptomics allowed expansion of 
the statistical data handling, whereby relevant data could also be extracted from
the 5 h data set. Out of the 31 differentially expressed core genes, some
represented specific 5 h responses, illustrating the value of comparative
transcriptomics when studying the dynamic nature of gene regulation in response
to infections.
CONCLUSION: Our hypothesis-free approach identified components of
infection-associated multi-cellular tissue responses and demonstrated how a
comparative analysis allows retrieval of relevant information from lower-quality 
data sets. The data further define marked representation of IFN-γ responsive
genes and a prompt inter-organ communication as a hallmark of an early local
tissue response to infection.

DOI: 10.1186/1471-2164-12-123 
PMCID: PMC3047304
PMID: 21338499  [Indexed for MEDLINE]


461. PLoS One. 2011 Feb 18;6(2):e16517. doi: 10.1371/journal.pone.0016517.

Phenotype sequencing: identifying the genes that cause a phenotype directly from 
pooled sequencing of independent mutants.

Harper MA(1), Chen Z, Toy T, Machado IM, Nelson SF, Liao JC, Lee CJ.

Author information: 
(1)Institute for Genomics and Proteomics, University of California Los Angeles,
Los Angeles, California, United States of America.

Random mutagenesis and phenotype screening provide a powerful method for
dissecting microbial functions, but their results can be laborious to analyze
experimentally. Each mutant strain may contain 50-100 random mutations,
necessitating extensive functional experiments to determine which one causes the 
selected phenotype. To solve this problem, we propose a "Phenotype Sequencing"
approach in which genes causing the phenotype can be identified directly from
sequencing of multiple independent mutants. We developed a new computational
analysis method showing that 1. causal genes can be identified with high
probability from even a modest number of mutant genomes; 2. costs can be cut
many-fold compared with a conventional genome sequencing approach via an
optimized strategy of library-pooling (multiple strains per library) and
tag-pooling (multiple tagged libraries per sequencing lane). We have performed
extensive validation experiments on a set of E. coli mutants with increased
isobutanol biofuel tolerance. We generated a range of sequencing experiments
varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our
statistical analysis of these data (4099 mutations from 32 mutant genomes)
successfully identified 3 genes (acrB, marC, acrA) that have been independently
validated as causing this experimental phenotype. It must be emphasized that our 
approach reduces mutant sequencing costs enormously. Whereas a conventional
genome sequencing experiment would have cost $7,200 in reagents alone, our
Phenotype Sequencing design yielded the same information value for only $1200. In
fact, our smallest experiments reliably identified acrB and marC at a cost of
only $110-$340.

DOI: 10.1371/journal.pone.0016517 
PMCID: PMC3041756
PMID: 21364744  [Indexed for MEDLINE]


462. BMC Bioinformatics. 2011 Feb 15;12 Suppl 1:S37. doi: 10.1186/1471-2105-12-S1-S37.

Query-based biclustering of gene expression data using Probabilistic Relational
Models.

Zhao H(1), Cloots L, Van den Bulcke T, Wu Y, De Smet R, Storms V, Meysman P,
Engelen K, Marchal K.

Author information: 
(1)Microbial and Molecular Systems, KU Leuven, Leuven 3001, Belgium.
hui.zhao@biw.kuleuven.be

BACKGROUND: With the availability of large scale expression compendia it is now
possible to view own findings in the light of what is already available and
retrieve genes with an expression profile similar to a set of genes of interest
(i.e., a query or seed set) for a subset of conditions. To that end, a
query-based strategy is needed that maximally exploits the coexpression behaviour
of the seed genes to guide the biclustering, but that at the same time is robust 
against the presence of noisy genes in the seed set as seed genes are often
assumed, but not guaranteed to be coexpressed in the queried compendium.
Therefore, we developed ProBic, a query-based biclustering strategy based on
Probabilistic Relational Models (PRMs) that exploits the use of prior
distributions to extract the information contained within the seed set.
RESULTS: We applied ProBic on a large scale Escherichia coli compendium to extend
partially described regulons with potentially novel members. We compared ProBic's
performance with previously published query-based biclustering algorithms, namely
ISA and QDB, from the perspective of bicluster expression quality, robustness of 
the outcome against noisy seed sets and biological relevance.This comparison
learns that ProBic is able to retrieve biologically relevant, high quality
biclusters that retain their seed genes and that it is particularly strong in
handling noisy seeds.
CONCLUSIONS: ProBic is a query-based biclustering algorithm developed in a
flexible framework, designed to detect biologically relevant, high quality
biclusters that retain relevant seed genes even in the presence of noise or when 
dealing with low quality seed sets.

DOI: 10.1186/1471-2105-12-S1-S37 
PMCID: PMC3044293
PMID: 21342568  [Indexed for MEDLINE]


463. J Bacteriol. 2011 Feb;193(3):649-59. doi: 10.1128/JB.01214-10. Epub 2010 Nov 29.

Novel members of the Cra regulon involved in carbon metabolism in Escherichia
coli.

Shimada T(1), Yamamoto K, Ishihama A.

Author information: 
(1)Hosei University, Department of Frontier Bioscience, Koganei, Tokyo 184-8584, 
Japan.

Cra (catabolite repressor activator) is a global regulator of the genes for
carbon metabolism in Escherichia coli. To gain insights into the regulatory roles
of Cra, attempts were made to identify the whole set of regulation targets using 
an improved genomic SELEX (systematic evolution of ligands by exponential
enrichment) system. Surprisingly, a total of 164 binding sites were identified
for Cra, 144 (88%) of which were newly identified. The majority of known targets 
were included in the SELEX chip pattern. The promoters examined by the lacZ
reporter assay in vivo were all regulated by Cra. These two lines of evidence
indicate that a total of as many as 178 promoters are under the control of Cra.
The majority of Cra targets are the genes coding for the enzymes involved in
central carbon metabolism, covering all the genes for the enzymes involved in
glycolysis and metabolism downstream of glycolysis, including the tricarboxylic
acid (TCA) cycle and aerobic respiration. Taken together, we propose that Cra
plays a key role in balancing the levels of the enzymes for carbon metabolism.

DOI: 10.1128/JB.01214-10 
PMCID: PMC3021228
PMID: 21115656  [Indexed for MEDLINE]


464. Mol Cell Probes. 2011 Feb;25(1):35-43. doi: 10.1016/j.mcp.2010.10.002. Epub 2010 
Oct 20.

A tool based on Ligation Detection Reaction-Universal Array (LDR-UA) for the
characterization of VTEC by identification of virulence-associated and
serogroup-specific genes.

Lauri A(1), Castiglioni B, Morabito S, Tozzoli R, Consolandi C, Mariani P.

Author information: 
(1)Parco Tecnologico Padano, Lodi, Italy. andrea.lauri@tecnoparco.org

Verocytoxigenic Escherichia coli (VTEC) are zoonotic pathogens whose natural
reservoir is represented by ruminants, particularly cattle. Infections are mainly
acquired by consumption of undercooked contaminated food of animal origin,
contact with infected animals and contaminated environment. VTEC O157 is the most
frequently isolated serogroup from cases of human disease, however, other VTEC
serogroups, such as O26, O111, O145 and O103, are increasingly reported as
causing Hemolytic Uremic Syndrome (HUS) worldwide. The identification of VTEC is 
troublesome, hindering the development of effective prevention strategies. In
fact, VTEC are morphologically indistinguishable from harmless E. coli and their 
pathogenic potential is not strictly dependent on the serogroup, but relies on
the presence of a collection of virulence genes. We developed a diagnostic tool
for VTEC based on the Ligation Detection Reaction coupled to Universal Array
(LDR-UA) for the simultaneous identification of virulence factors and
serogroup-associated genes. The method includes the investigation of 40 sites
located in 13 fragments from 12 genes (sodCF1/F2, adfO, terB, ehxA, eae, vtx1,
vtx2, ihp1, wzx, wbdI, rfbE, dnaK) and was evaluated by performing a trial on a
collection of 67 E. coli strains, both VTEC and VT-negative E. coli, as well as
on 25 isolates belonging to other related species. Results of this study showed
that the LDR-UA technique was specific in identifying the target microorganism.
Moreover, due to its higher throughput, the LDR-UA can be a valid and cheaper
alternative to real time PCR-based (rt-PCR) methods for VTEC identification.

Copyright © 2010 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.mcp.2010.10.002 
PMID: 20969949  [Indexed for MEDLINE]


465. Mol Microbiol. 2011 Feb;79(4):846-60. doi: 10.1111/j.1365-2958.2010.07491.x. Epub
2010 Dec 22.

The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is
modulated by oxidation.

Schook PO(1), Stohl EA, Criss AK, Seifert HS.

Author information: 
(1)Department of Microbiology-Immunology, Feinberg School of Medicine,
Northwestern University, Chicago, IL 60611, USA.

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to
UV light or chemicals but is likely to encounter reactive oxygen species during
infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed
that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack 
an SOS system, although NG1427 shows amino acid sequence similarity to the SOS
response regulator LexA from Escherichia coli. Similar to LexA and other S24
peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by
either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis
requires the active and cleavage site residues conserved between LexA and NG1427.
NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific,
putative integral membrane protein; and recN, a DNA repair gene known to be
required for oxidative damage survival. Full NG1427 regulon de-repression
requires RecA following methyl methanesulphonate or mitomycin C treatment, but is
largely RecA-independent following hydrogen peroxide treatment. NG1427 binds
specifically to the operator regions of the genes it controls, and DNA binding is
abolished by oxidation of the single cysteine residue encoded in NG1427. We
propose that NG1427 is inactivated independently of RecA by oxidation.

© 2010 Blackwell Publishing Ltd.

DOI: 10.1111/j.1365-2958.2010.07491.x 
PMCID: PMC3080098
PMID: 21299643  [Indexed for MEDLINE]


466. Mol Microbiol. 2011 Feb;79(4):830-45. doi: 10.1111/j.1365-2958.2010.07498.x. Epub
2010 Dec 30.

Discretely calibrated regulatory loops controlled by ppGpp partition gene
induction across the 'feast to famine' gradient in Escherichia coli.

Traxler MF(1), Zacharia VM, Marquardt S, Summers SM, Nguyen HT, Stark SE, Conway 
T.

Author information: 
(1)Advanced Center for Genome Technology, University of Oklahoma, Norman, OK
73019, USA.

Comment in
    Mol Microbiol. 2011 Feb;79(4):827-9.

Bacteria comprehensively reorganize their global gene expression when faced with 
starvation. The alarmone ppGpp facilitates this massive response by co-ordinating
the downregulation of genes of the translation apparatus, and the induction of
biosynthetic genes and the general stress response. Such a large reorientation
requires the activities of multiple regulators, yet the regulatory network
downstream of ppGpp remains poorly defined. Transcription profiling during
isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed
us to identify genes that required ppGpp, Lrp and RpoS for their induction and to
deduce the regulon response times. Although the Lrp and RpoS regulons required
ppGpp for their activation, they were not induced simultaneously. The data
suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low
level of ppGpp for their induction. In contrast, the RpoS regulon was induced
only when high levels of ppGpp accumulated. We tested several predictions of a
model that explains how bacteria allocate transcriptional resources between
metabolism and stress response by discretely tuning two regulatory circuits to
different levels of ppGpp. The emergent regulatory structure insures that stress 
survival circuits are only triggered if homeostatic metabolic networks fail to
compensate for environmental deficiencies.

© 2010 Blackwell Publishing Ltd.

DOI: 10.1111/j.1365-2958.2010.07498.x 
PMCID: PMC3073637
PMID: 21299642  [Indexed for MEDLINE]


467. BMC Genomics. 2011 Jan 14;12:34. doi: 10.1186/1471-2164-12-34.

Divergent responses to peptidoglycans derived from different E. coli serotypes
influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages.

Boltaña S(1), Reyes-Lopez F, Morera D, Goetz F, MacKenzie SA.

Author information: 
(1)Institute of Biotechnology and Biomedicine, Universitat Autónoma de Barcelona,
08193 Barcelona, Spain.

BACKGROUND: Pathogen-associated molecular patterns (PAMPs) are structural
components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) 
from bacterial cell walls. PAMP-recognition by the host results in an induction
of defence-related genes and often the generation of an inflammatory response. We
evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) 
macrophages in primary cell culture stimulated with DAP-PGN (DAP;
meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia
coli (PGN-K12 and PGN-O111:B4) over time.
RESULTS: Transcript profiling was assessed using function-targeted cDNA
microarray hybridisation (n = 36) and results show differential responses to both
PGNs that are both time and treatment dependent. Wild type E. coli (K12)
generated an increase in transcript number/diversity over time whereas
PGN-O111:B4 stimulation resulted in a more specific and intense response. In line
with this, Gene Ontology analysis (GO) highlights a specific transcriptomic
remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high
similarity to a generalised inflammatory priming response where multiple
functional classes are related to ribosome biogenesis or cellular metabolism.
Prostaglandin release was induced by both PGNs and macrophages were significantly
more sensitive to PGN-O111:B4 as suggested from microarray data.
CONCLUSION: Responses at the level of the transcriptome and the inflammatory
outcome (prostaglandin synthesis) highlight the different sensitivity of the
macrophage to slight differences (serotype) in peptidoglycan structure. Such
divergent responses are likely to involve differential receptor sensitivity to
ligands or indeed different receptor types. Such changes in biological response
will likely reflect upon pathogenicity of certain serotypes and the development
of disease.

DOI: 10.1186/1471-2164-12-34 
PMCID: PMC3087353
PMID: 21235753  [Indexed for MEDLINE]


468. Environ Microbiol. 2011 Jan;13(1):62-73. doi: 10.1111/j.1462-2920.2010.02308.x.

3-indolylacetonitrile decreases Escherichia coli O157:H7 biofilm formation and
Pseudomonas aeruginosa virulence.

Lee JH(1), Cho MH(1), Lee J(1).

Author information: 
(1)School of Display and Chemical Engineering, Yeungnam University, Gyeongsan-si,
Gyeongsangbuk-do 712-749, Korea.

Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia
coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and
bacterial indole derivatives are unstable in the microbial community because they
are quickly degraded by diverse bacterial oxygenases. Hence, this work sought to 
identify novel, non-toxic, stable and potent indole derivatives from plant
sources for inhibiting the biofilm formation of E. coli O157:H7 and P.
aeruginosa. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit
the biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting
its growth. IAN more effectively inhibited biofilms than indole for the two
pathogenic bacteria. Additionally, IAN decreased the production of virulence
factors including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), pyocyanin and
pyoverdine in P. aeruginosa. DNA microarray analysis indicated that IAN repressed
genes involved in curli formation and glycerol metabolism, whereas IAN induced
indole-related genes and prophage genes in E. coli O157:H7. It appeared that IAN 
inhibited the biofilm formation of E. coli by reducing curli formation and
inducing indole production. Also, corroborating phenotypic results of P.
aeruginosa, whole-transcriptomic data showed that IAN repressed virulence-related
genes and motility-related genes, while IAN induced several small molecule
transport genes. Furthermore, unlike bacterial indole derivatives,
plant-originated IAN was stable in the presence of either E. coli or P.
aeruginosa. Additionally, indole-3-carboxyaldehyde was another natural biofilm
inhibitor for both E. coli and P. aeruginosa.

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

DOI: 10.1111/j.1462-2920.2010.02308.x 
PMID: 20649646  [Indexed for MEDLINE]


469. Genome Biol. 2011;12(2):R17. doi: 10.1186/gb-2011-12-2-r17. Epub 2011 Feb 16.

Stress response regulators identified through genome-wide transcriptome analysis 
of the (p)ppGpp-dependent response in Rhizobium etli.

Vercruysse M(1), Fauvart M, Jans A, Beullens S, Braeken K, Cloots L, Engelen K,
Marchal K, Michiels J.

Author information: 
(1)Centre of Microbial and Plant Genetics, Katholiek Universiteit Leuven,
Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.

BACKGROUND: The alarmone (p)ppGpp mediates a global reprogramming of gene
expression upon nutrient limitation and other stresses to cope with these
unfavorable conditions. Synthesis of (p)ppGpp is, in most bacteria, controlled by
RelA/SpoT (Rsh) proteins. The role of (p)ppGpp has been characterized primarily
in Escherichia coli and several Gram-positive bacteria. Here, we report the first
in-depth analysis of the (p)ppGpp-regulon in an α-proteobacterium using a
high-resolution tiling array to better understand the pleiotropic stress
phenotype of a relA/rsh mutant.
RESULTS: We compared gene expression of the Rhizobium etli wild type and rsh
(previously rel) mutant during exponential and stationary phase, identifying
numerous (p)ppGpp targets, including small non-coding RNAs. The majority of the
834 (p)ppGpp-dependent genes were detected during stationary phase. Unexpectedly,
223 genes were expressed (p)ppGpp-dependently during early exponential phase,
indicating the hitherto unrecognized importance of (p)ppGpp during active growth.
Furthermore, we identified two (p)ppGpp-dependent key regulators for survival
during heat and oxidative stress and one regulator putatively involved in
metabolic adaptation, namely extracytoplasmic function sigma factor
EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA.
CONCLUSIONS: The regulatory role of (p)ppGpp in R. etli stress adaptation is
far-reaching in redirecting gene expression during all growth phases. Genome-wide
transcriptome analysis of a strain deficient in a global regulator, and
exhibiting a pleiotropic phenotype, enables the identification of more specific
regulators that control genes associated with a subset of stress phenotypes. This
work is an important step toward a full understanding of the regulatory network
underlying stress responses in α-proteobacteria.

DOI: 10.1186/gb-2011-12-2-r17 
PMCID: PMC3188799
PMID: 21324192  [Indexed for MEDLINE]


470. Int J Nanomedicine. 2011;6:1833-52. doi: 10.2147/IJN.S24019. Epub 2011 Sep 6.

Silver nanoparticles embedded in zeolite membranes: release of silver ions and
mechanism of antibacterial action.

Nagy A(1), Harrison A, Sabbani S, Munson RS Jr, Dutta PK, Waldman WJ.

Author information: 
(1)Department of Pathology, The Ohio State University, Columbus, OH, USA.

BACKGROUND: The focus of this study is on the antibacterial properties of silver 
nanoparticles embedded within a zeolite membrane (AgNP-ZM).
METHODS AND RESULTS: These membranes were effective in killing Escherichia coli
and were bacteriostatic against methicillin-resistant Staphylococcus aureus. E.
coli suspended in Luria Bertani (LB) broth and isolated from physical contact
with the membrane were also killed. Elemental analysis indicated slow release of 
Ag(+) from the AgNP-ZM into the LB broth. The E. coli killing efficiency of
AgNP-ZM was found to decrease with repeated use, and this was correlated with
decreased release of silver ions with each use of the support. Gene expression
microarrays revealed upregulation of several antioxidant genes as well as genes
coding for metal transport, metal reduction, and ATPase pumps in response to
silver ions released from AgNP-ZM. Gene expression of iron transporters was
reduced, and increased expression of ferrochelatase was observed. In addition,
upregulation of multiple antibiotic resistance genes was demonstrated. The
expression levels of multicopper oxidase, glutaredoxin, and thioredoxin decreased
with each support use, reflecting the lower amounts of Ag(+) released from the
membrane. The antibacterial mechanism of AgNP-ZM is proposed to be related to the
exhaustion of antioxidant capacity.
CONCLUSION: These results indicate that AgNP-ZM provide a novel matrix for
gradual release of Ag(+).

DOI: 10.2147/IJN.S24019 
PMCID: PMC3173047
PMID: 21931480  [Indexed for MEDLINE]


471. J Biomed Biotechnol. 2011;2011:560124. doi: 10.1155/2011/560124. Epub 2010 Oct
13.

Isolation of specific clones from nonarrayed BAC libraries through homologous
recombination.

Nefedov M(1), Carbone L, Field M, Schein J, de Jong PJ.

Author information: 
(1)BACPAC Resources, Children's Hospital Oakland Research Institute, 747-52nd St,
Oakland, CA 94609, USA. mnefedov@chori.org

We have developed a new approach to screen bacterial artificial chromosome (BAC) 
libraries by recombination selection. To test this method, we constructed an
orangutan BAC library using an E. coli strain (DY380) with temperature inducible 
homologous recombination (HR) capability. We amplified one library segment,
induced HR at 42°C to make it recombination proficient, and prepared
electrocompetent cells for transformation with a kanamycin cassette to target
sequences in the orangutan genome through terminal recombineering homologies.
Kanamycin-resistant colonies were tested for the presence of BACs containing the 
targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin
insertion. The results indicate that this is an effective approach for screening 
clones. The advantage of recombination screening is that it avoids the high costs
associated with the preparation, screening, and archival storage of arrayed BAC
libraries. In addition, the screening can be conceivably combined with genetic
engineering to create knockout and reporter constructs for functional studies.

DOI: 10.1155/2011/560124 
PMCID: PMC2957146
PMID: 20981149  [Indexed for MEDLINE]


472. J Biosci Bioeng. 2011 Jan;111(1):26-30. doi: 10.1016/j.jbiosc.2010.08.007. Epub
2010 Sep 9.

Long-term continuous adaptation of Escherichia coli to high succinate stress and 
transcriptome analysis of the tolerant strain.

Kwon YD(1), Kim S, Lee SY, Kim P.

Author information: 
(1)Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi
420-743, Republic of Korea.

To understand the responses of Escherichia coli to high succinate stress and to
determine the roles of upregulated genes in high succinate tolerance, a
continuous culture of wild-type E. coli W3110 was performed for 268 days in a
gradually increasing concentration of succinate. Growth of the final adapted
strain, designated DST160, proceeded growth rate of 0.20 h(-1) without a lag
phase in medium containing 0.592 M succinate, while the wild-type strain showed
0.02 h(-1) in 38 h. The growth rates of DST160 in media containing either 0.61 M 
NaCl, 0.61 M KCl, or at pH 4.5 were 25% higher, 18% lower, and 57% higher than
those of wild-type, respectively, implying DST160 acquired salt tolerance and pH 
shock tolerance as well as succinate tolerance. DNA microarray and real-time PCR 
results indicated that genes controlling active transport and biosynthesis of
osmoprotectants were upregulated in DST160 compared to W3110. When ygjE, encoding
a putative tartrate/succinate antiporter, and betA, encoding betaine
biosynthesis, were expressed in a wild-type E. coli as represent genes for active
transport and osmoprotectant synthesis, respectively, greater growth rates were
achieved under 0.592 M succinate stress conditions (seven times higher due to
ygjE expression and six times higher due to betA expression) than wild-type. The 
potential to design a metabolic engineering for microbial succinate production is
suggested based on the transcriptional regulation of the long-term adapted
DST160.

Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V.
All rights reserved.

DOI: 10.1016/j.jbiosc.2010.08.007 
PMID: 20829106  [Indexed for MEDLINE]


473. J Food Sci. 2011 Jan-Feb;76(1):M79-87. doi: 10.1111/j.1750-3841.2010.01952.x.
Epub 2011 Jan 6.

Differential gene expression of E. coli O157:H7 in ground beef extract compared
to tryptic soy broth.

Fratamico PM(1), Wang S, Yan X, Zhang W, Li Y.

Author information: 
(1)U.S. Dept. of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, Wyndmoor, PA 19038, USA. pina.fratamico@ars.usda.gov

E. coli O157:H7 is an important foodborne pathogen, and ground beef is a common
vehicle of infection. DNA microarrays have been used for transcriptomic studies
of E. coli O157:H7 using laboratory media; however, analysis of gene expression
in complex matrices such as food are lacking. This study compared gene expression
profiles of E. coli O157:H7 Sakai strain in raw ground beef extract (GBE) and
tryptic soy broth (TSB). Total RNA was isolated from GBE and TSB after 2 h of
incubation with E. coli O157:H7. Following reverse transcription (RT) of the RNA,
labeled cDNA was hybridized to microarrays representing 9608 open reading frames 
(Operon; Genome Array-Ready Oligo Set) corresponding to 4 genomes of E. coli
strains and 3 plasmids. There were 74 up-regulated (genes involved in protein and
polysaccharide biosynthesis, transcription factors, membrane transport proteins, 
and acid shock proteins) and 54 down-regulated (encoding proteins for energy
metabolism, biosynthesis of cofactors, transporters of small molecules, and
transcription factors and enzymes responsible for protein degradation) genes in
E. coli O157:H7 grown in GBE compared to TSB, respectively. Furthermore, compared
to incubation in TSB, E. coli O157:H7 incubated in GBE for 2 h showed
significantly increased survival when exposed to synthetic gastric fluid, pH 1.5.
This study demonstrated that microarray analyses can be performed using complex
food matrices, and gene expression of E. coli O157:H7 differs in TSB compared to 
GBE. The information will be useful for identification of genes that can be
employed as potential targets for interventions to control E. coli O157:H7.

DOI: 10.1111/j.1750-3841.2010.01952.x 
PMID: 21535697  [Indexed for MEDLINE]


474. Methods Enzymol. 2011;500:165-95. doi: 10.1016/B978-0-12-385118-5.00010-4.

Systems biology of recombinant protein production using Bacillus megaterium.

Biedendieck R(1), Borgmeier C, Bunk B, Stammen S, Scherling C, Meinhardt F,
Wittmann C, Jahn D.

Author information: 
(1)Institute of Microbiology, Technische Universität Braunschweig,
Spielmannstrasse 7, Braunschweig, Germany.

The Gram-negative bacterium Escherichia coli is the most widely used production
host for recombinant proteins in both academia and industry. The Gram-positive
bacterium Bacillus megaterium represents an increasingly used alternative for
high yield intra- and extracellular protein synthesis. During the past two
decades, multiple tools including gene expression plasmids and production strains
have been developed. Introduction of free replicating and integrative plasmids
into B. megaterium is possible via protoplasts transformation or
transconjugation. Using His(6)- and StrepII affinity tags, the intra- or
extracellular produced proteins can easily be purified in one-step procedures.
Different gene expression systems based on the xylose controlled promoter P(xylA)
and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B.
megaterium to produce up to 1.25g of recombinant protein per liter. Biomass
concentrations of up to 80g/l can be achieved by high cell density cultivations
in bioreactors. Gene knockouts and gene replacements in B. megaterium are
possible via an optimized gene disruption system. For a safe application in
industry, sporulation and protease-deficient as well as UV-sensitive mutants are 
available. With the help of the recently published B. megaterium genome sequence,
it is possible to characterize bottle necks in the protein production process via
systems biology approaches based on transcriptome, proteome, metabolome, and
fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de)
integrates obtained theoretical and experimental data.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/B978-0-12-385118-5.00010-4 
PMID: 21943898  [Indexed for MEDLINE]


475. Methods Enzymol. 2011;498:449-71. doi: 10.1016/B978-0-12-385120-8.00020-6.

Mapping E. coli RNA polymerase and associated transcription factors and
identifying promoters genome-wide.

Davis SE(1), Mooney RA, Kanin EI, Grass J, Landick R, Ansari AZ.

Author information: 
(1)Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA.

The ability to examine gene regulation in living cells has been greatly enabled
by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP
captures a snapshot of protein-DNA interactions in vivo and has been used to
study interactions in bacteria, yeast, and mammalian cell culture. ChIP
conditions vary depending upon the organism and the nature of the DNA-binding
proteins under study. Here, we describe a customized ChIP protocol to examine the
genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA
Polymerase (RNAP) as well as the factors that dynamically associate with RNAP
during different stages of transcription. We describe new data analysis methods
for determining the association of a broadly distributed DNA-binding complex.
Further, we describe our approach of combining small molecules and antibiotics
that perturb specific cellular events with ChIP and genomic platforms to dissect 
mechanisms of gene regulation in vivo. The chemical genomic methods can be
leveraged to map natural and cryptic promoters and transcription units, annotate 
genomes, and reveal coupling between different processes in regulation of genes. 
This approach provides the framework for engineering gene networks and
controlling biological output in a desired manner.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/B978-0-12-385120-8.00020-6 
PMID: 21601690  [Indexed for MEDLINE]


476. Methods Enzymol. 2011;497:207-20. doi: 10.1016/B978-0-12-385075-1.00009-3.

From SELEX to cell dual selections for synthetic riboswitches.

Sinha J(1), Topp S, Gallivan JP.

Author information: 
(1)Department of Chemistry and Center for Fundamental and Applied Molecular
Evolution, Emory University, Atlanta, Georgia, USA.

Synthetic riboswitches have emerged as useful tools for controlling gene
expression to reprogram cellular behavior. However, advancing beyond
proof-of-principle experiments requires the ability to quickly generate new
synthetic riboswitches from RNA libraries. In this chapter, we provide a
step-by-step overview of the process of obtaining synthetic riboswitches for use 
in Escherichia coli, starting from a randomized RNA library.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/B978-0-12-385075-1.00009-3 
PMID: 21601088  [Indexed for MEDLINE]


477. Methods Enzymol. 2011;497:135-55. doi: 10.1016/B978-0-12-385075-1.00006-8.

Directed evolution of promoters and tandem gene arrays for customizing RNA
synthesis rates and regulation.

Tyo KE(1), Nevoigt E, Stephanopoulos G.

Author information: 
(1)Department of Chemical and Biological Engineering, Northwestern University,
Evanston, Illinois, USA.

Manipulating RNA synthesis rates is a primary method the cell uses to adjust its 
physiological state. Therefore to design synthetic genetic networks and circuits,
precise control of RNA synthesis rates is of the utmost importance. Often,
however, a native promoter does not exist that has the precise characteristics
required for a given application. Here, we describe two methods to change the
rates and regulation of RNA synthesis in cells to create RNA synthesis of a
desired specification. First, error-prone PCR is discussed for diversifying the
properties of native promoters, that is, changing the rate of synthesis in
constitutive promoters and the induction properties for an inducible promoter.
Specifically, we describe techniques for generating diversified promoter
libraries of the constitutive promoters P(L)tetO-1 in Escherichia coli and TEF1
in Saccharomyces cerevisiae as well as the inducible, oxygen-repressed promoter
DAN1 in S. cerevisiae. Beyond generating promoter libraries, we discuss
techniques to quantify the parameters of each new promoter. Promoter
characteristics for each promoter in hand, the designer can then pick and choose 
the promoters needed for the specific genetic circuit described in silico.
Second, Chemically Induced Chromosomal Evolution (CIChE) is presented as an
alternative method to finely adjust RNA synthesis rates in E. coli by variation
of gene cassette copy numbers in tandem gene arrays. Both techniques result in
precisely defined RNA synthesis and should be of great utility in synthetic
biology.

Copyright © 2011 Elsevier Inc. All rights reserved.

DOI: 10.1016/B978-0-12-385075-1.00006-8 
PMID: 21601085  [Indexed for MEDLINE]


478. Methods Mol Biol. 2011;765:125-53. doi: 10.1007/978-1-61779-197-0_9.

Array-based synthetic genetic screens to map bacterial pathways and functional
networks in Escherichia coli.

Babu M(1), Gagarinova A, Greenblatt J, Emili A.

Author information: 
(1)Banting and Best Department of Medical Research, University of Toronto,
Toronto, ON, Canada.

Cellular processes are carried out through a series of molecular interactions.
Various experimental approaches can be used to investigate these functional
relationships on a large-scale. Recently, the power of investigating biological
systems from the perspective of genetic (gene-gene or epistatic) interactions has
been evidenced by the ability to elucidate novel functional relationships.
Examples of functionally related genes include genes that buffer each other's
function or impinge on the same biological process. Genetic interactions have
traditionally been investigated in bacteria by combining pairs of mutations
(e.g., gene deletions) and assessing deviation of the phenotype of each double
mutant from an expected neutral (or no interaction) phenotype. Fitness is a
particularly convenient phenotype to measure: when the double mutant grows faster
or slower than expected, the two mutated genes are said to show alleviating or
aggravating interactions, respectively. The most commonly used neutral model
assumes that the fitness of the double mutant is equal to the product of
individual single mutant fitness. A striking genetic interaction is exemplified
by the loss of two nonessential genes that buffer each other in performing an
essential biological function: deleting only one of these genes produces no
detectable fitness defect; however, loss of both genes simultaneously results in 
systems failure, leading to synthetic sickness or lethality. Systematic
large-scale genetic interaction screens have been used to generate functional
maps for model eukaryotic organisms, such as yeast, to describe the functional
organization of gene products into pathways and protein complexes within a cell. 
They also reveal the modular arrangement and cross talk of pathways and complexes
within broader functional neighborhoods (Dixon et al., Annu Rev Genet 43:601-625,
2009). Here, we present a high-throughput quantitative Escherichia coli Synthetic
Genetic Array (eSGA) screening procedure, which we developed to systematically
infer genetic interactions by scoring growth defects among large numbers of
double mutants in a classic Gram-negative bacterium. The eSGA method exploits the
rapid colony growth, ease of genetic manipulation, and natural efficient genetic 
exchange via conjugation of laboratory E. coli strains. Replica pinning is used
to grow and mate arrayed sets of single gene mutant strains and to select double 
mutants en masse. Strain fitness, which is used as the eSGA readout, is
quantified by the digital imaging of the plates and subsequent measuring and
comparing single and double mutant colony sizes. While eSGA can be used to screen
select mutants to probe the functions of individual genes, using eSGA more
broadly to collect genetic interaction data for many combinations of genes can
help reconstruct a functional interaction network to reveal novel links and
components of biological pathways as well as unexpected connections between
pathways. A variety of bacterial systems can be investigated, wherein the genes
impinge on a essential biological process (e.g., cell wall assembly, ribosome
biogenesis, chromosome replication) that are of interest from the perspective of 
drug development (Babu et al., Mol Biosyst 12:1439-1455, 2009). We also show how 
genetic interactions generated by high-throughput eSGA screens can be validated
by manual small-scale genetic crosses and by genetic complementation and gene
rescue experiments.

DOI: 10.1007/978-1-61779-197-0_9 
PMID: 21815091  [Indexed for MEDLINE]


479. OMICS. 2011 Jan-Feb;15(1-2):15-23. doi: 10.1089/omi.2010.0074. Epub 2010 Sep 23.

Dynamics of time-lagged gene-to-metabolite networks of Escherichia coli
elucidated by integrative omics approach.

Takahashi H(1), Morioka R, Ito R, Oshima T, Altaf-Ul-Amin M, Ogasawara N, Kanaya 
S.

Author information: 
(1)Department of Bioinformatics and Genomics, Graduate School of Information
Science, Nara Institute of Science and Technology, Nara, Japan.

In the postgenomics era, integrative analysis of several "omics" data is
absolutely required for understanding the cell as a system. Integrative analysis 
of transcriptomics and metabolomics can lead to elucidation of gene-to-metabolite
networks. When integrating different time series "omics" data, it is necessary to
take into consideration a time lag between those data. In the present study, we
conducted an integrative analysis of time series transcriptomics and metabolomics
data of Escherichia coli generated by cDNA microarray and Fourier transform ion
cyclotron resonance mass spectrometry (FT-ICR/MS), respectively. We identified a 
60-min time lag between transition points of transcriptomics and metabolomics
data by using a Linear Dynamical System. Furthermore, we investigated
gene-to-metabolite correlations in the context of time lag, obtained the maximum 
number of correlated pairs at transcripts leading 60-min time lag, and finally
revealed gene-to-metabolite relations in the phospholipid biosynthesis pathway.
Taking into consideration the time lag between transcriptomics and metabolomics
data in time series analysis could unravel novel gene-to-metabolite relations.
According to gene-to-metabolite correlations, phosphatidylglycerol plays a more
critical role for membrane balance than phosphatidylethanolamine in E. coli.

DOI: 10.1089/omi.2010.0074 
PMCID: PMC3125544
PMID: 20863252  [Indexed for MEDLINE]


480. PLoS One. 2011;6(12):e28452. doi: 10.1371/journal.pone.0028452. Epub 2011 Dec 2.

Uropathogenic E. coli induce different immune response in testicular and
peritoneal macrophages: implications for testicular immune privilege.

Bhushan S(1), Hossain H, Lu Y, Geisler A, Tchatalbachev S, Mikulski Z, Schuler G,
Klug J, Pilatz A, Wagenlehner F, Chakraborty T, Meinhardt A.

Author information: 
(1)Department of Anatomy and Cell Biology, Unit of Reproductive Biology,
Justus-Liebig-University Giessen, Giessen, Germany.

Infertility affects one in seven couples and ascending bacterial infections of
the male genitourinary tract by Escherichia coli are an important cause of male
factor infertility. Thus understanding mechanisms by which immunocompetent cells 
such as testicular macrophages (TM) respond to infection and how bacterial
pathogens manipulate defense pathways is of importance. Whole genome expression
profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E.
coli (UPEC) revealed major differences in regulated genes. However, a multitude
of genes implicated in calcium signaling pathways was a common feature which
indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) 
signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and
in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of
NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM
(IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity 
delineated to the pore forming toxin alpha-hemolysin by bacterial mutant
analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and 
caused differential activation of MAP kinase and AP-1 signaling pathways in TM
and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM
(IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition,
unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence
of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6,
TNF-α). Taken together, these results suggest a mechanism to the conundrum by
which TM initiate immune responses to bacteria, while maintaining testicular
immune privilege with its ability to tolerate neo-autoantigens expressed on
developing spermatogenic cells.

DOI: 10.1371/journal.pone.0028452 
PMCID: PMC3229579
PMID: 22164293  [Indexed for MEDLINE]


481. PLoS One. 2011;6(11):e26655. doi: 10.1371/journal.pone.0026655. Epub 2011 Nov 22.

Evaluation of high-throughput PCR and microarray-based assay in conjunction with 
automated DNA extraction instruments for diagnosis of sepsis.

Laakso S(1), Kirveskari J, Tissari P, Mäki M.

Author information: 
(1)Mobidiag Ltd, Helsinki, Finland.

BACKGROUND: High incidence of septic patients increases the pressure of faster
and more reliable bacterial identification methods to adapt patient management
towards focused and effective treatment options. The aim of this study was to
assess two automated DNA extraction solutions with the PCR and microarray-based
assay to enable rapid and reliable detection and speciation of causative agents
in the diagnosis of sepsis.
METHODOLOGY/PRINCIPAL FINDINGS: We evaluated two automated DNA instruments
NucliSENS® easyMAG® and NorDiag Arrow for the preparation of blood culture
samples. A set of 91 samples flagged as positive during incubation was analyzed
prospectively with the high-throughput generation of Prove-it™ Sepsis assay
designed to identify over 60 gram-negative and gram-positive bacterial species as
well as methicillin resistance marker from a blood culture. Bacterial findings
were accurately reported from 77 blood culture samples, whereas 14 samples were
reported as negative, containing bacteria not belonging to the pathogen panel of 
the assay. No difference was observed between the performance of NorDiag Arrow or
NucliSENS® easyMAG® with regard to the result reporting of Prove-it™ Sepsis. In
addition, we also assessed the quality and quantity of DNA extracted from the
clinical Escherichia coli isolate with DNA extraction instruments. We observed
only minor differences between the two instruments.
CONCLUSIONS: Use of automated and standardized sample preparation methods
together with rapid, multiplex pathogen detection offers a strategy to speed up
reliably the diagnostics of septic patients. Both tested DNA extraction devices
were shown to be feasible for blood culture samples and the Prove-it™ Sepsis
assay, providing an accurate identification of pathogen within 4.5 hours when the
detected pathogen was in the repertoire of the test.

DOI: 10.1371/journal.pone.0026655 
PMCID: PMC3222647
PMID: 22132076  [Indexed for MEDLINE]


482. PLoS One. 2011;6(8):e21969. doi: 10.1371/journal.pone.0021969. Epub 2011 Aug 12.

Inferring a transcriptional regulatory network from gene expression data using
nonlinear manifold embedding.

Zare H(1), Kaveh M, Khodursky A.

Author information: 
(1)National Institutes of Health, Bethesda, Maryland, United States of America.

Transcriptional networks consist of multiple regulatory layers corresponding to
the activity of global regulators, specialized repressors and activators as well 
as proteins and enzymes shaping the DNA template. Such intrinsic complexity makes
uncovering connections difficult and it calls for corresponding methodologies,
which are adapted to the available data. Here we present a new computational
method that predicts interactions between transcription factors and target genes 
using compendia of microarray gene expression data and documented interactions
between genes and transcription factors. The proposed method, called Kernel
Embedding of Regulatory Networks (KEREN), is based on the concept of gene-regulon
association, and captures hidden geometric patterns of the network via manifold
embedding. We applied KEREN to reconstruct transcription regulatory interactions 
on a genome-wide scale in the model bacteria Escherichia coli (E. coli).
Application of the method not only yielded accurate predictions of verifiable
interactions, which outperformed on certain metrics comparable methodologies, but
also demonstrated the utility of a geometric approach in the analysis of
high-dimensional biological data. We also described possible applications of
kernel embedding techniques to other function and network discovery algorithms.

DOI: 10.1371/journal.pone.0021969 
PMCID: PMC3155518
PMID: 21857910  [Indexed for MEDLINE]


483. PLoS One. 2011;6(7):e20938. doi: 10.1371/journal.pone.0020938. Epub 2011 Jul 14.

COLOMBOS: access port for cross-platform bacterial expression compendia.

Engelen K(1), Fu Q, Meysman P, Sánchez-Rodríguez A, De Smet R, Lemmens K, Fierro 
AC, Marchal K.

Author information: 
(1)Department of Microbial and Molecular Systems, Katholieke Universiteit Leuven,
Heverlee-Leuven, Belgium. kristof.engelen@biw.kuleuven.be

BACKGROUND: Microarrays are the main technology for large-scale transcriptional
gene expression profiling, but the large bodies of data available in public
databases are not useful due to the large heterogeneity. There are several
initiatives that attempt to bundle these data into expression compendia, but such
resources for bacterial organisms are scarce and limited to integration of
experiments from the same platform or to indirect integration of per experiment
analysis results.
METHODOLOGY/PRINCIPAL FINDINGS: We have constructed comprehensive
organism-specific cross-platform expression compendia for three bacterial model
organisms (Escherichia coli, Bacillus subtilis, and Salmonella enterica serovar
Typhimurium) together with an access portal, dubbed COLOMBOS, that not only
provides easy access to the compendia, but also includes a suite of tools for
exploring, analyzing, and visualizing the data within these compendia. It is
freely available at http://bioi.biw.kuleuven.be/colombos. The compendia are
unique in directly combining expression information from different microarray
platforms and experiments, and we illustrate the potential benefits of this
direct integration with a case study: extending the known regulon of the Fur
transcription factor of E. coli. The compendia also incorporate extensive
annotations for both genes and experimental conditions; these heterogeneous data 
are functionally integrated in the COLOMBOS analysis tools to interactively
browse and query the compendia not only for specific genes or experiments, but
also metabolic pathways, transcriptional regulation mechanisms, experimental
conditions, biological processes, etc.
CONCLUSIONS/SIGNIFICANCE: We have created cross-platform expression compendia for
several bacterial organisms and developed a complementary access port COLOMBOS,
that also serves as a convenient expression analysis tool to extract useful
biological information. This work is relevant to a large community of
microbiologists by facilitating the use of publicly available microarray
experiments to support their research.

DOI: 10.1371/journal.pone.0020938 
PMCID: PMC3136457
PMID: 21779320  [Indexed for MEDLINE]


484. PLoS One. 2011;6(6):e20081. doi: 10.1371/journal.pone.0020081. Epub 2011 Jun 1.

Novel roles of cAMP receptor protein (CRP) in regulation of transport and
metabolism of carbon sources.

Shimada T(1), Fujita N, Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

CRP (cAMP receptor protein), the global regulator of genes for carbon source
utilization in the absence of glucose, is the best-studied prokaryotic
transcription factor. A total of 195 target promoters on the Escherichia coli
genome have been proposed to be under the control of cAMP-bound CRP. Using the
newly developed Genomic SELEX screening system of transcription factor-binding
sequences, however, we have identified a total of at least 254 CRP-binding sites.
Based on their location on the E. coli genome, we predict a total of at least 183
novel regulation target operons, altogether with the 195 hitherto known targets, 
reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP.
All the promoters selected from the newly identified targets and examined by
using the lacZ reporter assay were found to be under the control of CRP,
indicating that the Genomic SELEX screening allowed to identify the CRP targets
with high accuracy. Based on the functions of novel target genes, we conclude
that CRP plays a key regulatory role in the whole processes from the selective
transport of carbon sources, the glycolysis-gluconeogenesis switching to the
metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle,
pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique
regulation mode is that a single and the same CRP molecule bound within
intergenic regions often regulates both of divergently transcribed operons.

DOI: 10.1371/journal.pone.0020081 
PMCID: PMC3105977
PMID: 21673794  [Indexed for MEDLINE]


485. Vet Immunol Immunopathol. 2011 Jan;139(1):67-72. doi:
10.1016/j.vetimm.2010.08.001. Epub 2010 Aug 10.

In vitro identification and verification of inflammatory biomarkers in swine.

Peters SM(1), Yancy H, Bremer E, Monroe J, Paul D, Stubbs JT 3rd, Myers MJ.

Author information: 
(1)US FDA/CVM, Laurel, MD 20708, USA.

Currently there are no non-steroidal anti-inflammatory drugs (NSAIDs) approved
for the control of inflammation in swine due to a lack of validated animal models
and suitable biomarkers to assess drug efficacy. This study investigates the
differential expression of genes altered in response to Escherichia coli
lipopolysaccharide (LPS) induced inflammation which may serve as indicators of
NSAID efficacy. Unstimulated whole blood from swine was mixed with tissue culture
media, stimulated with LPS, and RNA extracted at the following time points 0h,
1h, 3h, 24h and 48h. Total RNA was extracted and analyzed using a commercial
swine DNA microarray. The DNA microarray was utilized as a screen to determine
potential biomarkers, focusing on the genes that exhibited the greatest degree of
differential expression. A master list of 57 genes was formed based on the
differential expression as a result of the stimulation. Following analysis, 12
genes whose expressions were significantly altered (8 up- and 4 down-regulated)
were chosen for verification via quantitative RT-PCR (qRT-PCR). The qRT-PCR
analysis confirmed the differential expression of 11 of the 12 genes chosen via
the microarray analyses. Specifically, traditional genes such as SAA, G-CSF, and 
IL-10 were up-regulated, while CD4 was down-regulated; all of the genes were
altered by 24h or 48h post-stimulation. We demonstrate here that expression of
these 11 genes is altered as a direct result of LPS stimulation and consequently 
inflammation.

Published by Elsevier B.V.

DOI: 10.1016/j.vetimm.2010.08.001 
PMID: 20828834  [Indexed for MEDLINE]


486. Yi Chuan. 2011 Jan;33(1):60-6.

[cDNA microarray on differently expressed genes in duodenum in porcine sensitive 
or resistant to Escherichia coli F18].

[Article in Chinese]

Bao WB(1), Ye L, Pan ZY, Zhu J, DU ZD, Cai JJ, Huang XG, Zhu GQ, Wu SL.

Author information: 
(1)Animal Science and Technology College, Yangzhou University, Yangzhou 225009,
China. wbbao@yzu.edu.cn

Based on the paired full-sib individuals selected from the established resource
populations of Sutai pig that were characterized as resistant or sensitive to
ETEC F18, Agilent double labeled cDNA microarray was used to identify the gene
expression profiles in duodenum on purpose of investigating the genes related to 
Escherichia coli F18 receptor, which may cause edema disease and post-weaning
diarrhea in piglets, as well as exploring the molecular mechanism about the
differences involved in two different lineages. The results showed that thirteen 
differently expressed genes were found in one matched group including sensitive
ones with GG genotype comparing with resistant ones with AA genotype at a
two-fold filter, where there were 6 up-regulated genes and 7 down-regulated
genes. In the other matched group composed of sensitive ones with AG genotype, 4 
up-regulated genes and 2 down-regulated genes, 6 in total were screened out. GO
analy-sis revealed that the differently expressed genes participated in many
biological processes, such as immune response, ex-tracellular region, bacterial
binding, response to external stimulus and so on. Meanwhile, these genes were
mainly related to the Glycan Biosynthesis and Metabolism and Immune System
pathways. Actually, the roles that they may play in edema disease and
post-weaning diarrhea need further study and verification.


PMID: 21377960  [Indexed for MEDLINE]


487. N Engl J Med. 2010 Dec 30;363(27):2628-37. doi: 10.1056/NEJMoa1006565.

Homozygous BUB1B mutation and susceptibility to gastrointestinal neoplasia.

Rio Frio T(1), Lavoie J, Hamel N, Geyer FC, Kushner YB, Novak DJ, Wark L, Capelli
C, Reis-Filho JS, Mai S, Pastinen T, Tischkowitz MD, Marcus VA, Foulkes WD.

Author information: 
(1)Program in Cancer Genetics, Department of Oncology, McGill University,
Montreal, QC, Canada.

Comment in
    N Engl J Med. 2010 Dec 30;363(27):2665-6.
    N Engl J Med. 2011 Mar 31;364(13):1279-80.

A patient received a diagnosis of adenocarcinoma of the ampulla of Vater at 34
years of age. Two decades later, adenomatous polyps were found, followed by
multiple primary invasive adenocarcinomas of both the colon and the stomach.
Premature chromatid separation and mosaic variegated aneuploidy, combined with
structural chromosomal abnormalities, were detected in his cells. We identified a
germline homozygous intronic mutation, c.2386-11A→G, in the spindle-assembly
checkpoint gene BUB1B, which creates a de novo splice site that is favored over
the authentic (i.e., preferentially used) site. Our findings expand the phenotype
associated with BUB1B mutations and the mosaic variegated aneuploidy syndrome to 
include common adult-onset cancers and provide evidence for the interdependency
of the APC protein (encoded by the adenomatous polyposis coli gene) and the BUBR1
protein (encoded by BUB1B) in humans. (Funded by the Turner Family Cancer
Research Fund and others.).

DOI: 10.1056/NEJMoa1006565 
PMID: 21190457  [Indexed for MEDLINE]


488. PLoS One. 2010 Dec 23;5(12):e15356. doi: 10.1371/journal.pone.0015356.

Tiling array analysis of UV treated Escherichia coli predicts novel
differentially expressed small peptides.

Thomassen GO(1), Weel-Sneve R, Rowe AD, Booth JA, Lindvall JM, Lagesen K,
Kristiansen KI, Bjørås M, Rognes T.

Author information: 
(1)Centre for Molecular Biology and Neuroscience (CMBN) and Department of
Microbiology, Rikshospitalet, Oslo University Hospital, Oslo, Norway.

BACKGROUND: Despite comprehensive investigation, the Escherichia coli SOS
response system is not yet fully understood. We have applied custom designed
whole genome tiling arrays to measure UV invoked transcriptional changes in E.
coli. This study provides a more complete insight into the transcriptome and the 
UV irradiation response of this microorganism.
RESULTS: We detected a number of novel differentially expressed transcripts in
addition to the expected SOS response genes (such as sulA, recN, uvrA, lexA, umuC
and umuD) in the UV treated cells. Several of the differentially expressed
transcripts might play important roles in regulation of the cellular response to 
UV damage. We have predicted 23 novel small peptides from our set of detected
non-gene transcripts. Further, three of the predicted peptides were cloned into
protein expression vectors to test the biological activity. All three constructs 
expressed the predicted peptides, in which two of them were highly toxic to the
cell. Additionally, a remarkably high overlap with previously in-silico predicted
non-coding RNAs (ncRNAs) was detected. Generally we detected a far higher
transcriptional activity than the annotation suggests, and these findings
correspond with previous transcription mappings from E. coli and other organisms.
CONCLUSIONS: Here we demonstrate that the E. coli transcriptome consists of far
more transcripts than the present annotation suggests, of which many transcripts 
seem important to the bacterial stress response. Sequence alignment of promoter
regions suggest novel regulatory consensus sequences for some of the upregulated 
genes. Finally, several of the novel transcripts identified in this study encode 
putative small peptides, which are biologically active.

DOI: 10.1371/journal.pone.0015356 
PMCID: PMC3009722
PMID: 21203457  [Indexed for MEDLINE]


489. Infect Immun. 2010 Dec;78(12):4990-8. doi: 10.1128/IAI.00531-10. Epub 2010 Sep
20.

The type VI secretion system plays a role in type 1 fimbria expression and
pathogenesis of an avian pathogenic Escherichia coli strain.

de Pace F(1), Nakazato G, Pacheco A, de Paiva JB, Sperandio V, da Silveira WD.

Author information: 
(1)Department of Genetics, Evolution, and Bioagents, Campinas State University
(UNICAMP), Campinas, Sao Paulo, Brazil.

Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal
infections and are responsible for significant economic losses in the poultry
industry worldwide. APEC isolates are closely related to human extraintestinal
pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans.
Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, 
iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, 
isolated from a septicemic hen, expresses a type VI secretion system (T6SS);
causes cytoskeleton rearrangements; and invades epithelial cells, replicates
within macrophages, and causes lethal disease in chicks. To assess the
contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants, hcp
(which encodes a protein suggested to be both secreted and a structural component
of the T6SS) and clpV (encoding the T6SS ATPase). Both mutants showed decreased
adherence and actin rearrangement on epithelial cells. However, only the hcp
mutant presented a mild decrease in its ability to invade epithelial cells, and
none of these mutants were defective for intramacrophage replication.
Transcriptome studies showed that the level of expression of type 1 fimbriae was 
decreased in these mutants, which may account for the diminished adhesion and
invasion of epithelial cells. The T6SS seems to be important for the disease
process, given that both mutants were attenuated for infection in chicks. These
results suggest that the T6SS influences the expression of type 1 fimbriae and
contributes to APEC pathogenesis.

DOI: 10.1128/IAI.00531-10 
PMCID: PMC2981326
PMID: 20855516  [Indexed for MEDLINE]


490. Inflamm Bowel Dis. 2010 Dec;16(12):2034-42. doi: 10.1002/ibd.21319.

Dysbiosis of fecal microbiota in Crohn's disease patients as revealed by a custom
phylogenetic microarray.

Kang S(1), Denman SE, Morrison M, Yu Z, Dore J, Leclerc M, McSweeney CS.

Author information: 
(1)Preventative Health National Research Flagship, CSIRO, St. Lucia, Qld,
Australia. kansbio@gmail.com

BACKGROUND: A custom phylogenetic microarray composed of small subunit ribosomal 
RNA probes, representing ≈500 bacterial species from the human and animal gut,
was developed and evaluated for analysis of gut microbial diversity using fecal
samples from healthy subjects and Crohn's disease (CD) patients.
METHODS: Oligonucleotide probes (≈40 mer) used on the microarray were selected
from published articles or designed with the "GoArray" microarray probe design
program using selected bacterial 16S rRNA sequences. Fecal 16S rDNA from
individual samples of six healthy subjects and six CD patients were used as
template to generate fluorescently labeled cRNA that was hybridized to the
microarray. Differences revealed by the microarray in relative abundance of
microbial populations between healthy and diseased patients were verified using
quantitative real-time polymerase chain reaction (PCR) with species-specific
primer sets.
RESULTS: The microarray analyses showed that Eubacterium rectale, Bacteroides
fragilis group, B. vulgatus, Ruminococcus albus, R. callidus, R. bromii, and
Faecalibacterium prausnitzii were 5-10-fold more abundant in the healthy subjects
than in the CD patients, while Enterococcus sp., Clostridium difficile,
Escherichia coli, Shigella flexneri, and Listeria sp. were more abundant in the
CD group.
CONCLUSIONS: The microarray detected differences in abundance of bacterial
populations within the phylum Firmicutes that had been reported previously for
the same samples based on phylogenetic analysis of metagenomic clone libraries.
In addition, the microarray showed that Enterococcus sp. was in higher abundance 
in the CD patients. This microarray should be another useful tool to examine the 
diversity and abundance of human intestinal microbiota.

Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

DOI: 10.1002/ibd.21319 
PMID: 20848492  [Indexed for MEDLINE]


491. J Appl Microbiol. 2010 Dec;109(6):2118-27. doi: 10.1111/j.1365-2672.2010.04843.x.
Epub 2010 Sep 16.

Targeted microarray analysis of stationary phase Escherichia coli O157:H7
subjected to disparate nutrient conditions.

Allen KJ(1), Lepp D, McKellar RC, Griffiths MW.

Author information: 
(1)Food, Nutrition and Health, University of British Columbia, Vancouver, BC,
Canada. kevin.allen@ubc.ca

AIMS: To determine how stress response and virulence gene expression of
stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels.
METHODS AND RESULTS: A targeted microarray (n=125 genes) was used to determine
the impact of nutrient deprivation [15 min in 3-(N-Morpholino)propanesulfonic
acid buffer] on SP E. coli O157:H7. In total, 24 genes were significantly
affected (>1·5-fold; P <0·05) with 17 induced and seven attenuated. Additionally,
11 genes belonging to significantly affected stress response regulons were
significantly induced (P<0·05), though <1·5-fold. Induced genes included global
and specific stress response regulators, the mar operon, iron acquisition and
virulence genes. In contrast, transcript for major porins and replicative genes
were repressed. Comparison of the nutrient deprived transcriptome to that derived
from nutrient replenished cells revealed a disparate transcriptome, with 44 genes
expressed at significantly elevated levels in nutrient replenished cells,
including all queried global and specific stress response regulators and key
virulence genes. Genes expressed at elevated levels in nutrient deprived cells
were related to σ(S) . The microarray data were validated by qRT-PCR.
CONCLUSIONS:   SP E. coli O157:H7 were affected by nutrient deprivation, with
both starvation-related and unrelated networks induced, thereby demonstrating how
the E. coli O157:H7 stress response transcriptome is fine-tuned to environmental 
conditions. Further, by comparison of starved cells to cells provided with fresh 
nutrients, it is clear starved E. coli O157:H7 undergo massive physiological
reprogramming dominated initially by stress response induction to adapt to a
nutrient rich environment.
SIGNIFICANCE AND IMPACT OF THE STUDY:   This study demonstrated how σ(S) -induced
SP E. coli O157:H7 remain highly sensitive and adaptable to environmental
conditions. Further, by examining how starved cells respond to nutrient-rich
conditions, we show preliminary adaptation to a nutrient rich environment is
dominated by the induction of diverse stress response networks. Combined, this
provides E. coli O157:H7 stress physiology-based knowledge that can be used to
design more effective food safety interventions.

© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for
Applied Microbiology.

DOI: 10.1111/j.1365-2672.2010.04843.x 
PMID: 20846334  [Indexed for MEDLINE]


492. Mol Cell Probes. 2010 Dec;24(6):325-45. doi: 10.1016/j.mcp.2010.07.004. Epub 2010
Aug 3.

Comparison of phenotypic and genotypic antimicrobial profiles in Escherichia coli
and Salmonella enterica from the same dairy cattle farms.

Scaria J(1), Warnick LD, Kaneene JB, May K, Teng CH, Chang YF.

Author information: 
(1)Department of Population Medicine and Diagnostic Sciences, College of
Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Transmission of antimicrobial drug resistance from resistant bacteria to
non-resistant strains is an important public health issue. In this study, we have
examined the possibility of multiple resistance gene transfer between Escherichia
coli and Salmonella in the natural setting. Bacteria isolated from calves
concurrently shedding E. coli and Salmonella showed similar antimicrobial drug
resistance patterns as measured by a broth dilution method. However, microarray
analysis of the antibiotic resistance at the gene level revealed several
differences in resistance gene profile. Resistance profiles of E. coli isolated
from different farms were closer than the profile of E. coli and Salmonella
isolated from the same farm. This shows that the chance of multiple resistance
gene transfers between these species is unlikely.

Copyright © 2010 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.mcp.2010.07.004 
PMCID: PMC2967584
PMID: 20688154  [Indexed for MEDLINE]


493. Mol Genet Genomics. 2010 Dec;284(6):437-54. doi: 10.1007/s00438-010-0578-8. Epub 
2010 Oct 1.

Functional genomics of probiotic Escherichia coli Nissle 1917 and 83972, and UPEC
strain CFT073: comparison of transcriptomes, growth and biofilm formation.

Hancock V(1), Vejborg RM, Klemm P.

Author information: 
(1)Microbial Adhesion Group, DTU Food, Bldg. 204, Technical University of
Denmark, 2800 Lyngby, Denmark. vhan@food.dtu.dk

Strain CFT073 is a bona fide uropathogen, whereas strains 83972 and Nissle 1917
are harmless probiotic strains of urinary tract and faecal origin, respectively. 
Despite their different environmental origins and dispositions the three strains 
are very closely related and the ancestors of 83972 and Nissle 1917 must have
been very similar to CFT073. Here, we report the first functional genome
profiling of Nissle 1917 and the first biofilm profiling of a uropathogen.
Transcriptomic profiling revealed that Nissle 1917 expressed many UPEC-associated
genes and showed that the active genomic profiles of the three strains are
closely related. The data demonstrate that the distance from a pathogen to a
probiotic strain can be surprisingly short. We demonstrate that Nissle 1917, in
spite of its intestinal niche origin, grows well in urine, and is a good biofilm 
former in this medium in which it also out-competes CFT073 during planktonic
growth. The role in biofilm formation of three up-regulated genes, yhaK, yhcN and
ybiJ, was confirmed by knockout mutants in Nissle 1917 and CFT073. Two of these
mutants CFT073∆yhcN and CFT073∆ybiJ had significantly reduced motility compared
with the parent strain, arguably accounting for the impaired biofilm formation.
Although the three strains have very different strategies vis-à-vis the human
host their functional gene profiles are surprisingly similar. It is also
interesting to note that the only two Escherichia coli strains used as probiotics
are in fact deconstructed pathogens.

DOI: 10.1007/s00438-010-0578-8 
PMID: 20886356  [Indexed for MEDLINE]


494. BMC Genomics. 2010 Nov 25;11:666. doi: 10.1186/1471-2164-11-666.

Structure and dynamics of the operon map of Buchnera aphidicola sp. strain APS.

Brinza L(1), Calevro F, Duport G, Gaget K, Gautier C, Charles H.

Author information: 
(1)INSA-Lyon, UMR203 BF2I, INRA, Biologie Fonctionnelle Insectes et Interactions,
Bât, Louis Pasteur 20 ave, Albert Einstein, F-69621 Villeurbanne, France.
lilia.brinza@insa-lyon.fr

BACKGROUND: Gene expression regulation is still poorly documented in bacteria
with highly reduced genomes. Understanding the evolution and mechanisms
underlying the regulation of gene transcription in Buchnera aphidicola, the
primary endosymbiont of aphids, is expected both to enhance our understanding of 
this nutritionally based association and to provide an intriguing case-study of
the evolution of gene expression regulation in a reduced bacterial genome.
RESULTS: A Bayesian predictor was defined to infer the B. aphidicola
transcription units, which were further validated using transcriptomic data and
RT-PCR experiments. The characteristics of B. aphidicola predicted transcription 
units (TUs) were analyzed in order to evaluate the impact of operon map
organization on the regulation of gene transcription.On average, B. aphidicola
TUs contain more genes than those of E. coli. The global layout of B. aphidicola 
operon map was mainly shaped by the big reduction and the rearrangements events, 
which occurred at the early stage of the symbiosis. Our analysis suggests that
this operon map may evolve further only by small reorganizations around the
frontiers of B. aphidicola TUs, through promoter and/or terminator sequence
modifications and/or by pseudogenization events. We also found that the need for 
specific transcription regulation exerts some pressure on gene conservation, but 
not on gene assembling in the operon map in Buchnera. Our analysis of the TUs
spacing pointed out that a selection pressure is maintained on the length of the 
intergenic regions between divergent adjacent gene pairs.
CONCLUSIONS: B. aphidicola can seemingly only evolve towards a more polycistronic
operon map. This implies that gene transcription regulation is probably subject
to weak selection pressure in Buchnera conserving operons composed of genes with 
unrelated functions.

DOI: 10.1186/1471-2164-11-666 
PMCID: PMC3091783
PMID: 21108805  [Indexed for MEDLINE]


495. ACS Chem Biol. 2010 Nov 19;5(11):1065-74. doi: 10.1021/cb1001894.

Combining SELEX screening and rational design to develop light-up fluorophore-RNA
aptamer pairs for RNA tagging.

Lee J(1), Lee KH, Jeon J, Dragulescu-Andrasi A, Xiao F, Rao J.

Author information: 
(1)Department of Chemistry, Stanford University School of Medicine, 1210 Welch
Road, Stanford, California 94305-5484, USA.

We report here a new small molecule fluorogen and RNA aptamer pair for RNA
labeling. The small-molecule fluorogen is designed on the basis of fluorescently 
quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by
EXponential enrichment) procedure and fluorescence screening in E. coli have been
applied to discover the aptamer that can specifically activate the fluorogen with
micromolar binding affinity. The systematic mutation and truncation study on the 
aptamer structure determined the minimum binding domain of the aptamer. A series 
of rationally modified fluorogen analogues have been made to probe the
interacting groups of fluorogen with the aptamer. These results led to the design
of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the
binding affinity for the selected aptamer in comparison to the original ASR 1 and
an 88-fold increase in the fluorescence emission after the aptamer binding. This 
study demonstrates the value of combining in vitro SELEX and E. coli fluorescence
screening with rational modifications in discovering and optimizing new
fluorogen-RNA aptamer labeling pairs.

DOI: 10.1021/cb1001894 
PMCID: PMC3044212
PMID: 20809562  [Indexed for MEDLINE]


496. PLoS Pathog. 2010 Nov 11;6(11):e1001187. doi: 10.1371/journal.ppat.1001187.

Escherichia coli global gene expression in urine from women with urinary tract
infection.

Hagan EC(1), Lloyd AL, Rasko DA, Faerber GJ, Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, Michigan, United States of America.

Murine models of urinary tract infection (UTI) have provided substantial data
identifying uropathogenic E. coli (UPEC) virulence factors and assessing their
expression in vivo. However, it is unclear how gene expression in these animal
models compares to UPEC gene expression during UTI in humans. To address this, we
used a UPEC strain CFT073-specific microarray to measure global gene expression
in eight E. coli isolates monitored directly from the urine of eight women
presenting at a clinic with bacteriuria. The resulting gene expression profiles
were compared to those of the same E. coli isolates cultured statically to
exponential phase in pooled, sterilized human urine ex vivo. Known fitness
factors, including iron acquisition and peptide transport systems, were highly
expressed during human UTI and support a model in which UPEC replicates rapidly
in vivo. While these findings were often consistent with previous data obtained
from the murine UTI model, host-specific differences were observed. Most
strikingly, expression of type 1 fimbrial genes, which are among the most highly 
expressed genes during murine experimental UTI and encode an essential virulence 
factor for this experimental model, was undetectable in six of the eight E. coli 
strains from women with UTI. Despite the lack of type 1 fimbrial expression in
the urine samples, these E. coli isolates were generally capable of expressing
type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine
infection. The findings presented here provide insight into the metabolic and
pathogenic profile of UPEC in urine from women with UTI and represent the first
transcriptome analysis for any pathogenic E. coli during a naturally occurring
infection in humans.

DOI: 10.1371/journal.ppat.1001187 
PMCID: PMC2978726
PMID: 21085611  [Indexed for MEDLINE]


497. PLoS One. 2010 Nov 3;5(11):e13827. doi: 10.1371/journal.pone.0013827.

Mice lacking Alkbh1 display sex-ratio distortion and unilateral eye defects.

Nordstrand LM(1), Svärd J, Larsen E, Nilsen A, Ougland R, Furu K, Lien GF, Rognes
T, Namekawa SH, Lee JT, Klungland A.

Author information: 
(1)Centre for Molecular Biology and Neuroscience, Institute of Medical
Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.

BACKGROUND: Escherichia coli AlkB is a 2-oxoglutarate- and iron-dependent
dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse 
AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of
mammalian dioxygenases.
METHODS AND FINDINGS: In the present study we show non-Mendelian inheritance of
the Alkbh1 targeted allele in mice. Both Alkbh1(-/-) and heterozygous Alkbh1(+/-)
offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is
considerably skewed against female offspring, with one female born for every
three to four males. Most mechanisms that cause segregation distortion, act in
the male gametes and affect male fertility. The skewing of the sexes appears to
be of paternal origin, and might be set in the pachythene stage of meiosis during
spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes,
apoptotic spermatids were revealed in 5-10% of the tubules in Alkbh1(-/-) adults.
The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5
during embryonic development. This is consistent with the incompletely penetrant 
phenotypes observed, particularly recurrent unilateral eye defects and
craniofacial malformations.
CONCLUSIONS: Genetic and phenotypic assessment suggests that Alkbh1 mediates gene
regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio 
distribution and embryonic development in mice.

DOI: 10.1371/journal.pone.0013827 
PMCID: PMC2972218
PMID: 21072209  [Indexed for MEDLINE]


498. Anal Chem. 2010 Nov 1;82(21):9028-33. doi: 10.1021/ac102002k. Epub 2010 Oct 7.

Label-free voltammetric detection using individually addressable oligonucleotide 
microelectrode arrays.

Kalantari R(1), Cantor R, Chen H, Yu G, Janata J, Josowicz M.

Author information: 
(1)School of Chemistry and Biochemistry, Georgia Institute of Technology,
Atlanta, Georgia 30332, United States.

The utility and performance of label-free, oligonucleotide probes for reagentless
detection of dilute target analytes was examined using a voltammetric
transduction principle in an array format. Multistep, solid-state fabrication
yielded preproduction arrays of 16 individually addressable, 30 μm diameter
microelectrodes in a 30 mm × 6.5 mm × 0.5 mm dipstick disposable device. The
specificity of 16 nucleotide (nt) 2'-O-methylribonucleic acid and 22 nt DNA
backbone probes bound through Mg(2+)-phosphonate bridges to polypyrrole films on 
the microelectrodes were studied using microbial target RNAs of various lengths. 
Probe-specific interactions with Escherichia coli O157 H7 23S rRNA (2907 nt) and 
Candida albicans 18S rRNA (1788 nt) were detected at 65 and 58 fmol/mL,
respectively, in volumes as low as 0.5 mL. Specificity studies showed that, for a
given probe, "nontarget" transcripts can contribute to changes in the
voltammetric detection signal, though with responses that never exceed 70% of the
detection signal acquired for specifically designed complementary targets. These 
results statistically validate the use of the voltammetric microelectrode array
for obtaining a "yes-no" answer on complementary specific binding. The study also
identifies challenges and pitfalls for the selection strategies of
oligonucleotide probes.

DOI: 10.1021/ac102002k 
PMID: 20929247  [Indexed for MEDLINE]


499. Infect Immun. 2010 Nov;78(11):4697-704. doi: 10.1128/IAI.00512-10. Epub 2010 Sep 
7.

OI-57, a genomic island of Escherichia coli O157, is present in other
seropathotypes of Shiga toxin-producing E. coli associated with severe human
disease.

Imamovic L(1), Tozzoli R, Michelacci V, Minelli F, Marziano ML, Caprioli A,
Morabito S.

Author information: 
(1)European Union Reference Laboratory for Escherichia coli, Dipartimento di
Sanità Pubblica Veterinaria e Sicurezza Alimentare, Rome, Italy.

Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E.
coli group that may cause severe disease in humans. STEC have been categorized
into seropathotypes (SPTs) based on their phenotypic and molecular
characteristics and the clinical features of the associated diseases. SPTs range 
from A to E, according to a decreasing rank of pathogenicity. To define the
virulence gene asset ("virulome") characterizing the highly pathogenic SPTs, we
used microarray hybridization to compare the whole genomes of STEC belonging to
SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading
frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR
in a larger panel of STEC and in other E. coli strains. A genomic island termed
OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the
putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC 
O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR
analyses showed that OI-57 was present in its entirety in the majority of the
STEC genomes examined, indicating that it represents a stable acquisition of the 
positive clonal lineages. OI-57 was also present in a high proportion of the
human enteropathogenic E. coli genomes assayed, suggesting that it could be
involved in the attaching-and-effacing colonization of the intestinal mucosa. In 
conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and
further studies are needed to elucidate its role in the pathogenesis of STEC
infections.

DOI: 10.1128/IAI.00512-10 
PMCID: PMC2976316
PMID: 20823207  [Indexed for MEDLINE]


500. Infect Immun. 2010 Nov;78(11):4779-91. doi: 10.1128/IAI.00678-10. Epub 2010 Aug
30.

Characterization of the CpxRA regulon in Haemophilus ducreyi.

Labandeira-Rey M(1), Brautigam CA, Hansen EJ.

Author information: 
(1)Department of Microbiology, University of Texas Southwestern Medical Center,
Dallas, TX 75390-9048, USA.

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA
two-component cell envelope stress response system originally characterized in
Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously 
to be involved in repression of the expression of the lspB-lspA2 operon (M.
Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009).
In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely
resemble those of other well-studied bacterial species. A cpxA deletion mutant
and a CpxR-overexpressing strain were used to explore the extent of the CpxRA
regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses
indicated several potential regulatory targets for the H. ducreyi CpxRA
two-component regulatory system. Electrophoretic mobility shift assays (EMSAs)
were used to prove that H. ducreyi CpxR interacted with the promoter regions of
genes encoding both known and putative virulence factors of H. ducreyi, including
the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs 
also indicated that H. ducreyi CpxR did not bind to the promoter regions of
several genes predicted to encode factors involved in the cell envelope stress
response. Taken together, these data suggest that the CpxRA system in H. ducreyi,
in contrast to that in E. coli, may be involved primarily in controlling
expression of genes not involved in the cell envelope stress response.

DOI: 10.1128/IAI.00678-10 
PMCID: PMC2976327
PMID: 20805330  [Indexed for MEDLINE]


501. BMC Genomics. 2010 Oct 21;11:591. doi: 10.1186/1471-2164-11-591.

High-throughput microarray technology in diagnostics of enterobacteria based on
genome-wide probe selection and regression analysis.

Friedrich T(1), Rahmann S, Weigel W, Rabsch W, Fruth A, Ron E, Gunzer F, Dandekar
T, Hacker J, Müller T, Dobrindt U.

Author information: 
(1)University of Würzburg, Institute for Molecular Infection Biology, Würzburg,
Germany.

BACKGROUND: The Enterobacteriaceae comprise a large number of clinically relevant
species with several individual subspecies. Overlapping virulence-associated gene
pools and the high overall genome plasticity often interferes with correct
enterobacterial strain typing and risk assessment. Array technology offers a
fast, reproducible and standardisable means for bacterial typing and thus
provides many advantages for bacterial diagnostics, risk assessment and
surveillance. The development of highly discriminative broad-range microbial
diagnostic microarrays remains a challenge, because of marked genome plasticity
of many bacterial pathogens.
RESULTS: We developed a DNA microarray for strain typing and detection of major
antimicrobial resistance genes of clinically relevant enterobacteria. For this
purpose, we applied a global genome-wide probe selection strategy on 32 available
complete enterobacterial genomes combined with a regression model for pathogen
classification. The discriminative power of the probe set was further tested in
silico on 15 additional complete enterobacterial genome sequences. DNA
microarrays based on the selected probes were used to type 92 clinical
enterobacterial isolates. Phenotypic tests confirmed the array-based typing
results and corroborate that the selected probes allowed correct typing and
prediction of major antibiotic resistances of clinically relevant
Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction
of different E. coli pathotypes.
CONCLUSIONS: Our results demonstrate that the global probe selection approach
based on longest common factor statistics as well as the design of a DNA
microarray with a restricted set of discriminative probes enables robust
discrimination of different enterobacterial variants and represents a proof of
concept that can be adopted for diagnostics of a wide range of microbial
pathogens. Our approach circumvents misclassifications arising from the
application of virulence markers, which are highly affected by horizontal gene
transfer. Moreover, a broad range of pathogens have been covered by an efficient 
probe set size enabling the design of high-throughput diagnostics.

DOI: 10.1186/1471-2164-11-591 
PMCID: PMC3017858
PMID: 20964857  [Indexed for MEDLINE]


502. Biosens Bioelectron. 2010 Oct 15;26(2):850-3. doi: 10.1016/j.bios.2010.08.007.
Epub 2010 Aug 8.

Double recognition of oligonucleotide and protein in the detection of DNA
methylation with surface plasmon resonance biosensors.

Pan S(1), Xu J, Shu Y, Wang F, Xia W, Ding Q, Xu T, Zhao C, Zhang M, Huang P, Lu 
S.

Author information: 
(1)Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing
Medical University, 300 Guangzhou Road, Nanjing 210029, China. sypan@njmu.edu.cn

DNA methylation plays an essential role in maintenance of cellular function. A
growing number of human diseases have been found to be associated with aberrant
DNA methylation, especially cancer. However, current technologies used in DNA
methylation detection are complicated and time consuming. A promotor of the
Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was 
used as the detection target DNA sequence. The double recognition mechanism was
realized with oligonucleotide probe hybridization and specific protein binding.
First, complementary target DNA was captured by the probe immobilized onto a
surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG
binding domain (MBD) protein was passed over the surface to recognize and bind to
methylated CpG sites. Binding resulted in an increase in the refractive index,
and a detectable optical signal was generated. Five picomoles of methylated APC
promotor DNA could be easily detected with this method. The entire detection
could be completed within 1h. This work represents the first SPR based biosensor 
technology, which achieves simple and specific DNA methylation detection and
avoids complicated bisulfite treatment and methylation-sensitive restriction
digestion. It will improve our ability to detect DNA methylation specifically and
rapidly, and promote our understanding of the role of DNA methylation in gene
regulation and diseases.

Copyright © 2010. Published by Elsevier B.V.

DOI: 10.1016/j.bios.2010.08.007 
PMID: 20810273  [Indexed for MEDLINE]


503. J Proteomics. 2010 Oct 10;73(11):2054-63. doi: 10.1016/j.jprot.2010.08.002. Epub 
2010 Aug 19.

Model organisms--A historical perspective.

Müller B(1), Grossniklaus U.

Author information: 
(1)Institute of Plant Biology, University of Zürich, Zürich, Switzerland.
bmueller@access.uzh.ch

Much of our knowledge on heredity, development, physiology and the underlying
cellular and molecular processes is derived from the studies of model, or
reference, organisms. Despite the great variety of life, a common base of shared 
principles could be extracted by studying a few life forms, selected based on
their amenability to experimental studies. Very briefly, the origins of a few
model organisms are described, including E. coli, yeast, C. elegans, Drosophila, 
Xenopus, zebrafish, mouse, maize and Arabidopsis. These model organisms were
chosen because of their importance and wide use, which made them systems of
choice for genome-wide studies. Many of their genomes were between the first to
be fully sequenced, opening unprecedented opportunities for large-scale
transcriptomics and proteomics studies.

Copyright © 2010 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jprot.2010.08.002 
PMID: 20727995  [Indexed for MEDLINE]


504. PLoS One. 2010 Oct 8;5(10):e13080. doi: 10.1371/journal.pone.0013080.

Using effective subnetworks to predict selected properties of gene networks.

Gunaratne GH(1), Gunaratne PH, Seemann L, Török A.

Author information: 
(1)Department of Physics, University of Houston, Houston, Texas, United States of
America. gemunu@uh.edu

BACKGROUND: Difficulties associated with implementing gene therapy are caused by 
the complexity of the underlying regulatory networks. The forms of interactions
between the hundreds of genes, proteins, and metabolites in these networks are
not known very accurately. An alternative approach is to limit consideration to
genes on the network. Steady state measurements of these influence networks can
be obtained from DNA microarray experiments. However, since they contain a large 
number of nodes, the computation of influence networks requires a prohibitively
large set of microarray experiments. Furthermore, error estimates of the network 
make verifiable predictions impossible.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we propose an alternative approach. Rather 
than attempting to derive an accurate model of the network, we ask what questions
can be addressed using lower dimensional, highly simplified models. More
importantly, is it possible to use such robust features in applications? We first
identify a small group of genes that can be used to affect changes in other nodes
of the network. The reduced effective empirical subnetwork (EES) can be computed 
using steady state measurements on a small number of genetically perturbed
systems. We show that the EES can be used to make predictions on expression
profiles of other mutants, and to compute how to implement pre-specified changes 
in the steady state of the underlying biological process. These assertions are
verified in a synthetic influence network. We also use previously published
experimental data to compute the EES associated with an oxygen deprivation
network of E.coli, and use it to predict gene expression levels on a double
mutant. The predictions are significantly different from the experimental results
for less than of genes.
CONCLUSIONS/SIGNIFICANCE: The constraints imposed by gene expression levels of
mutants can be used to address a selected set of questions about a gene network.

DOI: 10.1371/journal.pone.0013080 
PMCID: PMC2951892
PMID: 20949025  [Indexed for MEDLINE]


505. BMC Bioinformatics. 2010 Oct 7;11 Suppl 6:S4. doi: 10.1186/1471-2105-11-S6-S4.

An FDA bioinformatics tool for microbial genomics research on molecular
characterization of bacterial foodborne pathogens using microarrays.

Fang H(1), Xu J, Ding D, Jackson SA, Patel IR, Frye JG, Zou W, Nayak R, Foley S, 
Chen J, Su Z, Ye Y, Turner S, Harris S, Zhou G, Cerniglia C, Tong W.

Author information: 
(1)Z-Tech Corporation, ICF International Company at National Center for
Toxicological Research, Food and Drug Administration, Jefferson, AR, USA.

BACKGROUND: Advances in microbial genomics and bioinformatics are offering
greater insights into the emergence and spread of foodborne pathogens in outbreak
scenarios. The Food and Drug Administration (FDA) has developed a genomics tool, 
ArrayTrack™, which provides extensive functionalities to manage, analyze, and
interpret genomic data for mammalian species. ArrayTrack™ has been widely adopted
by the research community and used for pharmacogenomics data review in the FDA's 
Voluntary Genomics Data Submission program.
RESULTS: ArrayTrack™ has been extended to manage and analyze genomics data from
bacterial pathogens of human, animal, and food origin. It was populated with
bioinformatics data from public databases such as NCBI, Swiss-Prot, KEGG Pathway,
and Gene Ontology to facilitate pathogen detection and characterization.
ArrayTrack™'s data processing and visualization tools were enhanced with analysis
capabilities designed specifically for microbial genomics including flag-based
hierarchical clustering analysis (HCA), flag concordance heat maps, and mixed
scatter plots. These specific functionalities were evaluated on data generated
from a custom Affymetrix array (FDA-ECSG) previously developed within the FDA.
The FDA-ECSG array represents 32 complete genomes of Escherichia coli and
Shigella. The new functions were also used to analyze microarray data focusing on
antimicrobial resistance genes from Salmonella isolates in a poultry production
environment using a universal antimicrobial resistance microarray developed by
the United States Department of Agriculture (USDA).
CONCLUSION: The application of ArrayTrack™ to different microarray platforms
demonstrates its utility in microbial genomics research, and thus will improve
the capabilities of the FDA to rapidly identify foodborne bacteria and their
genetic traits (e.g., antimicrobial resistance, virulence, etc.) during outbreak 
investigations. ArrayTrack™ is free to use and available to public, private, and 
academic researchers at http://www.fda.gov/ArrayTrack.

DOI: 10.1186/1471-2105-11-S6-S4 
PMCID: PMC3026378
PMID: 20946615  [Indexed for MEDLINE]


506. Aging Cell. 2010 Oct;9(5):868-81. doi: 10.1111/j.1474-9726.2010.00618.x.

Genome-wide screen identifies Escherichia coli TCA-cycle-related mutants with
extended chronological lifespan dependent on acetate metabolism and the
hypoxia-inducible transcription factor ArcA.

Gonidakis S(1), Finkel SE, Longo VD.

Author information: 
(1)Department of Biological Sciences, University of Southern California, Los
Angeles, CA 90089, USA.

Single-gene mutants with extended lifespan have been described in several model
organisms. We performed a genome-wide screen for long-lived mutants in
Escherichia coli, which revealed strains lacking tricarboxylic acid
(TCA)-cycle-related genes that exhibit longer stationary-phase survival and
increased resistance to heat stress compared to wild-type. Extended lifespan in
the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with
the reduced production of superoxide and increased stress resistance. On the
other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is
associated with reduced oxygen consumption and requires the acetate-producing
enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that
converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription
factor ArcA, acting independently of acetate metabolism, is also required for
maximum lifespan extension in the lipA and lpdA mutants, indicating that these
mutations promote entry into a mode normally associated with a low-oxygen
environment. Because analogous changes from respiration to fermentation have been
observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans
strains, such metabolic alterations may represent an evolutionarily conserved
strategy to extend lifespan.

© 2010 The Authors Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society 
of Great Britain and Ireland.

DOI: 10.1111/j.1474-9726.2010.00618.x 
PMCID: PMC2941539
PMID: 20707865  [Indexed for MEDLINE]


507. Immunology. 2010 Oct;131(2):268-81. doi: 10.1111/j.1365-2567.2010.03301.x.

Lactobacillus acidophilus induces virus immune defence genes in murine dendritic 
cells by a Toll-like receptor-2-dependent mechanism.

Weiss G(1), Rasmussen S, Zeuthen LH, Nielsen BN, Jarmer H, Jespersen L, Frøkiaer 
H.

Author information: 
(1)Faculty of Life Sciences, Department of Basic Sciences and Environment,
University of Copenhagen, Frederiksberg C, Denmark.

Lactobacilli are probiotics that, among other health-promoting effects, have been
ascribed immunostimulating and virus-preventive properties. Certain Lactobacillus
spp. have been shown to possess strong interleukin-12 (IL-12) -inducing
properties. As IL-12 production depends on the up-regulation of type I
interferons (IFNs), we hypothesized that the strong IL-12-inducing capacity of
Lactobacillus acidophilus NCFM in murine bone-marrow-derived dendritic cells
(DCs) is caused by an up-regulation of IFN-β, which subsequently induces IL-12
and the double-stranded RNA binding Toll-like receptor-3 (TLR-3). The expression 
of the genes encoding IFN-β, TLR-3, IL-12 and IL-10 in DCs upon stimulation with 
L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much
stronger expression of Ifn-β, Il-12 and Il-10 compared with the synthetic
double-stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr-3 were
similar. Whole genome microarray gene expression analysis revealed that other
genes related to viral defence were significantly up-regulated and among the
strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability
to induce IFN-β was also detected in another L. acidophilus strain (X37), but was
not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9
and Escherichia coli Nissle 1917. The IFN-β expression was markedly reduced in
TLR-2(-/-) DCs, dependent on endocytosis, and the major cause of the induction of
Il-12 and Tlr-3 in DCs stimulated with L. acidophilus NCFM. Collectively, our
results reveal that certain lactobacilli trigger the expression of viral defence 
genes in DCs in a TLR-2 manner dependent on IFN-β.

© 2010 The Authors. Immunology © 2010 Blackwell Publishing Ltd.

DOI: 10.1111/j.1365-2567.2010.03301.x 
PMCID: PMC2967272
PMID: 20545783  [Indexed for MEDLINE]


508. J Microbiol. 2010 Oct;48(5):682-8. doi: 10.1007/s12275-010-0119-5. Epub 2010 Nov 
3.

Detection of representative enteropathogenic bacteria, Vibrio spp., pathogenic
Escherichia coli, Salmonella spp., Shigella spp., and Yersinia enterocolitica,
using a virulence factor gene-based oligonucleotide microarray.

Kim DH(1), Lee BK, Kim YD, Rhee SK, Kim YC.

Author information: 
(1)Department of Microbiology, Chungbuk National University, Cheongju 361-763,
Republic of Korea.

Rapid identification of enteropathogenic bacteria in stool samples is critical
for clinical diagnosis and antimicrobial therapy. In this study, we describe the 
development of an approach that couples multiplex PCR with hybridization to a DNA
microarray, to allow the simultaneous detection of the 10 pathogens. The
microarray was synthesized with 20-mer oligonucleotide probes that were designed 
to be specific for virulence-factor genes of each strain. The detection limit for
genomic DNA from a single strain was approximately 10 fg. In the presence of
heterogeneous non-target DNA, the detection sensitivity of the array decreased to
approximately 100 fg. We did not observe any non-specific hybridization. In
addition, we successfully used this oligonucleotide-based DNA microarray to
identify the causative agents in clinical stool samples from patients with
food-borne enteritis.

DOI: 10.1007/s12275-010-0119-5 
PMID: 21046348  [Indexed for MEDLINE]


509. PLoS One. 2010 Sep 28;5(9). pii: e12776. doi: 10.1371/journal.pone.0012776.

Inferring regulatory networks from expression data using tree-based methods.

Huynh-Thu VA(1), Irrthum A, Wehenkel L, Geurts P.

Author information: 
(1)Department of Electrical Engineering and Computer Science, Systems and
Modeling, University of Liège, Liège, Belgium. vahuynh@ulg.ac.be

One of the pressing open problems of computational systems biology is the
elucidation of the topology of genetic regulatory networks (GRNs) using high
throughput genomic data, in particular microarray gene expression data. The
Dialogue for Reverse Engineering Assessments and Methods (DREAM) challenge aims
to evaluate the success of GRN inference algorithms on benchmarks of simulated
data. In this article, we present GENIE3, a new algorithm for the inference of
GRNs that was best performer in the DREAM4 In Silico Multifactorial challenge.
GENIE3 decomposes the prediction of a regulatory network between p genes into p
different regression problems. In each of the regression problems, the expression
pattern of one of the genes (target gene) is predicted from the expression
patterns of all the other genes (input genes), using tree-based ensemble methods 
Random Forests or Extra-Trees. The importance of an input gene in the prediction 
of the target gene expression pattern is taken as an indication of a putative
regulatory link. Putative regulatory links are then aggregated over all genes to 
provide a ranking of interactions from which the whole network is reconstructed. 
In addition to performing well on the DREAM4 In Silico Multifactorial challenge
simulated data, we show that GENIE3 compares favorably with existing algorithms
to decipher the genetic regulatory network of Escherichia coli. It doesn't make
any assumption about the nature of gene regulation, can deal with combinatorial
and non-linear interactions, produces directed GRNs, and is fast and scalable. In
conclusion, we propose a new algorithm for GRN inference that performs well on
both synthetic and real gene expression data. The algorithm, based on feature
selection with tree-based ensemble methods, is simple and generic, making it
adaptable to other types of genomic data and interactions.

DOI: 10.1371/journal.pone.0012776 
PMCID: PMC2946910
PMID: 20927193  [Indexed for MEDLINE]


510. PLoS Pathog. 2010 Sep 23;6(9):e1001120. doi: 10.1371/journal.ppat.1001120.

Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent
effects on Escherichia coli virulence.

Yadav M(1), Zhang J, Fischer H, Huang W, Lutay N, Cirl C, Lum J, Miethke T,
Svanborg C.

Author information: 
(1)Department of Microbiology, Institute of Laboratory Medicine, Lund University,
Sweden.

Toll-like receptor signaling requires functional Toll/interleukin-1 (IL-1)
receptor (TIR) domains to activate innate immunity. By producing TIR homologous
proteins, microbes inhibit host response induction and improve their own
survival. The TIR homologous protein TcpC was recently identified as a virulence 
factor in uropathogenic Escherichia coli (E. coli), suppressing innate immunity
by binding to MyD88. This study examined how the host MyD88 genotype modifies the
in vivo effects of TcpC and whether additional, TIR-domain containing proteins
might be targeted by TcpC. In wild type mice (wt), TcpC enhanced bacterial
virulence, increased acute mortality, bacterial persistence and tissue damage
after infection with E. coli CFT073 (TcpC+), compared to a ΔTcpC deletion mutant.
These effects were attenuated in Myd88(-/-) and Tlr4(-/-) mice. Transcriptomic
analysis confirmed that TcpC inhibits MYD88 dependent gene expression in CFT073
infected human uroepithelial cells but in addition the inhibitory effect included
targets in the TRIF and IL-6/IL-1 signaling pathways, where MYD88 dependent and
independent signaling may converge. The effects of TcpC on bacterial persistence 
were attenuated in Trif (-/-) or Il-1β (-/-) mice and innate immune responses to 
ΔTcpC were increased, confirming that Trif and Il-1β dependent targets might be
involved in vivo, in addition to Myd88. Furthermore, soluble TcpC inhibited Myd88
and Trif dependent TLR signaling in murine macrophages. Our results suggest that 
TcpC may promote UTI-associated pathology broadly, through inhibition of TIR
domain signaling and downstream pathways. Dysregulation of the host response by
microbial TcpC thus appears to impair the protective effects of innate immunity, 
while promoting inflammation and tissue damage.

DOI: 10.1371/journal.ppat.1001120 
PMCID: PMC2944809
PMID: 20886104  [Indexed for MEDLINE]


511. PLoS Pathog. 2010 Sep 23;6(9):e1001109. doi: 10.1371/journal.ppat.1001109.

Pathogen specific, IRF3-dependent signaling and innate resistance to human kidney
infection.

Fischer H(1), Lutay N, Ragnarsdóttir B, Yadav M, Jönsson K, Urbano A, Al Hadad A,
Rämisch S, Storm P, Dobrindt U, Salvador E, Karpman D, Jodal U, Svanborg C.

Author information: 
(1)Department of Microbiology, Immunology and Glycobiology, Institute of
Laboratory Medicine, Lund University, Lund, Sweden.

The mucosal immune system identifies and fights invading pathogens, while
allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen
discrimination are poorly understood, as is the contribution of human genetic
variation in disease susceptibility. We describe here a new, IRF3-dependent
signaling pathway that is critical for distinguishing pathogens from normal flora
at the mucosal barrier. Following uropathogenic E. coli infection, Irf3(-/-) mice
showed a pathogen-specific increase in acute mortality, bacterial burden, abscess
formation and renal damage compared to wild type mice. TLR4 signaling was
initiated after ceramide release from glycosphingolipid receptors, through TRAM, 
CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in
nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial
effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was
activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored
glycosphingolipid receptors. Relevance of this pathway for human disease was
supported by polymorphic IRF3 promoter sequences, differing between children with
severe, symptomatic kidney infection and children who were asymptomatic bacterial
carriers. IRF3 promoter activity was reduced by the disease-associated genotype, 
consistent with the pathology in Irf3(-/-) mice. Host susceptibility to common
infections like UTI may thus be strongly influenced by single gene modifications 
affecting the innate immune response.

DOI: 10.1371/journal.ppat.1001109 
PMCID: PMC2944801
PMID: 20886096  [Indexed for MEDLINE]


512. ACS Chem Biol. 2010 Sep 17;5(9):851-61. doi: 10.1021/cb100070j.

Engineering a direct and inducible protein-RNA interaction to regulate RNA
biology.

Belmont BJ(1), Niles JC.

Author information: 
(1)Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, 02139, USA.

The importance and pervasiveness of naturally occurring regulation of RNA
function in biology is increasingly being recognized. A common mechanism uses
inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate.
Recapitulating this regulatory mode in cells using a novel set of protein-RNA
interactions is appealing given the potential to subsequently modulate RNA
biology in a manner decoupled from endogenous cellular physiology. Achieving this
outcome, however, has previously proven challenging. Here, we describe a
ligand-responsive protein-RNA interaction module, which can be used to target a
specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands
by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial
Tet Repressor protein (TetR) with low- to subnanomolar affinities were obtained. 
This interaction is reversibly controlled by tetracycline in a manner analogous
to the interaction of TetR with its cognate DNA operator. Aptamer minimization
and mutational analyses support a functional role for two conserved sequence
motifs in TetR binding. As an initial illustration of using this system to
achieve protein-based regulation of RNA function in living cells, insertion of a 
TetR aptamer into the 5'-UTR of a reporter mRNA confers post-transcriptionally
regulated, ligand-inducible protein synthesis in E. coli. Altogether, these
results define and validate an inducible protein-RNA interaction module that
incorporates desirable aspects of a ubiquitous mechanism for regulating RNA
function in Nature and can be used as a foundational interaction for functionally
and reversibly controlling the multiple fates of RNA in cells.

DOI: 10.1021/cb100070j 
PMID: 20545348  [Indexed for MEDLINE]


513. BMC Bioinformatics. 2010 Sep 9;11:454. doi: 10.1186/1471-2105-11-454.

On the choice and number of microarrays for transcriptional regulatory network
inference.

Cosgrove EJ(1), Gardner TS, Kolaczyk ED.

Author information: 
(1)Department of Mathematics and Statistics, Boston University, Boston, MA, USA.

BACKGROUND: Transcriptional regulatory network inference (TRNI) from large
compendia of DNA microarrays has become a fundamental approach for discovering
transcription factor (TF)-gene interactions at the genome-wide level. In
correlation-based TRNI, network edges can in principle be evaluated using
standard statistical tests. However, while such tests nominally assume
independent microarray experiments, we expect dependency between the experiments 
in microarray compendia, due to both project-specific factors (e.g., microarray
preparation, environmental effects) in the multi-project compendium setting and
effective dependency induced by gene-gene correlations. Herein, we characterize
the nature of dependency in an Escherichia coli microarray compendium and explore
its consequences on the problem of determining which and how many arrays to use
in correlation-based TRNI.
RESULTS: We present evidence of substantial effective dependency among
microarrays in this compendium, and characterize that dependency with respect to 
experimental condition factors. We then introduce a measure neff of the effective
number of experiments in a compendium, and find that corresponding to the
dependency observed in this particular compendium there is a huge reduction in
effective sample size i.e., neff = 14.7 versus n = 376. Furthermore, we found
that the neff of select subsets of experiments actually exceeded neff of the full
compendium, suggesting that the adage 'less is more' applies here. Consistent
with this latter result, we observed improved performance in TRNI using subsets
of the data compared to results using the full compendium. We identified
experimental condition factors that trend with changes in TRNI performance and
neff , including growth phase and media type. Finally, using the set of known E. 
coli genetic regulatory interactions from RegulonDB, we demonstrated that false
discovery rates (FDR) derived from neff -adjusted p-values were well-matched to
FDR based on the RegulonDB truth set.
CONCLUSIONS: These results support utilization of neff as a potent descriptor of 
microarray compendia. In addition, they highlight a straightforward
correlation-based method for TRNI with demonstrated meaningful statistical
testing for significant edges, readily applicable to compendia from any species, 
even when a truth set is not available. This work facilitates a more refined
approach to construction and utilization of mRNA expression compendia in TRNI.

DOI: 10.1186/1471-2105-11-454 
PMCID: PMC2949888
PMID: 20825684  [Indexed for MEDLINE]


514. Arch Microbiol. 2010 Sep;192(9):715-28. doi: 10.1007/s00203-010-0599-z. Epub 2010
Jun 18.

Environmental and genetic factors that contribute to Escherichia coli K-12
biofilm formation.

Prüss BM(1), Verma K, Samanta P, Sule P, Kumar S, Wu J, Christianson D, Horne SM,
Stafslien SJ, Wolfe AJ, Denton A.

Author information: 
(1)Department of Veterinary and Microbiological Sciences, North Dakota State
University, Fargo, ND 58108-6050, USA. Birgit.Pruess@ndsu.edu

Biofilms are communities of bacteria whose formation on surfaces requires a large
portion of the bacteria's transcriptional network. To identify environmental
conditions and transcriptional regulators that contribute to sensing these
conditions, we used a high-throughput approach to monitor biofilm biomass
produced by an isogenic set of Escherichia coli K-12 strains grown under
combinations of environmental conditions. Of the environmental combinations,
growth in tryptic soy broth at 37 degrees C supported the most biofilm
production. To analyze the complex relationships between the diverse cell-surface
organelles, transcriptional regulators, and metabolic enzymes represented by the 
tested mutant set, we used a novel vector-item pattern-mining algorithm. The
algorithm related biofilm amounts to the functional annotations of each mutated
protein. The pattern with the best statistical significance was the gene ontology
'pyruvate catabolic process,' which is associated with enzymes of acetate
metabolism. Phenotype microarray experiments illustrated that carbon sources that
are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are
particularly supportive of biofilm formation. Scanning electron microscopy
revealed structural differences between mutants that lack acetate metabolism
enzymes and their parent and confirmed the quantitative differences. We conclude 
that acetate metabolism functions as a metabolic sensor, transmitting changes in 
environmental conditions to biofilm biomass and structure.

DOI: 10.1007/s00203-010-0599-z 
PMCID: PMC2923660
PMID: 20559621  [Indexed for MEDLINE]


515. FEMS Microbiol Rev. 2010 Sep;34(5):628-45. doi: 10.1111/j.1574-6976.2010.00227.x.
Epub 2010 Apr 14.

Prokaryotic genome regulation: multifactor promoters, multitarget regulators and 
hierarchic networks.

Ishihama A(1).

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan. aishiham@hosei.ac.jp

Comment in
    FEMS Microbiol Rev. 2010 Sep;34(5):611-27.

The vast majority of experimental data have been accumulated on the transcription
regulation of individual genes within a single model prokaryote, Escherichia
coli, which form the well-established on-off switch model of transcription by
DNA-binding regulatory proteins. After the development of modern high-throughput 
experimental systems such as microarray analysis of whole genome transcription
and the Genomic SELEX search for the whole set of regulation targets by
transcription factors, a number of E. coli promoters are now recognized to be
under the control of multiple transcription factors, as in the case of
eukaryotes. The number of regulation targets of a single transcription factor has
also been found to be more than hitherto recognized, ranging up to hundreds of
promoters, genes or operons for several global regulators. The multifactor
promoters and the multitarget transcription factors can be assembled into complex
networks of transcription regulation, forming hierarchical networks.

DOI: 10.1111/j.1574-6976.2010.00227.x 
PMID: 20491932  [Indexed for MEDLINE]


516. Foodborne Pathog Dis. 2010 Sep;7(9):1039-45. doi: 10.1089/fpd.2009.0505.

Fluoroquinolone resistance detection in Campylobacter coli and Campylobacter
jejuni by Luminex xMAP technology.

Barco L(1), Lettini AA, Dalla Pozza MC, Ramon E, Fasolato M, Ricci A.

Author information: 
(1)OIE, National Reference Laboratory for Salmonella, Istituto Zooprofilattico
Sperimentale delle Venezie, Legnaro, Padova, Italy.

The proportion of Campylobacter spp. isolates that are resistant to
fluoroquinolones, the drugs of choice for campylobacteriosis, has been increasing
worldwide. We developed an innovative method based on a Luminex xMAP DNA
suspension array that allows the identification of Campylobacter species and,
simultaneously, the detection of the most common point mutation in the gyrA gene 
(substitution from threonine 86 to isoleucine 86) that is responsible for
fluoroquinolone resistance. Ninety-six Campylobacter coli and Campylobacter
jejuni isolates collected from turkeys were first investigated by microdilution
test to characterize the antimicrobial resistance patterns. The isolates,
amplified for the quinolone resistance determining region of the gyrA gene, were 
then tested using Luminex suspension array. The reliability of the method was
demonstrated by the total concordance between the results obtained using Luminex 
and those of the sequencing of gyrA polymerase chain reaction products. The
genotypic characterization of fluoroquinolone resistance using Luminex was also
consistent with the data on phenotypical resistance obtained by microdilution
test. The results of this study strongly support the potential of Luminex xMAP
technology as an efficient molecular method for the rapid and accurate
identification of C. coli and C. jejuni isolates and the characterization of the 
major determinant of fluoroquinolone resistance.

DOI: 10.1089/fpd.2009.0505 
PMID: 20500084  [Indexed for MEDLINE]


517. Int J Food Microbiol. 2010 Sep 1;142(3):318-29. doi:
10.1016/j.ijfoodmicro.2010.07.010. Epub 2010 Jul 14.

Micro-array for the identification of Shiga toxin-producing Escherichia coli
(STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic
Syndrome in humans.

Bugarel M(1), Beutin L, Martin A, Gill A, Fach P.

Author information: 
(1)AFSSA (French Food Safety Agency), Laboratory for Study and Research on Food
Quality and Processes (LERQAP), 23 Av du Général De Gaulle, Fr-94706
Maisons-Alfort, France.

A micro-array has been developed, based on the GeneDisc(R) array, for the genetic
identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia
coli (STEC) including the most clinically relevant enterohemorrhagic E. coli
(EHEC) serotypes. The genes selected for determination of the O antigens
(rfbE(O157), wzx(O26), wzx(O103), wbd1(O111), ihp1(O145), wzx(O121), wzy(O113),
wzy(O91), wzx(O104), wzy(O118), wzx(O45), and wbgN(O55)) and H-types (fliC(H2),
fliC(H7), fliC(H8), fliC(H11), fliC(H19), fliC(H21), and fliC(H28)) showed a high
specificity and concordance with serology. The micro-array also had a high
specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2
(stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP),
catalase peroxidase (katP), the type II secretion system (etpD), subtilase
cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors
encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71
(nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains,
and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely 
associated with certain serotypes of typical EHEC and emerging EHEC strains.
Virulence plasmid associated genes such as katP, espP, and etpD were more common 
in EHEC than in STEC strains; this supports their association with virulence.
This array constitutes a valuable approach for the identification of STEC strains
with a high potential for human virulence.

Copyright 2010 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.ijfoodmicro.2010.07.010 
PMID: 20675003  [Indexed for MEDLINE]


518. J Bacteriol. 2010 Sep;192(18):4680-93. doi: 10.1128/JB.00543-10. Epub 2010 Jul
16.

In-depth profiling of the LiaR response of Bacillus subtilis.

Wolf D(1), Kalamorz F, Wecke T, Juszczak A, Mäder U, Homuth G, Jordan S, Kirstein
J, Hoppert M, Voigt B, Hecker M, Mascher T.

Author information: 
(1)Department of Biology I, Ludwig Maximilians University of Munich,
Grosshaderner Str 2-4, D-82152 Planegg-Martinsried, Germany.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is
comprised of the LiaRS two-component system and a membrane-anchored inhibitor
protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all
orthologs investigated so far are activated by cell wall antibiotics. In response
to envelope stress, the systems in Firmicutes cocci induce the expression of a
number of genes that are involved in conferring resistance against its inducers. 
In contrast, a complete picture of the LiaR regulon of B. subtilis is still
missing and no phenotypes could be associated with mutants lacking LiaRS. Here,
we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic
profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a
comprehensive picture of the Lia response of Bacillus subtilis. In addition to
the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene
affected by LiaR-dependent regulation. The results of detailed follow-up gene
expression studies, together with proteomic analysis, demonstrate that the liaIH 
operon represents the only relevant LiaR target locus in vivo. It encodes a small
membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms
large oligomeric rings reminiscent of those described for Escherichia coli PspA
or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies
demonstrated that the gene products of the liaIH operon are involved in
protecting the cell against oxidative stress and some cell wall antibiotics. Our 
data suggest that the LiaFSR system of B. subtilis and, presumably, other
Firmicutes bacilli coordinates a phage shock protein-like response.

DOI: 10.1128/JB.00543-10 
PMCID: PMC2937411
PMID: 20639339  [Indexed for MEDLINE]


519. Mol Microbiol. 2010 Sep;77(6):1380-93. doi: 10.1111/j.1365-2958.2010.07315.x.
Epub 2010 Aug 18.

H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be 
relieved by the transcription activator LeuO.

Westra ER(1), Pul U, Heidrich N, Jore MM, Lundgren M, Stratmann T, Wurm R, Raine 
A, Mescher M, Van Heereveld L, Mastop M, Wagner EG, Schnetz K, Van Der Oost J,
Wagner R, Brouns SJ.

Author information: 
(1)Laboratory of Microbiology, Department of Agrotechnology and Food Sciences,
Wageningen University, Dreijenplein 10, 6703 HB Wageningen, the Netherlands.

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity
against viral infections and plasmid conjugation. It has been demonstrated that
in Escherichia coli transcription of the Cascade genes (casABCDE) and to some
extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS)
protein, a global transcriptional repressor. Here we elaborate on the control of 
the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral
immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are
complementary to phage Lambda does not lead to detectable protection against
Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with
the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage
infection. In addition, it is demonstrated that LeuO, a LysR-type transcription
factor, binds to two sites flanking the casA promoter and the H-NS nucleation
site, resulting in derepression of casABCDE12 transcription. Overexpression of
LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced
protection against phage infection. This study demonstrates that in E. coli H-NS 
and LeuO are antagonistic regulators of CRISPR-based immunity.

© 2010 Blackwell Publishing Ltd.

DOI: 10.1111/j.1365-2958.2010.07315.x 
PMID: 20659289  [Indexed for MEDLINE]


520. Water Res. 2010 Sep;44(17):4924-31. doi: 10.1016/j.watres.2010.07.026. Epub 2010 
Jul 27.

Comparative transcriptomics of the response of Escherichia coli to the
disinfectant monochloramine and to growth conditions inducing monochloramine
resistance.

Berry D(1), Holder D, Xi C, Raskin L.

Author information: 
(1)Department of Civil and Environmental Engineering, University of Michigan,
1351 Beal Ave, 107 EWRE Bldg, Ann Arbor, MI 48109-2125, USA.

Escherichia coli growth in biofilms and growth at a suboptimal temperature of 20 
°C have been shown to decrease sensitivity to monochloramine (Berry, D., C. Xi,
L. Raskin. 2009. Environ. Sci. Technol. 43, 884-889). In order to better
understand why growth conditions affect sensitivity to monochloramine, a
comparative transcriptomic approach was used to identify common patterns of
differentially-expressed genes under these growth conditions and during
monochloramine exposure. This approach revealed a set of differentially-expressed
genes shared under multiple conditions (planktonic growth at 20 °C, biofilm
growth, and exposure of planktonic cells to monochloramine), with nine genes
shared under all three conditions. Functional gene categories enriched in the
shared gene sets included: general metabolic inhibition, redox and oxidoreductase
response, cell envelope integrity response, control of iron and sulfur transport 
metabolism and several genes of unknown function. Single gene deletion mutant
analyses verified that loss of 15 of the 24 genes up-regulated during
monochloramine exposure as well as during other tested conditions increased E.
coli sensitivity to monochloramine up to two fold. Constitutive expression of
down-regulated genes in single gene mutants yielded mixed results, indicating
that the expression of some down-regulated genes actually decreases sensitivity
to monochloramine. These results contribute to the understanding of the bacterial
response to disinfectants by characterizing the overlap between growth condition 
associated stress responses and monochloramine-associated stress responses. This 
characterization highlights the bacterial responses responsible for decreased
sensitivity to monochloramine under different growth conditions.

Copyright © 2010 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.watres.2010.07.026 
PMID: 20692677  [Indexed for MEDLINE]


521. PLoS Pathog. 2010 Aug 26;6(8):e1001078. doi: 10.1371/journal.ppat.1001078.

Host imprints on bacterial genomes--rapid, divergent evolution in individual
patients.

Zdziarski J(1), Brzuszkiewicz E, Wullt B, Liesegang H, Biran D, Voigt B,
Grönberg-Hernandez J, Ragnarsdottir B, Hecker M, Ron EZ, Daniel R, Gottschalk G, 
Hacker J, Svanborg C, Dobrindt U.

Author information: 
(1)Institute for Molecular Biology of Infectious Diseases,
Julius-Maximilians-University Würzburg, Würzburg, Germany.

Bacteria lose or gain genetic material and through selection, new variants become
fixed in the population. Here we provide the first, genome-wide example of a
single bacterial strain's evolution in different deliberately colonized patients 
and the surprising insight that hosts appear to personalize their microflora. By 
first obtaining the complete genome sequence of the prototype asymptomatic
bacteriuria strain E. coli 83972 and then resequencing its descendants after
therapeutic bladder colonization of different patients, we identified 34
mutations, which affected metabolic and virulence-related genes. Further
transcriptome and proteome analysis proved that these genome changes altered
bacterial gene expression resulting in unique adaptation patterns in each
patient. Our results provide evidence that, in addition to stochastic events,
adaptive bacterial evolution is driven by individual host environments. Ongoing
loss of gene function supports the hypothesis that evolution towards commensalism
rather than virulence is favored during asymptomatic bladder colonization.

DOI: 10.1371/journal.ppat.1001078 
PMCID: PMC2928814
PMID: 20865122  [Indexed for MEDLINE]


522. J Bacteriol. 2010 Aug;192(16):4181-91. doi: 10.1128/JB.00193-10. Epub 2010 Jun
18.

The LysR-type virulence activator AphB regulates the expression of genes in
Vibrio cholerae in response to low pH and anaerobiosis.

Kovacikova G(1), Lin W, Skorupski K.

Author information: 
(1)Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, 
NH 03755, USA.

AphB is a LysR-type activator that initiates the expression of the virulence
cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated
activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To
identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we
carried out a microarray analysis and show here that AphB influences the
expression of a number of genes that are not associated with the VPI. One gene
strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional
activator responsible for activating the expression of lysine decarboxylase,
which plays an important role in survival at low pH. Other genes activated by
AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC
family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB
influences each of these genes directly by recognizing a conserved binding site
within their promoters, as determined by gel mobility shift assays.
Transcriptional lacZ fusions indicate that AphB activates the expression of these
genes under aerobic conditions in response to low pH and also under anaerobic
conditions at neutral pH. Further experiments show that the regulation of cadC by
AphB in response to low pH and anaerobiosis is mirrored in the heterologous
organism Escherichia coli, is independent of the global regulators Fnr and ArcAB,
and depends upon the region of the promoter that contains the AphB binding site. 
These results raise the possibility that the activity of AphB is influenced by
the pH and oxygen tension of the environment.

DOI: 10.1128/JB.00193-10 
PMCID: PMC2916415
PMID: 20562308  [Indexed for MEDLINE]


523. Nat Biotechnol. 2010 Aug;28(8):856-62. doi: 10.1038/nbt.1653. Epub 2010 Jul 18.

Rapid profiling of a microbial genome using mixtures of barcoded
oligonucleotides.

Warner JR(1), Reeder PJ, Karimpour-Fard A, Woodruff LB, Gill RT.

Author information: 
(1)Department of Chemical and Biological Engineering, University of Colorado,
Boulder, Colorado, USA.

Comment in
    Nat Methods. 2010 Sep;7(9):674.

A fundamental goal in biotechnology and biology is the development of approaches 
to better understand the genetic basis of traits. Here we report a versatile
method, trackable multiplex recombineering (TRMR), whereby thousands of specific 
genetic modifications are created and evaluated simultaneously. To demonstrate
TRMR, in a single day we modified the expression of >95% of the genes in
Escherichia coli by inserting synthetic DNA cassettes and molecular barcodes
upstream of each gene. Barcode sequences and microarrays were then used to
quantify population dynamics. Within a week we mapped thousands of genes that
affect E. coli growth in various media (rich, minimal and cellulosic hydrolysate)
and in the presence of several growth inhibitors (beta-glucoside, D-fucose,
valine and methylglyoxal). This approach can be applied to a broad range of
traits to identify targets for future genome-engineering endeavors.

DOI: 10.1038/nbt.1653 
PMID: 20639866  [Indexed for MEDLINE]


524. BMC Bioinformatics. 2010 Jul 23;11:397. doi: 10.1186/1471-2105-11-397.

Simultaneous prediction of transcription factor binding sites in a group of
prokaryotic genomes.

Zhang S(1), Li S, Pham PT, Su Z.

Author information: 
(1)Department of Bioinformatics and Genomics, Center for Bioinformatics Research,
the University of North Carolina at Charlotte, Charlotte, NC 28223, USA.

BACKGROUND: Our current understanding of transcription factor binding sites
(TFBSs) in sequenced prokaryotic genomes is very limited due to the lack of an
accurate and efficient computational method for the prediction of TFBSs at a
genome scale. In an attempt to change this situation, we have recently developed 
a comparative genomics based algorithm called GLECLUBS for de novo genome-wide
prediction of TFBSs in a target genome. Although GLECLUBS has achieved rather
high prediction accuracy of TFBSs in a target genome, it is still not efficient
enough to be applied to all the sequenced prokaryotic genomes.
RESULTS: Here, we designed a new algorithm based on GLECLUBS called extended
GLECLUBS (eGLECLUBS) for simultaneous prediction of TFBSs in a group of related
prokaryotic genomes. When tested on a group of gamma-proteobacterial genomes
including E. coli K12, a group of firmicutes genomes including B. subtilis and a 
group of cyanobacterial genomes using the same parameter settings, eGLECLUBS
predicts more than 82% of known TFBSs in extracted inter-operonic sequences in
both E. coli K12 and B. subtilis. Because each genome in a group is equally
treated, it is highly likely that similar prediction accuracy has been achieved
for each genome in the group.
CONCLUSIONS: We have developed a new algorithm for genome-wide de novo prediction
of TFBSs in a group of related prokaryotic genomes. The algorithm has achieved
the same level of accuracy and robustness as its predecessor GLECLUBS, but can
work on dozens of genomes at the same time.

DOI: 10.1186/1471-2105-11-397 
PMCID: PMC2920276
PMID: 20653963  [Indexed for MEDLINE]


525. J Biol Chem. 2010 Jul 2;285(27):20724-31. doi: 10.1074/jbc.M109.085506. Epub 2010
Apr 28.

Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative
stress responses and protein nitration and nitrosylation.

McLean S(1), Bowman LA, Sanguinetti G, Read RC, Poole RK.

Author information: 
(1)Departments of Molecular Biology and Biotechnology, University of Sheffield,
Firth Court, Western Bank, Sheffield S10 2TN, UK. S.McLean@Sheffield.ac.uk

Peroxynitrite is formed in macrophages by the diffusion-limited reaction of
superoxide and nitric oxide. This highly reactive species is thought to
contribute to bacterial killing by interaction with diverse targets and nitration
of protein tyrosines. This work presents for the first time a comprehensive
analysis of transcriptional responses to peroxynitrite under tightly controlled
chemostat growth conditions. Up-regulation of the cysteine biosynthesis pathway
and an increase in S-nitrosothiol levels suggest S-nitrosylation to be a
consequence of peroxynitrite exposure. Genes involved in the assembly/repair of
iron-sulfur clusters also show enhanced transcription. Unexpectedly, arginine
biosynthesis gene transcription levels were also elevated after treatment with
peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed
that post-translational nitration of tyrosine residues within this protein is
responsible for its degradation in vitro. Further up-regulation was seen in
oxidative stress response genes, including katG and ahpCF. However, genes known
to be up-regulated by nitric oxide and nitrosating agents (e.g. hmp and norVW)
were unaffected. Probabilistic modeling of the transcriptomic data identified
five altered transcription factors in response to peroxynitrite exposure,
including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in
commercially available peroxynitrite preparations. Transcriptomic analysis of
cells treated with hydrogen peroxide alone also revealed up-regulation of
oxidative stress response genes but not of many other genes that are up-regulated
by peroxynitrite. Thus, the cellular responses to peroxynitrite and hydrogen
peroxide are distinct.

DOI: 10.1074/jbc.M109.085506 
PMCID: PMC2898335
PMID: 20427277  [Indexed for MEDLINE]


526. Foodborne Pathog Dis. 2010 Jul;7(7):763-73. doi: 10.1089/fpd.2009.0482.

Microarray analysis and draft genomes of two Escherichia coli O157:H7 lineage II 
cattle isolates FRIK966 and FRIK2000 investigating lack of Shiga toxin
expression.

Dowd SE(1), Crippen TL, Sun Y, Gontcharova V, Youn E, Muthaiyan A, Wolcott RD,
Callaway TR, Ricke SC.

Author information: 
(1)Research and Testing Laboratory, Lubbock, Texas 79407, USA.
sdowd@pathogenresearch.org

The existence of two separate genetic lineages of Escherichia coli O157:H7 has
previously been reported, and research indicates that lineage I could be more
pathogenic toward human hosts than lineage II. We have previously shown that
lineage I as a group expresses higher levels of Shiga toxin 2 (Stx2) than lineage
II. To help evaluate why lineage II strains do not express appreciable levels of 
this toxin, whole-genome microarrays were performed using Agilent custom
microarrays. Gene expression of the two representative bovine lineage II strains 
(FRIK966 and FRIK2000) were compared with gene expression of E. coli O157:H7
EDL933 (lineage I clinical type strain). Missing or differentially expressed
genes and pathways were identified. Quantitative reverse transcription-polymerase
chain reaction was performed to validate the microarray data. Draft genomes of
FRIK966 and FRIK2000 were sequenced using Roche Applied Science/454 GS-FLX
technology shotgun and paired-end approaches followed by de novo assembly. These 
assemblies were compared with the lineage I genome sequences from E. coli O157:H7
EDL933. The bacteriophage 933W, which encodes the Stx2 genes, showed a notable
repression in gene expression. Polymerase chain reaction primers, based upon
EDL933 genomic information, were also designed against all of the potentially
missing genes of this bacteriophage. Most of the structural genes associated with
the bacteriophage were found to be absent from the genome of the two bovine
strains. These analyses, combined with evaluation of the genomic information,
suggest that transposon (IS629) rearrangements may be associated with disruption 
of the bacteriophage genome in the FRIK strains. The results support the
hypothesis that lineage II strains may be less of a risk as human foodborne
pathogens. The microarray and genome data have been made available to the
scientific community to allow continuing analysis of these cattle-isolated
lineage II genomes and their gene expression.

DOI: 10.1089/fpd.2009.0482 
PMID: 20156085  [Indexed for MEDLINE]


527. J Bacteriol. 2010 Jul;192(13):3329-36. doi: 10.1128/JB.01352-09. Epub 2010 Apr
23.

YieJ (CbrC) mediates CreBC-dependent colicin E2 tolerance in Escherichia coli.

Cariss SJ(1), Constantinidou C, Patel MD, Takebayashi Y, Hobman JL, Penn CW,
Avison MB.

Author information: 
(1)Department of Cellular and Molecular Medicine, University of Bristol, School
of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.

Colicin E2-tolerant (known as Cet2) Escherichia coli K-12 mutants overproduce an 
inner membrane protein, CreD, which is believed to cause the Cet2 phenotype.
Here, we show that overproduction of CreD in a Cet2 strain results from
hyperactivation of the CreBC two-component regulator, but CreD overproduction is 
not responsible for the Cet2 phenotype. Through microarray analysis and gene
knockout and overexpression studies, we show that overexpression of another
CreBC-regulated gene, yieJ (also known as cbrC), causes the Cet2 phenotype.

DOI: 10.1128/JB.01352-09 
PMCID: PMC2897664
PMID: 20418396  [Indexed for MEDLINE]


528. J Clin Microbiol. 2010 Jul;48(7):2449-58. doi: 10.1128/JCM.01611-09. Epub 2010
May 5.

Escherichia coli isolates causing asymptomatic bacteriuria in catheterized and
noncatheterized individuals possess similar virulence properties.

Watts RE(1), Hancock V, Ong CL, Vejborg RM, Mabbett AN, Totsika M, Looke DF,
Nimmo GR, Klemm P, Schembri MA.

Author information: 
(1)School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, QLD, Australia.

Urinary tract infections (UTIs) are among the most common infectious diseases of 
humans, with Escherichia coli being responsible for >80% of all cases.
Asymptomatic bacteriuria (ABU) occurs when bacteria colonize the urinary tract
without causing clinical symptoms and can affect both catheterized patients
(catheter-associated ABU [CA-ABU]) and noncatheterized patients. Here, we
compared the virulence properties of a collection of ABU and CA-ABU nosocomial E.
coli isolates in terms of antibiotic resistance, phylogenetic grouping, specific 
UTI-associated virulence genes, hemagglutination characteristics, and biofilm
formation. CA-ABU isolates were similar to ABU isolates with regard to the
majority of these characteristics; exceptions were that CA-ABU isolates had a
higher prevalence of the polysaccharide capsule marker genes kpsMT II and kpsMT
K1, while more ABU strains were capable of mannose-resistant hemagglutination. To
examine biofilm growth in detail, we performed a global gene expression analysis 
with two CA-ABU strains that formed a strong biofilm and that possessed a limited
adhesin repertoire. The gene expression profile of the CA-ABU strains during
biofilm growth showed considerable overlap with that previously described for the
prototype ABU E. coli strain, 83972. This is the first global gene expression
analysis of E. coli CA-ABU strains. Overall, our data suggest that nosocomial ABU
and CA-ABU E. coli isolates possess similar virulence profiles.

DOI: 10.1128/JCM.01611-09 
PMCID: PMC2897502
PMID: 20444967  [Indexed for MEDLINE]


529. J Microbiol Methods. 2010 Jul;82(1):36-41. doi: 10.1016/j.mimet.2010.03.017. Epub
2010 Mar 31.

Development and validation of a resistance and virulence gene microarray
targeting Escherichia coli and Salmonella enterica.

Davis MA(1), Lim JY, Soyer Y, Harbottle H, Chang YF, New D, Orfe LH, Besser TE,
Call DR.

Author information: 
(1)Department of Veterinary Microbiology and Pathology, College of Veterinary
Medicine, Washington State University, Pullman, WA 99164-7040, United States.
madavis@vetmed.wsu.edu

A microarray was developed to simultaneously screen Escherichia coli and
Salmonella enterica for multiple genetic traits. The final array included 203
60-mer oligonucleotide probes, including 117 for resistance genes, 16 for
virulence genes, 25 for replicon markers, and 45 other markers. Validity of the
array was tested by assessing inter-laboratory agreement among four collaborating
groups using a blinded study design. Internal validation indicated that the assay
was reliable (area under the receiver-operator characteristic curve=0.97).
Inter-laboratory agreement, however, was poor when estimated using the intraclass
correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24,
0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and
procedure standardization will be needed before bacterial genotyping arrays can
be readily shared between laboratories.

Copyright (c) 2010 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.mimet.2010.03.017 
PMCID: PMC2882532
PMID: 20362014  [Indexed for MEDLINE]


530. Nucleic Acids Res. 2010 Jul;38(12):e135. doi: 10.1093/nar/gkq274. Epub 2010 May
3.

Inclusion of neighboring base interdependencies substantially improves
genome-wide prokaryotic transcription factor binding site prediction.

Salama RA(1), Stekel DJ.

Author information: 
(1)Centre of Systems Biology, School of Biosciences, University of Birmingham,
B15 2TT, UK.

Prediction of transcription factor binding sites is an important challenge in
genome analysis. The advent of next generation genome sequencing technologies
makes the development of effective computational approaches particularly
imperative. We have developed a novel training-based methodology intended for
prokaryotic transcription factor binding site prediction. Our methodology extends
existing models by taking into account base interdependencies between
neighbouring positions using conditional probabilities and includes genomic
background weighting. This has been tested against other existing and novel
methodologies including position-specific weight matrices, first-order Hidden
Markov Models and joint probability models. We have also tested the use of gapped
and ungapped alignments and the inclusion or exclusion of background weighting.
We show that our best method enhances binding site prediction for all of the 22
Escherichia coli transcription factors with at least 20 known binding sites, with
many showing substantial improvements. We highlight the advantage of using block 
alignments of binding sites over gapped alignments to capture neighbouring
position interdependencies. We also show that combining these methods with
ChIP-on-chip data has the potential to further improve binding site prediction.
Finally we have developed the ungapped likelihood under positional background
platform: a user friendly website that gives access to the prediction method
devised in this work.

DOI: 10.1093/nar/gkq274 
PMCID: PMC2896541
PMID: 20439311  [Indexed for MEDLINE]


531. PLoS Pathog. 2010 Jul 1;6:e1000971. doi: 10.1371/journal.ppat.1000971.

Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor.

Zhang Z(1), Selariu A, Warden C, Huang G, Huang Y, Zaccheus O, Cheng T, Xia N,
Zhu H.

Author information: 
(1)Department of Microbiology and Molecular Genetics, UMNDJ-Newark, Newark, New
Jersey, United States of America.

The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is 
the causative agent of chicken pox and shingles. Although an attenuated VZV
vaccine (v-Oka) has been widely used in children in the United States, chicken
pox outbreaks are still seen, and the shingles vaccine only reduces the risk of
shingles by 50%. Therefore, VZV still remains an important public health concern.
Knowledge of VZV replication and pathogenesis remains limited due to its highly
cell-associated nature in cultured cells, the difficulty of generating
recombinant viruses, and VZV's almost exclusive tropism for human cells and
tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka)
genome into a bacterial artificial chromosome that included a dual-reporter
system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the
homologous recombination system in the E. coli to individually delete each of the
genome's 70 unique ORFs. The collection of viral mutants obtained was
systematically examined both in MeWo cells and in cultured human fetal skin organ
samples. We use our genome-wide deletion library to provide novel functional
annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be
essential for viral replication. Among the 26 non-essential ORF deletion mutants,
eight have discernable growth defects in MeWo. Interestingly, four ORFs were
found to be required for viral replication in skin organ cultures, but not in
MeWo cells, suggesting their potential roles as skin tropism factors. One of the 
genes (ORF7) has never been described as a skin tropic factor. The global
profiling of the VZV genome gives further insights into the replication and
pathogenesis of this virus, which can lead to improved prevention and therapy of 
chicken pox and shingles.

DOI: 10.1371/journal.ppat.1000971 
PMCID: PMC2895648
PMID: 20617166  [Indexed for MEDLINE]


532. BMC Genomics. 2010 Jun 24;11:405. doi: 10.1186/1471-2164-11-405.

Genome-wide identification and expression analysis of serine proteases and
homologs in the silkworm Bombyx mori.

Zhao P(1), Wang GH, Dong ZM, Duan J, Xu PZ, Cheng TC, Xiang ZH, Xia QY.

Author information: 
(1)The Key Sericultural Laboratory of Agricultural Ministry, Southwest
University, Chongqing 400715, PR China.

BACKGROUND: Serine proteases (SPs) and serine proteases homologs (SPHs) are a
large group of proteolytic enzymes, with important roles in a variety of
physiological processes, such as cell signalling, defense and development.
Genome-wide identification and expression analysis of serine proteases and their 
homologs in the silkworm might provide valuable information about their
biological functions.
RESULTS: In this study, 51 SP genes and 92 SPH genes were systematically
identified in the genome of the silkworm Bombyx mori. Phylogenetic analysis
indicated that six gene families have been amplified species-specifically in the 
silkworm, and the members of them showed chromosomal distribution of tandem
repeats. Microarray analysis suggests that many silkworm-specific genes, such as 
members of SP_fam12, 13, 14 and 15, show expression patterns that are specific to
tissues or developmental stages. The roles of SPs and SPHs in resisting pathogens
were investigated in silkworms when they were infected by Escherichia coli,
Bacillus bombysepticus, Batrytis bassiana and B. mori nucleopolyhedrovirus,
respectively. Microarray experiment and real-time quantitative RT-PCR showed that
18 SP or SPH genes were significantly up-regulated after pathogen induction,
suggesting that SP and SPH genes might participate in pathogenic microorganism
resistance in B. mori.
CONCLUSION: Silkworm SP and SPH genes were identified. Comparative genomics
showed that SP and SPH genes belong to a large family, whose members are
generated mainly by tandem repeat evolution. We found that silkworm has
species-specific SP and SPH genes. Phylogenetic and microarray analyses provide
an overview of the silkworm SP and SPHs, and facilitate future functional studies
on these enzymes.

DOI: 10.1186/1471-2164-11-405 
PMCID: PMC2996933
PMID: 20576138  [Indexed for MEDLINE]


533. J Vis Exp. 2010 Jun 18;(40). pii: 2007. doi: 10.3791/2007.

Genome-wide analysis of aminoacylation (charging) levels of tRNA using
microarrays.

Zaborske J(1), Pan T.

Author information: 
(1)Department of Biochemistry and Molecular Biology, University of Chicago, IL,
USA.

tRNA aminoacylation, or charging, levels can rapidly change within a cell in
response to the environment[1]. Changes in tRNA charging levels in both
prokaryotic and eukaryotic cells lead to translational regulation which is a
major cellular mechanism of stress response. Familiar examples are the stringent 
response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent
work in E. coli and S. cerevisiae have shown that tRNA charging patterns are
highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. 
The highly dynamic, variable nature of tRNA charging makes it essential to
determine changes in tRNA charging levels at the genomic scale, in order to fully
elucidate cellular response to environmental variations. In this review we
present a method for simultaneously measuring the relative charging levels of all
tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this
protocol has been successfully applied for determining relative charging levels
in a wide variety of organisms including E. coli and human cell cultures[7, 8].

DOI: 10.3791/2007 
PMCID: PMC3153904
PMID: 20567214  [Indexed for MEDLINE]


534. J Biol Chem. 2010 Jun 4;285(23):17498-506. doi: 10.1074/jbc.M109.095570. Epub
2010 Mar 18.

Metabolic and transcriptional response to cofactor perturbations in Escherichia
coli.

Holm AK(1), Blank LM, Oldiges M, Schmid A, Solem C, Jensen PR, Vemuri GN.

Author information: 
(1)Department of Systems Biology, Center for Systems Microbiology, Technical
University of Denmark, Denmark.

Metabolic cofactors such as NADH and ATP play important roles in a large number
of cellular reactions, and it is of great interest to dissect the role of these
cofactors in different aspects of metabolism. Toward this goal, we overexpressed 
NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of 
NADH and ATP, respectively. We used a global interaction network, comprising of
protein interactions, transcriptional regulation, and metabolic networks, to
integrate data from transcription profiles, metabolic fluxes, and the metabolite 
levels. We identified high-scoring networks for the two strains. The results
revealed a smaller, but denser network for perturbations of ATP level, compared
with that of NADH level. The action of many global transcription factors such as 
ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others
responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in 
widespread aspects of metabolism involving the redox cofactors (NADH and NADPH), 
whereas ATPase has a more focused response to restore ATP level by enhancing
proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH
played a key role in restoring redox homeostasis through the concerted activity
of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled
network of regulation that illustrates the overlapping and distinct aspects of
metabolism controlled by NADH and ATP. Our study contributes to the general
understanding of redox and energy metabolism and should help in developing
metabolic engineering strategies in E. coli.

DOI: 10.1074/jbc.M109.095570 
PMCID: PMC2878514
PMID: 20299454  [Indexed for MEDLINE]


535. BMC Genomics. 2010 Jun 3;11:350. doi: 10.1186/1471-2164-11-350.

Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4
using high-resolution genome tiling arrays.

Kumar R(1), Shah P, Swiatlo E, Burgess SC, Lawrence ML, Nanduri B.

Author information: 
(1)Department of Basic sciences, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762, USA.

BACKGROUND: The identification of non-coding transcripts in human, mouse, and
Escherichia coli has revealed their widespread occurrence and functional
importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have 
shown that non-coding transcripts participate in a broad range of cellular
functions like gene regulation, stress and virulence. However, very little is
known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an
obligate human respiratory pathogen responsible for significant worldwide
morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as 
well as identification of novel transcripts at a high-resolution.
RESULTS: Here, we describe a high-resolution transcription map of the S.
pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results
indicate that approximately 66% of the genome is expressed under our experimental
conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the
intergenic regions, of which 36 had no predicted function. Half of the identified
sRNA sequences were found to be unique to S. pneumoniae genome. We identified
eight overrepresented sequence motifs among sRNA sequences that correspond to
sRNAs in different functional categories. Tiling arrays also identified
approximately 202 operon structures in the genome.
CONCLUSIONS: In summary, the pneumococcal operon structures and novel sRNAs
identified in this study enhance our understanding of the complexity and extent
of the pneumococcal 'expressed' genome. Furthermore, the results of this study
open up new avenues of research for understanding the complex RNA regulatory
network governing S. pneumoniae physiology and virulence.

DOI: 10.1186/1471-2164-11-350 
PMCID: PMC2887815
PMID: 20525227  [Indexed for MEDLINE]


536. Antimicrob Agents Chemother. 2010 Jun;54(6):2666-9. doi: 10.1128/AAC.01743-09.
Epub 2010 Apr 5.

Discovery of a gene conferring multiple-aminoglycoside resistance in Escherichia 
coli.

Davis MA(1), Baker KN, Orfe LH, Shah DH, Besser TE, Call DR.

Author information: 
(1)Department of Veterinary Microbiology and Pathology, Washington State
University, Pullman, WA 99164-7040, USA. madavis@vetmed.wsu.edu

Bovine-origin Escherichia coli isolates were tested for resistance phenotypes
using a disk diffusion assay and for resistance genotypes using a DNA microarray.
An isolate with gentamicin and amikacin resistance but with no corresponding
genes detected yielded a 1,056-bp DNA sequence with the closest homologues for
its inferred protein sequence among a family of 16S rRNA methyltransferase
enzymes. These enzymes confer high-level aminoglycoside resistance and have only 
recently been described in Gram-negative bacteria.

DOI: 10.1128/AAC.01743-09 
PMCID: PMC2876372
PMID: 20368404  [Indexed for MEDLINE]


537. DNA Res. 2010 Jun;17(3):185-96. doi: 10.1093/dnares/dsq012. Epub 2010 May 7.

Relative codon adaptation: a generic codon bias index for prediction of gene
expression.

Fox JM(1), Erill I.

Author information: 
(1)Department of Biological Sciences, University of Maryland Baltimore County
(UMBC), 1000 Hilltop Road, Baltimore, MD 21228, USA.

The development of codon bias indices (CBIs) remains an active field of research 
due to their myriad applications in computational biology. Recently, the relative
codon usage bias (RCBS) was introduced as a novel CBI able to estimate codon bias
without using a reference set. The results of this new index when applied to
Escherichia coli and Saccharomyces cerevisiae led the authors of the original
publications to conclude that natural selection favours higher expression and
enhanced codon usage optimization in short genes. Here, we show that this
conclusion was flawed and based on the systematic oversight of an intrinsic bias 
for short sequences in the RCBS index and of biases in the small data sets used
for validation in E. coli. Furthermore, we reveal that how the RCBS can be
corrected to produce useful results and how its underlying principle, which we
here term relative codon adaptation (RCA), can be made into a powerful
reference-set-based index that directly takes into account the genomic base
composition. Finally, we show that RCA outperforms the codon adaptation index
(CAI) as a predictor of gene expression when operating on the CAI reference set
and that this improvement is significantly larger when analysing genomes with
high mutational bias.

DOI: 10.1093/dnares/dsq012 
PMCID: PMC2885275
PMID: 20453079  [Indexed for MEDLINE]


538. Infect Immun. 2010 Jun;78(6):2454-65. doi: 10.1128/IAI.01341-09. Epub 2010 Mar
29.

Global transcriptional response of macrophage-like THP-1 cells to Shiga toxin
type 1.

Leyva-Illades D(1), Cherla RP, Galindo CL, Chopra AK, Tesh VL.

Author information: 
(1)Department of Microbial and Molecular Pathogenesis, 407 Reynolds Medical
Building, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.

Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens
Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause
bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to
studying the capacity of Stxs to alter gene expression in intoxicated cells have 
been limited to individual genes. However, it is known that many of the signaling
pathways activated by Stxs regulate the expression of multiple genes in mammalian
cells. To expand the scope of analysis of gene expression and to better
understand the underlying mechanisms for the various effects of Stxs on host cell
functions, we carried out comparative microarray analyses to characterize the
global transcriptional response of human macrophage-like THP-1 cells to Shiga
toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a
rigorous combinatorial approach with three separate statistical algorithms. A
total of 36 genes met the criteria of upregulated expression in response to Stx1 
treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were
validated by real-time reverse transcriptase PCR for genes encoding early growth 
response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2;
inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10
(regulation of mitogen-activated protein kinase signaling). Stx1-mediated
signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to 
be involved in the increased expression and production of the proinflammatory
mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the 
increased production of proinflammatory and vasoactive eicosanoids. However, the 
capacity of Stx1 to increase the expression of genes encoding phosphatases
suggests that mechanisms to dampen the macrophage proinflammatory response may be
built into host response to the toxins.

DOI: 10.1128/IAI.01341-09 
PMCID: PMC2876531
PMID: 20351145  [Indexed for MEDLINE]


539. Int J Antimicrob Agents. 2010 Jun;35(6):593-8. doi:
10.1016/j.ijantimicag.2010.02.011. Epub 2010 Mar 30.

Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial
resistance and virulence factor genes in clinical isolates of Enterobacteriaceae 
and non-Enterobacteriaceae.

Walsh F(1), Cooke NM, Smith SG, Moran GP, Cooke FJ, Ivens A, Wain J, Rogers TR.

Author information: 
(1)Department of Clinical Microbiology, Sir Patrick Dun Translational Research
Laboratory, School of Medicine, University of Dublin, Trinity College, St James's
Hospital Campus, Dublin 8, Ireland. fiona1walsh@gmail.com

A DNA microarray was developed to detect plasmid-mediated antimicrobial
resistance (AR) and virulence factor (VF) genes in clinical isolates of
Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the
following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); 
Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1);
Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and
Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were
identified by polymerase chain reaction (PCR). The DNA microarray consisted of
155 and 133 AR and VF gene probes, respectively. Results were compared with the
commercially available Identibac AMR-ve Array Tube. Hybridisation results
indicated that there was excellent correlation between PCR and array results for 
AR and VF genes. Genes conferring resistance to each antibiotic class were
identified by the DNA array. Unusual resistance genes were also identified, such 
as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The
phylogenetic group of each E. coli isolate was verified by the array. These data 
demonstrate that it is possible to screen simultaneously for all important
classes of mobile AR and VF genes in Enterobacteriaceae and
non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E.
coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria 
for all known AR genes and to provide important information regarding
pathogenicity simultaneously.

2010 Elsevier B.V. and the International Society of Chemotherapy. All rights
reserved.

DOI: 10.1016/j.ijantimicag.2010.02.011 
PMCID: PMC3820026
PMID: 20356716  [Indexed for MEDLINE]


540. J Antimicrob Chemother. 2010 Jun;65(6):1171-7. doi: 10.1093/jac/dkq114. Epub 2010
Apr 21.

Signature gene expression profile of triclosan-resistant Escherichia coli.

Yu BJ(1), Kim JA, Pan JG.

Author information: 
(1)Systems Microbiology Research Center, Korea Research Institute of Bioscience
and Biotechnology, Daejeon 305-806, Korea.

OBJECTIVES: To gain further insight into the defence mechanisms against triclosan
in a mutant derived from an Escherichia coli strain carrying the
triclosan-resistant target enzyme, FabI(G93V).
METHODS: An E. coli imp4231 FabI(G93V) strain was constructed by replacing intact
fabI with a linear DNA cassette, fabI(G93V)-CmR, that contains a single mutation,
GGT to GTT, at codon 93 of fabI(G93V) and a chloramphenicol resistance gene (CmR)
as a marker for the mutant allele by a Red-mediated recombination system. Using
this E. coli imp4231 FabI(G93V) strain, nitrosoguanidine (NTG) mutagenesis was
performed to generate E. coli IFNs [imp4231 FabI(G93V) treated with NTG]
displaying higher MICs of triclosan than its parent strain. The genes
overexpressed in E. coli IFN4 were identified by DNA microarray analysis.
RESULTS: An E. coli imp4231 FabI(G93V) strain displays approximately 400-fold
increased MICs of triclosan (MIC approximately 8 mg/L) compared with the parent
strain (MIC approximately 0.02 mg/L). Furthermore, E. coli IFN4 has the highest
MIC of triclosan (MIC approximately 80 mg/L). DNA microarray analysis of E. coli 
IFN4 shows that many genes involved in the biosynthesis of membrane proteins,
including transporters, reductases/dehydrogenases and stress response regulators,
were highly expressed in the mutant.
CONCLUSIONS: These results strongly indicate that E. coli IFN cells might protect
themselves from triclosan by activating various defence mechanisms, such as (i)
changing efflux activities; (ii) capturing the triclosan; and (iii) increasing
the expression of important regulators or metabolic enzymes.

DOI: 10.1093/jac/dkq114 
PMID: 20410062  [Indexed for MEDLINE]


541. J Appl Microbiol. 2010 Jun;108(6):2088-102. doi:
10.1111/j.1365-2672.2009.04611.x. Epub 2009 Oct 30.

Identification of stress-related proteins in Escherichia coli using the pollutant
cis-dichloroethylene.

Lee J(1), Hiibel SR, Reardon KF, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A & M University,
College Station, TX 77843-3122, USA.

AIMS: To complement our proteome study, whole-transcriptome analyses were
utilized here to identify proteins related to degrading cis-1,2-dichloroethylene 
(cis-DCE).
METHODS AND RESULTS: Metabolically engineered Escherichia coli strains were
utilized expressing an evolved toluene ortho-monooxygenase along with either (i) 
glutathione S-transferase and altered gamma-glutamylcysteine synthetase or (ii) a
rationally engineered epoxide hydrolase. cis-DCE degradation induced 30 known
stress genes and 32 uncharacterized genes. Because of the reactive cis-DCE
epoxides formed, we hypothesized that some of these uncharacterized genes may be 
related to a variety of stresses. Using isogenic mutants, IbpB, YchH, YdeI, YeaR,
YgiW, YoaG and YodD were related to hydrogen peroxide, cadmium and acid stress.
Additional whole-transcriptome studies with hydrogen peroxide stress using the
most hydrogen peroxide-sensitive mutants, ygiW and ychH, identified that FliS,
GalS, HcaR, MglA, SufE, SufS, Tap, TnaB, YhcN and YjaA are also involved in the
stress response of E. coli to hydrogen peroxide, cadmium and acid, as well as are
involved in biofilm formation.
CONCLUSION: Seventeen proteins are involved in the stress network for this
organism, and YhcN and YchH were shown to be important for the degradation of
cis-DCE.
SIGNIFICANCE AND IMPACT OF THE STUDY: Six previously uncharacterized proteins
(YchH, YdeI, YgiW, YhcN, YjaA and YodD) were shown to be stress proteins.

DOI: 10.1111/j.1365-2672.2009.04611.x 
PMID: 19919618  [Indexed for MEDLINE]


542. Mol Biosyst. 2010 Jun;6(6):988-98. doi: 10.1039/b917571g. Epub 2010 Feb 19.

Inferring large-scale gene regulatory networks using a low-order constraint-based
algorithm.

Wang M(1), Augusto Benedito V, Xuechun Zhao P, Udvardi M.

Author information: 
(1)Plant Biology Division, The Samuel Roberts Noble Foundation, Inc., 2510 Sam
Noble Parkway, Ardmore, OK 73401, USA.

Recently, simplified graphical modeling approaches based on low-order conditional
(in-)dependence calculations have received attention because of their potential
to model gene regulatory networks. Such methods are able to reconstruct
large-scale gene networks with a small number of experimental measurements, at
minimal computational cost. However, unlike Bayesian networks, current low-order 
graphical models provide no means to distinguish between cause and effect in gene
regulatory relationships. To address this problem, we developed a low-order
constraint-based algorithm for gene regulatory network inference. The method is
capable of inferring causal directions using limited-order conditional
independence tests and provides a computationally-feasible way to analyze
high-dimensional datasets while maintaining high reliability. To assess the
performance of our algorithm, we compared it to several existing graphical
models: relevance networks; graphical Gaussian models; ARACNE; Bayesian networks;
and the classical constraint-based algorithm, using realistic synthetic datasets.
Furthermore, we applied our algorithm to real microarray data from Escherichia
coli Affymetrix arrays and validated the results by comparison to known
regulatory interactions collected in RegulonDB. The algorithm was found to be
both effective and efficient at reconstructing gene regulatory networks from
microarray data.

DOI: 10.1039/b917571g 
PMID: 20485743  [Indexed for MEDLINE]


543. Nucleic Acids Res. 2010 Jun;38(11):3794-808. doi: 10.1093/nar/gkq032. Epub 2010
Mar 26.

Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in
antisense transcripts.

Lorenz C(1), Gesell T, Zimmermann B, Schoeberl U, Bilusic I, Rajkowitsch L,
Waldsich C, von Haeseler A, Schroeder R.

Author information: 
(1)Department of Biochemistry, Medical University of Vienna and University of
Veterinary Medicine, Vienna, Austria.

An unexpectedly high number of regulatory RNAs have been recently discovered that
fine-tune the function of genes at all levels of expression. We employed Genomic 
SELEX, a method to identify protein-binding RNAs encoded in the genome, to search
for further regulatory RNAs in Escherichia coli. We used the global regulator
protein Hfq as bait, because it can interact with a large number of RNAs,
promoting their interaction. The enriched SELEX pool was subjected to deep
sequencing, and 8865 sequences were mapped to the E. coli genome. These short
sequences represent genomic Hfq-aptamers and are part of potential regulatory
elements within RNA molecules. The motif 5'-AAYAAYAA-3' was enriched in the
selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed 
to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the
antisense strand of protein coding genes than on the sense strand. They were
enriched opposite to translation start sites or opposite to intervening sequences
between ORFs in operons. These results expand the repertoire of Hfq targets and
also suggest that Hfq might regulate the expression of a large number of genes
via interaction with cis-antisense RNAs.

DOI: 10.1093/nar/gkq032 
PMCID: PMC2887942
PMID: 20348540  [Indexed for MEDLINE]


544. Nucleic Acids Res. 2010 Jun;38(11):3605-18. doi: 10.1093/nar/gkq077. Epub 2010
Feb 15.

A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth
conditions.

Teramoto J(1), Yoshimura SH, Takeyasu K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan.

A systematic search was performed for DNA-binding sequences of YgiP, an
uncharacterized transcription factor of Escherichia coli, by using the Genomic
SELEX. A total of 688 YgiP-binding loci were identified after genome-wide
profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel
shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites
along DNA probes with a consensus GTTNATT sequence. Atomic force microscope
observation indicated that at low concentrations, YgiP associates at various
sites on DNA probes, but at high concentrations, YgiP covers the entire DNA
surface supposedly through protein-protein contact. The intracellular
concentration of YgiP is very low in growing E. coli cells under aerobic
conditions, but increases more than 100-fold to the level as high as the major
nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP
showed retarded growth under anaerobic conditions. High abundance and large
number of binding sites together indicate that YgiP is a nucleoid-associated
protein with both architectural and regulatory roles as the nucleoid proteins Fis
and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under
anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic
conditions).

DOI: 10.1093/nar/gkq077 
PMCID: PMC2887951
PMID: 20156994  [Indexed for MEDLINE]


545. Vet Microbiol. 2010 May 19;142(3-4):373-8. doi: 10.1016/j.vetmic.2009.10.019.
Epub 2009 Oct 30.

Development of a serogroup-specific DNA microarray for identification of
Escherichia coli strains associated with bovine septicemia and diarrhea.

Liu B(1), Wu F, Li D, Beutin L, Chen M, Cao B, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA,
Tianjin, PR China.

Escherichia coli strains belonging to serogroups O8, O9, O15, O26, O35, O78, O86,
O101, O115 and O119 are commonly associated with septicemia or diarrhea in calves
and pose a significant threat to the cattle industry worldwide. In this study, a 
microarray detection system targeting O-antigen-specific genes was developed for 
the identification of those serogroups. By testing against 186 E. coli and
Shigella O-serogroup reference strains, 36 E. coli clinical isolates, and 9
representative strains of other closely related bacterial species, the microarray
was shown to be specific and reproducible. The detection sensitivity was
determined to be 50 ng genomic DNA. The microarray assay developed here is
suitable for the detection and identification of relevant strains from
environmental and/or clinical samples, and is especially useful for epidemiologic
studies.

Copyright 2009 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.vetmic.2009.10.019 
PMID: 19932572  [Indexed for MEDLINE]


546. DNA Repair (Amst). 2010 May 4;9(5):567-78. doi: 10.1016/j.dnarep.2010.02.009.
Epub 2010 Mar 12.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

Siaud N(1), Dubois E, Massot S, Richaud A, Dray E, Collier J, Doutriaux MP.

Author information: 
(1)Institut de Biologie des Plantes, CNRS UMR8618, Bâtiment 630, Université Paris
Sud 11, 91405 Orsay Cedex, France. nicolas.siaud@u-psud.fr

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup
with the aim of identifying Arabidopsis functions that might potentially be
involved in the DNA metabolism. Such functions, when expressed in bacteria, are
prone to disturb replication and thus trigger the SOS response. Consistently,
expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even
affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA
library constructed from an Arabidopsis cell suspension that was found to highly 
express meiotic genes. A large proportion of these SOS(+) candidates are clearly 
related to the DNA metabolism, others could be involved in the RNA metabolism,
while the remaining cDNAs encode either totally unknown proteins or proteins that
were considered as irrelevant. Seven SOS(+) candidate genes are induced following
gamma irradiation. The in planta function of several of the SOS-inducing clones
was investigated using T-DNA insertional mutants or RNA interference. Only one
SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced
plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the
leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is
essential to prevent uracil incorporation in the course of DNA replication.

(c) 2010 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.dnarep.2010.02.009 
PMID: 20227352  [Indexed for MEDLINE]


547. Am J Obstet Gynecol. 2010 May;202(5):471.e1-11. doi: 10.1016/j.ajog.2010.02.031.

Innate inflammatory responses of human decidual cells to periodontopathic
bacteria.

Keelan JA(1), Wong PM, Bird PS, Mitchell MD.

Author information: 
(1)School of Women's and Infants' Health, University of Western Australia, Perth,
WA, Australia.

OBJECTIVE: The purpose of this study was to test the hypothesis that
periodontopathic bacteria exert potent proinflammatory effects in human decidua.
STUDY DESIGN: The immunostimulatory effects of Gram-positive and negative
periodontopathic bacteria and their lipopolysaccharides were tested in human
decidual cell cultures in comparison with Escherichia coli. Cytokine production
was measured by enzyme-linked immunosorbent assay; inflammatory gene expression
was measured by oligonucleotide arrays and quantitative real time-polymerase
chain reaction.
RESULTS: All bacteria that were tested elicited an inflammatory response,
although concentration-dependence and efficacy varied considerably with organism 
and culture. Lipopolysaccharides were more potent stimuli than intact bacterial
cells, although bacteria exerted greater effects at high concentrations. Of 112
genes on the arrays, 18 genes were stimulated significantly by one or more
lipopolysaccharide preparation.
CONCLUSION: The ability of periodontopathic bacteria to stimulate a decidual
inflammatory response is highly variable and partly dependent on the presence and
structure of constituent lipopolysaccharides. This adds to our understanding of
the causal association between periodontal disease and preterm birth.

Crown Copyright (c) 2010. Published by Mosby, Inc. All rights reserved.

DOI: 10.1016/j.ajog.2010.02.031 
PMID: 20452492  [Indexed for MEDLINE]


548. Biotechnol Lett. 2010 May;32(5):681-8. doi: 10.1007/s10529-010-0205-0. Epub 2010 
Apr 3.

Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli
using two-step ultra-rapid real-time PCR.

Kim IW(1), Kang MH, Kwon SH, Cho SH, Yoo BS, Han SH, Yoon BS.

Author information: 
(1)Department of Life Science, College of Natural Science, Kyonggi University,
Suwon, 443-760, Korea.

A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has
the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time
(URRT) PCR. URRT PCR was designed to detect the stx2 gene using a
microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6
microl total reaction volume with an extremely short denaturation step and
combined annealing/extension step (1 and 3 s, respectively) for each cycle.
Specific primers for the stx2 gene were designed to amplify a 100 bp region known
for genetic stability among the various EHEC strains. Using the URRT PCR method, 
stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis.
The detection limit for the stx2 gene for URRT-PCR was estimated to be 3
c.f.u./PCR with the amplification product having a consistent Tm of 85.2 +/- 0.4 
degrees C. This method was tested for the various applications relevant to the
different EHEC strains and was useful for the rapid detection of stx2-carrying
EHEC strains.

DOI: 10.1007/s10529-010-0205-0 
PMID: 20364295  [Indexed for MEDLINE]


549. Environ Microbiol. 2010 May;12(5):1105-21. doi: 10.1111/j.1462-2920.2009.02147.x.
Epub 2010 Jan 26.

Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD.

Kim Y(1), Wang X, Zhang XS, Grigoriu S, Page R, Peti W, Wood TK.

Author information: 
(1)Department of Chemical Engineering, Texas A&M University, College Station, TX 
77843-3122, USA.

Previously we identified that the Escherichia coli protein MqsR (YgiU) functions 
as a toxin and that it is involved in the regulation of motility by quorum
sensing signal autoinducer-2 (AI-2). Furthermore, MqsR is directly associated
with biofilm development and is linked to the development of persister cells.
Here we show that MqsR and MqsA (YgiT) are a toxin/antitoxin (TA) pair, which, in
significant difference to other TA pairs, regulates additional loci besides its
own. We have recently identified that MqsR functions as an RNase. However, using 
three sets of whole-transcriptome studies and two nickel-enrichment DNA binding
microarrays coupled with cell survival studies in which MqsR was overproduced in 
isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB,
relE and hokA) that are involved in a mode of MqsR toxicity in addition to its
RNase activity. Quantitative real-time reverse transcription polymerase chain
reaction (qRT-PCR) showed that (i) the MqsR/MqsA complex (and MqsA alone)
represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii)
stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are
overproduced or when mqsRA is deleted. Electrophoretic mobility shift assays show
that the MqsA/MqsR complex binds the promoter of cspD. In addition, proteases Lon
and ClpXP are necessary for MqsR toxicity. Together, these results indicate the
MqsR/MqsA complex represses cspD which may be derepressed by titrating MqsA with 
MqsR or by degrading MqsA via stress conditions through proteases Lon and ClpXP. 
Hence, we demonstrate that the MqsR/MqsA TA system controls cell physiology via
its own toxicity as well as through its regulation of another toxin, CspD.

DOI: 10.1111/j.1462-2920.2009.02147.x 
PMCID: PMC3980499
PMID: 20105222  [Indexed for MEDLINE]


550. J Clin Microbiol. 2010 May;48(5):1578-83. doi: 10.1128/JCM.01130-09. Epub 2010
Mar 17.

Development and evaluation of oligonucleotide chip based on the 16S-23S rRNA gene
spacer region for detection of pathogenic microorganisms associated with sepsis.

Kim CM(1), Song ES, Jang HJ, Kim HJ, Lee S, Shin JH, Kim SJ, Jeong SH, Jeong J,
Koh K, Choi GE, Lee EY, Chang CL.

Author information: 
(1)Department of Biochemistry, School of Medicine, College of Nano Science and
Nano Technology, Joint Research Center of Pusan National University-Fraunhofer
IGB Institute, Busan, South Korea.

Oligonucleotide chips targeting the bacterial internal transcribed spacer region 
(ITS) of the 16S-23S rRNA gene, which contains genus- and species-specific
regions, were developed and evaluated. Forty-three sequences were designed
consisting of 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30
species-specific probes. The specificity of the probes was confirmed using
bacterial type strains including 54 of 52 species belonging to 18 genera. The
performance of the probes was evaluated using 825 consecutive samples that were
positive by blood culture in broth medium. Among the 825 clinical specimens, 708 
(85.8%) were identified correctly by the oligonucleotide chip. Most (536
isolates, or 75.7%) were identified as staphylococci, Escherichia coli, or
Klebsiella pneumoniae. Thirty-seven isolates (4.5%) did not bind to the
corresponding specific probes. Most of these also were staphylococci, E. coli, or
K. pneumoniae and accounted for 6.3% of total number of the species. Sixty-two
specimens (7.5%) did not bind the genus- or species-specific probes because of
lack of corresponding specific probes. Among them, Acinetobacter baumannii was
the single most frequent isolate (26/62). The oligonucleotide chip was highly
specific and sensitive in detecting the causative agents of bacteremia directly
from positive blood cultures.

DOI: 10.1128/JCM.01130-09 
PMCID: PMC2863927
PMID: 20237100  [Indexed for MEDLINE]


551. J Clin Periodontol. 2010 May;37(5):412-8. doi: 10.1111/j.1600-051X.2010.01548.x.

Triclosan inhibition of acute and chronic inflammatory gene pathways.

Barros SP(1), Wirojchanasak S, Barrow DA, Panagakos FS, Devizio W, Offenbacher S.

Author information: 
(1)School of Dentistry, Center for Oral and Systemic Diseases, North Carolina
Oral Health Institute, University of North Carolina at Chapel Hill, Chapel Hill, 
NC 27709, USA. silvana_barros@dentistry.unc.edu

AIM: We sought to determine whether triclosan
(2,4,4'-trichloro-2'-hydroxydiphenylether), an extensively used anti-plaque agent
with broad-spectrum anti-microbial activity, with reported anti-inflammatory
effects via inhibition of prostaglandin E2 and interleukin 1 (IL-1)beta, could
also more broadly suppress multiple inflammatory gene pathways responsible for
the pathogenesis of gingivitis and periodontitis.
MATERIALS AND METHODS: As an exploratory study, the effects of triclosan on the
inflammatory gene expression profile were assessed ex vivo using peripheral whole
blood samples from eight periodontally healthy donors. Ten-millilitres whole
blood aliquots were incubated 2 h with 0.3 microg/ml Escherichia coli
lipopolysaccharide (LPS) with or without 0.5 microg/ml triclosan. Affymetrix
microarray gene expression profiles from isolated leucocytes and pathway-specific
quantitative polymerase chain reaction arrays were used to investigate changes in
expression of target cytokines and cell signalling molecules.
RESULTS: Ex vivo human whole blood assays indicated that triclosan significantly 
down-regulated the LPS-stimulated expression of Toll-like receptor signalling
molecules and other multiple inflammatory molecules including IL-1 and IL-6 and
the dampening of signals that activate the T-helper type 1 acquired immune
response via suppression of CD70 with concomitant up-regulation of growth factors
related to bone morphogenetic protein (BMP)2 and BMP6 synthesis.
CONCLUSIONS: Anti-inflammatory effects were found in this exploratory survey,
including suppression of microbial-pathogen recognition pathway molecules and the
suppression of acute and chronic mediators of inflammation.

DOI: 10.1111/j.1600-051X.2010.01548.x 
PMID: 20507366  [Indexed for MEDLINE]


552. J Microbiol Methods. 2010 May;81(2):96-100. doi: 10.1016/j.mimet.2010.01.024.
Epub 2010 Feb 4.

An oligonucleotide microarray to characterize multidrug resistant plasmids.

Lindsey RL(1), Frye JG, Fedorka-Cray PJ, Welch TJ, Meinersmann RJ.

Author information: 
(1)U.S. Department of Agriculture, Agricultural Research Service, Bacterial
Epidemiology and Antimicrobial Resistance Research Unit, Richard B. Russell
Agricultural Research Center, 950 College Station Road, Athens, GA 30604-2720,
United States.

Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on
large plasmids of replicon type Inc A/C and Inc H1. It is important to understand
the transmission of these MDR plasmids because the genes they carry can affect
the outcome of antimicrobial therapy. The aim of this study was to design a
microarray with oligonucleotide probes for every gene in the six Inc A/C and one 
Inc H1 plasmids of interest while representing all redundant sequences only once.
The microarray is printed in triplicate with 493 unique oligonucleotide probes 70
nucleotides in length. Salmonella enterica and Escherichia coli control strains
and test plasmids (in the parent strain and transformed into a known E. coli
background strain) were hybridized to the plasmid microarray. This hybridization 
arrays presents a rapid and cost effective method for high-density screening of
isolates to evaluate the gene content of Inc A/C and H1 plasmids and will show
how plasmids can change content with transmission.

Published by Elsevier B.V.

DOI: 10.1016/j.mimet.2010.01.024 
PMID: 20138094  [Indexed for MEDLINE]


553. Biotechnol Bioeng. 2010 Apr 15;105(6):1131-47. doi: 10.1002/bit.22628.

Metabolite stress and tolerance in the production of biofuels and chemicals:
gene-expression-based systems analysis of butanol, butyrate, and acetate stresses
in the anaerobe Clostridium acetobutylicum.

Alsaker KV(1), Paredes C, Papoutsakis ET.

Author information: 
(1)Department of Chemical and Biological Engineering, Northwestern University,
Evanston, IL, USA.

Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell
physiology, but such effects are not well understood at the molecular level.
Cells respond and adapt to metabolite stress by mechanisms largely unexplored,
especially in the context of multiple and simultaneous stresses. Solventogenic
and related clostridia have an inherent advantage for production of biofuels and 
chemicals directly from cellulosic material and other complex carbohydrates, but 
issues of product/metabolite tolerance and related culture productivities remain.
Using DNA microarray-based gene expression analysis, the transcriptional-stress
responses of Clostridium acetobutylicum to fermentation acids acetate and
butyrate and the solvent product butanol were analyzed and compared in the
context of cell physiology. Ontological analysis demonstrated that stress by all 
three metabolites resulted in upregulation of genes related to post-translational
modifications and chaperone activity, and downregulation of the
translation-machinery genes. Motility genes were downregulated by acetate-stress 
only. The general metabolite stress included upregulation of numerous stress
genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic
operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated
expression of the butyryl-CoA- and butyrate-formation genes, while butyrate
stress downregulated expression of acetate-formation genes.
Pyrimidine-biosynthesis genes were downregulated by most stresses, but
purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for 
thiamine and histidine biosynthesis. Methionine-biosynthesis genes were
upregulated by acetate stress, indicating a possibly conserved stress response
mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression
was upregulated by acetate stress. Butyrate stress upregulated many
iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related
to cellular repair from oxidative stress, such as perR and superoxide dismutases.
Butanol stress upregulated the glycerol metabolism genes glpA and glpF.
Surprisingly, metabolite stress had no apparent effect on the expression of the
sporulation-cascade genes. It is argued that the list of upregulated genes in
response to the three metabolite stresses includes several genes whose
overexpression would likely impart tolerance, thus making the information
generated in this study, a valuable source for the development of tolerant
recombinant strains.

(c) 2009 Wiley Periodicals, Inc.

DOI: 10.1002/bit.22628 
PMID: 19998280  [Indexed for MEDLINE]


554. J Biol Chem. 2010 Apr 2;285(14):10690-702. doi: 10.1074/jbc.M109.089755. Epub
2010 Jan 14.

Translational regulation of gene expression by an anaerobically induced small
non-coding RNA in Escherichia coli.

Boysen A(1), Møller-Jensen J, Kallipolitis B, Valentin-Hansen P, Overgaard M.

Author information: 
(1)Department of Biochemistry and Molecular Biology, University of Southern
Denmark, 5230 Odense M, Denmark.

Small non-coding RNAs (sRNA) have emerged as important elements of gene
regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella
many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to
mammalian Sm-like proteins and act in the post-transcriptional regulation of many
genes. A number of these highly conserved ribo-regulators are stringently
regulated at the level of transcription and are part of major regulons that deal 
with the immediate response to various stress conditions, indicating that every
major transcription factor may control the expression of at least one sRNA
regulator. Here, we extend this view by the identification and characterization
of a highly conserved, anaerobically induced small sRNA in E. coli, whose
expression is strictly dependent on the anaerobic transcriptional fumarate and
nitrate reductase regulator (FNR). The sRNA, named FnrS, possesses signatures of 
base-pairing RNAs, and we show by employing global proteomic and transcriptomic
profiling that the expression of multiple genes is negatively regulated by the
sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions
or enzymes linked to oxidative stress. Furthermore, in previous work most of the 
potential target genes have been shown to be repressed by FNR through an
undetermined mechanism. Collectively, our results provide insight into the
mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and 
metE, thereby demonstrating that adaptation to anaerobic growth involves the
action of a small regulatory RNA.

DOI: 10.1074/jbc.M109.089755 
PMCID: PMC2856277
PMID: 20075074  [Indexed for MEDLINE]


555. Biomed Microdevices. 2010 Apr;12(2):353-9. doi: 10.1007/s10544-009-9391-8.

An integrated disposable device for DNA extraction and helicase dependent
amplification.

Mahalanabis M(1), Do J, ALMuayad H, Zhang JY, Klapperich CM.

Author information: 
(1)Department of Biomedical Engineering, Boston University, Boston, MA 02215,
USA.

Erratum in
    Biomed Microdevices. 2011 Jun;13(3):599-602.

Here we report the demonstration of an integrated microfluidic chip that performs
helicase dependent amplification (HDA) on samples containing live bacteria.
Combined chip-based sample preparation and isothermal amplification are
attractive for world health applications, since the need for instrumentation to
control flow rate and temperature changes are reduced or eliminated. Bacteria
lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter
are incorporated into a disposable polymer cartridge format. Smart passive
fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE
membrane) with a simple on-chip mixer eliminates multiple user operations. The
device is able to detect as few as ten colony forming units (CFU) of E. coli in
growth medium.

DOI: 10.1007/s10544-009-9391-8 
PMCID: PMC2998058
PMID: 20066496  [Indexed for MEDLINE]


556. Mol Cell Probes. 2010 Apr;24(2):77-86. doi: 10.1016/j.mcp.2009.10.005. Epub 2009 
Oct 13.

Development of an oligonucleotide-based microarray to detect multiple foodborne
pathogens.

Suo B(1), He Y, Paoli G, Gehring A, Tu SI, Shi X.

Author information: 
(1)Joint Sino-US Food Safety Research Center & Bor Luh Food Safety Center, School
of Agriculture & Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd.,
Shanghai 200240, China.

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and
Campylobacter jejuni are considered important pathogens causing the most
food-related human illnesses worldwide. Current methods for pathogen detection
have limitations in the effectiveness of identifying multiple foodborne
pathogens. In this study, a pathogen detection microarray was developed using
various 70-mer oligonucleotides specifically targeting the above pathogens. To
reduce the cost of detection, each microarray chip was designed and fabricated to
accommodate 12 identical arrays which could be used for screening up to 12
different samples. To achieve high detection sensitivity and specificity,
target-specific DNA amplification instead of whole genome random amplification
was used prior to microarray analysis. Combined with 14-plex PCR amplification of
target sequences, the microarray unambiguously distinguished all 4 pathogens with
a detection sensitivity of 1 x 10(-4) ng (approximately 20 copies) of each
genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to
be contaminated by either 1 or 2 of these pathogens. The co-occurrences of
Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat 
samples were also observed. Overall, the microarray combined with multiplex PCR
method was able to effectively screen single or multiple pathogens in food
samples and to provide important genotypic information related to pathogen
virulence.

DOI: 10.1016/j.mcp.2009.10.005 
PMID: 19833198  [Indexed for MEDLINE]


557. BMC Bioinformatics. 2010 Mar 25;11:154. doi: 10.1186/1471-2105-11-154.

TimeDelay-ARACNE: Reverse engineering of gene networks from time-course data by
an information theoretic approach.

Zoppoli P(1), Morganella S, Ceccarelli M.

Author information: 
(1)Department of Biological and Environmental Studies, University of Sannio,
Benevento, I-82100, Italy.

BACKGROUND: One of main aims of Molecular Biology is the gain of knowledge about 
how molecular components interact each other and to understand gene function
regulations. Using microarray technology, it is possible to extract measurements 
of thousands of genes into a single analysis step having a picture of the cell
gene expression. Several methods have been developed to infer gene networks from 
steady-state data, much less literature is produced about time-course data, so
the development of algorithms to infer gene networks from time-series
measurements is a current challenge into bioinformatics research area. In order
to detect dependencies between genes at different time delays, we propose an
approach to infer gene regulatory networks from time-series measurements starting
from a well known algorithm based on information theory.
RESULTS: In this paper we show how the ARACNE (Algorithm for the Reconstruction
of Accurate Cellular Networks) algorithm can be used for gene regulatory network 
inference in the case of time-course expression profiles. The resulting method is
called TimeDelay-ARACNE. It just tries to extract dependencies between two genes 
at different time delays, providing a measure of these dependencies in terms of
mutual information. The basic idea of the proposed algorithm is to detect
time-delayed dependencies between the expression profiles by assuming as
underlying probabilistic model a stationary Markov Random Field. Less informative
dependencies are filtered out using an auto calculated threshold, retaining most 
reliable connections. TimeDelay-ARACNE can infer small local networks of time
regulated gene-gene interactions detecting their versus and also discovering
cyclic interactions also when only a medium-small number of measurements are
available. We test the algorithm both on synthetic networks and on microarray
expression profiles. Microarray measurements concern S. cerevisiae cell cycle, E.
coli SOS pathways and a recently developed network for in vivo assessment of
reverse engineering algorithms. Our results are compared with ARACNE itself and
with the ones of two previously published algorithms: Dynamic Bayesian Networks
and systems of ODEs, showing that TimeDelay-ARACNE has good accuracy, recall and 
F-score for the network reconstruction task.
CONCLUSIONS: Here we report the adaptation of the ARACNE algorithm to infer gene 
regulatory networks from time-course data, so that, the resulting network is
represented as a directed graph. The proposed algorithm is expected to be useful 
in reconstruction of small biological directed networks from time course data.

DOI: 10.1186/1471-2105-11-154 
PMCID: PMC2862045
PMID: 20338053  [Indexed for MEDLINE]


558. BMC Genomics. 2010 Mar 11;11:165. doi: 10.1186/1471-2164-11-165.

Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising
reduction in the expression of the translation machinery.

Neusser T(1), Polen T, Geissen R, Wagner R.

Author information: 
(1)Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf,
Universitätsstr 1, D-40225 Düsseldorf, Germany.

BACKGROUND: 6S RNA from E. coli is known to bind to RNA polymerase interfering
with transcription initiation. Because 6S RNA concentrations are maximal at
stationary phase and binding occurs preferentially to the holoenzyme associated
with sigma(70) (Esigma(70)) it is believed that 6S RNA supports adjustment to
stationary phase transcription. Previous studies have also suggested that
inhibition is specific for sigma(70)-dependent promoters characterized by a weak 
-35 recognition motif or extended -10 promoters. There are many exceptions to
this precept, showing that other types of promoters, including stationary
phase-specific (sigma(38)-dependent) promoters are inhibited.
RESULTS: To solve this apparent ambiguity and to better understand the role of 6S
RNA in stationary phase transition we have performed a genome-wide
transcriptional analysis of wild-type and 6S RNA deficient cells growing to
mid-log or early stationary phase. We found 245 genes at the exponential growth
phase and 273 genes at the early stationary phase to be > or = 1.5-fold
differentially expressed. Up- and down-regulated genes include many
transcriptional regulators, stress-related proteins, transporters and several
enzymes involved in purine metabolism. As the most striking result during
stationary phase, however, we obtained in the 6S RNA deficient strain a concerted
expression reduction of genes constituting the translational apparatus. In
accordance, primer extension analysis showed that transcription of ribosomal
RNAs, representing the key molecules for ribosome biogenesis, is also
significantly reduced under the same conditions. Consistent with this finding
biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S
RNA is apparently compensated by an increase of the basal ppGpp concentration,
known to affect growth adaptation and ribosome biogenesis.
CONCLUSIONS: The analysis demonstrated that the effect of 6S RNA on transcription
is not strictly confined to sigma(70)-dependent promoters. Moreover, the results 
indicate that 6S RNA is embedded in stationary phase adaptation, which is
governed by the capacity of the translational machinery.

DOI: 10.1186/1471-2164-11-165 
PMCID: PMC2848244
PMID: 20222947  [Indexed for MEDLINE]


559. PLoS One. 2010 Mar 3;5(3):e9513. doi: 10.1371/journal.pone.0009513.

Bayesian network expansion identifies new ROS and biofilm regulators.

Hodges AP(1), Dai D, Xiang Z, Woolf P, Xi C, He Y.

Author information: 
(1)Center for Computational Medicine and Bioinformatics, University of Michigan, 
Ann Arbor, Michigan, United States of America.

Signaling and regulatory pathways that guide gene expression have only been
partially defined for most organisms. However, given the increasing number of
microarray measurements, it may be possible to reconstruct such pathways and
uncover missing connections directly from experimental data. Using a compendium
of microarray gene expression data obtained from Escherichia coli, we constructed
a series of Bayesian network models for the reactive oxygen species (ROS) pathway
as defined by EcoCyc. A consensus Bayesian network model was generated using
those networks sharing the top recovered score. This microarray-based network
only partially agreed with the known ROS pathway curated from the literature and 
databases. A top network was then expanded to predict genes that could enhance
the Bayesian network model using an algorithm we termed 'BN+1'. This expansion
procedure predicted many stress-related genes (e.g., dusB and uspE), and their
possible interactions with other ROS pathway genes. A term enrichment method
discovered that biofilm-associated microarray data usually contained high
expression levels of both uspE and gadX. The predicted involvement of gene uspE
in the ROS pathway and interactions between uspE and gadX were confirmed
experimentally using E. coli reporter strains. Genes gadX and uspE showed a
feedback relationship in regulating each other's expression. Both genes were
verified to regulate biofilm formation through gene knockout experiments. These
data suggest that the BN+1 expansion method can faithfully uncover hidden or
unknown genes for a selected pathway with significant biological roles. The
presently reported BN+1 expansion method is a generalized approach applicable to 
the characterization and expansion of other biological pathways and living
systems.

DOI: 10.1371/journal.pone.0009513 
PMCID: PMC2831072
PMID: 20209085  [Indexed for MEDLINE]


560. Antimicrob Agents Chemother. 2010 Mar;54(3):1218-25. doi: 10.1128/AAC.00944-09.
Epub 2009 Dec 14.

Constitutive SoxS expression in a fluoroquinolone-resistant strain with a
truncated SoxR protein and identification of a new member of the marA-soxS-rob
regulon, mdtG.

Fàbrega A(1), Martin RG, Rosner JL, Tavio MM, Vila J.

Author information: 
(1)Department of Microbiology, Hospital Clínic, School of Medicine, University of
Barcelona, Barcelona, Spain.

Elevated levels of fluoroquinolone resistance are frequently found among
Escherichia coli clinical isolates. This study investigated the antibiotic
resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli
clinical isolate, PS5, to two selection steps with increasing concentrations of
norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in
PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by
Western blotting that NorE5 has a multidrug resistance phenotype resulting from
the overexpression of the antibiotic resistance efflux pump AcrAB-TolC.
Microarray and gene fusion analyses revealed significantly increased expression
in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS
activity is attributable to a frameshift mutation that truncates SoxR, rendering 
it a constitutive transcriptional activator of soxS. Furthermore, microarray and 
reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative
efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob
activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, 
showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox
sequence is in the backward or class I orientation within the promoter, and its
disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus,
chromosomal mutations in parC and soxR are responsible for the increased
antibiotic resistance of NorE5.

DOI: 10.1128/AAC.00944-09 
PMCID: PMC2825980
PMID: 20008776  [Indexed for MEDLINE]


561. Cell Mol Life Sci. 2010 Mar;67(6):965-71. doi: 10.1007/s00018-009-0225-x. Epub
2009 Dec 18.

A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein
immunolocalization.

Frassanito AM(1), Barsanti L, Passarelli V, Evangelista V, Gualtieri P.

Author information: 
(1)Istituto di Biofisica, CNR, Pisa, Italy.

Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora
paradoxa (Glaucophyta). The primers were designed according to the conserved
regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the
GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The
correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like
proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is
characterized by a region with no transmembrane alpha-helices (80 aa), followed
by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region.
The DNA sequence of the N-terminal region was expressed in E. coli and the
recombinant purified peptide was used as antigen in hens to obtain polyclonal
antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked
labeling of the muroplast (aka cyanelle) membrane.

DOI: 10.1007/s00018-009-0225-x 
PMID: 20016996  [Indexed for MEDLINE]


562. Funct Integr Genomics. 2010 Mar;10(1):21-38. doi: 10.1007/s10142-009-0133-z. Epub
2009 Aug 29.

A sentinel function for teat tissues in dairy cows: dominant innate immune
response elements define early response to E. coli mastitis.

Rinaldi M(1), Li RW, Bannerman DD, Daniels KM, Evock-Clover C, Silva MV, Paape
MJ, Van Ryssen B, Burvenich C, Capuco AV.

Author information: 
(1)USDA-ARS, Beltsville, MD 20705, USA.

Escherichia coli intramammary infection elicits localized and systemic responses,
some of which have been characterized in mammary secretory tissue. Our objective 
was to characterize gene expression patterns that become activated in different
regions of the mammary gland during the acute phase of experimentally induced E. 
coli mastitis. Tissues evaluated were from Fürstenburg's rosette, teat cistern
(TC), gland cistern (GC), and lobulo-alveolar (LA) regions of control and
infected mammary glands, 12 and 24 h after bacterial (or control) infusions. The 
main networks activated by E. coli infection pertained to immune and inflammatory
response, with marked induction of genes encoding proteins that function in
chemotaxis and leukocyte activation and signaling. Genomic response at 12 h
post-infection was greatest in tissues of the TC and GC. Only at 24 h
post-infection did tissue from the LA region respond, at which time the response 
was the greatest of all regions. Similar genetic networks were impacted in all
regions during early phases of intramammary infection, although regional
differences throughout the gland were noted. Data support an important sentinel
function for the teat, as these tissues responded rapidly and intensely, with
production of cytokines and antimicrobial peptides.

DOI: 10.1007/s10142-009-0133-z 
PMID: 19727872  [Indexed for MEDLINE]


563. Microb Drug Resist. 2010 Mar;16(1):9-19. doi: 10.1089/mdr.2009.0082.

Development of a DNA microarray to detect antimicrobial resistance genes
identified in the National Center for Biotechnology Information database.

Frye JG(1), Lindsey RL, Rondeau G, Porwollik S, Long F, McClelland M, Jackson CR,
Englen MD, Meinersmann RJ, Berrang ME, Davis JA, Barrett JB, Turpin JB, Thitaram 
SN, Fedorka-Cray PJ.

Author information: 
(1)Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Richard B. 
Russell Research Center, Agriculture Research Service, U.S. Department of
Agriculture, 950 College Station Road, Athens, GA 30605, USA.
jonathan.frye@ars.usda.gov

To understand the mechanisms and epidemiology of antimicrobial resistance (AR),
the genetic elements responsible must be identified. Due to the myriad of
possible genes, a high-density genotyping technique is needed for initial
screening. To achieve this, AR genes in the National Center for Biotechnology
Information GenBank database were identified by their annotations and compiled
into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer 
oligonucelotide probes designed to detect these genes encoding resistances to
aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals,
lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium
compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well
as resistance transfer genes. The microarray was validated with two fully
sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and
Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR 
plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no
resistance genes were detected in LT2. The microarray was also tested with a
variety of bacteria, including MDR Salmonella enterica serovars, Escherichia
coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus
aureus, Listeria spp., and Clostridium difficile. The results presented here
demonstrate that a microarray can be designed to detect virtually all AR genes
found in the National Center for Biotechnology Information database, thus
reducing the subsequent assays necessary to identify specific resistance gene
alleles.

DOI: 10.1089/mdr.2009.0082 
PMCID: PMC3116450
PMID: 19916789  [Indexed for MEDLINE]


564. Microbiology. 2010 Mar;156(Pt 3):719-30. doi: 10.1099/mic.0.032631-0. Epub 2009
Nov 26.

Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid
resistance, and alters locus of enterocyte effacement (LEE) expression in
Escherichia coli O157 : H7.

Riordan JT(1), Tietjen JA, Walsh CW, Gustafson JE, Whittam TS.

Author information: 
(1)Department of Cell Biology, Microbiology and Molecular Biology (CMMB),
University of South Florida, Tampa, FL 33620, USA. riordan@cas.usf.edu

Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance
and virulence genes in many bacterial species. In this study, we report on the
gene expression alterations that follow rpoN inactivation in Escherichia coli
O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the
acid resistance phenotype. Microarray gene expression profiling revealed the
differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This
included the growth-phase-dependent upregulation of genes required for
glutamate-dependent acid resistance (GDAR) ( gadA, gadB, gadC and gadE), and the 
downregulation of locus of enterocyte effacement (LEE) genes, which encode a type
III secretion system. Upregulation of gad genes in SakairpoN : : kan during
exponential growth correlated with increased GDAR and survival in a model stomach
system. Complementation of SakairpoN : : kan with a cloned version of rpoN
restored acid susceptibility. Genes involved in GDAR regulation, including rpoS
(sigma factor 38) and gadE (acid-responsive regulator), were shown to be required
for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes
the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals
RpoN to be an important regulator of stress resistance and virulence genes in E. 
coli O157 : H7.

DOI: 10.1099/mic.0.032631-0 
PMCID: PMC2889430
PMID: 19942657  [Indexed for MEDLINE]


565. Nan Fang Yi Ke Da Xue Xue Bao. 2010 Mar;30(3):417-21.

[Preparation of a 96-microwell plate DNA diagnostic chip for detection of
foodborne bacteria and its application in an incident of food poisoning].

[Article in Chinese]

Mo QH(1), Li Q, Lin JC, Tan H, Tu CN, Ye LQ, Liu ZM, Du J, Sun H, Li SX, Wang S, 
Yang Z.

Author information: 
(1)Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai, China.
mpcr@yahoo.cn

OBJECTIVE: To develop a 96-microwell plate DNA diagnostic chip for simultaneous
detection of 9 major foodborne bacteria.
METHODS: Type-specific PCR primers labeled with biotin and oligonucleotide probes
were designed according to the conservative genes of 9 major foodborne bacteria
Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7 (Stx1 and Stx2),
Shigella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, 
Vibrio cholerae and Vibrio parahaemolyticus. A one-tube multiplex PCR system for 
simultaneous amplification of these bacteria was established, and the DNA probes 
were spotted and immobilized in the wells of the plate in 5x5 array format.
Stable hybridization system between PCR products and oligonucleotide probes in
the microwell was established after condition optimization. Alkaline
phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the
hybridized PCR products.
RESULTS: Twenty standard bacteria strains were used to validate the 96 microwell 
plate DNA diagnostic chip and highly specific and stable experiment results were 
obtained. Using this chip assay, the causal pathogen Staphylococcus aureus was
identified within 12 h after the sampling from an incident of food poisoning, and
the result was consistent with that obtained using conventional bacterial culture
and biochemical identification.
CONCLUSION: The novel 96 microwell plate DNA diagnostic chip allows rapid,
accurate, automated and high-throughput bacterial detection and is especially
valuable for quick response to such public health emergencies as food poisoning.


PMID: 20335099  [Indexed for MEDLINE]


566. Nucleic Acids Res. 2010 Mar;38(5):1636-51. doi: 10.1093/nar/gkp1150. Epub 2009
Dec 14.

Imprecise transcription termination within Escherichia coli greA leader gives
rise to an array of short transcripts, GraL.

Potrykus K(1), Murphy H, Chen X, Epstein JA, Cashel M.

Author information: 
(1)Laboratory of Molecular Genetics, Program in Genomics of Development, Eunice
Kennedy Shriver NICHD, NIH, Bethesda, MD 20892-2785, USA. potrykuk@mail.nih.gov

We report that greA expression is driven by two strong, overlapping P1 and P2
promoters. The P1 promoter is sigma(70)-dependent and P2 is sigma(E)-dependent.
Two-thirds of transcripts terminate within the leader region and the remaining
third comprises greA mRNA. Termination efficiency seems to be unaffected by
growth phase. Two collections of small 40-50 (initiating from P2) and 50-60 nt
(from P1) RNA chains, termed GraL, are demonstrable in vivo and in vitro. We
document that GraL arrays arise from an intrinsic terminator with an 11 bp stem
followed by an AU(7)GCU(2) sequence. Atypical chain termination occurs at
multiple sites; the 3'-ends differ by 1 nt over a range of 10 nt. Transcripts
observed are shown to be insensitive to Gre factors and physically released from 
RNAP-DNA complexes. The abundance of individual chains within each cluster
displays a characteristic pattern, which can be differentially altered by
oligonucleotide probes. Multiple termination sites are particularly sensitive to 
changes at the bottom of the stem. Evolutionarily conserved GraL stem structures 
and fitness assays suggest a biological function for the RNA clusters themselves.
Although GraL overexpression induces >/=3-fold transcriptional changes of over
100 genes, a direct target remains elusive.

DOI: 10.1093/nar/gkp1150 
PMCID: PMC2836576
PMID: 20008510  [Indexed for MEDLINE]


567. Nucleic Acids Res. 2010 Mar;38(4):e26. doi: 10.1093/nar/gkp1090. Epub 2009 Dec 4.

A native chromatin purification system for epigenomic profiling in Caenorhabditis
elegans.

Ooi SL(1), Henikoff JG, Henikoff S.

Author information: 
(1)Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA, USA.

High-resolution mapping of chromatin features has emerged as an important
strategy for understanding gene regulation and epigenetic inheritance. We
describe an in vivo tagging system coupled to chromatin purification for
genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we
coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the
C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a
biotinylation substrate for BirA, in vivo in worms. We found that the fusion
BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to
isolate chromatin under different salt extraction conditions, followed by
affinity purification of biotinylated chromatin with streptavidin and genome-wide
profiling with microarrays. We found that embryonic chromatin is differentially
extracted with increasing salt concentrations. Interestingly, chromatin that
remains insoluble after washing in 600 mM salt is enriched at 5' and 3' ends,
suggesting the presence of large protein complexes that render chromatin
insoluble at transcriptional initiation and termination sites. We also found that
H3.3 landscapes from these salt fractions display consistent features that
correlate with gene activity: the most highly expressed genes contain the most
H3.3. This versatile two-component approach has the potential of facilitating
genome-wide chromatin dynamics and regulatory site identification in C. elegans.

DOI: 10.1093/nar/gkp1090 
PMCID: PMC2831312
PMID: 19966274  [Indexed for MEDLINE]


568. Plant Cell. 2010 Mar;22(3):850-66. doi: 10.1105/tpc.109.073270. Epub 2010 Mar 26.

Genomic and coexpression analyses predict multiple genes involved in triterpene
saponin biosynthesis in Medicago truncatula.

Naoumkina MA(1), Modolo LV, Huhman DV, Urbanczyk-Wochniak E, Tang Y, Sumner LW,
Dixon RA.

Author information: 
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401, USA.

Saponins, an important group of bioactive plant natural products, are glycosides 
of triterpenoid or steroidal aglycones (sapogenins). Saponins possess many
biological activities, including conferring potential health benefits for humans.
However, most of the steps specific for the biosynthesis of triterpene saponins
remain uncharacterized at the molecular level. Here, we use comprehensive gene
expression clustering analysis to identify candidate genes involved in the
elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the
model legume Medicago truncatula. Four candidate uridine diphosphate
glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3)
showed specificity for multiple sapogenins and was confirmed to glucosylate
hederagenin at the C28 position. Genetic loss-of-function studies in M.
truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis.
This report provides a basis for future studies to define genetically the roles
of multiple cytochromes P450 and glycosyltransferases in triterpene saponin
biosynthesis in Medicago.

DOI: 10.1105/tpc.109.073270 
PMCID: PMC2861471
PMID: 20348429  [Indexed for MEDLINE]


569. BMC Genomics. 2010 Feb 25;11:138. doi: 10.1186/1471-2164-11-138.

Escherichia coli infection induces distinct local and systemic transcriptome
responses in the mammary gland.

Mitterhuemer S(1), Petzl W, Krebs S, Mehne D, Klanner A, Wolf E, Zerbe H, Blum H.

Author information: 
(1)Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich,
Feodor-Lynen-Str 25, 81377 Munich, Germany.

BACKGROUND: Coliform bacteria are the most common etiologic agents in severe
mastitis of cows. Escherichia coli infections are mostly restricted to a single
udder quarter whereas neighboring quarters stay clinically inapparent,
implicating the presence of a systemic defense reaction. To address its
underlying mechanism, we performed a transcriptome study of mammary tissue from
udder quarters inoculated with E. coli (6 h and 24 h post infection), from
neighboring quarters of the same animals, and from untreated control animals.
RESULTS: After 6 h 13 probe sets of differentially expressed genes (DEG) were
detected in infected quarters versus control animals. Eighteen hours later 2154
and 476 DEG were found in infected and in neighboring quarters vs. control
animals. Cluster analysis revealed DEG found only in infected quarters (local
response) and DEG detected in both infected and neighboring quarters (systemic
response). The first group includes genes mainly involved in immune response and 
inflammation, while the systemic reaction comprises antigen processing and
presentation, cytokines, protein degradation and apoptosis. Enhanced expression
of antimicrobial genes (S100A8, S100A9, S100A12, CXCL2, GNLY), acute phase genes 
(LBP, SAA3, CP, BF, C6, C4BPA, IF), and indicators of oxidative stress (GPX3,
MT1A, MT2A, SOD2) point to an active defense reaction in infected and neighboring
healthy quarters. Its early onset is indicated by increased transcription of
NFIL3 at 6 h. NFIL3 is a predicted regulator of many genes of the systemic
response at 24 h. The significance of our transcriptome study was evidenced by
some recent findings with candidate gene based approaches.
CONCLUSIONS: The discovery and holistic analysis of an extensive systemic
reaction in the mammary gland significantly expands the knowledge of
host-pathogen interactions in mastitis which may be relevant for the development 
of novel therapies and for genetic selection towards mastitis resistance.

DOI: 10.1186/1471-2164-11-138 
PMCID: PMC2846913
PMID: 20184744  [Indexed for MEDLINE]


570. PLoS One. 2010 Feb 11;5(2):e9169. doi: 10.1371/journal.pone.0009169.

Monitoring genomic sequences during SELEX using high-throughput sequencing:
neutral SELEX.

Zimmermann B(1), Gesell T, Chen D, Lorenz C, Schroeder R.

Author information: 
(1)Max F Perutz Laboratories, Department of Biochemistry, University of Vienna,
Vienna, Austria.

BACKGROUND: SELEX is a well established in vitro selection tool to analyze the
structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX
transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA
sequences binding a ligand of interest, called genomic aptamers. Concerns have
been raised regarding requirements imposed on RNA sequences undergoing SELEX
selection.
METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these 
effects, we designed and performed a Neutral SELEX experiment omitting the
selection step, such that the sequences are under the sole selective pressure of 
SELEX's amplification steps. Using high-throughput sequencing, we obtained
thousands of full-length sequences from the initial genomic library and the pools
after each of the 10 rounds of Neutral SELEX. We compared these to sequences
obtained from a Genomic SELEX experiment deriving from the same initial library, 
but screening for RNAs binding with high affinity to the E. coli regulator
protein Hfq. With each round of Neutral SELEX, sequences became less stable and
changed in nucleotide content, but no sequences were enriched. In contrast, we
detected substantial enrichment in the Hfq-selected set with enriched sequences
having structural stability similar to the neutral sequences but with
significantly different nucleotide selection.
CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts
independently of the neutral selective requirements imposed on the sequences. We 
conclude that Genomic SELEX, when combined with high-throughput sequencing of
positively and neutrally selected pools, as well as the gnomic library, is a
powerful method to identify genomic aptamers.

DOI: 10.1371/journal.pone.0009169 
PMCID: PMC2820082
PMID: 20161784  [Indexed for MEDLINE]


571. Genes Chromosomes Cancer. 2010 Feb;49(2):99-106. doi: 10.1002/gcc.20724.

Genome-wide scan identifies a copy number variable region at 3q26 that regulates 
PPM1L in APC mutation-negative familial colorectal cancer patients.

Thean LF(1), Loi C, Ho KS, Koh PK, Eu KW, Cheah PY.

Author information: 
(1)Department of Colorectal Surgery, Singapore General Hospital, Singapore,
Republic of Singapore.

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited form of
colorectal cancer (CRC) caused by mutation in the adenomatous polyposis coli
(APC) gene. However, APC mutations are not detected in 10-50% of FAP patients. We
searched for a new cancer gene by performing genome-wide genotyping on members of
an APC mutation-negative FAP variant family and ethnicity-matched healthy
controls. No common copy number change was found in all affected members using
the unaffected members and healthy controls as baseline. A 111 kb copy number
variable (CNV) region at 3q26.1 was shown to have copy number loss in all eight
polyps compared to matched lymphocytes of two affected members. A common region
of loss in all polyps, which are precursors to CRC, is likely to harbor
disease-causing gene in accordance to Knudsen's "two-hit" hypothesis. There is,
however, no gene within the deleted region. A 2-Mb scan of the genomic region
encompassing the deleted region identified PPM1L, coding for a novel
serine-threonine phosphatase in the TGF-beta and BMP signaling pathways.
Real-time PCR analyses indicate that the 3'UTR of PPM1L transcript was
down-regulated more than two-folds in all six polyps and tumors compared to
matched mucosa of the affected member. This down-regulation was not observed in
APC mutation-positive FAP patients. Our results suggest that the CNV region at
3q26 harbors an element that regulates the expression of an upstream candidate
tumor suppressor, PPM1L, thus providing a novel mechanism for colorectal
tumorigenesis in APC mutation-negative familial CRC patients.

DOI: 10.1002/gcc.20724 
PMID: 19847890  [Indexed for MEDLINE]


572. Microbiology. 2010 Feb;156(Pt 2):408-19. doi: 10.1099/mic.0.033126-0. Epub 2009
Nov 5.

Differences in adherence and virulence gene expression between two outbreak
strains of enterohaemorrhagic Escherichia coli O157 : H7.

Abu-Ali GS(1), Ouellette LM, Henderson ST, Whittam TS, Manning SD.

Author information: 
(1)Microbial Evolution Laboratory, National Food Safety and Toxicology Center,
Michigan State University, East Lansing, MI 48824, USA.

The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state
outbreak in North America in 2006, which resulted in high rates of severe
disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest
O157 : H7 outbreak to date. Both strains were shown to represent divergent
phylogenetic lineages. Here we compared global gene expression patterns before
and after epithelial cell exposure, as well as the ability to adhere to and
invade epithelial cells, between the two outbreak strains. Epithelial cell assays
demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to
Sakai, while whole-genome microarrays detected significant differential
expression of 914 genes, 206 of which had a fold change >/=1.5. Interestingly,
most locus of enterocyte effacement (LEE) genes were upregulated in TW14359,
whereas flagellar and chemotaxis genes were primarily upregulated in Sakai,
suggesting discordant expression of these genes between the two strains. The
Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several 
pO157-encoded genes that promote adherence, including type II secretion genes and
their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression
differences detected in the microarray analysis, and expression levels were lower
for a subset of LEE genes before versus after exposure to epithelial cells. In
all, this study demonstrated the upregulation of major and ancillary virulence
genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions
that precede intimate bacterial attachment to epithelial cells. Differences in
the level of adherence to epithelial cells were also observed, implying that
these two phylogenetically divergent O157 : H7 outbreak strains vary in their
ability to colonize, or initiate the disease process.

DOI: 10.1099/mic.0.033126-0 
PMCID: PMC2890088
PMID: 19892762  [Indexed for MEDLINE]


573. BMC Bioinformatics. 2010 Jan 18;11 Suppl 1:S56. doi: 10.1186/1471-2105-11-S1-S56.

Reverse engineering gene regulatory network from microarray data using linear
time-variant model.

Kabir M(1), Noman N, Iba H.

Author information: 
(1)Department of Computer Science and Engineering, University of Dhaka, Dhaka,
Bangladesh. mitrakabir@gmail.com

BACKGROUND: Gene regulatory network is an abstract mapping of gene regulations in
living cells that can help to predict the system behavior of living organisms.
Such prediction capability can potentially lead to the development of improved
diagnostic tests and therapeutics. DNA microarrays, which measure the expression 
level of thousands of genes in parallel, constitute the numeric seed for the
inference of gene regulatory networks. In this paper, we have proposed a new
approach for inferring gene regulatory networks from time-series gene expression 
data using linear time-variant model. Here, Self-Adaptive Differential Evolution,
a versatile and robust Evolutionary Algorithm, is used as the learning paradigm.
RESULTS: To assess the potency of the proposed work, a well known nonlinear
synthetic network has been used. The reconstruction method has inferred this
synthetic network topology and the associated regulatory parameters with high
accuracy from both the noise-free and noisy time-series data. For validation
purposes, the proposed approach is also applied to the simulated expression
dataset of cAMP oscillations in Dictyostelium discoideum and has proved it's
strength in finding the correct regulations. The strength of this work has also
been verified by analyzing the real expression dataset of SOS DNA repair system
in Escherichia coli and it has succeeded in finding more correct and reasonable
regulations as compared to various existing works.
CONCLUSION: By the proposed approach, the gene interaction networks have been
inferred in an efficient manner from both the synthetic, simulated cAMP
oscillation expression data and real expression data. The computational time of
this approach is also considerably smaller, which makes it to be more suitable
for larger network reconstruction. Thus the proposed approach can serve as an
initiate for the future researches regarding the associated area.

DOI: 10.1186/1471-2105-11-S1-S56 
PMCID: PMC3009529
PMID: 20122231  [Indexed for MEDLINE]


574. Appl Environ Microbiol. 2010 Jan;76(1):60-8. doi: 10.1128/AEM.01666-09. Epub 2009
Oct 30.

Differential expression of virulence and stress fitness genes between Escherichia
coli O157:H7 strains with clinical or bovine-biased genotypes.

Vanaja SK(1), Springman AC, Besser TE, Whittam TS, Manning SD.

Author information: 
(1)Microbial Evolution Laboratory, National Food Safety and Toxicology Center,
165 Food Safety and Toxicology Building, Michigan State University, East Lansing,
MI 48824, USA.

Escherichia coli O157:H7 strains can be classified into different genotypes based
on the presence of specific Shiga toxin-encoding bacteriophage insertion sites.
Certain O157:H7 genotypes predominate among human clinical cases (clinical
genotypes), while others are more frequently found in bovines (bovine-biased
genotypes). To determine whether inherent differences in gene expression explain 
the variation in infectivity of these genotypes, we compared the expression
patterns of clinical genotype 1 strains with those of bovine-biased genotype 5
strains using microarrays. Important O157:H7 virulence factors, including locus
of enterocyte effacement genes, the enterohemolysin, and several pO157 genes,
showed increased expression in the clinical versus bovine-biased genotypes. In
contrast, genes essential for acid resistance (e.g., gadA, gadB, and gadC) and
stress fitness were upregulated in bovine-biased genotype 5 strains. Increased
expression of acid resistance genes was confirmed functionally using a model
stomach assay, in which strains of bovine-biased genotype 5 had a 2-fold-higher
survival rate than strains of clinical genotype 1. Overall, these results suggest
that the increased prevalence of O157:H7 illness caused by clinical genotype 1
strains is due in part to the overexpression of key virulence genes. The
bovine-biased genotype 5 strains, however, are more resistant to adverse
environmental conditions, a characteristic that likely facilitates O157:H7
colonization of bovines.

DOI: 10.1128/AEM.01666-09 
PMCID: PMC2798638
PMID: 19880650  [Indexed for MEDLINE]


575. Appl Environ Microbiol. 2010 Jan;76(1):203-11. doi: 10.1128/AEM.01921-09. Epub
2009 Oct 30.

Low-density macroarray targeting non-locus of enterocyte effacement effectors
(nle genes) and major virulence factors of Shiga toxin-producing Escherichia coli
(STEC): a new approach for molecular risk assessment of STEC isolates.

Bugarel M(1), Beutin L, Fach P.

Author information: 
(1)French Food Safety Agency, Food Quality and Agro-alimentary Processes Research
Laboratory, Avenue du Général De Gaulle, F-94706 Maisons-Alfort, France.

Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC)
strains with a high level of virulence for humans has become a priority for
public health authorities. This study reports on the development of a low-density
macroarray for simultaneously testing the genes stx1, stx2, eae, and ehxA and six
different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and 
OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the
environment, food, animals, and healthy children have been compared with clinical
isolates of various seropathotypes. The eae gene was detected in all
enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli
(EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more 
prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally 
distributed. The nle genes were detected only in some EPEC and EHEC strains but
with various distributions, showing that nle genes are strain and/or serotype
specific, probably reflecting adaptation of the strains to different hosts or
environmental niches. One characteristic nle gene distribution in EHEC O157:[H7],
O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H-, O55:H7, O123:H11, 
O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets 
indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2,
nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and 
O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear 
signature of STEC strains with high virulence for humans.

DOI: 10.1128/AEM.01921-09 
PMCID: PMC2798666
PMID: 19880649  [Indexed for MEDLINE]


576. Eur J Mass Spectrom (Chichester). 2010;16(2):227-35. doi: 10.1255/ejms.1068.

Identification and relative quantification of proteins in Escherichia coli
proteome by "up-front" collision-induced dissociation.

Arike L(1), Nahku R, Borrisova M, Adamberg K, Vilu R.

Author information: 
(1)Competence Centre of Food and Fermentation Technologies, 12618 Tallinn,
Estonia. liisa@tftak.eu

A method for identifying and quantifying proteins with relatively low-cost
orthogonal acceleration time-of- flight mass spectrometry (oa-ToF-MS) was tested.
Escherichia coli (E. coli) K12 MG1655 cell lysate was separated by 1D
gel-electrophoresis; fractions were digested and separated fast and reproducibly 
by ultra-performance liquid chromatography (UPLC). Peptides were identified using
oa-ToF-MS to measure exact masses of parent ions and the fragment ions generated 
by up-front collision-induced dissociation. Fragmentation of all compounds was
achieved by rapidly cycling between high- and low values of energy applied to
ions. More than 100 proteins from E. coli K12 proteome were identified and
relatively quantified. Results were found to correlate with transcriptome data
determined by DNA microarrays.

DOI: 10.1255/ejms.1068 
PMID: 20212332  [Indexed for MEDLINE]


577. Genome Inform. 2010;24:154-63.

Analyzing gene coexpression data by an evolutionary model.

Schütte M(1), Mutwil M, Persson S, Ebenhöh O.

Author information: 
(1)Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476
Potsdam-Golm, Germany. schuette@mpimp-golm.mpg.de.

Coexpressed genes are tentatively translated into proteins that are involved in
similar biological functions. Here, we constructed gene coexpression networks
from collected microarray data of the organisms Arabidopsis thaliana,
Saccharomyces cerevisiae, and Escherichia coli. Their degree distributions show
the common property of an overrepresentation of highly connected nodes followed
by a sudden truncation. In order to analyze this behavior, we present an
evolutionary model simulating the genetic evolution. This model assumes that new 
genes emerge by duplication from a small initial set of primordial genes. Our
model does not include the removal of unused genes but selective pressure is
indirectly taken into account by preferentially duplicating the old genes. Thus, 
gene duplication represents the emergence of a new gene and its successful
establishment. After a duplication event, all genes are slightly but iteratively 
mutated, thus altering their expression patterns. Our model is capable of
reproducing global properties of the investigated coexpression networks. We show 
that our model reflects the mean inter-node distances and especially the
characteristic humps in the degree distribution that, in the biological examples,
result from functionally related genes.


PMID: 22081597  [Indexed for MEDLINE]


578. J Bacteriol. 2010 Jan;192(1):59-67. doi: 10.1128/JB.00873-09.

Small RNAs and small proteins involved in resistance to cell envelope stress and 
acid shock in Escherichia coli: analysis of a bar-coded mutant collection.

Hobbs EC(1), Astarita JL, Storz G.

Author information: 
(1)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, Bethesda, Maryland 20892-5430, USA.

Comment in
    J Bacteriol. 2010 Jan;192(1):26-8.

More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino
acids (small proteins) are encoded in the Escherichia coli genome. The vast
majority of the corresponding genes have no known function. We screened 125 DNA
bar-coded mutants to identify novel cell envelope stress and acute acid shock
phenotypes associated with deletions of genes coding for sRNAs and small
proteins. Nine deletion mutants (ssrA, micA, ybaM, ryeF, yqcG, sroH, ybhT, yobF, 
and glmY) were sensitive to cell envelope stress and two were resistant (rybB and
blr). Deletion mutants of genes coding for four small proteins (yqgB, mgrB, yobF,
and yceO) were sensitive to acute acid stress. We confirmed each of these
phenotypes in one-on-one competition assays against otherwise-wild-type lacZ
mutant cells. A more detailed investigation of the SsrA phenotype suggests that
ribosome release is critical for resistance to cell envelope stress. The
bar-coded deletion collection we generated can be screened for sensitivity or
resistance to virtually any stress condition.

DOI: 10.1128/JB.00873-09 
PMCID: PMC2798238
PMID: 19734312  [Indexed for MEDLINE]


579. J Biotechnol. 2010 Jan 1;145(1):60-5. doi: 10.1016/j.jbiotec.2009.10.007.

Specific growth rate dependent transcriptome profiling of Escherichia coli K12
MG1655 in accelerostat cultures.

Nahku R(1), Valgepea K, Lahtvee PJ, Erm S, Abner K, Adamberg K, Vilu R.

Author information: 
(1)Tallinn University of Technology, Department of Chemistry, Estonia.
raivo@kbfi.ee

Specific growth rate dependent gene expression changes of Escherichia coli K12
MG1655 were studied by microarray and real-time PCR analyses. The bacteria were
cultivated on glucose limited minimal medium using the accelerostat method
(A-stat) where starting from steady state conditions (chemostat culture) dilution
rate is constantly increased. At specific growth rate (mu) 0.47h(-1), E. coli had
focused its metabolism to glucose utilization by down-regulation of alternative
substrate transporters expression compared to mu=0.3h(-1). It was found that
acetic acid accumulation began at mu=0.34+/-0.01h(-1) and two acetate synthesis
pathways - phosphotransacetylase-acetate kinase (pta-ackA) and pyruvate oxidase
(poxB) - contributed to the synthesis at the beginning of overflow metabolism,
i.e. onset of acetate excretion. On the other hand, poxB, pta and ackA expression
patterns suggest that pyruvate oxidase may be the only enzyme synthesizing
acetate at mu=0.47h(-1). Loss of glucose and acetate co-utilization represented
by down-regulation of acs-yjcH-actP operon between specific growth rates
0.3-0.42h(-1) and acetic acid accumulation from mu=0.34+/-0.01h(-1) allows one to
surmise that the acetate utilization operon expression might play an important
role in overflow metabolism.

DOI: 10.1016/j.jbiotec.2009.10.007 
PMID: 19861135  [Indexed for MEDLINE]


580. Mol Biol (Mosk). 2010 Jan-Feb;44(1):60-73.

[Lysogenic infection of a Shiga toxin 2-converting bacteriophage changes host
gene expression, enhances host acid resistance and motility].

[Article in Russian]

Su LK, Lu CP, Wang Y, Cao DM, Sun JH, Yan YX.

Shiga toxin 2 (Stx2)-converting bacteriophages can infect and lysogenize other
bacteria in vivo and in vitro, and, thus, contribute to a genotypic heterogeneity
of infected host. However, the global transcription patterns accompanying the
lysogenic infection of E. coli host have not been clearly resolved. In this
study, gene expression profiles of Stx2 phage phi Min27(delta stx::cat) converted
and native E. coli MG1655 hosts were compared using microarray assay. The phi
Min27(delta stx::cat) conversion had a direct effect on the global expression of 
bacterial host genes as 166 genes were found to be differentially expressed (104 
up-regulated and 62 downregulated). These genes were predominantly responsible
for bacterial central metabolism, transport and transcription. It was shown that 
in addition to the down-regulation of genes involved in synthesis of thiamine and
protein transporters, expression of genes associated with bacterial energy
production (e.g., fadABDEHIJL, aceK, and acnA) was also suppressed. Conversely,
most up-regulated genes were transport genes, flagellar synthesis genes
(fliDESTZ), and acid resistance genes (e.g., gadEW, hdeABD, and adiY).
Futhermore, conversion of phi Min27(delta stx::cat) was shown to change
physiological properties of the host cell. In comparison with the uninfected
cells the converted bacteria host had increased acid tolerance and promoted
swimming motility on a semisolid agar surface.


PMID: 20198860  [Indexed for MEDLINE]


581. Mol Immunol. 2010 Jan;47(4):849-60. doi: 10.1016/j.molimm.2009.10.019. Epub 2009 
Dec 3.

Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda,
Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma
paraensei (Digenea, Platyhelminthes).

Adema CM(1), Hanington PC, Lun CM, Rosenberg GH, Aragon AD, Stout BA, Lennard
Richard ML, Gross PS, Loker ES.

Author information: 
(1)Center for Evolutionary and Theoretical Immunology, Biology MSC03 2020, 1
University of New Mexico, Albuquerque, NM 87131, USA. coenadem@unm.edu

A 70-mer-oligonucleotide-based microarray (1152 features) that emphasizes stress 
and immune responses factors was constructed to study transcriptomic responses of
the snail Biomphalaria glabrata to different immune challenges. In addition to
sequences with relevant putative ID and Gene Ontology (GO) annotation, the array 
features non-immune factors and unknown B. glabrata ESTs for functional gene
discovery. The transcription profiles of B. glabrata (3 biological replicates,
each a pool of 5 snails) were recorded at 12h post-wounding, exposure to Gram
negative or Gram positive bacteria (Escherichia coli and Micrococcus luteus,
respectively), or infection with compatible trematode parasites (Schistosoma
mansoni or Echinostoma paraensei, 20 miracidia/snail), relative to controls,
using universal reference RNA. The data were subjected to Significance Analysis
for Microarrays (SAM), with a false positive rate (FPR) <or=10%. Wounding yielded
a modest differential expression profile (27 up/21 down) with affected features
mostly dissimilar from other treatments. Partially overlapping, yet distinct
expression profiles were recorded from snails challenged with E. coli (83 up/20
down) or M. luteus (120 up/42 down), mostly showing up-regulation of defense and 
stress-related features. Significantly altered expression of selected immune
features indicates that B. glabrata detects and responds differently to
compatible trematodes. Echinostoma paraensei infection was associated mostly with
down-regulation of many (immune-) transcripts (42 up/68 down), whereas S. mansoni
exposure yielded a preponderance of up-regulated features (140 up/23 down), with 
only few known immune genes affected. These observations may reflect the
divergent strategies developed by trematodes during their evolution as
specialized pathogens of snails to negate host defense responses. Clearly, the
immune defenses of B. glabrata distinguish and respond differently to various
immune challenges.

Copyright 2010 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.molimm.2009.10.019 
PMCID: PMC2814977
PMID: 19962194  [Indexed for MEDLINE]


582. Nucleic Acids Res. 2010 Jan;38(Database issue):D396-400. doi: 10.1093/nar/gkp919.
Epub 2009 Nov 11.

MicrobesOnline: an integrated portal for comparative and functional genomics.

Dehal PS(1), Joachimiak MP, Price MN, Bates JT, Baumohl JK, Chivian D, Friedland 
GD, Huang KH, Keller K, Novichkov PS, Dubchak IL, Alm EJ, Arkin AP.

Author information: 
(1)Virtual Institute for Microbial Stress and Survival, Lawrence Berkeley
National Laboratory, Berkeley, CA 94720, USA. psdehal@lbl.gov

Since 2003, MicrobesOnline (http://www.microbesonline.org) has been providing a
community resource for comparative and functional genome analysis. The portal
includes over 1000 complete genomes of bacteria, archaea and fungi and thousands 
of expression microarrays from diverse organisms ranging from model organisms
such as Escherichia coli and Saccharomyces cerevisiae to environmental microbes
such as Desulfovibrio vulgaris and Shewanella oneidensis. To assist in annotating
genes and in reconstructing their evolutionary history, MicrobesOnline includes a
comparative genome browser based on phylogenetic trees for every gene family as
well as a species tree. To identify co-regulated genes, MicrobesOnline can search
for genes based on their expression profile, and provides tools for identifying
regulatory motifs and seeing if they are conserved. MicrobesOnline also includes 
fast phylogenetic profile searches, comparative views of metabolic pathways,
operon predictions, a workbench for sequence analysis and integration with
RegTransBase and other microbial genome resources. The next update of
MicrobesOnline will contain significant new functionality, including comparative 
analysis of metagenomic sequence data. Programmatic access to the database, along
with source code and documentation, is available at
http://microbesonline.org/programmers.html.

DOI: 10.1093/nar/gkp919 
PMCID: PMC2808868
PMID: 19906701  [Indexed for MEDLINE]


583. Stat Appl Genet Mol Biol. 2010;9:Article 15. doi: 10.2202/1544-6115.1519. Epub
2010 Feb 1.

Weighted-LASSO for structured network inference from time course data.

Charbonnier C(1), Chiquet J, Ambroise C.

Author information: 
(1)University of Evry-Val-d'Essonne. camille.charbonnier@genopole.cnrs.fr

We present a weighted-LASSO method to infer the parameters of a first-order
vector auto-regressive model that describes time course expression data generated
by directed gene-to-gene regulation networks. These networks are assumed to own
prior internal structures of connectivity which drive the inference method. This 
prior structure can be either derived from prior biological knowledge or inferred
by the method itself. We illustrate the performance of this structure-based
penalization both on synthetic data and on two canonical regulatory networks (the
yeast cell cycle regulation network and the E. coli S.O.S. DNA repair network).

DOI: 10.2202/1544-6115.1519 
PMID: 20196750  [Indexed for MEDLINE]


584. Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22564-8. doi:
10.1073/pnas.0912673106. Epub 2009 Dec 14.

Oscillations in supercoiling drive circadian gene expression in cyanobacteria.

Vijayan V(1), Zuzow R, O'Shea EK.

Author information: 
(1)Graduate Program in Systems Biology, Howard Hughes Medical Institute, Harvard 
Faculty of Arts and Sciences Center for Systems Biology, Departments of Molecular
and Cellular Biology and Chemistry and Chemical Biology, Harvard University,
Cambridge, MA 02138, USA.

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA
transcript abundance with 24-h periodicity under continuous light conditions. The
mechanism underlying these oscillations remains elusive--neither cis nor
trans-factors controlling circadian gene expression phase have been identified.
Here, we show that the topological status of the chromosome is highly correlated 
with circadian gene expression state. We also demonstrate that DNA sequence
characteristics of genes that appear monotonically activated and monotonically
repressed by chromosomal relaxation during the circadian cycle are similar to
those of supercoiling-responsive genes in Escherichia coli. Furthermore,
perturbation of superhelical status within the physiological range elicits global
changes in gene expression similar to those that occur during the normal
circadian cycle.

DOI: 10.1073/pnas.0912673106 
PMCID: PMC2799730
PMID: 20018699  [Indexed for MEDLINE]


585. PLoS One. 2009 Dec 22;4(12):e8402. doi: 10.1371/journal.pone.0008402.

Loss-of-function mutations in Rab escort protein 1 (REP-1) affect intracellular
transport in fibroblasts and monocytes of choroideremia patients.

Strunnikova NV(1), Barb J, Sergeev YV, Thiagarajasubramanian A, Silvin C, Munson 
PJ, Macdonald IM.

Author information: 
(1)Ophthalmic Genetics and Visual Function Branch, National Eye Institute,
National Institutes of Health, Bethesda, Maryland, USA. Strunnikovan@nei.nih.gov

BACKGROUND: Choroideremia (CHM) is a progressive X-linked retinopathy caused by
mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort 
protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs,
as a result of loss of function mutations in REP-1, could affect vesicular
trafficking, exocytosis and secretion in peripheral cells of CHM patients.
METHODOLOGY/PRINCIPAL FINDINGS: To evaluate this hypothesis, intracellular
vesicle transport, lysosomal acidification and rates of proteolytic degradation
were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the 
nine age-matched controls and thirteen CHM patients carrying 10 different
loss-of-function mutations. With the use of pHrodo BioParticles conjugated with
E. coli, collagen I coated FluoSpheres beads and fluorescent DQ ovalbumin with
BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased
in monocytes of CHM patients and, as a consequence, the rates of proteolytic
degradation were slowed. Microarray analysis of gene expression revealed that
some genes involved in the immune response, small GTPase regulation,
transcription, cell adhesion and the regulation of exocytosis were significantly 
up and down regulated in cells from CHM patients compared to controls. Finally,
CHM fibroblasts secreted significantly lower levels of cytokine/growth factors
such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived 
factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) 
beta and interleukin (lL)-8.
CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time that peripheral
cells of CHM patients had increased pH levels in lysosomes, reduced rates of
proteolytic degradation and altered secretion of cytokines. Peripheral cells from
CHM patients expose characteristics that were not previously recognized and could
used as an alternative models to study the effects of different mutations in the 
REP-1 gene on mechanism of CHM development in human population.

DOI: 10.1371/journal.pone.0008402 
PMCID: PMC2793004
PMID: 20027300  [Indexed for MEDLINE]


586. Bioinformatics. 2009 Dec 15;25(24):3267-74. doi: 10.1093/bioinformatics/btp588.
Epub 2009 Oct 13.

Detailing regulatory networks through large scale data integration.

Huttenhower C(1), Mutungu KT, Indik N, Yang W, Schroeder M, Forman JJ,
Troyanskaya OG, Coller HA.

Author information: 
(1)Department of Computer Science, Princeton University, 35 Olden Street,
Princeton, NJ 08540, USA.

MOTIVATION: Much of a cell's regulatory response to changing environments occurs 
at the transcriptional level. Particularly in higher organisms, transcription
factors (TFs), microRNAs and epigenetic modifications can combine to form a
complex regulatory network. Part of this system can be modeled as a collection of
regulatory modules: co-regulated genes, the conditions under which they are
co-regulated and sequence-level regulatory motifs.
RESULTS: We present the Combinatorial Algorithm for Expression and Sequence-based
Cluster Extraction (COALESCE) system for regulatory module prediction. The
algorithm is efficient enough to discover expression biclusters and putative
regulatory motifs in metazoan genomes (>20,000 genes) and very large microarray
compendia (>10,000 conditions). Using Bayesian data integration, it can also
include diverse supporting data types such as evolutionary conservation or
nucleosome placement. We validate its performance using a functional evaluation
of co-clustered genes, known yeast and Escherichea coli TF targets, synthetic
data and various metazoan data compendia. In all cases, COALESCE performs as well
or better than current biclustering and motif prediction tools, with high
accuracy in functional and TF/target assignments and zero false positives on
synthetic data. COALESCE provides an efficient and flexible platform within which
large, diverse data collections can be integrated to predict metazoan regulatory 
networks.
AVAILABILITY: Source code (C++) is available at
http://function.princeton.edu/sleipnir, and supporting data and a web interface
are provided at http://function.princeton.edu/coalesce.
CONTACT: ogt@cs.princeton.edu; hcoller@princeton.edu.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online.

DOI: 10.1093/bioinformatics/btp588 
PMCID: PMC2788929
PMID: 19825796  [Indexed for MEDLINE]


587. J Bacteriol. 2009 Dec;191(23):7193-205. doi: 10.1128/JB.00970-09. Epub 2009 Oct
2.

CbpA: a polarly localized novel cyclic AMP-binding protein in Pseudomonas
aeruginosa.

Endoh T(1), Engel JN.

Author information: 
(1)Department of Medicine, University of California, San Francisco, California
941431, USA.

In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the
transcription of hundreds of genes encoding diverse virulence factors, including 
the type II secretion system (T2SS) and type III secretion system (T3SS) and
their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent 
transcriptional regulator that is homologous to the Escherichia coli catabolite
repressor protein, is thought to be the major cAMP-binding protein that regulates
these important virulence determinants. Using a bioinformatic approach, we have
identified a gene (PA4704) encoding an additional putative cAMP-binding protein
in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein
A. Structural modeling predicts that CbpA is composed of a C-terminal
cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is
structurally similar to eukaryotic protein kinase A regulatory subunits. We show 
that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using
in vitro trypsin protection assays, we demonstrate that CbpA undergoes a
conformational change upon cAMP binding. Reporter gene assays and electrophoresis
mobility shift assays defined the cbpA promoter and a Vfr-binding site that are
necessary for Vfr-dependent transcription. Although CbpA is highly regulated by
Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the
T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model 
of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green
fluorescent protein was found to be localized to the flagellated old cell pole in
a cAMP-dependent manner. These results suggest that polar localization of CbpA
may be important for its function.

DOI: 10.1128/JB.00970-09 
PMCID: PMC2786554
PMID: 19801409  [Indexed for MEDLINE]


588. Mol Biosyst. 2009 Dec;5(12):1439-55. doi: 10.1039/B907407d. Epub 2009 Jul 31.

Systems-level approaches for identifying and analyzing genetic interaction
networks in Escherichia coli and extensions to other prokaryotes.

Babu M(1), Musso G, Díaz-Mejía JJ, Butland G, Greenblatt JF, Emili A.

Author information: 
(1)Banting and Best Department of Medical Research, Terrence Donnelly Center for 
Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario,
Canada M5S 3E1.

Molecular interactions define the functional organization of the cell. Epistatic 
(genetic, or gene-gene) interactions, one of the most informative and commonly
encountered forms of functional relationships, are increasingly being used to map
process architecture in model eukaryotic organisms. In particular,
'systems-level' screens in yeast and worm aimed at elucidating genetic
interaction networks have led to the generation of models describing the global
modular organization of gene products and protein complexes within a cell.
However, comparable data for prokaryotic organisms have not been available. Given
its ease of growth and genetic manipulation, the Gram-negative bacterium
Escherichia coli appears to be an ideal model system for performing comprehensive
genome-scale examinations of genetic redundancy in bacteria. In this review, we
highlight emerging experimental and computational techniques that have been
developed recently to examine functional relationships and redundancy in E. coli 
at a systems-level, and their potential application to prokaryotes in general.
Additionally, we have scanned PubMed abstracts and full-text published articles
to manually curate a list of approximately 200 previously reported synthetic sick
or lethal genetic interactions in E. coli derived from small-scale experimental
studies.

DOI: 10.1039/B907407d 
PMID: 19763343  [Indexed for MEDLINE]


589. Mol Microbiol. 2009 Dec;74(6):1314-30. doi: 10.1111/j.1365-2958.2009.06944.x.
Epub 2009 Nov 2.

A PhoQ/P-regulated small RNA regulates sensitivity of Escherichia coli to
antimicrobial peptides.

Moon K(1), Gottesman S.

Author information: 
(1)Laboratory of Molecular Biology, Center for Cancer Research, National Cancer
Institute, Bethesda, MD 20892, USA.

Comment in
    Mol Microbiol. 2009 Dec;74(6):1289-94.

Non-coding small RNAs (sRNAs) play a major role in post-transcriptional
regulation of gene expression. Of the 80 sRNAs that have been identified in E.
coli, one-third bind to the RNA chaperone Hfq. Hfq both stabilizes these sRNAs in
vivo and stimulates pairing to targets in vitro. A novel Hfq-dependent RNA,
called here MgrR, was identified by its ability to bind Hfq. Expression of MgrR
requires the PhoQ/PhoP two-component system; the PhoP response regulator is
active under low Mg2+ concentrations and is an important virulence regulator in
Salmonella; mgrR is also found in Salmonella species. Negatively regulated
targets of MgrR identified using microarrays include eptB, involved in
lipopolysaccharide (LPS) modification, and ygdQ, encoding a hypothetical protein.
Cell sensitivity to the antimicrobial polymyxin B is affected by LPS
modifications, and cells carrying an mgrR deletion were approximately 10 times
more resistant than wild-type cells to polymyxin B. Thus, lower Mg2+
concentrations, sensed by PhoQ/PhoP, lead to expression of MgrR, changing LPS.
sRNAs have previously been shown to regulate many outer membrane proteins. This
work demonstrates that LPS, a major contributor of bacterial interactions with
mammalian cells, is also subject to regulation by sRNAs.

DOI: 10.1111/j.1365-2958.2009.06944.x 
PMCID: PMC2841474
PMID: 19889087  [Indexed for MEDLINE]


590. Plant Physiol. 2009 Dec;151(4):1952-64. doi: 10.1104/pp.109.146415. Epub 2009 Oct
2.

Multiple biochemical and morphological factors underlie the production of
methylketones in tomato trichomes.

Ben-Israel I(1), Yu G, Austin MB, Bhuiyan N, Auldridge M, Nguyen T, Schauvinhold 
I, Noel JP, Pichersky E, Fridman E.

Author information: 
(1)Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture,
Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem,
Rehovot 76100, Israel.

Genetic analysis of interspecific populations derived from crosses between the
wild tomato species Solanum habrochaites f. sp. glabratum, which synthesizes and 
accumulates insecticidal methylketones (MK), mostly 2-undecanone and
2-tridecanone, in glandular trichomes, and cultivated tomato (Solanum
lycopersicum), which does not, demonstrated that several genetic loci contribute 
to MK metabolism in the wild species. A strong correlation was found between the 
shape of the glandular trichomes and their MK content, and significant
associations were seen between allelic states of three genes and the amount of MK
produced by the plant. Two genes belong to the fatty acid biosynthetic pathway,
and the third is the previously identified Methylketone Synthase1 (MKS1) that
mediates conversion to MK of beta-ketoacyl intermediates. Comparative
transcriptome analysis of the glandular trichomes of F2 progeny grouped into low-
and high-MK-containing plants identified several additional genes whose
transcripts were either more or less abundant in the high-MK bulk. In particular,
a wild species-specific transcript for a gene that we named MKS2, encoding a
protein with some similarity to a well-characterized bacterial thioesterase, was 
approximately 300-fold more highly expressed in F2 plants with high MK content
than in those with low MK content. Genetic analysis in the segregating population
showed that MKS2's significant contribution to MK accumulation is mediated by an 
epistatic relationship with MKS1. Furthermore, heterologous expression of MKS2 in
Escherichia coli resulted in the production of methylketones in this host.

DOI: 10.1104/pp.109.146415 
PMCID: PMC2785994
PMID: 19801397  [Indexed for MEDLINE]


591. Yeast. 2009 Dec;26(12):663-73. doi: 10.1002/yea.1725.

Cellular and transcriptional responses of yeast to the cleavage of cytosolic
tRNAs induced by colicin D.

Shigematsu M(1), Ogawa T, Kido A, Kitamoto HK, Hidaka M, Masaki H.

Author information: 
(1)Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan.

Colicin D is a plasmid-encoded antibacterial protein that specifically cleaves
the anticodon loops of four Escherichia coli tRNA(Arg) species. Here, we report
that the catalytic domain of colicin D, which is expressed in Saccharomyces
cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray
analysis revealed that mating-related genes were upregulated, while genes
involved in a range of metabolic processes were downregulated, thereby impairing 
cell growth. The pheromone-signalling pathway was activated only in alpha cells
by tRNA cleavage, which was not observed in 'a' cells or diploid cells. On the
basis of these results and on the recent identification of two killer toxins that
cleave specific tRNAs, the relationship between tRNA depletion and the resultant 
cellular response is discussed.

DOI: 10.1002/yea.1725 
PMID: 19877125  [Indexed for MEDLINE]


592. Zhonghua Gan Zang Bing Za Zhi. 2009 Dec;17(12):925-9.

[Function of TTG1A in hepatic stellate cells].

[Article in Chinese]

Xiao L(1), Cheng J, Zhang LY, Guo J, Hong Y, Zhang LJ, Lun YZ, Lan XY, Wu HJ,
Zhang YX, Zhang JL, Li Y.

Author information: 
(1)Institute of Infectious Diseases, Beijing Ditan Hospital, Beijing 100015,
China.

OBJECTIVE: To investigate the biological functions of TTG1A in liver fibrosis.
METHODS: Yeast two-hybrid system was used to screen proteins associated with
TTG1A. Briefly, the coding sequence of TTG1A was cloned into pGBKT7 vector, and
the recombinant plasmid was transformed into yeast cells AH109 ( a type), then
these cells were mated with yeast cells Y187 (a type) transformed with human
leukocyte cDNA library plasmid pACT2. The obtained diploid yeast cells were
plated on synthetic dropout nutrient medium containing X-alpha-gal for double
selection. The plasmids from positive colonies were transformed into E.coli and
sequenced.
RESULTS: The recombinant yeast expression vector pGBKT7-TTG1A was successfully
constructed. Nineteen TTG1A binding proteins, including Homo sapiens major
histocompatibility complex, class II DP beta 1 (HLA-DPb1), Homo sapiens ribosomal
protein L30 (RPL30), Homo sapiens nucleophosmin Homo sapiens nucleobindin 2
(NUCB2), Homo sapiens ash2, variant Gaucher disease and variant metachromatic
leukodystrophy, MORF4L1, Homo sapiens ubiquitin-conjugating enzyme E2L3 (UBE2L3),
APOA1, Homo sapiens lectin, and galectin 1, were identified.
CONCLUSIONS: This study may help to elucidate the molecular function of TTG1A.


PMID: 20038335  [Indexed for MEDLINE]


593. BMC Syst Biol. 2009 Nov 16;3:108. doi: 10.1186/1752-0509-3-108.

Network analysis of the transcriptional pattern of young and old cells of
Escherichia coli during lag phase.

Pin C(1), Rolfe MD, Muñoz-Cuevas M, Hinton JC, Peck MW, Walton NJ, Baranyi J.

Author information: 
(1)Institute of Food Research, Norwich NR4 7UA, UK. carmen.pin@bbsrc.ac.uk

BACKGROUND: The aging process of bacteria in stationary phase is halted if cells 
are subcultured and enter lag phase and it is then followed by cellular division.
Network science has been applied to analyse the transcriptional response, during 
lag phase, of bacterial cells starved previously in stationary phase for 1 day
(young cells) and 16 days (old cells).
RESULTS: A genome scale network was constructed for E. coli K-12 by connecting
genes with operons, transcription and sigma factors, metabolic pathways and cell 
functional categories. Most of the transcriptional changes were detected
immediately upon entering lag phase and were maintained throughout this period.
The lag period was longer for older cells and the analysis of the transcriptome
revealed different intracellular activity in young and old cells. The number of
genes differentially expressed was smaller in old cells (186) than in young cells
(467). Relatively, few genes (62) were up- or down-regulated in both cultures.
Transcription of genes related to osmotolerance, acid resistance, oxidative
stress and adaptation to other stresses was down-regulated in both young and old 
cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were
up-regulated in young cells while old cells up-regulated the Entner Doudoroff and
gluconate pathways and down-regulated the pentose phosphate pathway. In both old 
and young cells, anaerobic respiration and fermentation pathways were
down-regulated, but only young cells up-regulated aerobic respiration while there
was no evidence of aerobic respiration in old cells.Numerous genes related to DNA
maintenance and replication, translation, ribosomal biosynthesis and RNA
processing as well as biosynthesis of the cell envelope and flagellum and several
components of the chemotaxis signal transduction complex were up-regulated only
in young cells. The genes for several transport proteins for iron compounds were 
up-regulated in both young and old cells. Numerous genes encoding transporters
for carbohydrates and organic alcohols and acids were down-regulated in old cells
only.
CONCLUSION: Network analysis revealed very different transcriptional activities
during the lag period in old and young cells. Rejuvenation seems to take place
during exponential growth by replicative dilution of old cellular components.

DOI: 10.1186/1752-0509-3-108 
PMCID: PMC2780417
PMID: 19917103  [Indexed for MEDLINE]


594. Appl Environ Microbiol. 2009 Nov;75(22):6955-62. doi: 10.1128/AEM.00375-09. Epub 
2009 Sep 11.

Pathotype and antibiotic resistance gene distributions of Escherichia coli
isolates from broiler chickens raised on antimicrobial-supplemented diets.

Bonnet C(1), Diarrassouba F, Brousseau R, Masson L, Topp E, Diarra MS.

Author information: 
(1)National Research Council of Canada, Biotechnology Research Institute,
Montreal, Quebec H4P 2R2, Canada.

The impact of feed supplementation with bambermycin, monensin, narasin,
virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the 
distribution of Escherichia coli pathotypes in broiler chickens was investigated 
using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined,
59 (23%) were classified as potentially extraintestinal pathogenic E. coli
(ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline 
treatment, the pathotype distribution was not significantly different among
treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to 
be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%)
considered potentially "other" ExPEC isolates. The distribution within
phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups
B2 and D, with the majority of APEC isolates classified as group D and most
commensal isolates (170, 86%) as group A or B1. Indirect assessment of the
presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of
the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at
least one antimicrobial resistance gene, with nearly half (107, 42%) showing
multiple resistance genes. The majority of resistance genes were distributed
among commensal isolates. Considering that the simultaneous detection of
antimicrobial resistance tet(A), sulI, and bla(TEM) genes and the integron class 
I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the
results revealed that 35 (14%) of the isolates, all commensals, possessed this
multigene resistance plasmid. The virulence plasmid was never found in
combination with the antimicrobial resistance plasmid. The presence of the ColV
plasmid or the combination of iss and tsh genes in the majority of APEC isolates 
supports the notion that when found together, the plasmid, iss, and tsh serve as 
good markers for APEC. These data indicate that different resistant E. coli
pathotypes can be found in broiler chickens and that the distribution of such
pathotypes and certain virulence determinants could be modulated by antimicrobial
agent feed supplementation.

DOI: 10.1128/AEM.00375-09 
PMCID: PMC2786528
PMID: 19749070  [Indexed for MEDLINE]


595. J Antimicrob Chemother. 2009 Nov;64(5):973-85. doi: 10.1093/jac/dkp320. Epub 2009
Sep 16.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to
triclosan induces a species-specific response, including drug detoxification.

Bailey AM(1), Constantinidou C, Ivens A, Garvey MI, Webber MA, Coldham N, Hobman 
JL, Wain J, Woodward MJ, Piddock LJ.

Author information: 
(1)Antimicrobial Agents Research Group, School of Immunity and Infection, The
Medical School, The University of Birmingham, Birmingham, UK.

OBJECTIVES: The use of triclosan within various environments has been linked to
the development of multiple drug resistance (MDR) through the increased
expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the
effect of triclosan exposure in order to ascertain the response of two species to
the presence of this widely used biocide.
METHODS: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and
Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L)
were determined in microarray experiments. Phenotypic validation of the
transcriptomic data included RT-PCR, ability to form a biofilm and motility
assays.
RESULTS: Despite important differences in the triclosan-dependent transcriptomes 
of the two species, increased expression of efflux pump component genes was seen 
in both. Increased expression of soxS was observed in Salmonella Typhimurium,
however, within E. coli, decreased expression was seen. Expression of fabBAGI in 
Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH
was increased. Increased expression of ompR and genes within this regulon (e.g.
ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An 
unexpected response of E. coli was the differential expression of genes within
operons involved in iron homeostasis; these included fhu, fep and ent.
CONCLUSIONS: These data indicate that whilst a core response to triclosan
exposure exists, the differential transcriptome of each species was different.
This suggests that E. coli K-12 should not be considered the paradigm for the
Enterobacteriaceae when exploring the effects of antimicrobial agents.

DOI: 10.1093/jac/dkp320 
PMID: 19759044  [Indexed for MEDLINE]


596. J Leukoc Biol. 2009 Nov;86(5):1217-26. doi: 10.1189/jlb.0908517. Epub 2009 Jul
22.

Human mast cells synthesize and release angiogenin, a member of the ribonuclease 
A (RNase A) superfamily.

Kulka M(1), Fukuishi N, Metcalfe DD.

Author information: 
(1)National Research Council, 550 University Ave., Charlottetown, PE, Canada.
marianna.kulka@nrc.ca

ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated
in tumor growth, heart failure, wound healing, asthma, and the composition of the
adult gut microflora. Human mast cells (HuMC) are similarly associated with
modulation of vascular permeability, angiogenic processes, wound healing, and
asthma. We hypothesized that HuMC express and secrete ANG in response to
divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and
CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or 
neuropeptides and following FcepsilonRI aggregation. Expression and production of
ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and
ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed 
to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC
constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. 
coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small 
amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin
activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that
HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived
ANG is available in the subsequent inflammatory response.

DOI: 10.1189/jlb.0908517 
PMCID: PMC2774883
PMID: 19625371  [Indexed for MEDLINE]


597. Microbiology. 2009 Nov;155(Pt 11):3758-74. doi: 10.1099/mic.0.027953-0. Epub 2009
Aug 14.

Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves
increased consumption of formate and reduced utilization of lactate.

Lewis JP(1), Iyer D, Anaya-Bergman C.

Author information: 
(1)The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia
Commonwealth University, Richmond, VA, USA. jplewis@vcu.edu

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in
the presence of low concentrations of oxygen. Microarray analysis revealed
alteration in gene expression in the presence of 6 % oxygen. During the
exponential growth phase, 96 genes were upregulated and 79 genes were
downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative
stress protection were upregulated, including alkyl hydroperoxide reductase
(ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant
changes in gene expression of proteins that mediate oxidative metabolism, such as
cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The 
expression of genes encoding formate uptake transporter (PG0209) and formate
tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that
formate metabolism plays a major role under aerobic conditions. The concomitant
reduction of expression of a gene encoding the lactate transporter PG1340
suggests decreased utilization of this nutrient. The concentrations of both
formate and lactate were assessed in culture supernatants and cells, and they
were in agreement with the results obtained at the transcriptional level. Also,
genes encoding gingipain protease secretion/maturation regulator (porR) and
protease transporter (porT) had reduced expression in the presence of oxygen,
which also correlated with reduced protease activities under aerobic conditions. 
In addition, metal transport was affected, and while iron-uptake genes such as
the genes encoding the haemin uptake locus (hmu) were downregulated, expression
of manganese transporter genes, such as feoB2, was elevated in the presence of
oxygen. Finally, genes encoding putative regulatory proteins such as
extracellular function (ECF) sigma factors as well as small proteins had elevated
expression levels in the presence of oxygen. As P. gingivalis is distantly
related to the well-studied model organism Escherichia coli, results from our
work may provide further understanding of oxygen metabolism and protection in
other related bacteria belonging to the phylum Bacteroidetes.

DOI: 10.1099/mic.0.027953-0 
PMCID: PMC2888126
PMID: 19684063  [Indexed for MEDLINE]


598. Plant Physiol. 2009 Nov;151(3):1096-113. doi: 10.1104/pp.109.141481. Epub 2009
Jul 1.

Integrated metabolite and transcript profiling identify a biosynthetic mechanism 
for hispidol in Medicago truncatula cell cultures.

Farag MA(1), Deavours BE, de Fátima A, Naoumkina M, Dixon RA, Sumner LW.

Author information: 
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401, USA.

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures 
using high-performance liquid chromatography coupled to photodiode and mass
spectrometry detection revealed the accumulation of the aurone hispidol
(6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major
response to yeast elicitor. Parallel, large-scale transcriptome profiling
indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately
induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone
synthase activity based upon in vitro substrate specificity and product profiles 
of recombinant proteins expressed in Escherichia coli. Hispidol possessed
significant antifungal activity relative to other M. truncatula phenylpropanoids 
tested but has not been reported in this species before and was not found in
differentiated roots in which high levels of the peroxidase transcripts
accumulated. We propose that hispidol is formed in cell cultures by metabolic
spillover when the pool of its precursor, isoliquiritigenin, builds up as a
result of an imbalance between the upstream and downstream segments of the
phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism.
The results illustrate that integration of metabolomics and transcriptomics in
genetically reprogrammed plant cell cultures is a powerful approach for the
discovery of novel bioactive secondary metabolites and the mechanisms underlying 
their generation.

DOI: 10.1104/pp.109.141481 
PMCID: PMC2773099
PMID: 19571306  [Indexed for MEDLINE]


599. PLoS Genet. 2009 Nov;5(11):e1000713. doi: 10.1371/journal.pgen.1000713. Epub 2009
Nov 6.

E Unibus Plurum: genomic analysis of an experimentally evolved polymorphism in
Escherichia coli.

Kinnersley MA(1), Holben WE, Rosenzweig F.

Author information: 
(1)Division of Biological Sciences, University of Montana, Missoula, Montana,
United States of America.

Microbial populations founded by a single clone and propagated under resource
limitation can become polymorphic. We sought to elucidate genetic mechanisms
whereby a polymorphism evolved in Escherichia coli under glucose limitation and
persisted because of cross-feeding among multiple adaptive clones. Apart from a
29 kb deletion in the dominant clone, no large-scale genomic changes
distinguished evolved clones from their common ancestor. Using transcriptional
profiling on co-evolved clones cultured separately under glucose-limitation we
identified 180 genes significantly altered in expression relative to the common
ancestor grown under similar conditions. Ninety of these were similarly expressed
in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are 
in operons coordinately regulated by CRP and/or rpoS. While the remaining
significant expression differences were clone-specific, 93% were exhibited by the
majority clone, many of which are controlled by global regulators, CRP and CpxR. 
When transcriptional profiling was performed on adaptive clones cultured
together, many expression differences that distinguished the majority clone
cultured in isolation were absent, suggesting that CpxR may be activated by
overflow metabolites removed by cross-feeding strains in co-culture. Relative to 
their common ancestor, shared expression differences among adaptive clones were
partly attributable to early-arising shared mutations in the trans-acting global 
regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences 
that distinguished clones may in part be explained by mutations in trans-acting
regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a
cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation 
in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists 
that thrive on overflow metabolites. Later-arising mutations that led to
specialization emphasize the importance of compensatory rather than
gain-of-function mutations in this system. Taken together, these findings
underscore the importance of regulatory change, founder genotype, and the biotic 
environment in the adaptive evolution of microbes.

DOI: 10.1371/journal.pgen.1000713 
PMCID: PMC2763269
PMID: 19893610  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no competing
interests exist.


600. J Biol Chem. 2009 Oct 23;284(43):29480-8. doi: 10.1074/jbc.M109.005868. Epub 2009
Aug 18.

Genome scale reconstruction of a Salmonella metabolic model: comparison of
similarity and differences with a commensal Escherichia coli strain.

AbuOun M(1), Suthers PF, Jones GI, Carter BR, Saunders MP, Maranas CD, Woodward
MJ, Anjum MF.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories Agency
(Weybridge), Addlestone, Surrey KT153NB, United Kingdom.
m.abuoun@vla.defra.gsi.gov.uk

Salmonella are closely related to commensal Escherichia coli but have gained
virulence factors enabling them to behave as enteric pathogens. Less well studied
are the similarities and differences that exist between the metabolic properties 
of these organisms that may contribute toward niche adaptation of Salmonella
pathogens. To address this, we have constructed a genome scale Salmonella
metabolic model (iMA945). The model comprises 945 open reading frames or genes,
1964 reactions, and 1036 metabolites. There was significant overlap with genes
present in E. coli MG1655 model iAF1260. In silico growth predictions were
simulated using the model on different carbon, nitrogen, phosphorous, and sulfur 
sources. These were compared with substrate utilization data gathered from high
throughput phenotyping microarrays revealing good agreement. Of the compounds
tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless
a number of differences were identified both between Salmonella and E. coli and
also within the Salmonella strains included. These differences provide valuable
insight into differences between a commensal and a closely related pathogen and
within different pathogenic strains opening new avenues for future explorations.

DOI: 10.1074/jbc.M109.005868 
PMCID: PMC2785581
PMID: 19690172  [Indexed for MEDLINE]


601. PLoS One. 2009 Oct 6;4(10):e7316. doi: 10.1371/journal.pone.0007316.

Insights into the molecular basis of L-form formation and survival in Escherichia
coli.

Glover WA(1), Yang Y, Zhang Y.

Author information: 
(1)Department of Molecular Microbiology and Immunology, Bloomberg School of
Public Health, Johns Hopkins University, Baltimore, Maryland, United States of
America.

L-forms have been shown to occur among many species of bacteria and are suspected
to be involved in persistent infections. Since their discovery in 1935, numerous 
studies characterizing L-form morphology, growth, and pathogenic potential have
been conducted. However, the molecular mechanisms underlying the formation and
survival of L-forms remain unknown. Using unstable L-form colonies of Escherichia
coli as a model, we performed genome-wide transcriptome analysis and screened a
deletion mutant library to study the molecular mechanisms involved in formation
and survival of L-forms. Microarray analysis of L-form versus classical colonies 
revealed many up-regulated genes of unknown function as well as multiple
over-expressed stress pathways shared in common with persister cells and
biofilms. Mutant screens identified three groups of mutants which displayed
varying degrees of defects in L-form colony formation. Group 1 mutants, which
showed the strongest defect in L-form colony formation, belonged to pathways
involved in cell envelope stress, DNA repair, iron homeostasis, outer membrane
biogenesis, and drug efflux/ABC transporters. Four (Group 1) mutants, rcsB, a
positive response regulator of colanic acid capsule synthesis, ruvA, a
recombinational junction binding protein, fur, a ferric uptake regulator and smpA
a small membrane lipoprotein were selected for complementation. Complementation
of the mutants using a high-copy overexpression vector failed, while utilization 
of a low-copy inducible vector successfully restored L-form formation. This work 
represents the first systematic genetic evaluation of genes and pathways involved
in the formation and survival of unstable L-form bacteria. Our findings provide
new insights into the molecular mechanisms underlying L-form formation and
survival and have implications for understanding the emergence of antibiotic
resistance, bacterial persistence and latent infections and designing novel drugs
and vaccines.

DOI: 10.1371/journal.pone.0007316 
PMCID: PMC2752164
PMID: 19806199  [Indexed for MEDLINE]


602. Curr Microbiol. 2009 Oct;59(4):425-31. doi: 10.1007/s00284-009-9455-2. Epub 2009 
Jul 21.

Cloning and characterization of a novel tuf promoter from Lactococcus lactis
subsp. lactis IL1403.

Kim EB(1), Piao da C, Son JS, Choi YJ.

Author information: 
(1)Department of Agricultural Biotechnology, Seoul National University, Seoul,
South Korea.

Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene
expression system. Especially, a stable promoter is an important genetic element 
to induce gene expression in such a system. We report on a novel tuf promoter
(Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected 
through analysis of previously published microarray data. Ptuf activity was
examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA,
and Pusp45) using different bacteria as expression hosts. Each promoter was,
respectively, fused to the promoterless and modified bmpB gene as a reporter, and
we estimated promoter activity through BmpB expression. All promoters were active
in IL1403, and Ptuf activity was strongest among them. The activity of each
promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus
reuteri ATCC23272, and Escherichia coli Top10F'). Ptuf had the highest activity
in IL1403 when growth reached late log phase. The activity of each promoter
correlated with the expression of each cognate gene in the microarray data (R (2)
= 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such 
as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms
and Ptuf can be used to develop a reliable gene expression system in L. lactis.

DOI: 10.1007/s00284-009-9455-2 
PMID: 19626364  [Indexed for MEDLINE]


603. Curr Opin Microbiol. 2009 Oct;12(5):536-46. doi: 10.1016/j.mib.2009.07.006. Epub 
2009 Sep 14.

Experimental approaches for the discovery and characterization of regulatory
small RNA.

Sharma CM(1), Vogel J.

Author information: 
(1)RNA Biology Group, Max Planck Institute for Infection Biology, Charitéplatz 1,
D-10117 Berlin, Germany.

Following the pioneering screens for small regulatory RNAs (sRNAs) in Escherichia
coli in 2001, sRNAs are now being identified in almost every branch of the
eubacterial kingdom. Experimental strategies have become increasingly important
for sRNA discovery, thanks to increased availability of tiling arrays and fast
progress in the development of high-throughput cDNA sequencing (RNA-Seq). The new
technologies also facilitate genome-wide discovery of potential target mRNAs by
sRNA pulse-expression coupled to transcriptomics, and immunoprecipitation with
RNA-binding proteins such as Hfq. Moreover, the staggering rate of new sRNAs
demands mechanistic analysis of target regulation. We will also review the
available toolbox for wet lab-based research, including in vivo and in vitro
reporter systems, genetic methods and biochemical co-purification of sRNA
interaction partners.

DOI: 10.1016/j.mib.2009.07.006 
PMID: 19758836  [Indexed for MEDLINE]


604. J Bacteriol. 2009 Oct;191(19):6136-44. doi: 10.1128/JB.00663-09. Epub 2009 Jul
24.

Participation of regulator AscG of the beta-glucoside utilization operon in
regulation of the propionate catabolism operon.

Ishida Y(1), Kori A, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Tokyo, Japan.

The asc operon of Escherichia coli is one of the cryptic genetic systems for
beta-D-galactoside utilization as a carbon source. The ascFB genes for
beta-D-galactoside transport and catabolism are repressed by the AscG regulator. 
After genomic SELEX screening, AscG was found to recognize and bind the consensus
palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites
upstream of the ascFB promoter and at three sites upstream of the prpBC operon
for propionate catabolism. In an ascG-disrupted mutant, transcription of ascFB
was enhanced, in agreement with the repressor model of AscG. This repression was 
indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box, 
which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase
to the promoter. Under conditions of steady-state E. coli growth in a rich
medium, the intracellular level of AscG stayed constant at a level supposedly
leading to tight repression of the ascFB operon. The level of prpR, encoding the 
activator of prpBCDE, was also increased in the absence of AscG, indicating the
involvement of AscG in repression of prpR. Taken together, these data suggest a
metabolic link through interplay between the asc and prp operons.

DOI: 10.1128/JB.00663-09 
PMCID: PMC2747900
PMID: 19633077  [Indexed for MEDLINE]


605. J Clin Microbiol. 2009 Oct;47(10):3178-84. doi: 10.1128/JCM.00366-09. Epub 2009
Jul 29.

Detection of Enterobacter sakazakii and other pathogens associated with infant
formula powder by use of a DNA microarray.

Wang M(1), Cao B, Gao Q, Sun Y, Liu P, Feng L, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, 23
HongDa St., TEDA, Tianjin 300457, China.

Pathogen detection is critical to the process of generating and testing powdered 
infant formula (PIF). An obstacle associated with PIF microbial surveillance is
that most current procedures are time-consuming and labor-intensive. We have
developed a rapid, DNA microarray-based detection technique to identify 10
different pathogenic bacteria associated with PIF contamination based on the
16S-23S rRNA gene internal transcribed spacer (ITS) sequences and wzy (O antigen 
polymerase) gene. Using this procedure, Enterobacter sakazakii, Salmonella
enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,
Acinetobacter baumannii, Bacillus cereus, Listeria monocytogenes, Staphylococcus 
aureus, and Escherichia coli O157 were identified. One hundred eighty-five
strains were used to validate the microarray assay (including 134 target pathogen
strains and 51 closely related bacteria). Twenty-seven probes reproducibly
detected multiple pathogens with high specificity and sensitivity (0.100 ng
genomic DNA or 10(4) CFU/ml). Twenty-one real PIF samples were tested by the
microarray with 100% accuracy. The data presented reveal that the designed
oligonucleotide microarray is a promising method for basic microbiology, clinical
diagnosis, food safety, and epidemiological surveillance.

DOI: 10.1128/JCM.00366-09 
PMCID: PMC2756907
PMID: 19641057  [Indexed for MEDLINE]


606. Mol Microbiol. 2009 Oct;74(2):347-63. doi: 10.1111/j.1365-2958.2009.06856.x. Epub
2009 Aug 24.

Hfq negatively regulates type III secretion in EHEC and several other pathogens.

Shakhnovich EA(1), Davis BM, Waldor MK.

Author information: 
(1)Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and 
Howard Hughes Medical Institute, Boston, MA 02115, USA.

Hfq is a conserved RNA-binding protein that regulates diverse cellular processes 
through post-transcriptional control of gene expression, often by functioning as 
a chaperone for regulatory sRNAs. Here, we explored the role of Hfq in
enterohaemorrhagic Escherichia coli (EHEC), a group of non-invasive intestinal
pathogens. EHEC virulence is dependent on a Type III secretion system encoded in 
the LEE pathogenicity island. The abundance of transcripts for all 41 LEE genes
and more than half of confirmed non-LEE-encoded T3 effectors were elevated in an 
EHEC hfq deletion mutant. Thus, Hfq promotes co-ordinated expression of the
LEE-encoded T3S apparatus and both LEE- and non-LEE-encoded effectors. Increased 
transcript levels led to the formation of functional secretion complexes capable 
of secreting high quantities of effectors into the supernatant. The increase in
LEE-derived transcripts and proteins was dependent on Ler, the LEE-encoded
transcriptional activator, and the ler transcript appears to be a direct target
of Hfq-mediated negative regulation. Finally, we found that Hfq contributes to
the negative regulation of T3SSs in several other pathogens, suggesting that Hfq,
potentially along with species-specific sRNAs, underlies a common means to
prevent unfettered expression of T3SSs.

DOI: 10.1111/j.1365-2958.2009.06856.x 
PMCID: PMC2765575
PMID: 19703108  [Indexed for MEDLINE]


607. BMC Microbiol. 2009 Sep 3;9:186. doi: 10.1186/1471-2180-9-186.

Transcriptome and proteome analyses of adaptive responses to methyl
methanesulfonate in Escherichia coli K-12 and ada mutant strains.

Baek JH(1), Han MJ, Lee SY, Yoo JS.

Author information: 
(1)Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess
Engineering Research Center, Center for Systems and Synthetic Biotechnology and
Institute for the BioCentury, KAIST, Daejeon, Republic of Korea.
swcry@kaist.ac.kr

BACKGROUND: The Ada-dependent adaptive response system in Escherichia coli is
important for increasing resistance to alkylation damage. However, the global
transcriptional and translational changes during this response have not been
reported. Here we present time-dependent global gene and protein expression
profiles following treatment with methyl methanesulfonate (MMS) in E. coli W3110 
and its ada mutant strains.
RESULTS: Transcriptome profiling showed that 1138 and 2177 genes were
differentially expressed in response to MMS treatment in the wild-type and mutant
strains, respectively. A total of 81 protein spots representing 76 nonredundant
proteins differentially expressed were identified using 2-DE and LC-MS/MS. In the
wild-type strain, many genes were differentially expressed upon long-exposure to 
MMS, due to both adaptive responses and stationary phase responses. In the ada
mutant strain, the genes involved in DNA replication, recombination, modification
and repair were up-regulated 0.5 h after MMS treatment, indicating its connection
to the SOS and other DNA repair systems. Interestingly, expression of the genes
involved in flagellar biosynthesis, chemotaxis, and two-component regulatory
systems related to drug or antibiotic resistance, was found to be controlled by
Ada.
CONCLUSION: These results show in detail the regulatory components and pathways
controlling adaptive response and how the related genes including the Ada regulon
are expressed with this response.

DOI: 10.1186/1471-2180-9-186 
PMCID: PMC2753364
PMID: 19728878  [Indexed for MEDLINE]


608. J Bacteriol. 2009 Sep;191(18):5592-602. doi: 10.1128/JB.00157-09. Epub 2009 Jul
10.

Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of
transcriptional regulators, including MetR.

Yeung AT(1), Torfs EC, Jamshidi F, Bains M, Wiegand I, Hancock RE, Overhage J.

Author information: 
(1)Center for Microbial Diseases and Immunity Research, University of British
Columbia, 2259 Lower Mall, Vancouver, BC, Canada.

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to
0.7% agar). Swarming is a more than just a form of locomotion and represents a
complex adaptation resulting in changes in virulence gene expression and
antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 
transposon mutant library to investigate how the complex swarming adaptation
process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were
verified to have alterations in swarming motility. The swarming-associated genes 
functioned not only in flagellar or type IV pilus biosynthesis but also in
processes as diverse as transport, secretion, and metabolism. Thirty-three
swarming-deficient and two hyperswarming mutants had transposon insertions in
transcriptional regulator genes, including genes encoding two-component sensors
and response regulators; 27 of these insertions were newly identified. Of the 25 
regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 
1 (a PA1458 mutant) had a major defect in swimming, suggesting that this
regulator might influence flagellar synthesis or function. Twitching motility,
which requires type IV pili, was strongly affected in only two regulatory mutants
(pilH and PA2571 mutants) and was moderately affected in three other mutants
(algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the
gene expression profile of a swarming-deficient PA3587 mutant to that of the
wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to
metR, which encodes a regulator of methionine biosynthesis in Escherichia coli.
The observed dysregulation in the metR mutant of nine different genes required
for swarming motility provided a possible explanation for the swarming-deficient 
phenotype of this mutant.

DOI: 10.1128/JB.00157-09 
PMCID: PMC2737960
PMID: 19592586  [Indexed for MEDLINE]


609. J Exp Med. 2009 Aug 31;206(9):1883-97. doi: 10.1084/jem.20091233. Epub 2009 Aug
3.

Disordered macrophage cytokine secretion underlies impaired acute inflammation
and bacterial clearance in Crohn's disease.

Smith AM(1), Rahman FZ, Hayee B, Graham SJ, Marks DJ, Sewell GW, Palmer CD, Wilde
J, Foxwell BM, Gloger IS, Sweeting T, Marsh M, Walker AP, Bloom SL, Segal AW.

Author information: 
(1)Department of Medicine, University College London, London WC1E 6BT, England,
UK.

Erratum in
    J Exp Med. 2009 Sep 28;206(10):2301.

Comment in
    Gastroenterology. 2010 Mar;138(3):1204-6; discussion 1207.

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, 
these patients possess an impaired acute inflammatory response, which could
result in delayed clearance of bacteria penetrating the lining of the bowel and
predispose to granuloma formation and chronicity. We tested this hypothesis in
human subjects by monitoring responses to killed Escherichia coli injected
subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at
these sites and clearance of (32)P-labeled bacteria from them were markedly
impaired in CD. Locally increased blood flow and bacterial clearance were
dependent on the numbers of bacteria injected. Secretion of proinflammatory
cytokines by CD macrophages was grossly impaired in response to E. coli or
specific Toll-like receptor agonists. Despite normal levels and stability of
cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were 
abnormally low in CD macrophages. Coupled with reduced secretion, these findings 
indicate accelerated intracellular breakdown. Differential transcription profiles
identified disease-specific genes, notably including those encoding proteins
involved in vesicle trafficking. Intracellular destruction of TNF was decreased
by inhibitors of lysosomal function. Together, our findings suggest that in CD
macrophages, an abnormal proportion of cytokines are routed to lysosomes and
degraded rather than being released through the normal secretory pathway.

DOI: 10.1084/jem.20091233 
PMCID: PMC2737162
PMID: 19652016  [Indexed for MEDLINE]


610. BMC Genomics. 2009 Aug 26;10:402. doi: 10.1186/1471-2164-10-402.

Two distinct groups of porcine enteropathogenic Escherichia coli strains of
serogroup O45 are revealed by comparative genomic hybridization and virulence
gene microarray.

Bruant G(1), Zhang Y, Garneau P, Wong J, Laing C, Fairbrother JM, Gannon VP,
Harel J.

Author information: 
(1)Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de médecine
vétérinaire, Université de Montréal, 3200 rue Sicotte, Saint-Hyacinthe, Québec
J2S 7C6, Canada. guillaume.bruant@nrc-cnrc.gc.ca

BACKGROUND: Porcine enteropathogenic Escherichia coli (PEPEC) strains of
serogroup O45 cause post-weaning diarrhea and produce characteristic attaching
and effacing (A/E) lesions. Most O45 PEPEC strains possess the locus of
enterocyte effacement (LEE), encoding the virulence factors required for
production of A/E lesions, and often possess the paa gene, which is thought to
contribute to the early stages of PEPEC pathogenicity. In this study, nine O45
PEPEC strains and a rabbit enteropathogenic (REPEC) strain, known to produce A/E 
lesions in vivo, were characterized using an E. coli O157-E. coli K12 whole
genome microarray and a virulence gene-specific microarray, and by PCR
experiments.
RESULTS: Based on their virulence gene profiles, the 10 strains were considered
to be atypical EPEC. The differences in their genomes pointed to the
identification of two distinct evolutionary groups of O45 PEPEC, Groups I and II,
and provided evidence for a contribution of these genetic differences to their
virulence in pigs. Group I included the REPEC strain and four O45 PEPEC strains
known to induce severe A/E lesions in challenged pigs whereas Group II was
composed of the five other O45 PEPEC strains, which induced less severe or no A/E
lesions in challenged pigs. Significant differences between Groups I and II were 
found with respect to the presence or absence of 50 O-Islands (OIs) or S-loops
and 13 K-islands (KIs) or K-loops, including the virulence-associated islands
OI#1 (S-loop#1), OI#47 (S-loop#71), OI#57 (S-loop#85), OI#71 (S-loop#108),
OI#115, OI#122, and OI#154 (S-loop#253).
CONCLUSION: We have genetically characterized a collection of O45 PEPEC strains
and classified them into two distinct groups. The differences in their virulence 
gene and genomic island content may influence the pathogenicity of O45 PEPEC
strains, and explain why Group I O45 PEPEC strains induced more severe A/E
lesions in explants and challenged pigs than Group II strains.

DOI: 10.1186/1471-2164-10-402 
PMCID: PMC2749873
PMID: 19709428  [Indexed for MEDLINE]


611. BMC Genomics. 2009 Aug 25;10:397. doi: 10.1186/1471-2164-10-397.

Implications of high level pseudogene transcription in Mycobacterium leprae.

Williams DL(1), Slayden RA, Amin A, Martinez AN, Pittman TL, Mira A, Mitra A,
Nagaraja V, Morrison NE, Moraes M, Gillis TP.

Author information: 
(1)HRSA, BPHC, Division of National Hansen's Disease Programs, Laboratory
Research Branch, Molecular Biology Research Department, School of Veterinary
Medicine, Louisiana State University, Baton Rouge, LA, USA. dwill21@lsu.edu

BACKGROUND: The Mycobacterium leprae genome has less than 50% coding capacity and
1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are
expressed. Therefore, defining pseudogene transcriptional and translational
potentials of this genome should increase our understanding of their impact on M.
leprae physiology.
RESULTS: Gene expression analysis identified transcripts from 49% of all M.
leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. 
Transcribed pseudogenes were randomly distributed throughout the chromosome.
Factors resulting in pseudogene transcription included: 1) co-orientation of
transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like 
structures; 2) the paucity of intrinsic stem-loop transcriptional terminators
between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene 
promoters. Mechanisms for translational "silencing" of pseudogene transcripts
included the lack of both translational start codons and strong Shine-Dalgarno
(SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop
codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and
ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong 
SD site, a start codon, but containing two in frame stop codons yielded a protein
product when expressed in E. coli.
CONCLUSION: Approximately half of M. leprae's transcriptome consists of inactive 
gene products consuming energy and resources without potential benefit to M.
leprae. Presently it is unclear what additional detrimental affect(s) this large 
number of inactive mRNAs has on the functional capability of this organism.
Translation of these pseudogenes may play an important role in overall energy
consumption and resultant pathophysiological characteristics of M. leprae.
However, this study also demonstrated that multiple translational "silencing"
mechanisms are present, reducing additional energy and resource expenditure
required for protein production from the vast majority of these transcripts.

DOI: 10.1186/1471-2164-10-397 
PMCID: PMC2753549
PMID: 19706172  [Indexed for MEDLINE]


612. BMC Microbiol. 2009 Aug 24;9:176. doi: 10.1186/1471-2180-9-176.

Identification of network topological units coordinating the global expression
response to glucose in Bacillus subtilis and its comparison to Escherichia coli.

Vázquez CD(1), Freyre-González JA, Gosset G, Loza JA, Gutiérrez-Ríos RM.

Author information: 
(1)Departamentos de Microbiología Molecular, Instituto de Biotecnología,
Universidad Nacional Autónoma de México, Apdo, Postal 510-3, Cuernavaca, Morelos 
62250, México. cvazquez@ibt.unam.mx

BACKGROUND: Glucose is the preferred carbon and energy source for Bacillus
subtilis and Escherichia coli. A complex regulatory network coordinates gene
expression, transport and enzymatic activities, in response to the presence of
this sugar. We present a comparison of the cellular response to glucose in these 
two model organisms, using an approach combining global transcriptome and
regulatory network analyses.
RESULTS: Transcriptome data from strains grown in Luria-Bertani medium (LB) or
LB+glucose (LB+G) were analyzed, in order to identify differentially transcribed 
genes in B. subtilis. We detected 503 genes in B. subtilis that change their
relative transcript levels in the presence of glucose. A similar previous study
identified 380 genes in E. coli, which respond to glucose. Catabolic repression
was detected in the case of transport and metabolic interconversion activities
for both bacteria in LB+G. We detected an increased capacity for de novo
synthesis of nucleotides, amino acids and proteins. A comparison between
orthologous genes revealed that global regulatory functions such as
transcription, translation, replication and genes relating to the central carbon 
metabolism, presented similar changes in their levels of expression. An analysis 
of the regulatory network of a subset of genes in both organisms revealed that
the set of regulatory proteins responsible for similar physiological responses
observed in the transcriptome analysis are not orthologous. An example of this
observation is that of transcription factors mediating catabolic repression for
most of the genes that displayed reduced transcript levels in the case of both
organisms. In terms of topological functional units in both these bacteria, we
found interconnected modules that cluster together genes relating to heat shock, 
respiratory functions, carbon and peroxide metabolism. Interestingly, B. subtilis
functions not found in E. coli, such as sporulation and competence were shown to 
be interconnected, forming modules subject to catabolic repression at the level
of transcription.
CONCLUSION: Our results demonstrate that the response to glucose is partially
conserved in model organisms E. coli and B. subtilis, including genes encoding
basic functions such as transcription, translation, replication and genes
involved in the central carbon metabolism.

DOI: 10.1186/1471-2180-9-176 
PMCID: PMC2749860
PMID: 19703276  [Indexed for MEDLINE]


613. BMC Microbiol. 2009 Aug 15;9:168. doi: 10.1186/1471-2180-9-168.

Genomic diversity of citrate fermentation in Klebsiella pneumoniae.

Chen YT(1), Liao TL, Wu KM, Lauderdale TL, Yan JJ, Huang IW, Lu MC, Lai YC, Liu
YM, Shu HY, Wang JT, Su IJ, Tsai SF.

Author information: 
(1)Division of Molecular and Genomic Medicine, National Health Research
Institutes, Miaoli, Taiwan, Republic of China. onion@nhri.org.tw

BACKGROUND: It has long been recognized that Klebsiella pneumoniae can grow
anaerobically on citrate. Genes responsible for citrate fermentation of K.
pneumoniae were known to be located in a 13-kb gene cluster on the chromosome. By
whole genome comparison of the available K. pneumoniae sequences (MGH 78578, 342,
and NTUH-K2044), however, we discovered that the fermentation gene cluster was
present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the
previously unknown genome diversity of citrate fermentation among K. pneumoniae
clinical isolates was investigated.
RESULTS: Using a genomic microarray containing probe sequences from multiple K.
pneumoniae strains, we investigated genetic diversity among K. pneumoniae
clinical isolates and found that a genomic region containing the citrate
fermentation genes was not universally present in all strains. We confirmed by
PCR analysis that the gene cluster was detectable in about half of the strains
tested. To demonstrate the metabolic function of the genomic region, anaerobic
growth of K. pneumoniae in artificial urine medium (AUM) was examined for ten
strains with different clinical histories and genomic backgrounds, and the
citrate fermentation potential was found correlated with the genomic region. PCR 
detection of the genomic region yielded high positive rates among a variety of
clinical isolates collected from urine, blood, wound infection, and pneumonia.
Conserved genetic organizations in the vicinity of the citrate fermentation gene 
clusters among K. pneumoniae, Salmonella enterica, and Escherichia coli suggest
that the 13-kb genomic region were not independently acquired.
CONCLUSION: Not all, but nearly half of the K. pneumoniae clinical isolates carry
the genes responsible for anaerobic growth on citrate. Genomic variation of
citrate fermentation genes in K. pneumoniae may contribute to metabolic diversity
and adaptation to variable nutrient conditions in different environments.

DOI: 10.1186/1471-2180-9-168 
PMCID: PMC2735749
PMID: 19682387  [Indexed for MEDLINE]


614. J Mol Biol. 2009 Aug 7;391(1):42-56. doi: 10.1016/j.jmb.2009.05.064. Epub 2009
May 27.

ATP regulates calcium efflux and growth in E. coli.

Naseem R(1), Wann KT, Holland IB, Campbell AK.

Author information: 
(1)Department of Medical Biochemistry and Immunology, Cardiff University, Tenovus
Building, Heath Park, Cardiff, Wales, UK.

Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through 
influx and efflux. Herein, we show for the first time that ATP is essential for
Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome
analysis identified 110 genes whose expression responded to an increase in
cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and 
4 membrane proteins were identified as potential Ca(2+) transport pathways.
Expression of a further 943 genes was modified after 1 h in growth medium
containing Ca(2+) relative to time zero. Based on the microarray results and
other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+) 
was measured in selected mutants from the Keio knockout collection using
intracellular aequorin. In this way, we identified a knockout of atpD, coding for
a component of the F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other
putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect
in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One
millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout
cells to that of the wild type and restored Ca(2+) efflux. One millimolar
2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells.
Under these conditions, a similar defect in Ca(2+) efflux in wild type was
observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) resulted in a
30% elevation in ATP in wild type and was accompanied by a 10% reduction in
generation time under these conditions. Knockouts of pitB, a potential Ca(2+)
transporter, atoA, the beta subunit of acetate CoA-transferase likely to be
involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate
kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP
is most likely to regulate Ca(2+) efflux in E. coli through an ATPase.

DOI: 10.1016/j.jmb.2009.05.064 
PMID: 19481094  [Indexed for MEDLINE]


615. BMC Genomics. 2009 Aug 3;10:349. doi: 10.1186/1471-2164-10-349.

Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7
strain EDL933.

Dong T(1), Schellhorn HE.

Author information: 
(1)Department of Biology Life Sciences Building, Rm, 433, McMaster University,
1280 Main Street, West Hamilton, ON L8S 4K1, Canada. dongt2@mcmaster.ca

BACKGROUND: RpoS is a conserved stress regulator that plays a critical role in
survival under stress conditions in Escherichia coli and other
gamma-proteobacteria. RpoS is also involved in virulence of many pathogens
including Salmonella and Vibrio species. Though well characterized in
non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome
expression has not been examined in pathogenic isolates. E. coli O157:H7 is a
serious human enteropathogen, possessing a genome 20% larger than that of E. coli
K12, and many of the additional genes are required for virulence. The genomic
difference may result in substantial changes in RpoS-regulated gene expression.
To test this, we compared the transcriptional profile of wild type and rpoS
mutants of the E. coli O157:H7 EDL933 type strain.
RESULTS: The rpoS mutation had a pronounced effect on gene expression in
stationary phase, and more than 1,000 genes were differentially expressed
(twofold, P<0.05). By contrast, we found 11 genes expressed differently in
exponential phase. Western blot analysis revealed that, as expected, RpoS level
was low in exponential phase and substantially increased in stationary phase. The
defect in rpoS resulted in impaired expression of genes responsible for stress
response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine
degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler,
espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes
expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS
mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors,
were elevated in the rpoS mutants under LEE induction conditions.
CONCLUSION: Our results show that RpoS has a profound effect on global gene
expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS
regulon, including many EDL933-specific genes, differs substantially from that of
laboratory K12 strains.

DOI: 10.1186/1471-2164-10-349 
PMCID: PMC2907692
PMID: 19650909  [Indexed for MEDLINE]


616. Biotechnol Bioeng. 2009 Aug 1;103(5):975-83. doi: 10.1002/bit.22305.

Localized expression profiles of rpoS in Escherichia coli biofilms.

Ito A(1), May T, Taniuchi A, Kawata K, Okabe S.

Author information: 
(1)Department of Urban and Environmental Engineering, Graduate School of
Engineering, Hokkaido University, North-13, West-8, Kita-ku, Sapporo 060-8628,
Japan.

Although importance of the rpoS gene on biofilm formation by Escherichia coli has
been suggested, there has not been any report showing where the rpoS is expressed
during biofilm formation process. Since physiological state of the cells in the
biofilms is considerably heterogeneous, the expression of the rpoS gene must be
heterogeneous. In this study, in situ spatial expression of the rpoS gene during 
biofilm formation was investigated with an rpoS-gfp transcriptional fusion mutant
strain. A ribosomal binding site and a gene encoding a green fluorescent protein 
were introduced into the downstream of the rpoS gene, which enabled us to observe
the in situ spatial expression of the rpoS gene during biofilm formation
processes without any disturbance of the rpoS expression. In the early stages of 
the biofilm formation process, the rpoS gene was expressed in the most of the
cells. On the other hand, the rpoS expression was observed only at the outside of
the biofilms during the late stages of the biofilm formation process. The in situ
spatial expression of the rpoS gene in the biofilm was verified by quantifying
the expression levels of the rpoS at the outside and the inside of the biofilms
with the real time RT-PCR. In addition, global gene expression analysis was
performed with DNA microarray to investigate physiological difference between the
outside and the inside of the biofilms. This heterogeneous rpoS expression
profile suggested that the cells at the outside of the biofilm need to express
the rpoS to shift the physiological state to the stationary growth mode such as
induction of various stress responses and suppression of the motility.

DOI: 10.1002/bit.22305 
PMID: 19288441  [Indexed for MEDLINE]


617. DNA Cell Biol. 2009 Aug;28(8):413-22. doi: 10.1089/dna.2009.0873.

A Mesocosm of Lactobacillus johnsonii, Bifidobacterium longum, and Escherichia
coli in the mouse gut.

Denou E(1), Rezzonico E, Panoff JM, Arigoni F, Brüssow H.

Author information: 
(1)Nutrition and Health Department, Nestlé Research Center, Lausanne,
Switzerland.

Comment in
    DNA Cell Biol. 2009 Aug;28(8):359-60.

The relative contribution of competition and cooperation at the microbe-microbe
level is not well understood for the bacteria constituting the gut microbiota.
The high number and variability of human gut commensals have hampered the
analysis. To get some insight into the question how so many different bacterial
species can coexist in the mammalian gut, we studied the interaction between
three human gut commensals (Escherichia coli K-12, Lactobacillus johnsonii
NCC533, and Bifidobacterium longum NCC2705) in the intestine of gnotobiotic mice.
The bacterial titers and their anatomical distribution were studied in the
colonized mice. L. johnsonii achieved the highest cell counts in the stomach,
while B. longum dominated the colon. The colon was also the intestinal location
in which B. longum displayed the highest number of expressed genes, followed by
the cecum and the small intestine. Addition of further bacterial strains led to
strikingly different results. A Lactobacillus paracasei strain coexisted, while a
second B. longum strain was excluded from the system. Notably, this strain lacked
an operon involved in the degradation, import, and metabolism of mannosylated
glycans. Subsequent introduction of the E. coli Nissle strain resulted in the
elimination of L. johnsonii NCC533 and E. coli K-12, while B. longum NCC2705
showed a transient decrease in population size, demonstrating the dynamic nature 
of microbe-microbe interactions. The study of such simple interacting bacterial
systems might help to derive some basic rules governing microbial ecology within 
the mammalian gut.

DOI: 10.1089/dna.2009.0873 
PMID: 19534605  [Indexed for MEDLINE]


618. J Bacteriol. 2009 Aug;191(16):5180-95. doi: 10.1128/JB.00476-09. Epub 2009 Jun 5.

Sxy induces a CRP-S regulon in Escherichia coli.

Sinha S(1), Cameron AD, Redfield RJ.

Author information: 
(1)Department of Zoology, University of British Columbia, Life Sciences Center,
Vancouver, Canada. sinha@zoology.ubc.ca

Escherichia coli is not considered naturally competent, yet it has homologues of 
the genes that most competent bacteria use for DNA uptake and processing. In
Haemophilus influenzae and Vibrio cholerae, these genes are regulated by the Sxy 
and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether
similar regulation occurs in E. coli. Expression of sxy strongly induced 63
transcriptional units, 34 of which required CRP for transcriptional activation
and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif
previously characterized in H. influenzae. As previously reported, sxy expression
also induced the sigma-H regulon. Flagellar operons were downregulated by sxy
expression, although motility remained unaffected. The CRP-S regulon included all
of E. coli's known competence gene homologues, so we investigated Sxy's effect on
competence-associated phenotypes. A sxy knockout reduced both "natural" plasmid
transformation and competitive fitness in long-term culture. In addition,
expression of plasmid-borne sxy led to production of type IV pilin, the main
subunit of the DNA uptake machinery of most bacteria. Although H. influenzae Sxy 
only weakly activated the E. coli Sxy regulon, induction was dramatically
improved when it was coexpressed with its cognate CRP, suggesting that intimate
interactions between Sxy and CRP are required for transcriptional activation at
CRP-S sites.

DOI: 10.1128/JB.00476-09 
PMCID: PMC2725579
PMID: 19502395  [Indexed for MEDLINE]


619. J Microbiol Methods. 2009 Aug;78(2):181-8. doi: 10.1016/j.mimet.2009.05.015. Epub
2009 May 29.

A DNA array based assay for the characterization of microbial community in raw
milk.

Giannino ML(1), Aliprandi M, Feligini M, Vanoni L, Brasca M, Fracchetti F.

Author information: 
(1)Istituto Sperimentale Italiano Lazzaro Spallanzani, via Einstein, località
Cascina Codazza, 26900 Lodi, Italy. laura.giannino@istitutospallanzani.it

An oligonucleotide array based on PCR method containing a combination of probes
for taxonomic markers and species-specific virulence genes was developed for the 
simultaneous identification of 14 Lactic Acid Bacteria (LAB) and food-borne
pathogenic bacteria in raw milk. The hypervariable regions V3 and V6 of 16S,
together with a variable region of 23S rRNA genes and several genes specific for 
virulence factors were selected. Universal primers and multiplex PCR were used
for the rapid differentiation of bacterial species and low concentration of
specific pathogenic and spoilage bacteria were detected in milk. The dominant
species such as Streptococcus thermophilus, Enterococcus faecalis, Lactococcus
lactis, andLeuconostoc lactis were identified by indirect-labelling reactions
based upon incorporation of amino-allyl dUTPs. The results regarding food-borne
pathogens detection showed highly specific hybridisation patterns with the
genomic DNA from Campylobacter jejuni, Escherichia coli, Salmonella thyphimurium,
Listeria monocytogenes and Yersinia enterocolitica. A clear differentiation of
dominant species present in a complex microbial community such as raw milk was
achieved by the application of short oligonucleotide probes which discriminate
sequences differing by few nucleotides.

DOI: 10.1016/j.mimet.2009.05.015 
PMID: 19482050  [Indexed for MEDLINE]


620. PLoS One. 2009 Jul 21;4(7):e6279. doi: 10.1371/journal.pone.0006279.

How the intestinal peptide transporter PEPT-1 contributes to an obesity phenotype
in Caenorhabditits elegans.

Spanier B(1), Lasch K, Marsch S, Benner J, Liao W, Hu H, Kienberger H, Eisenreich
W, Daniel H.

Author information: 
(1)Abteilung Biochemie, ZIEL Research Center of Nutrition and Food Sciences,
Technische Universität München, Freising, Germany. spanier@wzw.tum.de

BACKGROUND: Amino acid absorption in the form of di- and tripeptides is mediated 
by the intestinal proton-coupled peptide transporter PEPT-1 (formally OPT-2) in
Caenorhabditits elegans. Transporter-deficient animals (pept-1(lg601)) show
impaired growth, slowed postembryonal development and major changes in amino acid
status.
PRINCIPAL FINDINGS: Here we demonstrate that abolished intestinal peptide
transport also leads to major metabolic alterations that culminate in a two fold 
increase in total body fat content. Feeding of C. elegans with [U-(13)C]-labelled
E. coli revealed a decreased de novo synthesis of long-chain fatty acids in
pept-1(lg601) and reduced levels of polyunsaturated fatty acids. mRNA profiling
revealed increased transcript levels of enzymes/transporters needed for
peroxisomal beta-oxidation and decreased levels for those required for fatty acid
synthesis, elongation and desaturation. As a prime and most fundamental process
that may account for the increased fat content in pept-1(lg601) we identified a
highly accelerated absorption of free fatty acids from the bacterial food in the 
intestine.
CONCLUSIONS: The influx of free fatty acids into intestinal epithelial cells is
strongly dependent on alterations in intracellular pH which is regulated by the
interplay of PEPT-1 and the sodium-proton exchanger NHX-2. We here provide
evidence for a central mechanism by which the PEPT-1/NHX-2 system strongly
influences the in vivo fat content of C. elegans. Loss of PEPT-1 decreases
intestinal proton influx leading to a higher uptake of free fatty acids with fat 
accumulation whereas loss of NHX-2 causes intracellular acidification by the
PEPT-1 mediated proton/dipeptide symport with an almost abolished uptake of fatty
acids and a lean phenotype.

DOI: 10.1371/journal.pone.0006279 
PMCID: PMC2708923
PMID: 19621081  [Indexed for MEDLINE]


621. Biochemistry. 2009 Jul 7;48(26):6278-84. doi: 10.1021/bi802373d.

In vitro selection of RNA aptamers derived from a genomic human library against
the TAR RNA element of HIV-1.

Watrin M(1), Von Pelchrzim F, Dausse E, Schroeder R, Toulmé JJ.

Author information: 
(1)Inserm U869, European Institute of Chemistry and Biology, Pessac, France.

The transactivating responsive (TAR) element is a RNA hairpin located in the 5'
untranslated region of HIV-1 mRNA. It is essential for full-length transcription 
of the retroviral genome and therefore for HIV-1 replication. Hairpin aptamers
that generate highly stable and specific complexes with TAR were previously
identified, thus decreasing the level of TAR-dependent expression in cultured
cells [Kolb, G., et al. (2006) RNA Biol. 3, 150-156]. We performed genomic SELEX 
against TAR using a human RNA library to identify human transcripts that might
interact with the retroviral genome through loop-loop interactions and
potentially contribute to the regulation of TAR-mediated processes. We identified
a genomic aptamer termed a1 that folds as a hairpin with an apical loop
complementary to five nucleotides of the TAR hexanucleotide loop. Surface plasmon
resonance experiments performed on a truncated or mutated version of the a1
aptamer, in the presence of the Rop protein of Escherichia coli, indicate the
formation of a highly stable a1-TAR kissing complex. The 5' ACCCAG loop of a1
constitutes a new motif of interaction with the TAR loop.

DOI: 10.1021/bi802373d 
PMID: 19496624  [Indexed for MEDLINE]


622. BMC Genomics. 2009 Jul 3;10:296. doi: 10.1186/1471-2164-10-296.

Genomic diversity of pathogenic Escherichia coli of the EHEC 2 clonal complex.

Abu-Ali GS(1), Lacher DW, Wick LM, Qi W, Whittam TS.

Author information: 
(1)Microbial Evolution Laboratory, National Food Safety & Toxicology Center, 165 
Food Safety & Toxicology Building, Michigan State University, East Lansing,
Michigan 48824, USA. abualiga@cvm.msu.edu

BACKGROUND: Evolutionary analyses of enterohemorrhagic Escherichia coli (EHEC)
have identified two distantly related clonal groups: EHEC 1, including serotype
O157:H7 and its inferred ancestor O55:H7; and EHEC 2, comprised of several
serogroups (O26, O111, O118, etc.). These two clonal groups differ in their
virulence and global distribution. Although several fully annotated genomic
sequences exist for strains of serotype O157:H7, much less is known about the
genomic composition of EHEC 2. In this study, we analyzed a set of 24 clinical
EHEC 2 strains representing serotypes O26:H11, O111:H8/H11, O118:H16, O153:H11
and O15:H11 from humans and animals by comparative genomic hybridization (CGH) on
an oligoarray based on the O157:H7 Sakai genome.
RESULTS: Backbone genes, defined as genes shared by Sakai and K-12, were highly
conserved in EHEC 2. The proportion of Sakai phage genes in EHEC 2 was
substantially greater than that of Sakai-specific bacterial (non-phage) genes.
This proportion was inverted in O55:H7, reiterating that a subset of Sakai
bacterial genes is specific to EHEC 1. Split decomposition analysis of gene
content revealed that O111:H8 was more genetically uniform and distinct from
other EHEC 2 strains, with respect to the Sakai O157:H7 gene distribution.
Serotype O26:H11 was the most heterogeneous EHEC 2 subpopulation, comprised of
strains with the highest as well as the lowest levels of Sakai gene content
conservation. Of the 979 parsimoniously informative genes, 15% were found to be
compatible and their distribution in EHEC 2 clustered O111:H8 and O118:H16
strains by serotype. CGH data suggested divergence of the LEE island from the
LEE1 to the LEE4 operon, and also between animal and human isolates irrespective 
of serotype. No correlation was found between gene contents and geographic
locations of EHEC 2 strains.
CONCLUSION: The gene content variation of phage-related genes in EHEC 2 strains
supports the hypothesis that extensive modular shuffling of mobile DNA elements
has occurred among EHEC strains. These results suggest that EHEC 2 is a multiform
pathogenic clonal complex, characterized by substantial intra-serotype genetic
variation. The heterogeneous distribution of mobile elements has impacted the
diversification of O26:H11 more than other EHEC 2 serotypes.

DOI: 10.1186/1471-2164-10-296 
PMCID: PMC2713265
PMID: 19575794  [Indexed for MEDLINE]


623. J Biol Chem. 2009 Jul 3;284(27):18377-89. doi: 10.1074/jbc.M109.001503. Epub 2009
Apr 19.

Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding
by ZinT, zinc transport and zinc-independent proteins.

Graham AI(1), Hunt S, Stokes SL, Bramall N, Bunch J, Cox AG, McLeod CW, Poole RK.

Author information: 
(1)Department of Molecular Biology and Biotechnology, University of Sheffield,
Western Bank, Sheffield S10 2TN, United Kingdom.

Zinc ions play indispensable roles in biological chemistry. However, bacteria
have an impressive ability to acquire Zn(2+) from the environment, making it
exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive
understanding of the importance of Zn(2+) has not been attained. Reduction of the
Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported
here for the first time, without recourse to chelators of poor specificity. Cells
grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to
five times less cellular Zn(2+). To understand global responses to Zn(2+)
deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete
and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were
up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient
chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc
uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold
lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein 
possessed a single, high affinity metal-binding site that can accommodate Zn(2+) 
or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) 
finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene 
encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression.
During both batch and chemostat growth, cells "found" more Zn(2+) than was
originally added to the culture, presumably because of leaching from the culture 
vessel. Zn(2+) elimination is shown to be a more precise method of depleting
Zn(2+) than by using the chelator
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

DOI: 10.1074/jbc.M109.001503 
PMCID: PMC2709383
PMID: 19377097  [Indexed for MEDLINE]


624. J Bacteriol. 2009 Jul;191(14):4562-71. doi: 10.1128/JB.00108-09. Epub 2009 May 8.

Involvement of the leucine response transcription factor LeuO in regulation of
the genes for sulfa drug efflux.

Shimada T(1), Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Tokyo, Japan.

LeuO, a LysR family transcription factor, exists in a wide variety of bacteria of
the family Enterobacteriaceae and is involved in the regulation of as yet
unidentified genes affecting the stress response and pathogenesis expression.
Using genomic screening by systematic evolution of ligands by exponential
enrichment (SELEX) in vitro, a total of 106 DNA sequences were isolated from 12
different regions of the Escherichia coli genome. All of the SELEX fragments
formed complexes in vitro with purified LeuO. After Northern blot analysis of the
putative target genes located downstream of the respective LeuO-binding sequence,
a total of nine genes were found to be activated by LeuO, while three genes were 
repressed by LeuO. The LeuO target gene collection included several multidrug
resistance genes. A phenotype microarray assay was conducted to identify the
gene(s) responsible for drug resistance and the drug species that are under the
control of the LeuO target gene(s). The results described herein indicate that
the yjcRQP operon, one of the LeuO targets, is involved in sensitivity control
against sulfa drugs. We propose to rename the yjcRQP genes the sdsRQP genes
(sulfa drug sensitivity determinant).

DOI: 10.1128/JB.00108-09 
PMCID: PMC2704711
PMID: 19429622  [Indexed for MEDLINE]


625. J Bacteriol. 2009 Jul;191(13):4298-306. doi: 10.1128/JB.01829-08. Epub 2009 Apr
24.

Involvement of a membrane-bound class III adenylate cyclase in regulation of
anaerobic respiration in Shewanella oneidensis MR-1.

Charania MA(1), Brockman KL, Zhang Y, Banerjee A, Pinchuk GE, Fredrickson JK,
Beliaev AS, Saffarini DA.

Author information: 
(1)Department of Biological Sciences, University of Wisconsin-Milwaukee,
Milwaukee, Wisconsin 53211, USA.

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella 
oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose.
Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class
IV, and class III adenylate cyclases, respectively, have been identified in the
genome sequence of this bacterium. Functional validation through complementation 
of an Escherichia coli cya mutant confirmed that these genes encode proteins with
adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not
affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or
Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO
and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that
of a crp mutant, which lacks the ability to grow anaerobically with DMSO,
fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted.
Microarray analysis of gene expression in the crp and cyaC mutants revealed the
involvement of both genes in the regulation of key respiratory pathways, such as 
DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated
with plasmid replication, flagellum biosynthesis, and electron transport were
differentially expressed in the cyaC mutant but not in the crp mutant. Our
results indicated that CyaC plays a major role in regulating anaerobic
respiration and may contribute to additional signaling pathways independent of
CRP.

DOI: 10.1128/JB.01829-08 
PMCID: PMC2698484
PMID: 19395492  [Indexed for MEDLINE]


626. J Dairy Sci. 2009 Jul;92(7):3027-39. doi: 10.3168/jds.2008-1773.

Pathogen detection in milk samples by ligation detection reaction-mediated
universal array method.

Cremonesi P(1), Pisoni G, Severgnini M, Consolandi C, Moroni P, Raschetti M,
Castiglioni B.

Author information: 
(1)Institute of Agricultural Biology and Biotechnology-Italian National Research 
Council, Via Bassini 15, 20133 Milan, Italy. paola.cremonesi@unimi.it

This paper describes a new DNA chip, based on the use of a ligation detection
reaction coupled to a universal array, developed to detect and analyze, directly 
from milk samples, microbial pathogens known to cause bovine, ovine, and caprine 
mastitis or to be responsible for foodborne intoxication or infection, or both.
Probes were designed for the identification of 15 different bacterial groups:
Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci,
Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus
dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus
pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp.,
and Escherichia coli and related species. These groups were identified based on
the 16S rRNA gene. For microarray validation, 22 strains from the American Type
Culture Collection or other culture collections and 50 milk samples were tested. 
The results demonstrated high specificity, with sensitivity as low as 6 fmol.
Moreover, the ligation detection reaction-universal array assay allowed for the
identification of Mycoplasma spp. in a few hours, avoiding the long incubation
times of traditional microbiological identification methods. The universal array 
described here is a versatile tool able to identify milk pathogens efficiently
and rapidly.

DOI: 10.3168/jds.2008-1773 
PMID: 19528580  [Indexed for MEDLINE]


627. Nucleic Acids Res. 2009 Jul;37(13):4407-19. doi: 10.1093/nar/gkp378. Epub 2009
May 25.

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins
revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB.

Yokoyama K(1), Nogami H, Kabasawa M, Ebihara S, Shimowasa A, Hashimoto K,
Kawashima T, Ishijima SA, Suzuki M.

Author information: 
(1)National Institute of Advanced Industrial Science and Technology, Tsukuba
Center 6-10, Tsukuba 305-8566, Japan.

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e.
paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was 
studied. Using the method of systematic evolution of ligands by exponential
enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11
and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC,
CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into
the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases
complementary to each other. Apparent equilibrium binding constants of the FFRPs 
and various DNA duplexes were determined, thereby confirming the DNA-binding
specificities of the FFRPs. It is likely that these FFRPs recognize DNA in
essentially the same way, since their DNA-binding specificities were all
explained by the same pattern of relationship between amino-acid positions and
base positions to form chemical interactions. As predicted from this
relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal 
DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the 
b base changed from T to G/A. DNA-binding characteristics of other archaeal
FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.

DOI: 10.1093/nar/gkp378 
PMCID: PMC2715240
PMID: 19468044  [Indexed for MEDLINE]


628. BMC Genomics. 2009 Jun 29;10:287. doi: 10.1186/1471-2164-10-287.

In silico genomic analyses reveal three distinct lineages of Escherichia coli
O157:H7, one of which is associated with hyper-virulence.

Laing CR(1), Buchanan C, Taboada EN, Zhang Y, Karmali MA, Thomas JE, Gannon VP.

Author information: 
(1)Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Lethbridge,
AB, Canada. chad_r_laing@phac-aspc.gc.ca

BACKGROUND: Many approaches have been used to study the evolution, population
structure and genetic diversity of Escherichia coli O157:H7; however,
observations made with different genotyping systems are not easily relatable to
each other. Three genetic lineages of E. coli O157:H7 designated I, II and I/II
have been identified using octamer-based genome scanning and microarray
comparative genomic hybridization (mCGH). Each lineage contains significant
phenotypic differences, with lineage I strains being the most commonly associated
with human infections. Similarly, a clade of hyper-virulent O157:H7 strains
implicated in the 2006 spinach and lettuce outbreaks has been defined using
single-nucleotide polymorphism (SNP) typing. In this study an in silico
comparison of six different genotyping approaches was performed on 19 E. coli
genome sequences from 17 O157:H7 strains and single O145:NM and K12 MG1655
strains to provide an overall picture of diversity of the E. coli O157:H7
population, and to compare genotyping methods for O157:H7 strains.
RESULTS: In silico determination of lineage, Shiga-toxin bacteriophage
integration site, comparative genomic fingerprint, mCGH profile, novel region
distribution profile, SNP type and multi-locus variable number tandem repeat
analysis type was performed and a supernetwork based on the combination of these 
methods was produced. This supernetwork showed three distinct clusters of strains
that were O157:H7 lineage-specific, with the SNP-based hyper-virulent clade 8
synonymous with O157:H7 lineage I/II. Lineage I/II/clade 8 strains clustered
closest on the supernetwork to E. coli K12 and E. coli O55:H7, O145:NM and
sorbitol-fermenting O157 strains.
CONCLUSION: The results of this study highlight the similarities in relationships
derived from multi-locus genome sampling methods and suggest a "common genotyping
language" may be devised for population genetics and epidemiological studies.
Future genotyping methods should provide data that can be stored centrally and
accessed locally in an easily transferable, informative and extensible format
based on comparative genomic analyses.

DOI: 10.1186/1471-2164-10-287 
PMCID: PMC2719669
PMID: 19563677  [Indexed for MEDLINE]


629. Appl Environ Microbiol. 2009 Jun;75(11):3542-53. doi: 10.1128/AEM.02841-08. Epub 
2009 Apr 3.

Gene expression induced in Escherichia coli O157:H7 upon exposure to model apple 
juice.

Bergholz TM(1), Vanaja SK, Whittam TS.

Author information: 
(1)National Food Safety and Toxicology Center, Michigan State University, East
Lansing, 48824, USA. tmb224@cornell.edu

Escherichia coli O157:H7 has caused serious outbreaks of food-borne illness via
transmission in a variety of food vehicles, including unpasteurized apple juice, 
dried salami, and spinach. To understand how this pathogen responds to the
multiple stresses of the food environment, we compared global transcription
patterns before and after exposure to model apple juice. Transcriptomes of
mid-exponential- and stationary-phase cells were evaluated after 10 min in model 
apple juice (pH 3.5) using microarrays probing 4,886 open reading frames. A total
of 331 genes were significantly induced upon exposure of cells to model apple
juice, including genes involved in the acid, osmotic, and oxidative stress
responses as well as the envelope stress response. Acid and osmotic stress
response genes, including asr, osmC, osmB, and osmY, were significantly induced
in response to model apple juice. Multiple envelope stress responses were
activated as evidenced by increased expression of CpxR and Rcs
phosphorelay-controlled genes. Genes controlled by CpxR (cpxP, degP, and htpX)
were significantly induced 2- to 15-fold upon exposure to apple juice.
Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7
in model apple juice compared to the isogenic parent strain. Of the 331 genes
induced in model apple juice, 104 are O157-specific genes, including those
encoding type three secretion effectors (espJ, espB, espM2, espL3, and espZ).
Elucidating the response of O157:H7 to acidic foods provides insight into how
this pathogen is able to survive in food matrices and how exposure to foods
influences subsequent transmission and virulence.

DOI: 10.1128/AEM.02841-08 
PMCID: PMC2687279
PMID: 19346340  [Indexed for MEDLINE]


630. Can J Microbiol. 2009 Jun;55(6):714-28. doi: 10.1139/w09-016.

Global responses of Escherichia coli to adverse conditions determined by
microarrays and FT-IR spectroscopy.

Moen B(1), Janbu AO, Langsrud S, Langsrud O, Hobman JL, Constantinidou C, Kohler 
A, Rudi K.

Author information: 
(1)Nofima Mat, N-1430 As, Norway. birgitte.moen@nofima.no

The global gene expression and biomolecular composition in an Escherichia coli
model strain exposed to 10 adverse conditions (sodium chloride, ethanol,
glycerol, hydrochloric and acetic acid, sodium hydroxide, heat (46 degrees C),
and cold (15 degrees C), as well as ethidium bromide and the disinfectant
benzalkonium chloride) were determined using DNA microarrays and Fourier
transform infrared (FT-IR) spectroscopy. In total, approximately 40% of all
investigated genes (1682/4279 genes) significantly changed expression, compared
with a nonstressed control. There were, however, only 3 genes (ygaW (unknown
function), rmf (encoding a ribosomal modification factor), and ghrA (encoding a
glyoxylate/hydroxypyruvate reductase)) that significantly changed expression
under all conditions (not including benzalkonium chloride). The FT-IR analysis
showed an increase in unsaturated fatty acids during ethanol and cold exposure,
and a decrease during acid and heat exposure. Cold conditions induced changes in 
the carbohydrate composition of the cell, possibly related to the upregulation of
outer membrane genes (glgAP and rcsA). Although some covariance was observed
between the 2 data sets, principle component analysis and regression analyses
revealed that the gene expression and the biomolecular responses are not well
correlated in stressed populations of E. coli, underlining the importance of
multiple strategies to begin to understand the effect on the whole cell.

DOI: 10.1139/w09-016 
PMID: 19767843  [Indexed for MEDLINE]


631. FEMS Microbiol Lett. 2009 Jun;295(1):96-102. doi:
10.1111/j.1574-6968.2009.01587.x.

Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock.

Carruthers MD(1), Minion C.

Author information: 
(1)Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, IA, USA.

The response to elevated temperature [heat shock (HS)] is highly conserved. The
transcriptome of Escherichia coli K-12 has been studied under a variety of
conditions while such studies involving E. coli O157:H7 are only now being
conducted. To better understand the impact of HS on E. coli O157:H7, global
transcript levels of strain EDL933 cells shifted from 37 to 50 degrees C for 15
min were compared with cells held at 37 degrees C by microarray. Using a mixed
model analysis, 193 genes were found to be differentially transcribed at P<0.0042
with a q value <0.1. The 111 downregulated genes include the curli
pili-associated genes csgABCDEFG, maltose transport-associated proteins malEFK,
and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes upregulated
include the HS-induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with 
two LEE-encoded genes: hypothetical gene Z5121 and sepZ. Twenty-three additional 
genes located in O-islands were found to be differentially expressed.
Quantitative real-time PCR (qRT-PCR) was performed to validate the microarray
results. Also, samples subjected to a 30-42 degrees C shift were examined by
qRT-PCR to confirm differential transcription of selected genes. These results
indicate that this pathogen may regulate its virulence factors in response to
temperature changes.

DOI: 10.1111/j.1574-6968.2009.01587.x 
PMID: 19473256  [Indexed for MEDLINE]


632. J Appl Physiol (1985). 2009 Jun;106(6):1935-42. doi:
10.1152/japplphysiol.91361.2008. Epub 2009 Apr 2.

Effects of spaceflight on innate immune function and antioxidant gene expression.

Baqai FP(1), Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, Chapes
SK, Pecaut MJ.

Author information: 
(1)Department of Radiation Medicine, Loma Linda University, Loma Linda, CA 92354,
USA. fpirayesh05b@llu.edu

Spaceflight conditions have a significant impact on a number of physiological
functions due to psychological stress, radiation, and reduced gravity. To explore
the effect of the flight environment on immunity, C57BL/6NTac mice were flown on 
a 13-day space shuttle mission (STS-118). In response to flight, animals had a
reduction in liver, spleen, and thymus masses compared with ground (GRD) controls
(P < 0.005). Splenic lymphocyte, monocyte/macrophage, and granulocyte counts were
significantly reduced in the flight (FLT) mice (P < 0.05). Although spontaneous
blastogenesis of splenocytes in FLT mice was increased, response to
lipopolysaccharide (LPS), a B-cell mitogen derived from Escherichia coli, was
decreased compared with GRD mice (P < 0.05). Secretion of IL-6 and IL-10, but not
TNF-alpha, by LPS-stimulated splenocytes was increased in FLT mice (P < 0.05).
Finally, many of the genes responsible for scavenging reactive oxygen species
were upregulated after flight. These data indicate that exposure to the
spaceflight environment can increase anti-inflammatory mechanisms and change the 
ex vivo response to LPS, a bacterial product associated with septic shock and a
prominent Th1 response.

DOI: 10.1152/japplphysiol.91361.2008 
PMCID: PMC2692779
PMID: 19342437  [Indexed for MEDLINE]


633. J Bacteriol. 2009 Jun;191(11):3553-68. doi: 10.1128/JB.01738-08. Epub 2009 Mar
27.

Genomic instability in regions adjacent to a highly conserved pch prophage in
Escherichia coli O157:H7 generates diversity in expression patterns of the LEE
pathogenicity island.

Yang Z(1), Kim J, Zhang C, Zhang M, Nietfeldt J, Southward CM, Surette MG,
Kachman SD, Benson AK.

Author information: 
(1)Department of Food Science and Technology, University of Nebraska, Lincoln, NE
68583-0919, USA.

The LEE pathogenicity island has been acquired on multiple occasions within the
different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In
each lineage, LEE expression is regulated by complex networks of pathways,
including core pathways shared by all lineages and lineage-specific pathways.
Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain
variation in LEE expression has been observed, implying that expression patterns 
can diversify even within highly related subpopulations. Using comparative
genomics of E. coli O157:H7 subpopulations, we have identified one source of
strain-level variation affecting LEE expression. The variation occurs in
prophage-dense regions of the genome that lie immediately adjacent to the late
regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch
gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB,
pchC, and pchX genes of their respective prophage are highly conserved but are
nonetheless embedded within adjacent genomic segments that are extraordinarily
variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable
degree of variation, the pattern of variation in pch AGR is highly correlated
with the distribution of phylogenetic markers on the backbone of the genome.
Quantitative analysis of transcription from the LEE1 promoter further revealed
that variation in the pch AGR has substantial effects on absolute levels and
patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a
mechanism to diversify LEE expression patterns, and the lineage-specific pattern 
of pch AGR variation could ultimately influence ecological or virulence
characteristics of subpopulations within each lineage.

DOI: 10.1128/JB.01738-08 
PMCID: PMC2681921
PMID: 19329643  [Indexed for MEDLINE]


634. Microbiology. 2009 Jun;155(Pt 6):1901-11. doi: 10.1099/mic.0.026062-0. Epub 2009 
Apr 21.

Effect of FliK mutation on the transcriptional activity of the {sigma}54 sigma
factor RpoN in Helicobacter pylori.

Douillard FP(1), Ryan KA, Hinds J, O'Toole PW.

Author information: 
(1)Department of Microbiology and Alimentary Pharmabiotic Centre, University
College Cork, Western Road, Cork, Ireland.

Helicobacter pylori is a motile Gram-negative bacterium that colonizes and
persists in the human gastric mucosa. The flagellum gene regulatory circuitry of 
H. pylori is unique in many aspects compared with the Salmonella/Escherichia coli
paradigms, and some regulatory checkpoints remain unclear. FliK controls the hook
length during flagellar assembly. Microarray analysis of a fliK-null mutant
revealed increased transcription of genes under the control of the sigma(54)
sigma factor RpoN. This sigma factor has been shown to be responsible for
transcription of the class II flagellar genes, including flgE and flaB. No genes 
higher in the flagellar hierarchy had altered expression, suggesting specific and
localized FliK-dependent feedback on the RpoN regulon. FliK thus appears to be
involved in three processes: hook-length control, export substrate specificity
and control of RpoN transcriptional activity.

DOI: 10.1099/mic.0.026062-0 
PMCID: PMC3145110
PMID: 19383688  [Indexed for MEDLINE]


635. PLoS One. 2009 May 28;4(5):e5713. doi: 10.1371/journal.pone.0005713.

How do human cells react to the absence of mitochondrial DNA?

Mineri R(1), Pavelka N, Fernandez-Vizarra E, Ricciardi-Castagnoli P, Zeviani M,
Tiranti V.

Author information: 
(1)Unit of Molecular Neurogenetics-Pierfranco and Luisa Mariani Center for the
study of Mitochondrial Disorders in Children, IRCCS Foundation Neurological
Institute C. Besta, Milan, Italy.

BACKGROUND: Mitochondrial biogenesis is under the control of two different
genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). 
The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90
subunits that form the respiratory chain, the remaining ones being encoded by the
nDNA. Eukaryotic cells are able to monitor and respond to changes in
mitochondrial function through alterations in nuclear gene expression, a
phenomenon first defined in yeast and known as retrograde regulation. To
investigate how the cellular transcriptome is modified in response to the absence
of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene
expression profile of two human cell lines, 143BTK(-) and A549, which had been
entirely depleted of mtDNA (rho(o) cells), and compared it with that of
corresponding undepleted parental cells (rho(+) cells).
RESULTS: Our data indicate that absence of mtDNA is associated with: i) a
down-regulation of cell cycle control genes and a reduction of cell replication
rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the
respiratory chain and iii) a down-regulation of a gene described as the human
homolog of ELAC2 of E. coli, which encodes a protein that we show to also target 
to the mitochondrial compartment.
CONCLUSIONS: Our results indicate a strong correlation between mitochondrial
biogenesis and cell cycle control and suggest that some proteins could have a
double role: for instance in controlling both cell cycle progression and
mitochondrial functions. In addition, the finding that ELAC2 and maybe other
transcripts that are located into mitochondria, are down-regulated in rho(o)
cells, make them good candidates for human disorders associated with defective
replication and expression of mtDNA.

DOI: 10.1371/journal.pone.0005713 
PMCID: PMC2683933
PMID: 19492094  [Indexed for MEDLINE]


636. Biosens Bioelectron. 2009 May 15;24(9):2818-24. doi: 10.1016/j.bios.2009.02.009. 
Epub 2009 Feb 21.

Wafer-scale fabrication of patterned carbon nanofiber nanoelectrode arrays: a
route for development of multiplexed, ultrasensitive disposable biosensors.

Arumugam PU(1), Chen H, Siddiqui S, Weinrich JA, Jejelowo A, Li J, Meyyappan M.

Author information: 
(1)NASA Ames Research Center, Moffett Field, CA 94035, United States.
prabhu.u.arumugam@nasa.gov

One of the major limitations in the development of ultrasensitive electrochemical
biosensors based on one-dimensional nanostructures is the difficulty involved
with reliably fabricating nanoelectrode arrays (NEAs). In this work, we describe 
a simple, robust and scalable wafer-scale fabrication method to produce
multiplexed biosensors. Each sensor chip consists of nine individually
addressable arrays that uses electron beam patterned vertically aligned carbon
nanofibers (VACNFs) as the sensing element. To ensure nanoelectrode behavior with
higher sensitivity, VACNFs were precisely grown on 100 nm Ni dots with 1 microm
spacing on each micro pad. Pretreatments by the combination of soaking in 1.0 M
HNO(3) and electrochemical etching in 1.0M NaOH dramatically improved the
electrode performance, indicated by the decrease of redox peak separation in
cyclic voltammogram (DeltaE(p)) to approximately 100 mV and an approximately 200%
increase in steady-state currents. The electrochemical detection of the
hybridization of DNA targets from E. coli O157:H7 onto oligonucleotide probes
were successfully demonstrated. The 9 arrays within the chip were divided into
three groups with triplicate sensors for positive control, negative control and
specific hybridization. The proposed method has the potential to be scaled up to 
NxN arrays with N up to 10, which is ideal for detecting a myriad of organisms.
In addition, such sensors can be used as a generic platform for many
electroanalysis applications.

DOI: 10.1016/j.bios.2009.02.009 
PMID: 19303281  [Indexed for MEDLINE]


637. Vet Immunol Immunopathol. 2009 May 15;129(1-2):36-48. doi:
10.1016/j.vetimm.2008.12.001. Epub 2008 Dec 6.

Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells
stimulated with lipopolysaccharide and lipoteichoic acid.

Daly KA(1), Mailer SL, Digby MR, Lefévre C, Thomson P, Deane E, Nicholas KR,
Williamson P.

Author information: 
(1)Faculty of Veterinary Science, B19, University of Sydney, Camperdown, NSW,
Australia.

The immunological function of the metatherian mammary gland plays a crucial part 
in neonatal survival of the marsupial young. Marsupial pouch young do not develop
adult like immune responses until just prior to leaving the pouch. The immune
components of the maternal milk secretions are important during this vulnerable
early post-partum period. In addition, infection of the mammary gland has not
been recognized in metatherians, despite the ready availability of pathogens in
the pouch. Regardless of which, little is known about the immunobiology of the
mammary gland and the immune responses of mammary epithelial cells in
metatherians. In this study, a molecular approach was utilized to examine the
response of tammar (Macropus eugenii) mammary epithelial cells to Escherichia
coli derived lipopolysaccharide (LPS) and Staphylococcus aureus derived
lipoteichoic acid (LTA). Using custom-made cDNA microarrays, candidate genes were
identified in the transciptome, which were involved in antigen presentation,
inflammation, cell growth and proliferation, cellular damage and apoptosis.
Quantification of mRNA expression of several of these candidate genes, along with
seven other genes (TLR4, CD14, TNF-alpha, cathelicidin, PRDX1, IL-5 and ABCG2)
associated with innate immunity in LPS and LTA challenged mammary epithelial
cells and leukocytes, was assessed for up to 24 h. Differences in genes
associated with cellular damage and pro-inflammatory cytokine production were
seen between stimulated mammary epithelial cells and leukocytes. LTA challenge
tended to result in lower level induction of pro-inflammatory cytokines,
increased PRDX1 mRNA levels, suggesting increased oxidative stress, and increased
CD14 expression, but in a non-TLR4-dependent manner. The use of functional
genomic tools in the tammar identified differences in the response of tammar
mammary epithelial cells (MEC) and leukocytes to challenge with LPS and LTA, and 
validates the utility of the approach. The results of this study are consistent
with a model in which tammar mammary epithelial cells have the capacity to elicit
a complex and robust immune response to pathogens.

DOI: 10.1016/j.vetimm.2008.12.001 
PMID: 19157568  [Indexed for MEDLINE]


638. Anal Sci. 2009 May;25(5):669-74.

Quantification of E. coli DNA on a flow-through chemiluminescence microarray
readout system after PCR amplification.

Donhauser SC(1), Niessner R, Seidel M.

Author information: 
(1)Institute of Hydrochemistry, Technische Universität München, Germany.

We report on a hybridization assay using DNA microarrays for the quantification
of amplification products of the uidA gene of E. coli. Using the stopped-PCR
strategy, the amplified target DNA was strongly dependent on the applied gene
copies. The quantification was carried out by a flow-through chemiluminescence
microarray readout system. The DNA microarrays were based on a poly(ethylene
glycol)-modified glass substrate. The probes on the surface were 18 or 25
nucleotides long and the quantified PCR product was 60 nucleotides. The
amplification was stopped after 25 cycles; at this point amplification was in the
middle of the logarithmical phase, and the spread between different DNA starting 
concentrations reached the maximum. A conjugate of streptavidin and horseradish
peroxidase (HRP) bound to the biotinylated strands on the microarray surface and 
catalyzed the reaction of luminol and hydrogen peroxide. The generated light
emission was recorded by a sensitive charge-coupled device (CCD) camera. The
detection limit for the gene uidA (beta-galactosidase) of E. coli was 1.1 x 10(5)
copies/mL. This system allowed for a sensitive detection and quantification of E.
coli in a concentration range from 10(6) to 10(9) copies/mL.


PMID: 19430151  [Indexed for MEDLINE]


639. Antimicrob Agents Chemother. 2009 May;53(5):2110-9. doi: 10.1128/AAC.01440-08.
Epub 2009 Mar 9.

In vivo and in vitro patterns of the activity of simocyclinone D8, an
angucyclinone antibiotic from Streptomyces antibioticus.

Oppegard LM(1), Hamann BL, Streck KR, Ellis KC, Fiedler HP, Khodursky AB, Hiasa
H.

Author information: 
(1)Department of Pharmacology, University of Minnesota Medical School-Twin
Cities, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.

Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive
bacteria but not against gram-negative bacteria. The molecular basis of the
cytotoxicity of SD8 is not fully understood, although SD8 has been shown to
inhibit the supercoiling activity of Escherichia coli gyrase. To understand the
mechanism of SD8, we have employed biochemical assays to directly measure the
sensitivities of E. coli and Staphylococcus aureus type II topoisomerases to SD8 
and microarray analysis to monitor the cellular responses to SD8 treatment. SD8
is a potent inhibitor of either E. coli or S. aureus gyrase. In contrast, SD8
exhibits only a moderate inhibitory effect on S. aureus topoisomerase IV, and E. 
coli topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect
of SD8 against E. coli has become evident in the absence of the AcrB multidrug
efflux pump. As expected, SD8 treatment exhibits the signature responses to the
loss of supercoiling activity in E. coli: upregulation of gyrase genes and
downregulation of the topoisomerase I gene. Unlike quinolone treatment, however, 
SD8 treatment does not induce the SOS response. These results suggest that DNA
gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and 
that the lack of the antibacterial effect against gram-negative bacteria is due, 
in part, to the activity of the AcrB efflux pump.

DOI: 10.1128/AAC.01440-08 
PMCID: PMC2681514
PMID: 19273673  [Indexed for MEDLINE]


640. IET Syst Biol. 2009 May;3(3):155-66. doi: 10.1049/iet-syb.2008.0130.

Genetic network identification using convex programming.

Julius A(1), Zavlanos M, Boyd S, Pappas GJ.

Author information: 
(1)University of Pennsylvania, Department of Electrical and Systems Engineering, 
USA. agung@seas.upenn.edu

Gene regulatory networks capture interactions between genes and other cell
substances, resulting in various models for the fundamental biological process of
transcription and translation. The expression levels of the genes are typically
measured as mRNA concentration in micro-array experiments. In a so-called genetic
perturbation experiment, small perturbations are applied to equilibrium states
and the resulting changes in expression activity are measured. One of the most
important problems in systems biology is to use these data to identify the
interaction pattern between genes in a regulatory network, especially in a large 
scale network. The authors develop a novel algorithm for identifying the smallest
genetic network that explains genetic perturbation experimental data. By
construction, our identification algorithm is able to incorporate and respect a
priori knowledge known about the network structure. A priori biological knowledge
is typically qualitative, encoding whether one gene affects another gene or not, 
or whether the effect is positive or negative. The method is based on a convex
programming relaxation of the combinatorially hard problem of L(0) minimisation. 
The authors apply the proposed method to the identification of a subnetwork of
the SOS pathway in Escherichia coli, the segmentation polarity network in
Drosophila melanogaster, and an artificial network for measuring the performance 
of the method.

DOI: 10.1049/iet-syb.2008.0130 
PMID: 19449976  [Indexed for MEDLINE]


641. Infect Immun. 2009 May;77(5):2104-12. doi: 10.1128/IAI.01200-08. Epub 2009 Mar 2.

Conditioning of uropathogenic Escherichia coli for enhanced colonization of host.

Bower JM(1), Gordon-Raagas HB, Mulvey MA.

Author information: 
(1)Pathology Department, Division of Cell Biology and Immunology, University of
Utah, 15 North Medical Drive East, Salt Lake City, UT 84112-0565, USA.

While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) 
encounters multiple stresses, including substantial levels of nitric oxide and
reactive nitrogen intermediates. Here we show that UPEC, the primary cause of
urinary tract infections, can be conditioned to grow at higher rates in the
presence of acidified sodium nitrite (ASN), a model system used to generate
nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned 
UPEC bacteria are far more likely to establish an infection than nonconditioned
bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several
NsrR-regulated genes and other stress- and polyamine-responsive factors may be
partially responsible for this effect. Compared to K-12 reference strains, most
UPEC isolates have increased resistance to ASN, and this resistance can be
substantially enhanced by addition of the polyamine cadaverine. Nitrosative
stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and
growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also
promote UPEC colonization of the bladder. These results suggest that UPEC
interactions with polyamines or stresses such as reactive nitrogen intermediates 
can in effect reprogram the bacteria, enabling them to better colonize the host.

DOI: 10.1128/IAI.01200-08 
PMCID: PMC2681758
PMID: 19255192  [Indexed for MEDLINE]


642. Int Urogynecol J Pelvic Floor Dysfunct. 2009 May;20(5):509-13. doi:
10.1007/s00192-008-0801-x. Epub 2009 Jan 20.

Genome-based expression profiles as a single standardized microarray platform for
the diagnosis of experimental interstitial cystitis: an array of 75 genes model.

Tseng LH(1), Chen I, Chen MY, Lee CL, Lo TS, Lloyd LK.

Author information: 
(1)Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and
University of Chang Gung School of Medicine, Tao-Yuan, Taiwan.
3g7330@yahoo.com.tw

INTRODUCTION AND HYPOTHESIS: To investigate the molecular signature underlying
experimental interstitial cystitis (IC) using cDNA microarray.
METHODS: Microarray gene expression profiles are studied in bladder epithelium of
C57BL/6 mice with ovalbumin or substance P-induced experimental IC versus
Escherichia coli lipopolysaccharide-induced bacterial cystitis.
RESULTS: Main findings are summarized as follows: firstly, a "75-gene" model was 
discovered to contain high expressions of bladder epithelium which feature in
experimental IC. Secondly, glucose, lipid, nucleotide, xenobiotics, and amino
acid metabolisms are involved in. Thirdly, T-cell-mediated immune and
inflammatory responses are observed. Fourthly, Wnt, Tgf-beta, Mapk, and insulin
growth factor receptor signaling pathways are also involved in. In addition,
experimental IC leads to Ephrin- and Semaphorin-mediated axon guidance promoting 
parasympathetic inflammatory reflexes.
CONCLUSIONS: Further characterization of human IC-induced gene expression
profiles would enable the use of genome-based expression profiling for the
therapeutic targets and diagnosis of IC.

DOI: 10.1007/s00192-008-0801-x 
PMID: 19153633  [Indexed for MEDLINE]


643. J Bacteriol. 2009 May;191(10):3226-36. doi: 10.1128/JB.01410-08. Epub 2009 Feb
27.

Similar and divergent effects of ppGpp and DksA deficiencies on transcription in 
Escherichia coli.

Aberg A(1), Fernández-Vázquez J, Cabrer-Panes JD, Sánchez A, Balsalobre C.

Author information: 
(1)Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.

The concerted action of ppGpp and DksA in transcription has been widely
documented. In disparity with this model, phenotypic studies showed that ppGpp
and DksA might also have independent and opposing roles in gene expression in
Escherichia coli. In this study we used a transcriptomic approach to compare the 
global transcriptional patterns of gene expression in strains deficient in ppGpp 
(ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were
significantly affected by more than twofold in ppGpp- and DksA-deficient strains,
respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain.
Although the data indicate that most of the affected genes were copositively or
conegatively regulated by ppGpp and DksA, some genes that were independently
and/or differentially regulated by the two factors were found. The large
functional group of chemotaxis and flagellum synthesis genes were notably
differentially affected, with all genes being upregulated in the DksA-deficient
strain but 60% of them being downregulated in the ppGpp-deficient strain.
Revealingly, mutations in the antipausing Gre factors suppress the upregulation
observed in the DksA-deficient strain, emphasizing the importance of the
secondary channel of the RNA polymerase for regulation and fine-tuning of gene
expression in E. coli.

DOI: 10.1128/JB.01410-08 
PMCID: PMC2687150
PMID: 19251846  [Indexed for MEDLINE]


644. PLoS Pathog. 2009 May;5(5):e1000432. doi: 10.1371/journal.ppat.1000432. Epub 2009
May 15.

Genetic dissection of an exogenously induced biofilm in laboratory and clinical
isolates of E. coli.

Amini S(1), Goodarzi H, Tavazoie S.

Author information: 
(1)Department of Molecular Biology and Lewis-Sigler Institute for Integrative
Genomics, Princeton University, Princeton, NJ, USA.

Microbial biofilms are a dominant feature of many human infections. However,
developing effective strategies for controlling biofilms requires an
understanding of the underlying biology well beyond what currently exists. Using 
a novel strategy, we have induced formation of a robust biofilm in Escherichia
coli by utilizing an exogenous source of poly-N-acetylglucosamine (PNAG) polymer,
a major virulence factor of many pathogens. Through microarray profiling of
competitive selections, carried out in both transposon insertion and
over-expression libraries, we have revealed the genetic basis of PNAG-based
biofilm formation. Our observations reveal the dominance of electrostatic
interactions between PNAG and surface structures such as lipopolysaccharides. We 
show that regulatory modulation of these surface structures has significant
impact on biofilm formation behavior of the cell. Furthermore, the majority of
clinical isolates which produced PNAG also showed the capacity to respond to the 
exogenously produced version of the polymer.

DOI: 10.1371/journal.ppat.1000432 
PMCID: PMC2675270
PMID: 19436718  [Indexed for MEDLINE]


645. PLoS Pathog. 2009 May;5(5):e1000408. doi: 10.1371/journal.ppat.1000408. Epub 2009
May 1.

The defective prophage pool of Escherichia coli O157: prophage-prophage
interactions potentiate horizontal transfer of virulence determinants.

Asadulghani M(1), Ogura Y, Ooka T, Itoh T, Sawaguchi A, Iguchi A, Nakayama K,
Hayashi T.

Author information: 
(1)Division of Bioenvironmental Sciences, Frontier Science Research Center,
University of Miyazaki, Miyazaki, Japan.

Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT)
between bacteria. Their roles in dynamic bacterial genome evolution have been
increasingly highlighted by the fact that many sequenced bacterial genomes
contain multiple prophages carrying a wide range of genes. Enterohemorrhagic
Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) 
possesses 18 prophages (Sp1-Sp18) that encode numerous genes related to O157
virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2.
However, most of these prophages appeared to contain multiple genetic defects. To
understand whether these defective prophages have the potential to act as mobile 
genetic elements to spread virulence determinants, we looked closely at the
Sp1-Sp18 sequences, defined the genetic defects of each Sp, and then
systematically analyzed all Sps for their biological activities. We show that
many of the defective prophages, including the Stx1 phage, are inducible and
released from O157 cells as particulate DNA. In fact, some prophages can even be 
transferred to other E. coli strains. We also show that new Stx1 phages are
generated by recombination between the Stx1 and Stx2 phage genomes. The results
indicate that these defective prophages are not simply genetic remnants generated
in the course of O157 evolution, but rather genetic elements with a high
potential for disseminating virulence-related genes and other genetic traits to
other bacteria. We speculate that recombination and various other types of
inter-prophage interactions in the O157 prophage pool potentiate such activities.
Our data provide new insights into the potential activities of the defective
prophages embedded in bacterial genomes and lead to the formulation of a novel
concept of inter-prophage interactions in defective prophage communities.

DOI: 10.1371/journal.ppat.1000408 
PMCID: PMC2669165
PMID: 19412337  [Indexed for MEDLINE]


646. BMC Syst Biol. 2009 Apr 14;3:39. doi: 10.1186/1752-0509-3-39.

Reconstruction of Escherichia coli transcriptional regulatory networks via
regulon-based associations.

Zare H(1), Sangurdekar D, Srivastava P, Kaveh M, Khodursky A.

Author information: 
(1)Department of Biochemistry, Biophysics and Molecular Biology, The University
of Minnesota, St, Paul, MN, USA. hossein@umn.edu

BACKGROUND: Network reconstruction methods that rely on covariance of expression 
of transcription regulators and their targets ignore the fact that transcription 
of regulators and their targets can be controlled differently and/or
independently. Such oversight would result in many erroneous predictions.
However, accurate prediction of gene regulatory interactions can be made possible
through modeling and estimation of transcriptional activity of groups of
co-regulated genes.
RESULTS: Incomplete regulatory connectivity and expression data are used here to 
construct a consensus network of transcriptional regulation in Escherichia coli
(E. coli). The network is updated via a covariance model describing the activity 
of gene sets controlled by common regulators. The proposed model-selection
algorithm was used to annotate the likeliest regulatory interactions in E. coli
on the basis of two independent sets of expression data, each containing many
microarray experiments under a variety of conditions. The key regulatory
predictions have been verified by an experiment and literature survey. In
addition, the estimated activity profiles of transcription factors were used to
describe their responses to environmental and genetic perturbations as well as
drug treatments.
CONCLUSION: Information about transcriptional activity of documented co-regulated
genes (a core regulon) should be sufficient for discovering new target genes,
whose transcriptional activities significantly co-vary with the activity of the
core regulon members. Our ability to derive a highly significant consensus
network by applying the regulon-based approach to two very different data sets
demonstrated the efficiency of this strategy. We believe that this approach can
be used to reconstruct gene regulatory networks of other organisms for which
partial sets of known interactions are available.

DOI: 10.1186/1752-0509-3-39 
PMCID: PMC2689187
PMID: 19366454  [Indexed for MEDLINE]


647. FEMS Microbiol Lett. 2009 Apr;293(1):58-64. doi:
10.1111/j.1574-6968.2009.01508.x. Epub 2009 Feb 7.

Environmental pH affects transcriptional responses to cadmium toxicity in
Escherichia coli K-12 (MG1655).

Worden CR(1), Kovac WK, Dorn LA, Sandrin TR.

Author information: 
(1)Department of Biology and Microbiology, University of Wisconsin-Oshkosh,
Oshkosh, WI, USA.

It has been widely reported that pH mediates cadmium toxicity to bacteria. We
used a tripartite approach to investigate mechanisms by which pH affects cadmium 
toxicity that included analyses of: (1) growth kinetics, (2) global gene
expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7,
but not at pH 5. DNA microarray analysis revealed that stress response genes
including hdeA, otsA, and yjbJ were more highly expressed at pH 5 than at pH 7
after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly
induced genes that confer cadmium resistance. In addition, genes involved in
transport and many hypothetical genes were more highly expressed at pH 5 than at 
pH 7 in the presence of cadmium. Concentrations of two cadmium species, including
one previously implicated in the mechanism by which pH mediates cadmium toxicity 
(CdOH+), increased with pH. Our data demonstrate that transcriptional responses
of Escherichia coli to cadmium are substantially affected by pH and suggest that 
several stress response, transport, and hypothetical genes play roles in the
mechanism by which pH mediates cadmium toxicity.

DOI: 10.1111/j.1574-6968.2009.01508.x 
PMID: 19220470  [Indexed for MEDLINE]


648. J Bacteriol. 2009 Apr;191(8):2691-702. doi: 10.1128/JB.01182-08. Epub 2009 Feb
13.

The 3'-to-5' exoribonuclease (encoded by HP1248) of Helicobacter pylori regulates
motility and apoptosis-inducing genes.

Tsao MY(1), Lin TL, Hsieh PF, Wang JT.

Author information: 
(1)Department of Microbiology, National Taiwan University College of Medicine, 1,
Sec 1, Jen-Ai Rd., Taipei, 10016 Taiwan, ROC.

The human gastric pathogen Helicobacter pylori has many virulence factors
involved in pathogenesis, but the mechanisms regulating these virulence factors
are not yet fully understood. In this study, we cloned HP1248, which is similar
in sequence to Escherichia coli vacB, which was previously shown to be associated
with the expression of virulence in Shigella and enteroinvasive E. coli. E. coli 
vacB encodes RNase R. RNase R is involved in the posttranscriptional regulation
of mRNA stability. By global transcriptional microarray profiling of an H. pylori
HP1248 deletion mutant, we defined six virulence-related genes which were
posttranscriptionally downregulated by HP1248, including the motility-related
genes HP1192 and flaB, the chemotaxis-related gene cheY, and the
apoptosis-inducing genes HP0175, cagA, and gtt. In this study, recombinant HP1248
protein expressed in E. coli showed 3'-to-5' exoribonuclease activity. Motility
and apoptosis induction were increased in the H. pylori HP1248 deletion mutant.
We also showed that HP1192 is associated with H. pylori motility, possibly
through HP1248 regulation. Further, we suggested and studied the possible
mechanisms of this specific regulation of virulent genes by HP1248. In addition, 
the expression level of HP1248 mRNA changed dramatically in response to a variety
of altered environmental conditions, including pH and temperature. Hence, HP1248 
in H. pylori seems to play a role in environmental sensing and in regulation of
virulent phenotypes, such as motility and host apoptosis induction.

DOI: 10.1128/JB.01182-08 
PMCID: PMC2668412
PMID: 19218383  [Indexed for MEDLINE]


649. J Bacteriol. 2009 Apr;191(8):2447-60. doi: 10.1128/JB.01746-08. Epub 2009 Feb 6.

A Burkholderia cenocepacia orphan LuxR homolog is involved in quorum-sensing
regulation.

Malott RJ(1), O'Grady EP, Toller J, Inhülsen S, Eberl L, Sokol PA.

Author information: 
(1)Department of Microbiology and Infectious Diseases, Faculty of Medicine,
University of Calgary, Calgary, Alberta, Canada.

Burkholderia cenocepacia utilizes quorum sensing to control gene expression,
including the expression of genes involved in virulence. In addition to CepR and 
CciR, a third LuxR homolog, CepR2, was found to regulate gene expression and
virulence factor production. All B. cenocepacia strains examined contained this
orphan LuxR homolog, which was not associated with an adjacent N-acyl-homoserine 
lactone synthase gene. Expression of cepR2 was negatively autoregulated and was
negatively regulated by CciR in strain K56-2. Microarray analysis and
quantitative reverse transcription-PCR determined that CepR2 did not influence
expression of cepIR or cciIR. However, in strain K56-2, CepR2 negatively
regulated expression of several known quorum-sensing-controlled genes, including 
genes encoding zinc metalloproteases. CepR2 exerted positive and negative
regulation on genes on three chromosomes, including strong negative regulation of
a gene cluster located adjacent to cepR2. In strain H111, which lacks the CciIR
quorum-sensing system, CepR2 positively regulated pyochelin production by
controlling transcription of one of the operons required for the biosynthesis of 
the siderophore in an N-acyl-homoserine lactone-independent manner. CepR2
activation of a luxI promoter was demonstrated in a heterologous Escherichia coli
host, providing further evidence that CepR2 can function in the absence of
signaling molecules. This study demonstrates that the orphan LuxR homolog CepR2
contributes to the quorum-sensing regulatory network in two distinct strains of
B. cenocepacia.

DOI: 10.1128/JB.01746-08 
PMCID: PMC2668411
PMID: 19201791  [Indexed for MEDLINE]


650. J Bacteriol. 2009 Apr;191(7):2388-91. doi: 10.1128/JB.01594-08. Epub 2009 Jan 16.

Differential binding profiles of StpA in wild-type and h-ns mutant cells: a
comparative analysis of cooperative partners by chromatin
immunoprecipitation-microarray analysis.

Uyar E(1), Kurokawa K, Yoshimura M, Ishikawa S, Ogasawara N, Oshima T.

Author information: 
(1)Graduate School of Information Science, Nara Institute of Science and
Technology, Japan.

We performed chromatin immunoprecipitation-microarray analysis to investigate
differences in function between StpA and H-NS in Escherichia coli cells. StpA
binding regions essentially overlap those of H-NS in wild-type cells, while they 
are reduced to one-third in the hns mutant. The H-NS binding profile was
unaffected by stpA inactivation.

DOI: 10.1128/JB.01594-08 
PMCID: PMC2655504
PMID: 19151137  [Indexed for MEDLINE]


651. Nucleic Acids Res. 2009 Apr;37(5):e38. doi: 10.1093/nar/gkp022. Epub 2009 Feb 2.

Model-based redesign of global transcription regulation.

Carrera J(1), Rodrigo G, Jaramillo A.

Author information: 
(1)Instituto de Biologia Molecular y Celular de Plantas, CSIC, Instituto de
Aplicaciones en Tecnologias de la Informacion y las Comunicaciones Avanzadas,
Universidad Politecnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.

Synthetic biology aims to the design or redesign of biological systems. In
particular, one possible goal could be the rewiring of the transcription
regulation network by exchanging the endogenous promoters. To achieve this
objective, we have adapted current methods to the inference of a model based on
ordinary differential equations that is able to predict the network response
after a major change in its topology. Our procedure utilizes microarray data for 
training. We have experimentally validated our inferred global regulatory model
in Escherichia coli by predicting transcriptomic profiles under new
perturbations. We have also tested our methodology in silico by providing
accurate predictions of the underlying networks from expression data generated
with artificial genomes. In addition, we have shown the predictive power of our
methodology by obtaining the gene profile in experimental redesigns of the E.
coli genome, where rewiring the transcriptional network by means of knockouts of 
master regulators or by upregulating transcription factors controlled by
different promoters. Our approach is compatible with most network inference
methods, allowing to explore computationally future genome-wide redesign
experiments in synthetic biology.

DOI: 10.1093/nar/gkp022 
PMCID: PMC2655681
PMID: 19188257  [Indexed for MEDLINE]


652. Plant Physiol. 2009 Apr;149(4):1860-71. doi: 10.1104/pp.108.133934. Epub 2009 Feb
4.

Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic
pathways by trehalose-6-phosphate.

Zhang Y(1), Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RA, Powers SJ,
Schluepmann H, Delatte T, Wingler A, Paul MJ.

Author information: 
(1)Plant Science, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United
Kingdom.

Comment in
    Plant Signal Behav. 2010 Apr;5(4):386-92.

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how
it signals was not clear. Here, we provide evidence that T6P functions as an
inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the
SNF1-related group of protein kinases. T6P, but not other sugars and sugar
phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling
extracts strongly (50%) at low concentrations (1-20 microM). Inhibition was
noncompetitive with respect to ATP. In immunoprecipitation studies using
antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were
physically separable, with T6P inhibition of SnRK1 dependent on an intermediary
factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues
analyzed except those of mature leaves, which did not contain the intermediary
factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression 
was determined in seedlings expressing Escherichia coli otsA encoding T6P
synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1
target genes showed opposite regulation, consistent with the regulation of SnRK1 
by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes 
involved in biosynthetic reactions, such as genes for amino acid, protein, and
nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron
transport, which are normally down-regulated by SnRK1. In contrast, genes
involved in photosynthesis and degradation processes, which are normally
up-regulated by SnRK1, were down-regulated by T6P. These experiments provide
strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in
growing tissues.

DOI: 10.1104/pp.108.133934 
PMCID: PMC2663748
PMID: 19193861  [Indexed for MEDLINE]


653. Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):554-9.

[Development of single base extension-tags microarray for the detection of
food-borne pathogens].

[Article in Chinese]

Lu C(1), Shi C, Zhang C, Chen J, Shi X.

Author information: 
(1)Department of Food Science and Bor Luh Food Safety Center, School
ofAgriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

We developed single base extension-tags (SBE-tags) microarray to detect eight
common food-borne pathogens, including Staphylococcus aureus, Vibrio
parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki,
Shigella, Escherichia coli O157:H7 and Campylobacter jejuni. With specific PCR
primers identified and integrated for eight food-borne pathogens, target
sequences were amplified and purified as template DNA of single base
extension-tags reaction. The products were hybridized to microarrays and scanned 
for fluorescence intensity. The experiment showed a specific and simultaneous
detection of eight food-borne pathogens. The system limits is 0.1 pg for a
genomic DNA and 5x10(2) CFU/mL for Salmonella typhimurium cultures. The single
base extension-tags assay can be used to detect food-borne pathogens rapidly and 
accurately with a high sensitivity, and provide an efficient way for diagnosis
and control of disease caused by food-borne pathogens.


PMID: 19637631  [Indexed for MEDLINE]


654. Biosens Bioelectron. 2009 Mar 15;24(7):2232-8. doi: 10.1016/j.bios.2008.11.030.
Epub 2008 Dec 11.

STD sensor based on nucleic acid functionalized nanostructured polyaniline.

Singh R(1), Prasad R, Sumana G, Arora K, Sood S, Gupta RK, Malhotra BD.

Author information: 
(1)Biomolecular Electronics and Conducting Polymer Research Group, National
Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India.

STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe
(from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized
nanostructured-polyaniline coated onto indium-tin-oxide-coated glass plate has
been fabricated using avidin-biotin as cross-linking agent. This DNA
functionalized electrode can specifically detect upto 0.5 x 10(-15)M of
complementary target within 60s of hybridization time at 25 degrees C by
differential pulse voltammetry (DPV) using methylene blue as electro-active DNA
hybridization indicator. This highly sensitive and specific nucleic acid
functionalized nanostructured-polyaniline electrode can distinguish presence of
N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and
spiked samples from the urethral swabs of the patients.

DOI: 10.1016/j.bios.2008.11.030 
PMID: 19162465  [Indexed for MEDLINE]


655. Biosens Bioelectron. 2009 Mar 15;24(7):1893-8. doi: 10.1016/j.bios.2008.09.023.
Epub 2008 Oct 8.

Rapid DNA multi-analyte immunoassay on a magneto-resistance biosensor.

Koets M(1), van der Wijk T, van Eemeren JT, van Amerongen A, Prins MW.

Author information: 
(1)Philips Research, High Tech Campus 11, 5656 AE Eindhoven, The Netherlands.

We present the rapid and sensitive detection of amplified DNA on a giant
magneto-resistance sensor using superparamagnetic particles as a detection label.
The one-step assay is performed on an integrated and miniaturized detection
platform suitable for application into point-of-care devices. A double-tagged PCR
amplification product of the LamB gene of the Escherichia coli bacterium was used
to investigate binding kinetics of the assay. We applied magnetic actuation to
concentrate the target-particle complexes at the sensor surface and to remove
unbound particles from the sensor surface. We achieved biological dose-response
curves detecting 4-250pM amplicon concentrations in a one-step format in total
assay times of less than 3min. Using various tag-antibody combinations specific
for one of the individual genes, multi-analyte detection is shown of several
antibiotic resistance genes of the food pathogen Salmonella.

DOI: 10.1016/j.bios.2008.09.023 
PMID: 19028086  [Indexed for MEDLINE]


656. Anal Bioanal Chem. 2009 Mar;393(6-7):1639-47. doi: 10.1007/s00216-008-2574-y.
Epub 2008 Dec 25.

Microarray of DNA-protein complexes on poly-3-hydroxybutyrate surface for
pathogen detection.

Park TJ(1), Yoo SM, Keum KC, Lee SY.

Author information: 
(1)Department of Chemical and Biomolecular Engineering (BK21 program), Center for
Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 335
Gwahangno, Yuseong-gu, Daejeon, 305-701, Republic of Korea.

A novel strategy was developed for the specific immobilization of DNA probes on
poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) 
of PHB depolymerase as an active binding motif. To demonstrate whether this
method can be used for the detection of clinical pathogens, the pathogen-specific
biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to
the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for
four model pathogens, while the target DNAs were prepared by PCR using universal 
primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was
immobilized on the PHB-coated slide by microspotting. This DNA-protein complex
microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia
coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the
specific pathogens could be diagnosed in the presence of other microorganisms.
Thus, the DNA-protein complex microarray platform technology employing PHB and
the SBD reported here can be widely used for the detection of DNA-DNA and
DNA-biomolecule interactions without synthetic or chemical modification of
biomolecules or solid surface.

DOI: 10.1007/s00216-008-2574-y 
PMID: 19107467  [Indexed for MEDLINE]


657. Ann N Y Acad Sci. 2009 Mar;1158:302-13. doi: 10.1111/j.1749-6632.2008.03757.x.

Inference of regulatory gene interactions from expression data using three-way
mutual information.

Watkinson J(1), Liang KC, Wang X, Zheng T, Anastassiou D.

Author information: 
(1)Department of Electrical Engineering.

This paper describes the technique designated best performer in the 2nd
conference on Dialogue for Reverse Engineering Assessments and Methods (DREAM2)
Challenge 5 (unsigned genome-scale network prediction from blinded microarray
data). Existing algorithms use the pairwise correlations of the expression levels
of genes, which provide valuable but insufficient information for the inference
of regulatory interactions. Here we present a computational approach based on the
recently developed context likelihood of related (CLR) algorithm, extracting
additional complementary information using the information theoretic measure of
synergy and assigning a score to each ordered pair of genes measuring the degree 
of confidence that the first gene regulates the second. When tested on a set of
publicly available Escherichia coli gene-expression data with known assumed
ground truth, the synergy augmented CLR (SA-CLR) algorithm had significantly
improved prediction performance when compared to CLR. There is also enhanced
potential for biological discovery as a result of the identification of the most 
likely synergistic partner genes involved in the interactions.

DOI: 10.1111/j.1749-6632.2008.03757.x 
PMID: 19348651  [Indexed for MEDLINE]


658. Ann N Y Acad Sci. 2009 Mar;1158:29-35. doi: 10.1111/j.1749-6632.2008.03746.x.

The condition-dependent transcriptional network in Escherichia coli.

Lemmens K(1), De Bie T, Dhollander T, Monsieurs P, De Moor B, Collado-Vides J,
Engelen K, Marchal K.

Author information: 
(1)Department of Electrical Engineering, Katholieke Universiteit Leuven, Leuven, 
Belgium.

Thanks to the availability of high-throughput omics data, bioinformatics
approaches are able to hypothesize thus-far undocumented genetic interactions.
However, due to the amount of noise in these data, inferences based on a single
data source are often unreliable. A popular approach to overcome this problem is 
to integrate different data sources. In this study, we describe DISTILLER, a
novel framework for data integration that simultaneously analyzes microarray and 
motif information to find modules that consist of genes that are co-expressed in 
a subset of conditions, and their corresponding regulators. By applying our
method on publicly available data, we evaluated the condition-specific
transcriptional network of Escherichia coli. DISTILLER confirmed 62% of 736
interactions described in RegulonDB, and 278 novel interactions were predicted.

DOI: 10.1111/j.1749-6632.2008.03746.x 
PMID: 19348629  [Indexed for MEDLINE]


659. Appl Environ Microbiol. 2009 Mar;75(6):1703-16. doi: 10.1128/AEM.02081-08. Epub
2009 Jan 23.

Reconfiguring the quorum-sensing regulator SdiA of Escherichia coli to control
biofilm formation via indole and N-acylhomoserine lactones.

Lee J(1), Maeda T, Hong SH, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A&M University,
College Station, TX 77843-3122, USA.

SdiA is a homolog of quorum-sensing regulators that detects N-acylhomoserine
lactone (AHL) signals from other bacteria. Escherichia coli uses SdiA to reduce
its biofilm formation in the presence of both AHLs and its own signal indole.
Here we reconfigured SdiA (240 amino acids) to control biofilm formation using
protein engineering. Four SdiA variants were obtained with altered biofilm
formation, including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and
K153X) and SdiA14C3 (W9R, P49T, N87T, frameshift at N96, and L123X), which
reduced biofilm formation by 5- to 20-fold compared to wild-type SdiA in the
presence of endogenous indole. Whole-transcriptome profiling revealed that
wild-type SdiA reduced biofilm formation by repressing genes related to indole
synthesis and curli synthesis compared to when no SdiA was expressed, while
variant SdiA1E11 induced genes related to indole synthesis in comparison to
wild-type SdiA. These results suggested altered indole metabolism, and
corroborating the DNA microarray results in regard to indole synthesis, variant
SdiA1E11 produced ninefold more indole, which led to reduced swimming motility
and cell density. Also, wild-type SdiA decreased curli production and tnaA
transcription, while SdiA1E11 increased tnaA transcription (tnaA encodes
tryptophanase, which forms indole) compared to wild-type SdiA. Hence, wild-type
SdiA decreased biofilm formation by reducing curli production and motility, and
SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive
variant (SdiA2D10, having four mutations at E31G, Y42F, R116H, and L165Q)
increased biofilm formation sevenfold in the presence of N-octanoyl-DL-homoserine
lactone and N-(3-oxododecatanoyl)-L-homoserine lactone. Therefore, SdiA can be
evolved to increase or decrease biofilm formation, and biofilm formation may be
controlled by altering sensors rather than signals.

DOI: 10.1128/AEM.02081-08 
PMCID: PMC2655446
PMID: 19168658  [Indexed for MEDLINE]


660. J Bacteriol. 2009 Mar;191(5):1556-64. doi: 10.1128/JB.01493-08. Epub 2008 Dec 12.

Characterization of YvcJ, a conserved P-loop-containing protein, and its
implication in competence in Bacillus subtilis.

Luciano J(1), Foulquier E, Fantino JR, Galinier A, Pompeo F.

Author information: 
(1)UPR, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille,
France.

The uncharacterized protein family UPF0042 of the Swiss-Prot database is
predicted to be a member of the conserved group of bacterium-specific
P-loop-containing proteins. Here we show that two of its members, YvcJ from
Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and 
hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In
contrast to results recently obtained for E. coli, which indicated that yhbJ
mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison
of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression 
was quite similar in the two strains. However, in mutants defective in yvcJ, the 
transformation efficiency and the fraction of cells that expressed competence
were reduced. Furthermore, our data show that YvcJ positively controls the
expression of late competence genes. The overexpression of comK or comS
compensates for the decrease in competence of the yvcJ mutant. Our results show
that even if YvcJ and YhbJ belong to the same family of P-loop-containing
proteins, the deletion of corresponding genes has different consequences in B.
subtilis and in E. coli.

DOI: 10.1128/JB.01493-08 
PMCID: PMC2648190
PMID: 19074378  [Indexed for MEDLINE]


661. Microbiology. 2009 Mar;155(Pt 3):891-902. doi: 10.1099/mic.0.022277-0.

lmo1273, a novel gene involved in Listeria monocytogenes virulence.

Bigot A(1), Raynaud C, Dubail I, Dupuis M, Hossain H, Hain T, Chakraborty T,
Charbit A.

Author information: 
(1)Université Paris Descartes, Faculté de Médecine René Descartes, Paris F-75015,
France.

Listeria monocytogenes is a foodborne pathogen able to infect humans and many
other mammalian species, leading to serious, often fatal disease. We have
previously identified a five-gene locus in the genome of L. monocytogenes EGD-e
which comprised three contiguous genes encoding paralogous type I signal
peptidases. In the present study, we focused on the two distal genes of the locus
(lmo1272 and lmo1273), encoding proteins sharing significant similarities with
the YlqF and RnhB proteins, respectively, of Bacillus subtilis. lmo1273 could
complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype,
suggesting that it encodes a functional RNase H. Strikingly, inactivation of
lmo1273 provoked a strong attenuation of virulence in the mouse model, and
kinetic studies in infected mice revealed that multiplication of the lmo1273
mutant in target organs was significantly impaired. However, the mutation did not
impair L. monocytogenes intracellular multiplication or cell-to-cell spread in
cell culture models. Transcriptional profiles obtained with an
lmo1273-overexpressing strain were compared to those of the wild-type strain,
using microarray analyses. The data obtained suggest a pleiotropic regulatory
role of Lmo1273 and possible links with amino acid uptake.

DOI: 10.1099/mic.0.022277-0 
PMID: 19246760  [Indexed for MEDLINE]


662. Microbiology. 2009 Mar;155(Pt 3):813-24. doi: 10.1099/mic.0.023911-0.

Exploring the antimicrobial action of a carbon monoxide-releasing compound
through whole-genome transcription profiling of Escherichia coli.

Nobre LS(1), Al-Shahrour F, Dopazo J, Saraiva LM.

Author information: 
(1)Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
Avenida da República (EAN), 2780-157 Oeiras, Portugal.

We recently reported that carbon monoxide (CO) has bactericidal activity. To
understand its mode of action we analysed the gene expression changes occurring
when Escherichia coli, grown aerobically and anaerobically, is treated with the
CO-releasing molecule CORM-2 (tricarbonyldichlororuthenium(II) dimer). Microarray
analysis shows that the E. coli CORM-2 response is multifaceted, with a high
number of differentially regulated genes spread through several functional
categories, namely genes involved in inorganic ion transport and metabolism,
regulators, and genes implicated in post-translational modification, such as
chaperones. CORM-2 has a higher impact in E. coli cells grown anaerobically, as
judged by the repression of genes belonging to eight functional classes which are
not seen in the response of aerobically CORM-2-treated cells. The biological
relevance of the variations caused by CORM-2 was substantiated by studying the
CORM-2 sensitivity of selected E. coli mutants. The results show that the
deletion of redox-sensing regulators SoxS and OxyR increased the sensitivity to
CORM-2 and suggest that while SoxS plays an important role in protection against 
CORM-2 under both growth conditions, OxyR seems to participate only in the
aerobic CORM-2 response. Under anaerobic conditions, we found that the heat-shock
proteins IbpA and IbpB contribute to CORM-2 defence since the deletion of these
genes increases the sensitivity of the strain. The induction of several met genes
and the hypersensitivity to CORM-2 of the DeltametR, DeltametI and DeltametN
mutant strains suggest that CO has effects on the methionine metabolism of E.
coli. CORM-2 also affects the transcription of several E. coli biofilm-related
genes and increases biofilm formation in E. coli. In particular, the absence of
tqsA or bhsA increases the resistance of E. coli to CORM-2, and deletion of tsqA 
leads to a strain that has lost its capacity to form biofilm upon treatment with 
CORM-2. In spite of the relatively stable nature of the CO molecule, our results 
show that CO is able to trigger a significant alteration in the transcriptome of 
E. coli which necessarily has effects in several key metabolic pathways.

DOI: 10.1099/mic.0.023911-0 
PMID: 19246752  [Indexed for MEDLINE]


663. BMC Microbiol. 2009 Feb 23;9:42. doi: 10.1186/1471-2180-9-42.

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica 
serovar Typhimurium.

Merighi M(1), Septer AN, Carroll-Portillo A, Bhatiya A, Porwollik S, McClelland
M, Gunn JS.

Author information: 
(1)Department of Molecular Virology, Center for Microbial Interface Biology, The 
Ohio State University, Columbus, OH 43210, USA. gunn.43@osu.edu

BACKGROUND: The Salmonella PreA/PreB two-component system (TCS) is an ortholog of
the QseBC TCS of Escherichia coli. In both Salmonella and E. coli, this system
has been shown to affect motility and virulence in response to quorum-sensing and
hormonal signals, and to affect the transcription of the Salmonella enterica
serovar Typhimurium (S. Typhimurium) pmrAB operon, which encodes an important
virulence-associated TCS.
RESULTS: To determine the PreA/PreB regulon in S. Typhimurium, we performed DNA
microarrays comparing the wild type strain and various preA and/or preB mutants
in the presence of ectopically expressed preA (qseB). These data confirmed our
previous findings of the negative effect of PreB on PreA gene regulation and
identified candidate PreA-regulated genes. A proportion of the activated loci
were previously identified as PmrA-activated genes (yibD, pmrAB, cptA, etc.) or
were genes located in the local region around preA, including the preAB operon.
The transcriptional units were defined in this local region by RT-PCR, suggesting
three PreA activated operons composed of preA-preB, mdaB-ygiN, and ygiW-STM3175. 
Several putative virulence-related phenotypes were examined for preAB mutants,
resulting in the observation of a host cell invasion and slight virulence defect 
of a preAB mutant. Contrary to previous reports on this TCS, we were unable to
show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of
epinephrine on S. Typhimurium with regard to bacterial motility.
CONCLUSION: This work further characterizes this unorthadox OmpR/EnvZ class TCS
and provides novel candidate regulated genes for further study. This first
in-depth study of the PreA/PreB regulatory system phenotypes and regulation
suggests significant comparative differences to the reported function of the
orthologous QseB/QseC in E. coli.

DOI: 10.1186/1471-2180-9-42 
PMCID: PMC2653508
PMID: 19236707  [Indexed for MEDLINE]


664. Biotechnol Bioeng. 2009 Feb 15;102(3):902-9. doi: 10.1002/bit.22098.

Cell cycle progression in Escherichia coli B/r affects transcription of certain
genes: Implications for synthetic genome design.

Echtenkamp PL(1), Wilson DB, Shuler ML.

Author information: 
(1)School of Chemical and Biomolecular Engineering, Cornell University, Ithaca,
New York 14853-5201, USA.

We propose that transcript levels for some genes are affected by the bacterial
cell division cycle and this may be an important factor to consider when
designing synthetic bacterial genomes. To test this hypothesis, transcript levels
of 58 genes in Escherichia coli B/r A were determined at five times during the
cell division cycle. A two-step ANOVA technique was used to analyze data from
custom oligonucleotide microarrays containing genes involved in important
cellular processes including central metabolism, macromolecular synthesis, and
transport and secretion. Consistent with results previously found in Caulobacter,
approximately 17% of the transcript levels were cell cycle dependent. Cell cycle 
regulation can be divided into two classes: genes displaying increased transcript
concentrations following gene replication and genes displaying an increased
transcript concentration prior to replication initiation. Transcripts levels for 
hns, uspA, and zwf were affected by the cell division cycle, but did not fit well
into either class. These results indicate that transcription of a significant
fraction of the genome is affected by replication cycle progression. The results 
also show that both physical gene position and the physiological function of a
gene affect when it is transcribed. In addition to the simple association with
replication fork progression, other phenomena must be occurring to account for
some of our observations. In conclusion, gene position, with regard to the C
period, and gene function are important factors to incorporate into design
criteria for synthetic bacterial genomes.

DOI: 10.1002/bit.22098 
PMID: 18823050  [Indexed for MEDLINE]


665. J Biol Chem. 2009 Feb 13;284(7):4516-24. doi: 10.1074/jbc.M808210200. Epub 2008
Dec 17.

Carbon monoxide-releasing antibacterial molecules target respiration and global
transcriptional regulators.

Davidge KS(1), Sanguinetti G, Yee CH, Cox AG, McLeod CW, Monk CE, Mann BE,
Motterlini R, Poole RK.

Author information: 
(1)Department of Molecular Biology and Biotechnology, The University of
Sheffield, Firth Ct., Western Bank, Sheffield S10 2TN, United Kingdom.

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory,
anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have
therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality.
Here we identify for the first time the bacterial targets of
Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO
rapidly under physiological conditions. Contrary to the expectation that CO would
be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia
coli cultures were also sensitive to CORM-3 at concentrations equimolar with
oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly
delivered CO intracellularly to terminal oxidases. Microarray analysis of
CORM-3-treated cells revealed extensively modified gene expression, notably
down-regulation of genes encoding key aerobic respiratory complexes. Genes
involved in metal metabolism, homeostasis, or transport were also differentially 
expressed, and free intracellular zinc levels were elevated. Probabilistic
modeling of transcriptomic data identified the global transcription regulators
ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO
sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator
of gene expression, especially under aerobic conditions, has important
implications for administration of CO-releasing agents in sepsis and
inflammation.

DOI: 10.1074/jbc.M808210200 
PMID: 19091747  [Indexed for MEDLINE]


666. Biol Chem. 2009 Feb;390(2):137-44. doi: 10.1515/BC.2009.022.

Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered
intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.

Liu Y(1), Kuan CT, Mi J, Zhang X, Clary BM, Bigner DD, Sullenger BA.

Author information: 
(1)Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.

Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein
uniquely expressed in glioblastoma, but not in normal brain tissues. To develop
targeted therapies for brain tumors, we selected RNA aptamers against the
histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for
protein expression and purification. Representative aptamer E21 has a
dissociation constant (Kd) of 33x10(-9) m, and exhibits high affinity and
specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, 
selected aptamers cannot bind the same protein expressed from eukaryotic cells
because glycosylation, a post-translational modification present only in
eukaryotic systems, significantly alters the structure of the target protein. By 
transfecting EGFRvIII aptamers into cells, we find that membrane-bound,
glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis
is increased. We postulate that transfected aptamers can interact with newly
synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of
mature EGFRvIII reaching the cell surface. Our work establishes the feasibility
of disrupting protein post-translational modifications in situ with aptamers.
This finding is useful for elucidating the function of proteins of interest with 
various modifications, as well as dissecting signal transduction pathways.

DOI: 10.1515/BC.2009.022 
PMCID: PMC3816755
PMID: 19040357  [Indexed for MEDLINE]


667. J Bacteriol. 2009 Feb;191(3):844-50. doi: 10.1128/JB.01081-08. Epub 2008 Nov 21.

The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides
general stress protection.

Rodríguez-Rojas A(1), Blázquez J.

Author information: 
(1)Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones
Científicas (CSIC), Madrid, Spain.

Hypermutator Pseudomonas aeruginosa strains, characterized by an increased
spontaneous-mutation rate, are found at high frequencies in chronic lung
infections. Hypermutability is associated with the loss of antimutator genes
related to DNA repair or damage avoidance systems. Only a few antimutator genes
have been described in P. aeruginosa, although there is some evidence that
additional genes may be involved in naturally occurring hypermutability. In order
to find new P. aeruginosa antimutator genes, we constructed and screened a
library of random insertions in the PA14 strain. Some previously described P.
aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD,
mutT, ung, and mutY, were detected, indicating a good coverage of our insertional
library. One additional mutant contained an insertion in the P. aeruginosa
PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI
superfamily, which includes chaperones, peptidases, and the Parkinson's disease
protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher
spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays
a key role in DNA protection under nonstress conditions. Moreover, the
inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced
mutant frequency. Global transcription studies showed the induction of
bacteriophage Pf1 genes and the repression of genes related to iron metabolism,
suggesting that the increased spontaneous-mutant frequency may be due to reduced 
protection against the basal level of reactive oxygen species. Finally, pfpI
mutants are more sensitive to different types of stress and are affected in
biofilm formation.

DOI: 10.1128/JB.01081-08 
PMCID: PMC2632063
PMID: 19028889  [Indexed for MEDLINE]


668. J Bacteriol. 2009 Feb;191(3):1106-10. doi: 10.1128/JB.00599-08. Epub 2008 Nov 14.

Genome-wide identification of H-NS-controlled, temperature-regulated genes in
Escherichia coli K-12.

White-Ziegler CA(1), Davis TR.

Author information: 
(1)Department of Biological Sciences, Smith College, Northampton, MA 01063, USA. 
cwhitezi@smith.edu

DNA microarrays demonstrate that H-NS controls 69% of the temperature regulated
genes in Escherichia coli K-12. H-NS is shown to be a common regulator of
multiple iron and other nutrient acquisition systems preferentially expressed at 
37 degrees C and of general stress response, biofilm formation, and cold shock
genes highly expressed at 23 degrees C.

DOI: 10.1128/JB.00599-08 
PMCID: PMC2632076
PMID: 19011022  [Indexed for MEDLINE]


669. J Comput Biol. 2009 Feb;16(2):213-27. doi: 10.1089/cmb.2008.08TT.

Reverse engineering molecular regulatory networks from microarray data with
qp-graphs.

Castelo R(1), Roverato A.

Author information: 
(1)Department of Experimental and Health Sciences, Universitat Pompeu Fabra,
Barcelona, Spain. robert.castelo@upf.edu

Reverse engineering bioinformatic procedures applied to high-throughput
experimental data have become instrumental in generating new hypotheses about
molecular regulatory mechanisms. This has been particularly the case for gene
expression microarray data, where a large number of statistical and computational
methodologies have been developed in order to assist in building network models
of transcriptional regulation. A major challenge faced by every different
procedure is that the number of available samples n for estimating the network
model is much smaller than the number of genes p forming the system under study. 
This compromises many of the assumptions on which the statistics of the methods
rely, often leading to unstable performance figures. In this work, we apply a
recently developed novel methodology based in the so-called q-order limited
partial correlation graphs, qp-graphs, which is specifically tailored towards
molecular network discovery from microarray expression data with p >> n. Using
experimental and functional annotation data from Escherichia coli, here we show
how qp-graphs yield more stable performance figures than other state-of-the-art
methods when the ratio of genes to experiments exceeds one order of magnitude.
More importantly, we also show that the better performance of the qp-graph method
on such a gene-to-sample ratio has a decisive impact on the functional coherence 
of the reverse-engineered transcriptional regulatory modules and becomes crucial 
in such a challenging situation in order to enable the discovery of a network of 
reasonable confidence that includes a substantial number of genes relevant to the
essayed conditions. An R package, called qpgraph implementing this method is part
of the Bioconductor project and can be downloaded from (www.bioconductor.org). A 
parallel standalone version for the most computationally expensive calculations
is available from (http://functionalgenomics.upf.xsedu/qpgraph).

DOI: 10.1089/cmb.2008.08TT 
PMID: 19178140  [Indexed for MEDLINE]


670. Mol Cell Probes. 2009 Feb;23(1):20-8. doi: 10.1016/j.mcp.2008.10.006. Epub 2008
Nov 18.

Selection, characterization, and application of DNA aptamers for the capture and 
detection of Salmonella enterica serovars.

Joshi R(1), Janagama H, Dwivedi HP, Senthil Kumar TM, Jaykus LA, Schefers J,
Sreevatsan S.

Author information: 
(1)Veterinary Population Medicine Department, College of Veterinary Medicine,
University of Minnesota, St. Paul, MN 55108, United States.

Sensitive and specific pre-analytical sample processing methods are needed to
enhance our ability to detect and quantify food borne pathogens from complex food
and environmental samples. In this study, DNA aptamers were selected and
evaluated for the capture and detection of Salmonella enterica serovar.
Typhimurium. A total of 66 candidate sequences were enriched against S.
Typhimurium outer membrane proteins (OMPs) with counter-selection against
Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected 
aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five
Salmonella-specific aptamer candidates were selected for further
characterization. A dilution-to-extinction capture protocol using pure cultures
of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and
45) which showed low-end detection limits of 10-40CFU. DNase protection assays
applied to these aptamers confirmed sequence-specific binding to S. Typhimurium
OMP preparations, while South-Western blot analysis combined with mass
spectrometry identified putative membrane proteins as targets for aptamer
binding. Aptamer 33 was bound to magnetic beads and used for the capture of S.
Typhimurium seeded into whole carcass chicken rinse samples, followed by
detection using quantitative real-time RT-PCR. In a pull-down assay format,
detection limits were 10(1)-10(2)CFU S. Typhimurium/9mL rinsate, while in a
recirculation format, detection limits were 10(2)-10(3)CFU/25mL rinsate.
Reproducible detection at <10(1)S. typhimurium CFU/g was also achieved in
spike-and-recovery experiments using bovine feces. The pull-down analysis using
aptamer 33 was validated on 3 naturally infected chicken litter samples
confirming their applicability in the field. This study demonstrates the
applicability of Salmonella specific aptamers for pre-analytical sample
processing as applied to food and environmental sample matrices.

DOI: 10.1016/j.mcp.2008.10.006 
PMID: 19049862  [Indexed for MEDLINE]


671. Plant J. 2009 Feb;57(3):463-72. doi: 10.1111/j.1365-313X.2008.03697.x. Epub 2008 
Sep 26.

Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced
disease resistance in rice plants.

Umemura K(1), Satou J, Iwata M, Uozumi N, Koga J, Kawano T, Koshiba T, Anzai H,
Mitomi M.

Author information: 
(1)Agricultural & Veterinary Research Laboratories, Meiji Seika Kaisha Ltd,
Yokohama 222-8567, Japan. kenji_umemura@meiji.co.jp

Systemic acquired resistance (SAR), a natural disease response in plants, can be 
induced chemically. Salicylic acid (SA) acts as a key endogenous signaling
molecule that mediates SAR in dicotyledonous plants. However, the role of SA in
monocotyledonous plants has yet to be elucidated. In this study, the mode of
action of the agrochemical protectant chemical probenazole was assessed by
microarray-based determination of gene expression. Cloning and characterization
of the most highly activated probenazole-responsive gene revealed that it encodes
UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of
free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice
leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic
acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and
the gene product expressed in Escherichia coli was characterized, and the results
suggested that probenazole-responsive OsSGT1 is involved in the production of
SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly
reduced the probenazole-dependent development of resistance against blast
disease, further supporting the suggestion that OsSGT1 is a key mediator of
development of chemically induced disease resistance. The OsSGT1 gene may
contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice
plants.

DOI: 10.1111/j.1365-313X.2008.03697.x 
PMID: 18826428  [Indexed for MEDLINE]


672. Platelets. 2009 Feb;20(1):50-7. doi: 10.1080/09537100802503368.

Anti-platelet drugs and outcome in severe infection: clinical impact and
underlying mechanisms.

Winning J(1), Reichel J, Eisenhut Y, Hamacher J, Kohl M, Deigner HP, Claus RA,
Bauer M, Lösche W.

Author information: 
(1)Anaesthesiology and Intensive Care Medicine, University Hospital Jena, Jena,
Germany.

Platelet activation contributes to microvascular thrombosis and organ failure in 
systemic inflammation. We tested the hypothesis whether anti-platelet drugs might
favourably affect outcome in patients at risk for organ failure as well as in a
mouse model of endotoxin shock. Two hundred twenty-four consecutive patients who 
were admitted for community acquired pneumonia over a time period of 5 years to a
University Hospital were enrolled; about 20% of whom received anti-platelet drugs
(acetylsalicylic acid, thienopyridines) for secondary prevention of
cardiovascular disease. Patients with anti-platelet drugs were about 12 years old
but did not differ in SOFA score and routine laboratory parameters at admission. 
Logistic regression and 2 x 2 table analysis in age-matched subgroups indicated
that anti-platelet drugs may reduce the need of intensive care treatment (odds
ratio (OR) 0.32 [95% confidential interval: 0.10-1.00] and 0.19 [0.04-0.87],
respectively). In age-matched subgroups, the use of anti-platelet drugs was also 
associated with a shorter stay in hospital (13.9 +/- 6.2 vs. 18.2 +/- 10.2 days; 
p < 0.02). In the animal model Balb/c mice were pre-treated with clopidogrel
(added to drinking water) for 4 days prior to intraperitoneal (i.p.)
administration of endotoxin (lipopolsaccharide (LPS) from Escherichia coli
0111:B4). Within the first 48 hours after LPS there were no differences between
clopidogrel and control animals (n = 26 each) in macro-haemodynamics. However,
clopidogrel abolished the LPS-induced drop in platelet count and reduced fibrin
deposition in lung tissue. Using DNA microarray technology, we could show that
clopidogrel suppressed endotoxin-induced up-regulation of inflammation-relevant
genes, including arachidonate-5-lipoxygenase activating protein and leukotriene
B4 receptor 1. According to our data a possible benefit of anti-platelet drugs in
patients on risk for systemic inflammation and organ failure should be tested in 
a prospective trial.

DOI: 10.1080/09537100802503368 
PMID: 19172522  [Indexed for MEDLINE]


673. Zhongguo Zhong Yao Za Zhi. 2009 Feb;34(4):454-7.

[Construction of subtractive cDNA library of apoptosis-related genes in NB4 cells
treated by arsenic trioxide].

[Article in Chinese]

Di C(1), Gu S, Tan X, Xian L, Wu Q, Yang L.

Author information: 
(1)Medical Laboratory of The First Affiliated Hospital of Medical College,
Shihezi University, Shihezi 832008, China. chunhongdi@163.com

OBJECTIVE: Construct the gene library of apoptosis related genes in acute
promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to
clarify the apoptotic mechanism of NB4 cells.
METHOD: APL cell line NB4 cells treated with or without arsenic trioxide for 24
hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was
conducted according to the manual. With the cDNA of the apoptosis cells as the
tester and that of control cells as the driver, forward and reverse hybridization
was performed. Differentially expressed genes were linked with pGEM-Teasy cloning
vector and transformed into E. coli DH5alpha. The positive clones were screened
by blue and white spot. PCR were used to amplify these genes.
RESULT: The subtractive cDNA libraries related with apoptosis of NB4 cells were
successfully constructed.
CONCLUSION: The constructed subtractive libraries are suitable for further study 
on the functional genes associated with apoptosis ofNB4 cells induced by arsenic 
trioxide.


PMID: 19459311  [Indexed for MEDLINE]


674. Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):864-9. doi: 10.1073/pnas.0810205106.
Epub 2009 Jan 14.

Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia 
coli.

Singh D(1), Chang SJ, Lin PH, Averina OV, Kaberdin VR, Lin-Chao S.

Author information: 
(1)Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.

Whereas ribosomal proteins (r-proteins) are known primarily as components of the 
translational machinery, certain of these r-proteins have been found to also have
extraribosomal functions. Here we report the novel ability of an r-protein, L4,
to regulate RNA degradation in Escherichia coli. We show by affinity
purification, immunoprecipitation analysis, and E. coli two-hybrid screening that
L4 interacts with a site outside of the catalytic domain of RNase E to regulate
the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific
cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling
plasmid DNA replication by stabilizing an antisense regulatory RNA normally
attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli
transcripts were shown by DNA microarray analysis, which revealed changes in the 
abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA,
YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid
metabolism and transport, transcription/translation, and DNA/RNA synthesis.
Analysis of mRNA stability showed that the half lives of stress-responsive
transcripts were increased by ectopic expression of L4, which normally increases 
along with other r-proteins in E. coli under stress conditions, and also by
inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay 
may account at least in part for the elevated production of stress-induced
proteins during bacterial adaptation to adverse environments.

DOI: 10.1073/pnas.0810205106 
PMCID: PMC2626609
PMID: 19144914  [Indexed for MEDLINE]


675. Appl Environ Microbiol. 2009 Jan;75(2):483-92. doi: 10.1128/AEM.01616-08. Epub
2008 Nov 7.

Overproduction of exopolysaccharides by an Escherichia coli K-12 rpoS mutant in
response to osmotic stress.

Ionescu M(1), Belkin S.

Author information: 
(1)Department of Plant and Environmental Sciences, Institute of Life Sciences,
The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

The yjbEFGH operon is implicated in the production of an exopolysaccharide of an 
unknown function and is induced by osmotic stress and negatively regulated by the
general stress response sigma factor RpoS. Despite the obvious importance of
RpoS, negative selection for rpoS has been reported to take place in starved
cultures, suggesting an adaptive occurrence allowing the overexpression of
RpoD-dependent uptake and nutrient-scavenging systems. The trade-off of the
RpoS-dependent functions for improved nutrient utilization abilities makes the
bacterium more sensitive to environmental stressors, e.g., osmotic stress. In
this work, we addressed the hypothesis that overinduction of genes in
rpoS-deficient strains indicates their essentiality. Using DNA microarrays,
real-time PCR, and transcriptional fusions, we show that genes of the wca operon,
implicated in the production of the colanic acid exopolysaccharide, previously
shown to be induced by osmotic stress, are also negatively controlled by RpoS.
Both exopolysaccharides in the synthesis of which yjb and wca are involved are
overproduced in an rpoS mutant during osmotic stress. We also show that both
operons are essential in an rpoS-deficient strain but not in the wild type;
promoters of both operons are constitutively active in yjb rpoS mutants; this
strain produces extremely mucoid colonies, forms long filaments, and exhibits a
reduced growth capability. In addition, the wca rpoS mutant's growth is inhibited
by osmotic stress. These results indicate that although induced in the wild type,
both operons are much more valuable for an rpoS-deficient strain, suggesting that
the overproduction of both exopolysaccharides is an adaptive action.

DOI: 10.1128/AEM.01616-08 
PMCID: PMC2620720
PMID: 18997023  [Indexed for MEDLINE]


676. Biosens Bioelectron. 2009 Jan 1;24(5):1405-10. doi: 10.1016/j.bios.2008.08.008.
Epub 2008 Aug 13.

Nano-silver-modified PQC/DNA biosensor for detecting E. coli in environmental
water.

Sun H(1), Choy TS, Zhu DR, Yam WC, Fung YS.

Author information: 
(1)The Department of Chemistry, The University of Hong Kong, Hong Kong SAR,
China.

To meet the requirement of World Health Organization for zero tolerance of E.
coli cell in 100mL drinking water, a new procedure based on photodeposition of
nano-Ag at TiO(2)-coated piezoelectric quartz crystal (PQC) electrode was
developed to fabricate a highly sensitive PQC/DNA biosensor. Enhancement of 3.3
times for binding of complementary DNA has been shown and attributed to the
following effects arising from the nano-Ag coating. First, a large increase in
the active surface area and packing density of neutravidin enhances the maximum
neutravidin load to 1.8 times of a normal electrode. Second, the functional
activity of neutravidin is enhanced by chemical interaction with nano-Ag to give 
rise to an increase in the binding ratio between neutravidin and biotinylated DNA
probe from 1.00:1.76 to 1.00:3.01. Third, the stronger binding leads to a higher 
stability of the biotinylated DNA probes bound and increase in hybridization with
the complementary DNA. Under the optimized conditions for flow analysis with
online PCR product denaturing and hybridization, a detection limit of eight E.
coli cells are obtained which require sampling at least 800mL water to detect a
single E. coli cell in 100mL water.

DOI: 10.1016/j.bios.2008.08.008 
PMID: 19022649  [Indexed for MEDLINE]


677. Biosens Bioelectron. 2009 Jan 1;24(5):1399-404. doi: 10.1016/j.bios.2008.08.012. 
Epub 2008 Aug 15.

High-throughput SPR sensor for food safety.

Piliarik M(1), Párová L, Homola J.

Author information: 
(1)Institute of Photonics and Electronics, Chaberská 57, 182 51 Prague, Czech
Republic.

High-throughput surface plasmon resonance (SPR) biosensor for rapid and
parallelized detection of nucleic acids identifying specific bacterial pathogens 
is reported. The biosensor consists of a high-performance SPR imaging sensor with
polarization contrast and internal referencing (refractive index resolution 2 x
10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR
sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases)
characteristic for bacterial pathogens such as Brucella abortus, Escherichia
coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of
specific DNA or RNA sequences can be performed in less than 15 min by the
reported SPR sensor.

DOI: 10.1016/j.bios.2008.08.012 
PMID: 18809310  [Indexed for MEDLINE]


678. Drug Chem Toxicol. 2009;32(3):258-67. doi: 10.1080/01480540902882192.

Antigenotoxic and antioxidant activities of isorhamnetin 3-O neohesperidoside
from Acacia salicina.

Bouhlel I(1), Skandrani I, Nefatti A, Valenti K, Ghedira K, Mariotte AM,
Hininger-Favier I, Laporte F, Dijoux-Franca MG, Chekir-Ghedira L.

Author information: 
(1)Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de
Pharmacie/Médecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisie.

Antioxidant activity of isorhamnetin 3-O neohesperidoside (I3ON), isolated from
the leaves of Acacia salicina, was determined by the ability of this compound to 
inhibit lipid peroxidation and to protect against hydroxyl radical-induced DNA
damage in pKS plasmid DNA and Escherichia coli cultures. Antigenotoxic activity
was assessed by using the comet assay. The IC(50) value of the inhibitory
activity toward lipid peroxidation by I3ON is 0.6 mM. This compound was also able
to protect against hydroxyl radical-induced DNA damage in pKS plasmid DNA.
Moreover, this compound induced an inhibitory activity toward H2O2-induced
genotoxicity. The protective effect exhibited by this molecule was also
determined by analysis of gene expression as a response to an oxidative stress,
using a cDNA microarray. Transcription of several genes related to the
antioxidant system (HMOX2 and TXNL) and to the DNA repair pathway (XPC, POLD1,
POLD2, PCNA, DDIT3, APEX, and LIG4) were upregulated after incubation with I3ON. 
Taken together, these observations provide evidence that the I3ON, isolated from 
the leaves of A. salicina, is able to protect cells against oxidative stress.

DOI: 10.1080/01480540902882192 
PMID: 19538023  [Indexed for MEDLINE]


679. Environ Microbiol. 2009 Jan;11(1):137-48. doi: 10.1111/j.1462-2920.2008.01748.x. 
Epub 2008 Sep 12.

Ion transport and osmotic adjustment in Escherichia coli in response to ionic and
non-ionic osmotica.

Shabala L(1), Bowman J, Brown J, Ross T, McMeekin T, Shabala S.

Author information: 
(1)School of Agricultural Science and Tasmanian Institute of Agricultural
Research, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia.

Bacteria respond to osmotic stress by a substantial increase in the intracellular
osmolality, adjusting their cell turgor for altered growth conditions. Using
Escherichia coli as a model organism we demonstrate here that bacterial responses
to hyperosmotic stress specifically depend on the nature of osmoticum used. We
show that increasing acute hyperosmotic NaCl stress above approximately 1.0 Os
kg(-1) causes a dose-dependent K(+) leak from the cell, resulting in a
substantial decrease in cytosolic K(+) content and a concurrent accumulation of
Na(+) in the cell. At the same time, isotonic sucrose or mannitol treatment
(non-ionic osmotica) results in a gradual increase of the net K(+) uptake. Ion
flux data are consistent with growth experiments showing that bacterial growth is
impaired by NaCl at the concentration resulting in a switch from net K(+) uptake 
to efflux. Microarray experiments reveal that about 40% of upregulated genes
shared no similarity in their responses to NaCl and sucrose treatment, further
suggesting specificity of osmotic adjustment in E. coli to ionic and non-ionic
osmotica. The observed differences are explained by the specificity of the
stress-induced changes in the membrane potential of bacterial cells highlighting 
the importance of voltage-gated K(+) transporters for bacterial adaptation to
hyperosmotic stress.

DOI: 10.1111/j.1462-2920.2008.01748.x 
PMID: 18793315  [Indexed for MEDLINE]


680. J Bacteriol. 2009 Jan;191(1):238-48. doi: 10.1128/JB.00915-08. Epub 2008 Oct 24.

The small RNA GcvB regulates sstT mRNA expression in Escherichia coli.

Pulvermacher SC(1), Stauffer LT, Stauffer GV.

Author information: 
(1)Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.

In Escherichia coli, the gcvB gene encodes a nontranslated RNA (referred to as
GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the
oligopeptide and dipeptide transport systems. An additional regulatory target of 
GcvB, sstT, was found by microarray analysis of RNA isolated from a wild-type
strain and a gcvB deletion strain grown to mid-log phase in Luria-Bertani broth. 
The SstT protein functions to transport L-serine and L-threonine by sodium
transport into the cell. Reverse transcription-PCR and translational fusions
confirmed that GcvB negatively regulates sstT mRNA levels in cells grown in
Luria-Bertani broth. A series of transcriptional fusions identified a region of
sstT mRNA upstream of the ribosome binding site needed for negative regulation by
GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region
of the sstT mRNA. The region of GcvB complementary to sstT mRNA is the same
region of GcvB identified to regulate the dppA and oppA mRNAs. Mutations
predicted to disrupt base pairing between sstT mRNA and GcvB were made in gcvB,
which resulted in the identification of a small region of GcvB necessary for
negative regulation of sstT-lacZ. Additionally, the RNA chaperone protein Hfq was
found to be necessary for GcvB to negatively regulate sstT-lacZ in Luria-Bertani 
broth and glucose minimal medium supplemented with glycine. The sstT mRNA is the 
first target found to be regulated by GcvB in glucose minimal medium supplemented
with glycine.

DOI: 10.1128/JB.00915-08 
PMCID: PMC2612445
PMID: 18952787  [Indexed for MEDLINE]


681. J Med Microbiol. 2009 Jan;58(Pt 1):69-81. doi: 10.1099/jmm.0.000794-0.

Molecular detection of all 34 distinct O-antigen forms of Shigella.

Li Y(1), Cao B, Liu B, Liu D, Gao Q, Peng X, Wu J, Bastin DA, Feng L, Wang L.

Author information: 
(1)Tianjin Biochip Corporation, 23 Hongda Street, TEDA, Tianjin 300457, PR China.

Shigella is the cause of shigellosis or bacillary dysentery, the occurrence of
which is estimated to be 165 million cases per year worldwide, resulting in 1.1
million deaths. Rapid and reliable assays for detecting and identifying Shigella 
in food, environmental and clinical samples are therefore necessary. Shigella
species are traditionally identified by their O antigens. This study developed a 
DNA microarray targeting O-serotype-specific genes to detect all 34 distinct
O-antigen forms of Shigella, including Shigella boydii types 1-18, Shigella
dysenteriae types 1-13, Shigella flexneri types 1-5 and 6, and Shigella sonnei. A
total of 282 strains were used to test the specificity of the microarray,
including 186 Shigella and Escherichia coli representative strains, 86 Shigella
clinical isolates and ten strains of other bacterial species that are commonly
isolated from food or clinical stool specimens. The oligonucleotide probes were
printed on the microarray in concentrations from 1 to 100 muM, and 10 muM proved 
to be the optimal probe concentration. The detection sensitivity for each
serotype was 50 ng genomic DNA or 1 c.f.u. in 25 g milk powder sample following a
6 h enrichment in broth. The microarray is specific, sensitive and reproducible, 
and, to our knowledge, is the first report of a microarray for serotyping all
O-antigen forms of Shigella.

DOI: 10.1099/jmm.0.000794-0 
PMID: 19074655  [Indexed for MEDLINE]


682. Methods Mol Biol. 2009;504:441-58. doi: 10.1007/978-1-60327-569-9_24.

Rapid DNA amplification using a battery-powered thin-film resistive thermocycler.

Herold KE(1), Sergeev N, Matviyenko A, Rasooly A.

Author information: 
(1)Fischell Department of Bioengineering, University of Maryland, College Park,
MD, USA.

A prototype handheld, compact, rapid thermocycler was developed for multiplex
analysis of nucleic acids in an inexpensive, portable configuration. Instead of
the commonly used Peltier heating/cooling element, electric thin-film resistive
heater and a miniature fan enable rapid heating and cooling of glass capillaries 
leading to a simple, low-cost Thin-Film Resistive Thermocycler (TFRT).
Computer-based pulse width modulation control yields heating rates of 6-7 K/s and
cooling rates of 5 K/s. The four capillaries are closely coupled to the heater,
resulting in low power consumption. The energy required by a nominal PCR cycle
(20 s at each temperature) was found to be 57+/-2 J yielding an average power of 
approximately 1.0 W (not including the computer and the control system). Thus the
device can be powered by a standard 9 V alkaline battery (or other 9 V power
supply). The prototype TFRT was demonstrated (in a benchtop configuration) for
detection of three important food pathogens (E. coli ETEC, Shigella dysenteriae, 
and Salmonella enterica). PCR amplicons were analyzed by gel electrophoresis. The
35 cycle PCR protocol using a single channel was completed in less then 18 min.
Simple and efficient heating/cooling, low cost, rapid amplification, and low
power consumption make the device suitable for portable DNA amplification
applications including clinical point of care diagnostics and field use.

DOI: 10.1007/978-1-60327-569-9_24 
PMCID: PMC2844723
PMID: 19159110  [Indexed for MEDLINE]


683. Microbiology. 2009 Jan;155(Pt 1):106-14. doi: 10.1099/mic.0.023598-0.

Role of the sRNA GcvB in regulation of cycA in Escherichia coli.

Pulvermacher SC(1), Stauffer LT, Stauffer GV.

Author information: 
(1)Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that
regulates several genes involved in transport of amino acids and peptides
(including sstT, oppA and dppA). Microarray analysis identified cycA as an
additional regulatory target of GcvB. The cycA gene encodes a permease for the
transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed
that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in
Luria-Bertani broth. In addition, deletion of the gcvB gene resulted in increased
sensitivity to d-cycloserine, consistent with increased expression of cycA. A
cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA 
expression in Luria-Bertani broth and that Hfq is required for the GcvB effect.
GcvB had no effect on cycA : : lacZ expression in glucose minimal medium
supplemented with glycine. However, Hfq still negatively regulated the fusion in 
the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified 
a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified 
a region complementary to this region of cycA mRNA. However, mutations predicted 
to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of
cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

DOI: 10.1099/mic.0.023598-0 
PMID: 19118351  [Indexed for MEDLINE]


684. Mol Genet Genomics. 2009 Jan;281(1):19-33. doi: 10.1007/s00438-008-0389-3. Epub
2008 Oct 9.

Control of RpoS in global gene expression of Escherichia coli in minimal media.

Dong T(1), Schellhorn HE.

Author information: 
(1)Department of Biology, McMaster University, Life Sciences Building, Rm. 433,
1280 Main Street West, Hamilton, ON, L8S 4K1, Canada. dongt2@mcmaster.ca

RpoS, an alternative sigma factor, is critical for stress response in Escherichia
coli. The RpoS regulon expression has been well characterized in rich media that 
support fast growth and high growth yields. In contrast, though RpoS levels are
high in minimal media, how RpoS functions under such conditions has not been
clearly resolved. In this study, we compared the global transcriptional profiles 
of wild type and an rpoS mutant of E. coli grown in glucose minimal media using
microarray analyses. The expression of over 200 genes was altered by loss of RpoS
in exponential and stationary phases, with only 48 genes common to both
conditions. The nature of the RpoS-controlled regulon in minimal media was
substantially different from that expressed in rich media. Specifically, the
expression of many genes encoding regulatory factors (e.g., hfq, csrA, and rpoE) 
and genes in metabolic pathways (e.g., lysA, lysC, and hisD) were regulated by
RpoS in minimal media. In early exponential phase, protein levels of RpoS in
minimal media were much higher than that in Luria-Bertani media, which may at
least partly account for the observed difference in the expression of
RpoS-controlled genes. Expression of genes required for flagellar function and
chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the
flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT
in all phase of growth. Consistent with this, the motility of rpoS mutants was
enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a
complex regulatory network that mediates the expression of a large regulon in
minimal media.

DOI: 10.1007/s00438-008-0389-3 
PMID: 18843507  [Indexed for MEDLINE]


685. PLoS One. 2009;4(3):e4889. doi: 10.1371/journal.pone.0004889. Epub 2009 Mar 18.

Enterohemorrhagic Escherichia coli O157:H7 gene expression profiling in response 
to growth in the presence of host epithelia.

Jandu N(1), Ho NK, Donato KA, Karmali MA, Mascarenhas M, Duffy SP, Tailor C,
Sherman PM.

Author information: 
(1)Department of Pathology, Stanford University School of Medicine, Stanford, CA,
USA.

BACKGROUND: The pathogenesis of enterohemorrhagic Escherichia coli (EHEC) O157:H7
infection is attributed to virulence factors encoded on multiple pathogenicity
islands. Previous studies have shown that EHEC O157:H7 modulates host cell signal
transduction cascades, independent of toxins and rearrangement of the
cytoskeleton. However, the virulence factors and mechanisms responsible for
EHEC-mediated subversion of signal transduction remain to be determined.
Therefore, the purpose of this study was to first identify differentially
regulated genes in response to EHEC O157:H7 grown in the presence of epithelial
cells, compared to growth in the absence of epithelial cells (that is, growth in 
minimal essential tissue culture medium alone, minimal essential tissue culture
medium in the presence of 5% CO(2), and Penassay broth alone) and, second, to
identify EHEC virulence factors responsible for pathogen modulation of host cell 
signal transduction.
METHODOLOGY/PRINCIPAL FINDINGS: Overnight cultures of EHEC O157:H7 were incubated
for 6 hr at 37 degrees C in the presence or absence of confluent epithelial
(HEp-2) cells. Total RNA was then extracted and used for microarray analyses
(Affymetrix E. coli Genome 2.0 gene chips). Relative to bacteria grown in each of
the other conditions, EHEC O157:H7 cultured in the presence of cultured
epithelial cells displayed a distinct gene-expression profile. A 2.0-fold
increase in the expression of 71 genes and a 2.0-fold decrease in expression of
60 other genes were identified in EHEC O157:H7 grown in the presence of
epithelial cells, compared to bacteria grown in media alone.
CONCLUSION/SIGNIFICANCE: Microarray analyses and gene deletion identified a
protease on O-island 50, gene Z1787, as a potential virulence factor responsible 
for mediating EHEC inhibition of the interferon (IFN)-gamma-Jak1,2-STAT-1 signal 
transduction cascade. Up-regulated genes provide novel targets for use in
developing strategies to interrupt the infectious process.

DOI: 10.1371/journal.pone.0004889 
PMCID: PMC2654852
PMID: 19293938  [Indexed for MEDLINE]


686. PLoS One. 2009;4(2):e4495. doi: 10.1371/journal.pone.0004495. Epub 2009 Feb 13.

Query large scale microarray compendium datasets using a model-based bayesian
approach with variable selection.

Hu M(1), Qin ZS.

Author information: 
(1)Department of Biostatistics, Center for Statistical Genetics, School of Public
Health, University of Michigan, Ann Arbor, Michigan, United States of America.

In microarray gene expression data analysis, it is often of interest to identify 
genes that share similar expression profiles with a particular gene such as a key
regulatory protein. Multiple studies have been conducted using various
correlation measures to identify co-expressed genes. While working well for small
datasets, the heterogeneity introduced from increased sample size inevitably
reduces the sensitivity and specificity of these approaches. This is because most
co-expression relationships do not extend to all experimental conditions. With
the rapid increase in the size of microarray datasets, identifying functionally
related genes from large and diverse microarray gene expression datasets is a key
challenge. We develop a model-based gene expression query algorithm built under
the Bayesian model selection framework. It is capable of detecting co-expression 
profiles under a subset of samples/experimental conditions. In addition, it
allows linearly transformed expression patterns to be recognized and is robust
against sporadic outliers in the data. Both features are critically important for
increasing the power of identifying co-expressed genes in large scale gene
expression datasets. Our simulation studies suggest that this method outperforms 
existing correlation coefficients or mutual information-based query tools. When
we apply this new method to the Escherichia coli microarray compendium data, it
identifies a majority of known regulons as well as novel potential target genes
of numerous key transcription factors.

DOI: 10.1371/journal.pone.0004495 
PMCID: PMC2637418
PMID: 19214232  [Indexed for MEDLINE]


687. PLoS One. 2009;4(2):e4367. doi: 10.1371/journal.pone.0004367. Epub 2009 Feb 4.

The HU regulon is composed of genes responding to anaerobiosis, acid stress, high
osmolarity and SOS induction.

Oberto J(1), Nabti S, Jooste V, Mignot H, Rouviere-Yaniv J.

Author information: 
(1)Laboratoire de Physiologie Bactérienne, CNRS, UPR 9073, Institut de Biologie
Physico-Chimique, Paris, France. jacques.oberto@igmors.u-psud.fr

BACKGROUND: The Escherichia coli heterodimeric HU protein is a small DNA-bending 
protein associated with the bacterial nucleoid. It can introduce negative
supercoils into closed circular DNA in the presence of topoisomerase I. Cells
lacking HU grow very poorly and display many phenotypes.
METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the transcription profile of every
Escherichia coli gene in the absence of one or both HU subunits. This genome-wide
in silico transcriptomic approach, performed in parallel with in vivo genetic
experimentation, defined the HU regulon. This large regulon, which comprises 8%
of the genome, is composed of four biologically relevant gene classes whose
regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS
induction.
CONCLUSIONS/SIGNIFICANCE: The regulation a large number of genes encoding enzymes
involved in energy metabolism and catabolism pathways by HU explains the highly
pleiotropic phenotype of HU-deficient cells. The uniform chromosomal distribution
of the many operons regulated by HU strongly suggests that the transcriptional
and nucleoid architectural functions of HU constitute two aspects of a unique
protein-DNA interaction mechanism.

DOI: 10.1371/journal.pone.0004367 
PMCID: PMC2634741
PMID: 19194530  [Indexed for MEDLINE]


688. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Jan;25(1):9-12.

[Expression of cytokines in murine BMDCs induced by recombinant E.coli LLO/OVA
via TLR4 and NOD1 receptors].

[Article in Chinese]

Xu M(1), Dai MS, Mi C.

Author information: 
(1)Department of Pathology, Chongqing University of Medical Sciences, China.
manxucqmu@yahoo.com

AIM: To investigate the relationship between the activation of pattern
recognition receptors and the cytokines expression of dendritic cells.
METHODS: After bone marrow-derived dendritic cells (BMDCs) were pulsed by E.coli 
LLO/OVA, the mRNA expression of pattern recognition receptors and the downstream 
NF-kappaB signal pathway associated molecules were detected by microarray
hybridization and RT-PCR; the expression of costimulatory molecules, MHC class II
and cytokines were determined by flow cytometry and ELISA.
RESULTS: After BMDCs were pulsed by E.coli LLO/OVA for 2 h, the levels of TLR4,
Myd88, Rip2, Irak1, Irak2, Ikkalpha, NF-kappaB1 and NF-kappaB2 mRNA up-regulated;
and the levels of Icam1, IL-1a, IL-1b, IL-6 and TNF-alpha mRNA up-regulated also;
after BMDCs were pulsed by E.coli LLO/OVA for 4 h, the levels of Card4(Nod1),
Rip2, Ikkbeta, NF-kappaB1 and NF-kappaB2 mRNA up-regulated, meanwhile, the levels
of TNF-gamma, TNF-beta and CD40 mRNA up-regulated. The expressions of
costimulatory molecules and MHC class II of the BMDCs up-regulated and the
concentration of IL-12 and IFN-gamma increased in the supernatant of BMDCs pulsed
by E.coli LLO/OVA at 24 h.
CONCLUSION: Recombinant E.coli LLO/OVA induces maturation and expression of
cytokines, especially IL-12 and IFN-gamma, of murine BMDCs through activation of 
NF-kappaB signal pathway with TLR4 and NOD1 receptors.


PMID: 19126379  [Indexed for MEDLINE]


689. BMC Biotechnol. 2008 Dec 23;8:94. doi: 10.1186/1472-6750-8-94.

Better estimation of protein-DNA interaction parameters improve prediction of
functional sites.

Nagaraj VH(1), O'Flanagan RA, Sengupta AM.

Author information: 
(1)BioMaPS Institute, Rutgers University, Piscataway, NJ 08854-8020, USA.
viji@waksman.rutgers.edu

BACKGROUND: Characterizing transcription factor binding motifs is a common
bioinformatics task. For transcription factors with variable binding sites, we
need to get many suboptimal binding sites in our training dataset to get accurate
estimates of free energy penalties for deviating from the consensus DNA sequence.
One procedure to do that involves a modified SELEX (Systematic Evolution of
Ligands by Exponential Enrichment) method designed to produce many such
sequences.
RESULTS: We analyzed low stringency SELEX data for E. coli Catabolic Activator
Protein (CAP), and we show here that appropriate quantitative analysis improves
our ability to predict in vitro affinity. To obtain large number of sequences
required for this analysis we used a SELEX SAGE protocol developed by Roulet et
al. The sequences obtained from here were subjected to bioinformatic analysis.
The resulting bioinformatic model characterizes the sequence specificity of the
protein more accurately than those sequence specificities predicted from previous
analysis just by using a few known binding sites available in the literature. The
consequences of this increase in accuracy for prediction of in vivo binding sites
(and especially functional ones) in the E. coli genome are also discussed. We
measured the dissociation constants of several putative CAP binding sites by EMSA
(Electrophoretic Mobility Shift Assay) and compared the affinities to the
bioinformatics scores provided by methods like the weight matrix method and
QPMEME (Quadratic Programming Method of Energy Matrix Estimation) trained on
known binding sites as well as on the new sites from SELEX SAGE data. We also
checked predicted genome sites for conservation in the related species S.
typhimurium. We found that bioinformatics scores based on SELEX SAGE data does
better in terms of prediction of physical binding energies as well as in
detecting functional sites.
CONCLUSION: We think that training binding site detection algorithms on datasets 
from binding assays lead to better prediction. The improvements in accuracy came 
from the unbiased nature of the SELEX dataset rather than from the number of
sites available. We believe that with progress in short-read sequencing
technology, one could use SELEX methods to characterize binding affinities of
many low specificity transcription factors.

DOI: 10.1186/1472-6750-8-94 
PMCID: PMC2654563
PMID: 19105805  [Indexed for MEDLINE]


690. Infect Immun. 2008 Dec;76(12):5760-7. doi: 10.1128/IAI.00618-08. Epub 2008 Oct 6.

Uropathogenic Escherichia coli CFT073 is adapted to acetatogenic growth but does 
not require acetate during murine urinary tract infection.

Anfora AT(1), Halladin DK, Haugen BJ, Welch RA.

Author information: 
(1)Department of Medical Microbiology and Immunology, University of
Wisconsin-Madison, Madison, WI 53706, USA.

In vivo accumulation of D-serine by Escherichia coli CFT073 leads to elevated
expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of
D-serine catabolism by CFT073 leads to a competitive advantage during murine
urinary tract infection (UTI), but loss of both D- and L-serine catabolism
results in attenuation. Serine is the first amino acid to be consumed in closed
tryptone broth cultures and precedes the production of acetyl phosphate, a
high-energy molecule involved in intracellular signaling, and the eventual
secretion of acetate. We propose that the colonization defect associated with the
loss of serine catabolism is due to perturbations of acetate metabolism. CFT073
grows more rapidly on acetogenic substrates than does E. coli K-12 isolate
MG1655. As shown by transcription microarray results, D-serine is catabolized
into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes 
while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, 
which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys
in the murine model of UTI indistinguishably from the wild type. Both pta and
ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073
pta and ackA mutants were screened to investigate the role of acetyl phosphate in
UTI pathogenesis. Both single mutants are at a competitive disadvantage relative 
to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA
pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that
CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling
of the acetyl phosphate pathway for colonization of the upper urinary tract.

DOI: 10.1128/IAI.00618-08 
PMCID: PMC2583553
PMID: 18838520  [Indexed for MEDLINE]


691. J Biomed Inform. 2008 Dec;41(6):914-26. doi: 10.1016/j.jbi.2008.01.011. Epub 2008
Feb 6.

Consensus and Meta-analysis regulatory networks for combining multiple microarray
gene expression datasets.

Steele E(1), Tucker A.

Author information: 
(1)Centre for Intelligent Data Analysis, Department of Information Systems and
Computing, Brunel University, Kingston Lane, Uxbridge Middlesex UB8 3PH, UK.
emma.steele@brunel.ac.uk

Microarray data is a key source of experimental data for modelling gene
regulatory interactions from expression levels. With the rapid increase of
publicly available microarray data comes the opportunity to produce regulatory
network models based on multiple datasets. Such models are potentially more
robust with greater confidence, and place less reliance on a single dataset.
However, combining datasets directly can be difficult as experiments are often
conducted on different microarray platforms, and in different laboratories
leading to inherent biases in the data that are not always removed through
pre-processing such as normalisation. In this paper we compare two frameworks for
combining microarray datasets to model regulatory networks: pre- and
post-learning aggregation. In pre-learning approaches, such as using simple
scale-normalisation prior to the concatenation of datasets, a model is learnt
from a combined dataset, whilst in post-learning aggregation individual models
are learnt from each dataset and the models are combined. We present two novel
approaches for post-learning aggregation, each based on aggregating high-level
features of Bayesian network models that have been generated from different
microarray expression datasets. Meta-analysis Bayesian networks are based on
combining statistical confidences attached to network edges whilst Consensus
Bayesian networks identify consistent network features across all datasets. We
apply both approaches to multiple datasets from synthetic and real (Escherichia
coli and yeast) networks and demonstrate that both methods can improve on
networks learnt from a single dataset or an aggregated dataset formed using a
standard scale-normalisation.

DOI: 10.1016/j.jbi.2008.01.011 
PMID: 18337190  [Indexed for MEDLINE]


692. J Microbiol Methods. 2008 Dec;75(3):566-71. doi: 10.1016/j.mimet.2008.09.007.
Epub 2008 Sep 14.

A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool 
specimens of patients with diarrhea.

You Y(1), Fu C, Zeng X, Fang D, Yan X, Sun B, Xiao D, Zhang J.

Author information: 
(1)National Institute for Communicable Disease Control and Prevention, Chinese
Center for Disease Control and Prevention, Beijing, PR China.

A microarray technique for the detection and identification of enteropathogenic
bacteria at the species and subspecies levels was developed in this study, and
the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio
parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia 
enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen
was chosen as the amplification target, labeled with a fluorescence dye by
multiplex polymerase chain reaction (PCR), and hybridized to the specific
virulence gene probes that had been immobilized on a microchip. Stool specimens
from 34 patients with diarrhea were tested in this study. Five were positive for 
multiple genera. Nested PCRs and sequencing were used to amplify and identify the
related genes, which were found to share 95.8% to 100% of the nucleotide identity
with the corresponding regions in the Genbank database. Real-time PCR was used to
determine the number of gene copies to determine the sensitivity of this
technique, which was shown to be 58 copies/microl. The results indicated that the
microarray technique which targets multiple virulence genes of enteropathogenic
bacteria at the species and subspecies levels is an attractive diagnostic tool
for rapidly and simultaneously identifying multiple enteropathogenic pathogens in
clinical practice, especially in patients with infectious diarrhea.

DOI: 10.1016/j.mimet.2008.09.007 
PMID: 18834908  [Indexed for MEDLINE]


693. J Microbiol Methods. 2008 Dec;75(3):523-30. doi: 10.1016/j.mimet.2008.08.003.
Epub 2008 Aug 17.

Statistical superiority of genome-probing microarrays as genomic DNA-DNA
hybridization in revealing the bacterial phylogenetic relationship compared to
conventional methods.

Chang HW(1), Nam YD, Jung MY, Kim KH, Roh SW, Kim MS, Jeon CO, Yoon JH, Bae JW.

Author information: 
(1)Biological Resource Center, KRIBB, Daejeon 305-806, Republic of Korea.

The genomic DNA-DNA hybridization (DDH) method has been widely used as a
practical method for the determination of phylogenetic relationships between
closely related biological strains. Traditional DDH methods have serious
limitations including low reproducibility, a high background and a time-consuming
procedure. The DDH method using a genome-probing microarray (GPM) has been
recently developed to complement conventional methods and could be used to
overcome the limitations that are typically encountered. It is necessary to
compare the GPM-based DDH method to the conventional methods before using the GPM
for the estimation of genomic similarities since all of the previous scientific
data have been entirely dependent on conventional DDH methods. In order to
address this issue we compared the DDH values obtained using the GPM, microplate 
and nylon membrane methods to multi-locus sequence typing (MLST) data for 9
Salmonella genomes and an Escherichia coli type strain. The results showed that
the genome similarity values and the degrees of standard deviation obtained using
the GPM method were lower than those obtained with the microplate and nylon
membrane methods. The dendrogram from the cluster analysis of GPM DDH values was 
consistent with the phylogenetic tree obtained from the multi-locus sequence
typing (MLST) data but was not similar to those obtained using the microplate and
nylon membrane methods. Although the signal intensity had to be maximal when the 
targets were hybridized to their own probe, the methods using membranes and
microplates frequently produced higher signals in the heterologous hybridizations
than those obtained in the homologous hybridizations. Only the GPM method
produced the highest signal intensity in homologous hybridizations. These results
show that the GPM method can be used to obtain results that are more accurate
than those generated by the other methods tested.

DOI: 10.1016/j.mimet.2008.08.003 
PMID: 18782592  [Indexed for MEDLINE]


694. Mol Microbiol. 2008 Dec;70(5):1076-93. doi: 10.1111/j.1365-2958.2008.06394.x.
Epub 2008 Aug 14.

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs.

Fozo EM(1), Kawano M, Fontaine F, Kaya Y, Mendieta KS, Jones KL, Ocampo A, Rudd
KE, Storz G.

Author information: 
(1)Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute
of Child Health and Human Development, Bethesda, MD, USA.

Erratum in
    Mol Microbiol. 2008 Dec;70(5):1305.

The sequences encoding the QUAD1 RNAs were initially identified as four repeats
in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely 
related bacteria, although the number of repeats varies. All five Sib RNAs in E. 
coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion 
strain. However, a phenotype reminiscent of plasmid addiction was observed for
overexpression of the Sib RNAs, and further examination of the SIB repeat
sequences revealed conserved open reading frames encoding highly hydrophobic 18- 
to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were
found to be toxic when overexpressed and this toxicity could be prevented by
coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in 
the yfhL-acpS intergenic region were similarly found to encode a small
hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression 
of both IbsC and ShoB led to immediate changes in membrane potential suggesting
both proteins affect the cell envelope. Whole genome expression analysis showed
that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and
TisB proteins, has both overlapping and unique consequences for the cell.

DOI: 10.1111/j.1365-2958.2008.06394.x 
PMCID: PMC2597788
PMID: 18710431  [Indexed for MEDLINE]


695. Plant Mol Biol. 2008 Dec;68(6):633-51. doi: 10.1007/s11103-008-9399-0. Epub 2008 
Sep 26.

Transcriptome analysis approaches for the isolation of trichome-specific genes
from the medicinal plant Cistus creticus subsp. creticus.

Falara V(1), Fotopoulos V, Margaritis T, Anastasaki T, Pateraki I, Bosabalidis
AM, Kafetzopoulos D, Demetzos C, Pichersky E, Kanellis AK.

Author information: 
(1)Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki,
541 24, Thessaloniki, Greece.

Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due
to the distinctive pharmaceutical properties of its resin. Labdane-type
diterpenes, the main constituents of the resin, exhibit considerable
antibacterial and cytotoxic activities. In this study chemical analysis of
isolated trichomes from different developmental stages revealed that young leaves
of 1-2 cm length displayed the highest content of labdane-type diterpenes (80
mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited 
gradual decreased concentrations. A cDNA library was constructed enriched in
transcripts from trichomes isolated from young leaves, which are characterized by
high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed 
sequence tags (ESTs) from the trichome cDNA library based on homology to A.
thaliana genes suggested that 8% of the putative identified sequences were
secondary metabolism-related and involved primarily in flavonoid and terpenoid
biosynthesis. A significant proportion of the ESTs (38%) displayed no significant
similarity to any other DNA deposited in databases, indicating a yet unknown
function. Custom DNA microarrays constructed with 1,248 individual clones from
the cDNA library facilitated transcriptome comparisons between trichomes and
trichome-free tissues. In addition, gene expression studies in various Cistus
tissues and organs for one of the genes highlighted as the most differentially
expressed by the microarray experiments revealed a putative sesquiterpene
synthase with a trichome-specific expression pattern. Full length cDNA isolation 
and heterologous expression in E. coli followed by biochemical analysis, led to
the characterization of the produced protein as germacrene B synthase.

DOI: 10.1007/s11103-008-9399-0 
PMID: 18819010  [Indexed for MEDLINE]


696. BMC Genomics. 2008 Nov 28;9:568. doi: 10.1186/1471-2164-9-568.

Genome-wide transcriptional response of an avian pathogenic Escherichia coli
(APEC) pst mutant.

Crépin S(1), Lamarche MG, Garneau P, Séguin J, Proulx J, Dozois CM, Harel J.

Author information: 
(1)Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Université
de Montréal, Faculté de Médecine Vétérinaire, Saint-Hyacinthe, Québec, Canada.
sebastien.crepin@iaf.inrs.ca

BACKGROUND: Avian pathogenic E. coli (APEC) are associated with extraintestinal
diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and
encodes the phosphate specific transport (Pst) system. A functional Pst system is
required for full virulence in APEC and other bacteria and contributes to
resistance of APEC to serum, to cationic antimicrobial peptides and acid shock.
The global mechanisms contributing to the attenuation and decreased resistance of
the APEC pst mutant to environmental stresses have not been investigated at the
transcriptional level. To determine the global effect of a pst mutation on gene
expression, we compared the transcriptomes of APEC strain chi7122 and its
isogenic pst mutant (K3) grown in phosphate-rich medium.
RESULTS: Overall, 470 genes were differentially expressed by at least 1.5-fold.
Interestingly, the pst mutant not only induced systems involved in phosphate
acquisition and metabolism, despite phosphate availability, but also modulated
stress response mechanisms. Indeed, transcriptional changes in genes associated
with the general stress responses, including the oxidative stress response were
among the major differences observed. Accordingly, the K3 strain was less
resistant to reactive oxygen species (ROS) than the wild-type strain. In
addition, the pst mutant demonstrated reduced expression of genes involved in
lipopolysaccharide modifications and coding for cell surface components such as
type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was
impaired in its capacity to produce type 1 fimbriae, as demonstrated by western
blotting and agglutination of yeast cells, when compared to wild-type APEC strain
chi7122.
CONCLUSION: Overall, our data elucidated the effects of a pst mutation on the
transcriptional response, and further support the role of the Pho regulon as part
of a complex network contributing to phosphate homeostasis, adaptive stress
responses, and E. coli virulence.

DOI: 10.1186/1471-2164-9-568 
PMCID: PMC2648988
PMID: 19038054  [Indexed for MEDLINE]


697. Carbohydr Res. 2008 Nov 24;343(17):2924-31. doi: 10.1016/j.carres.2008.08.018.
Epub 2008 Aug 22.

Studying aminoglycoside modification by the acetyltransferase class of
resistance-causing enzymes via microarray.

Barrett OJ(1), Pushechnikov A, Wu M, Disney MD.

Author information: 
(1)Department of Chemistry, The Center for Excellence in Bioinformatics and Life 
Sciences, University at Buffalo, 657 Natural Sciences Complex, Buffalo, NY 14260,
USA.

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have
acquired resistance. The most common mode of resistance to aminoglycosides is
enzymatic modification of the drug by different classes of enzymes including
acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2')
from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied
using aminoglycoside microarrays. Results show that both enzymes modify their
substrates displayed on an array surface in a manner that mimics their relative
levels of modification in solution. Because aminoglycosides that are modified by 
resistance-causing enzymes have reduced affinities for binding their therapeutic 
target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for
binding to a fluorescently labeled oligonucleotide mimic of the A-site after
modification. A decrease in binding was observed when aminoglycosides were
modified by AAC(3). In contrast, a decrease in binding of the A-site is not
observed when aminoglycosides are modified by AAC(2'). Interestingly, these
effects mirror the biological functions of the enzymes: the AAC(3) used in this
study is known to confer aminoglycoside resistance, while the AAC(2') is
chromosomally encoded and unlikely to play a role in resistance. These studies
lay a direct foundation for studying resistance to aminoglycosides and can also
have more broad applications in identifying and studying non-aminoglycoside
carbohydrates or proteins as substrates for acetyltransferase enzymes.

DOI: 10.1016/j.carres.2008.08.018 
PMCID: PMC2783256
PMID: 18774127  [Indexed for MEDLINE]


698. Arch Microbiol. 2008 Nov;190(5):559-71. doi: 10.1007/s00203-008-0406-2. Epub 2008
Jul 22.

Effect of cra gene knockout together with edd and iclR genes knockout on the
metabolism in Escherichia coli.

Sarkar D(1), Siddiquee KA, Araúzo-Bravo MJ, Oba T, Shimizu K.

Author information: 
(1)Department of Bioscience and Bioinformatics, Kyushu Institute of Technology,
680-4, Kawazu, Iizuka, Fukuoka 820-8502, Japan.

To elucidate the physiological adaptation of Escherichia coli due to cra gene
knockout, a total of 3,911 gene expressions were investigated by DNA microarray
for continuous culture. About 50 genes were differentially regulated for the cra 
mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose
phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the
cra mutant. The glucose uptake rate and the acetate production rate were
increased with less acetate consumption for the cra mutant. To identify the genes
controlled by Cra protein, the Cra recognition weight matrix from foot-printing
data was developed and used to scan the whole genome. Several new Cra-binding
sites were found, and some of the result was consistent with the DNA microarray
data. The ED pathway was active in the cra mutant; we constructed cra.edd double 
genes knockout mutant to block this pathway, where the acetate overflowed due to 
the down-regulation of aceA,B and icd gene expressions. Then we further
constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow
through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate 
production, resulting in the highest cell yield together with the activation of
the glycolysis pathway, but the glucose consumption rate could not be improved.

DOI: 10.1007/s00203-008-0406-2 
PMID: 18648770  [Indexed for MEDLINE]


699. DNA Repair (Amst). 2008 Nov 1;7(11):1855-68. doi: 10.1016/j.dnarep.2008.07.013.
Epub 2008 Sep 10.

A molecular bar-coded DNA repair resource for pooled toxicogenomic screens.

Rooney JP(1), Patil A, Zappala MR, Conklin DS, Cunningham RP, Begley TJ.

Author information: 
(1)Department of Biomedical Sciences, Gen*NY*sis Center for Excellence in Cancer 
Genomics, University at Albany, State University of New York, Rensselaer, NY
12144, USA.

DNA damage from exogenous and endogenous sources can promote mutations and cell
death. Fortunately, cells contain DNA repair and damage signaling pathways to
reduce the mutagenic and cytotoxic effects of DNA damage. The identification of
specific DNA repair proteins and the coordination of DNA repair pathways after
damage has been a central theme to the field of genetic toxicology and we have
developed a tool for use in this area. We have produced 99 molecular bar-coded
Escherichia coli gene-deletion mutants specific to DNA repair and damage
signaling pathways, and each bar-coded mutant can be tracked in pooled format
using bar-code specific microarrays. Our design adapted bar-codes developed for
the Saccharomyces cerevisiae gene-deletion project, which allowed us to utilize
an available microarray product for pooled gene-exposure studies.
Microarray-based screens were used for en masse identification of individual
mutants sensitive to methyl methanesulfonate (MMS). As expected, gene-deletion
mutants specific to direct, base excision, and recombinational DNA repair
pathways were identified as MMS-sensitive in our pooled assay, thus validating
our resource. We have demonstrated that molecular bar-codes designed for S.
cerevisiae are transferable to E. coli, and that they can be used with
pre-existing microarrays to perform competitive growth experiments. Further, when
comparing microarray to traditional plate-based screens both overlapping and
distinct results were obtained, which is a novel technical finding, with
discrepancies between the two approaches explained by differences in output
measurements (DNA content versus cell mass). The microarray-based classification 
of Deltatag and DeltadinG cells as depleted after MMS exposure, contrary to
plate-based methods, led to the discovery that Deltatag and DeltadinG cells show 
a filamentation phenotype after MMS exposure, thus accounting for the
discrepancy. A novel biological finding is the observation that while DeltadinG
cells filament in response to MMS they exhibit wild-type sulA expression after
exposure. This decoupling of filamentation from SulA levels suggests that DinG is
associated with the SulA-independent filamentation pathway.

DOI: 10.1016/j.dnarep.2008.07.013 
PMCID: PMC2613943
PMID: 18723126  [Indexed for MEDLINE]


700. Genome Res. 2008 Nov;18(11):1798-805. doi: 10.1101/gr.080358.108. Epub 2008 Aug
13.

Transcription of foreign DNA in Escherichia coli.

Warren RL(1), Freeman JD, Levesque RC, Smailus DE, Flibotte S, Holt RA.

Author information: 
(1)BC Cancer Agency, Canada's Michael Smith Genome Sciences Centre, Vancouver,
British Columbia V5Z 4S6, Canada.

Propagation of heterologous DNA in E. coli host cells is central to molecular
biology. DNA constructs are often engineered for expression of recombinant
protein in E. coli, but the extent of incidental transcription arising from
natural regulatory sequences in cloned DNA remains underexplored. Here, we have
used programmable microarrays and RT-PCR to measure, comprehensively, the
transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E.
coli as bacterial artificial chromosomes. We find evidence that at least half of 
all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are 
principally involved in energy metabolism, and their proximal promoter regions
are significantly enriched with E. coli sigma(70) (also known as RpoD) binding
sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion
are also highly transcribed. Compared with H. influenzae, a smaller proportion of
P. aeruginosa genes are transcribed in E. coli, and in E. coli there is
punctuated transcription of human DNA. The presence of foreign DNA in E. coli
disturbs the host transcriptional profile, with expression of the E. coli phage
shock protein operon and the flagellar gene cluster being particularly strongly
up-regulated. While cross-species transcriptional activation is expected to be
enabling for horizontal gene transfer in bacteria, incidental expression of toxic
genes can be problematic for DNA cloning. Ongoing characterization of
cross-expression will help inform the design of biosynthetic gene clusters and
synthetic microbial genomes.

DOI: 10.1101/gr.080358.108 
PMCID: PMC2577866
PMID: 18701636  [Indexed for MEDLINE]


701. Infect Immun. 2008 Nov;76(11):5072-81. doi: 10.1128/IAI.00677-08. Epub 2008 Sep
15.

CadA negatively regulates Escherichia coli O157:H7 adherence and intestinal
colonization.

Vazquez-Juarez RC(1), Kuriakose JA, Rasko DA, Ritchie JM, Kendall MM, Slater TM, 
Sinha M, Luxon BA, Popov VL, Waldor MK, Sperandio V, Torres AG.

Author information: 
(1)Department of Microbiology and Immunology, University of Texas Medical Branch,
Galveston, TX 77555-1070, USA.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is
essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7,
we have previously demonstrated that multiple factors govern this pathogen's
adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun.
71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and
this protein has been proposed to negatively regulate virulence in several
enteric pathogens. In the case of EHEC strains, CadA modulates expression of the 
intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated
cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC
adhesion to tissue-cultured monolayers, global gene expression patterns, and
colonization of the infant rabbit intestine. The cadA mutant did not possess
lysine decarboxylation activity and was hyperadherent to tissue-cultured cells.
Adherence of the cadA mutant was nearly twofold greater than that of the wild
type, and the adherence phenotype was independent of pH, lysine, or cadaverine in
the media. Additionally, complementation of the cadA defect reduced adherence
back to wild-type levels, and it was found that the mutation affected the
expression of the intimin protein. Disruption of the eae gene (intimin-encoding
gene) in the cadA mutant significantly reduced its adherence to tissue-cultured
cells. However, adherence of the cadA eae double mutant was greater than that of 
an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is
not entirely attributable to enhanced expression of intimin in this background.
Gene array analysis revealed that the cadA mutation significantly altered EHEC
gene expression patterns; expression of 1,332 genes was downregulated and that of
132 genes was upregulated in the mutant compared to the wild-type strain.
Interestingly, the gene expression variation shows an EHEC-biased gene alteration
including intergenic regions. Two putative adhesins, flagella and F9 fimbria,
were upregulated in the cadA mutant, suggestive of their association with
adherence in the absence of the Cad regulatory mechanism. In the infant rabbit
model, the cadA mutant outcompeted the wild-type strain in the ileum but not in
the cecum or mid-colon, raising the possibility that CadA negatively regulates
EHEC pathogenicity in a tissue-specific fashion.

DOI: 10.1128/IAI.00677-08 
PMCID: PMC2573373
PMID: 18794292  [Indexed for MEDLINE]


702. Infect Immun. 2008 Nov;76(11):5247-56. doi: 10.1128/IAI.00770-08. Epub 2008 Sep
2.

RegA, an AraC-like protein, is a global transcriptional regulator that controls
virulence gene expression in Citrobacter rodentium.

Hart E(1), Yang J, Tauschek M, Kelly M, Wakefield MJ, Frankel G, Hartland EL,
Robins-Browne RM.

Author information: 
(1)Department of Microbiology and Immunology, The University of Melbourne,
Victoria 3010, Australia.

Citrobacter rodentium is an attaching and effacing pathogen which causes
transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as 
a model for infection of humans with enteropathogenic and enterohemorrhagic
Escherichia coli. To identify novel colonization factors of C. rodentium, we
screened a signature-tagged mutant library of C. rodentium in mice. One
noncolonizing mutant had a single transposon insertion in an open reading frame
(ORF) which we designated regA because of its homology to genes encoding members 
of the AraC family of transcriptional regulators. Deletion of regA in C.
rodentium resulted in markedly reduced colonization of the mouse intestine.
Examination of lacZ transcriptional fusions using promoter regions of known and
putative virulence-associated genes of C. rodentium revealed that RegA strongly
stimulated transcription of two newly identified genes located close to regA,
which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation
and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation
led to a reduction in the duration of intestinal colonization, but the kfc mutant
was far less attenuated than the regA mutant. These results indicated that other 
genes of C. rodentium whose expression required activation by RegA were required 
for colonization. Microarray analysis revealed a number of RegA-regulated ORFs
encoding proteins homologous to known colonization factors. Transcription of
these putative virulence determinants was activated by RegA only in the presence 
of sodium bicarbonate. Taken together, these results show that RegA is a global
regulator of virulence in C. rodentium which activates factors that are required 
for intestinal colonization.

DOI: 10.1128/IAI.00770-08 
PMCID: PMC2573378
PMID: 18765720  [Indexed for MEDLINE]


703. Infect Immun. 2008 Nov;76(11):4814-22. doi: 10.1128/IAI.00446-08. Epub 2008 Aug
25.

Escherichia coli O157:H7 survives within human macrophages: global gene
expression profile and involvement of the Shiga toxins.

Poirier K(1), Faucher SP, Béland M, Brousseau R, Gannon V, Martin C, Harel J,
Daigle F.

Author information: 
(1)Department of Microbiology and Immunology, University of Montreal, C.P. 6128
Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.

Escherichia coli O157:H7 is an important food-borne pathogen that specifically
binds to the follicle-associated epithelium in the intestine, which rapidly
brings this bacterial pathogen in contact with underlying human macrophages. Very
little information is available about the interaction between E. coli O157:H7 and
human macrophages. We evaluated the uptake and survival of strain EDL933 during
infection of human macrophages. Surprisingly, EDL933 survived and multiplied in
human macrophages at 24 h postinfection. The global gene expression profile of
this pathogen during macrophage infection was determined. Inside human
macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as 
pagC, the genes for both of the Shiga toxins, and the two iron transport system
operons fit and chu) was observed. Genes involved in acid resistance and in the
SOS response were upregulated. However, genes of the locus of enterocyte
effacement or genes involved in peroxide resistance were not differentially
expressed. Many genes with putative or unknown functions were upregulated inside 
human macrophages and may be newly discovered virulence factors. As the Shiga
toxin genes were upregulated in macrophages, survival and cytotoxicity assays
were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga
toxins mutants was higher than that of the wild type; however, the survival rates
were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated
in the interaction between E. coli O157:H7 and human macrophages. Understanding
the molecular mechanisms used by E. coli to survive within macrophages may help
in the identification of targets for new therapeutic agents.

DOI: 10.1128/IAI.00446-08 
PMCID: PMC2573336
PMID: 18725421  [Indexed for MEDLINE]


704. Infect Immun. 2008 Nov;76(11):4833-41. doi: 10.1128/IAI.00630-08. Epub 2008 Aug
18.

PapX, a P fimbrial operon-encoded inhibitor of motility in uropathogenic
Escherichia coli.

Simms AN(1), Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109, USA.

Motility and adherence are two integral aspects of bacterial pathogenesis.
Adherence, often mediated by fimbriae, permits bacteria to attach to host cells
and establish infection, whereas flagellum-driven motility allows bacteria to
disseminate to sites more advantageous for colonization. Both fimbriae and
flagella have been proven important for virulence of uropathogenic Escherichia
coli (UPEC). Reciprocal regulation is one mechanism by which bacteria may
reconcile the contradictory actions of adherence and motility. PapX, a P fimbrial
gene product of UPEC strain CFT073, is a functional homolog of MrpJ of Proteus
mirabilis; ectopic expression of papX in P. mirabilis reduces motility. To define
the connection between P fimbria expression and motility in UPEC, the role of
papX in the regulation of motility of strain CFT073 was examined. Overexpression 
of papX decreased motility of CFT073, which correlated with both a significant
reduction in flagellin protein synthesized and flagella assembled on the cell
surface. Conversely, an increase in motility and flagellin production was seen in
an isogenic papX deletion mutant of CFT073. Microarray and quantitative reverse
transcription-PCR analysis indicated that repression of motility of CFT073 by
PapX appears to occur at the transcriptional level; expression of many
motility-associated genes, including flhDC, the master regulator of motility, is 
decreased when papX is overexpressed. Transcription of motility genes is
increased in the papX mutant compared to wild type. Electrophoretic mobility gel 
shift analysis revealed that PapX binds to the flhD promoter. We conclude that
synthesis of P fimbriae regulates flagellum synthesis to repress motility via
PapX.

DOI: 10.1128/IAI.00630-08 
PMCID: PMC2573324
PMID: 18710869  [Indexed for MEDLINE]


705. J Bacteriol. 2008 Nov;190(22):7532-47. doi: 10.1128/JB.01002-08. Epub 2008 Sep
19.

Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host
interactions.

Tiaden A(1), Spirig T, Carranza P, Brüggemann H, Riedel K, Eberl L, Buchrieser C,
Hilbi H.

Author information: 
(1)Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich,
Switzerland.

The causative agent of Legionnaires' disease, Legionella pneumophila, is a
natural parasite of environmental protozoa and employs a biphasic life style to
switch between a replicative and a transmissive (virulent) phase. L. pneumophila 
harbors the lqs (Legionella quorum sensing) cluster, which includes genes
encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response
regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in 
Escherichia coli. LqsR promotes host-cell interactions as an element of the
stationary-phase virulence regulatory network. Here, we characterize L.
pneumophila mutant strains lacking all four genes of the lqs cluster or only the 
hdeD gene. While an hdeD mutant strain did not have overt physiological or
virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary
growth phase and was defective for intracellular growth, efficient phagocytosis, 
and cytotoxicity against host cells. Cytotoxicity was restored upon
reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The 
deletion of the lqs cluster caused more-severe phenotypes than deletion of only
lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome
analysis indicated that in the stationary phase more than 380 genes were
differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes
involved in protein production, metabolism, and bioenergetics were upregulated in
the lqs mutant, whereas genes encoding virulence factors, such as effectors
secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome 
analysis revealed that a set of Icm/Dot substrates is not produced in the absence
of the lqs gene cluster, which confirms the findings from DNA microarray assays
and mirrors the virulence phenotype of the lqs mutant strain.

DOI: 10.1128/JB.01002-08 
PMCID: PMC2576672
PMID: 18805977  [Indexed for MEDLINE]


706. J Bacteriol. 2008 Nov;190(22):7479-90. doi: 10.1128/JB.00823-08. Epub 2008 Sep
12.

Escherichia coli harboring a natural IncF conjugative F plasmid develops complex 
mature biofilms by stimulating synthesis of colanic acid and Curli.

May T(1), Okabe S.

Author information: 
(1)Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8,
Kita-Ku, Sapporo 060-8628, Japan.

It has been shown that Escherichia coli harboring the derepressed IncFI and
IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus
connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore,
in this study we investigated the contribution of a natural IncF conjugative F
plasmid to the formation of E. coli biofilms. Unlike the presence of a
derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm 
formation by generating the cell-to-cell mating F pili between pairs of F(+)
cells (approximately two to four pili per cell) and by stimulating the formation 
of colanic acid and curli meshwork. Formation of colanic acid and curli was
required after the initial deposition of F-pilus connections to generate a
three-dimensional mushroom-type biofilm. In addition, we demonstrated that the
conjugative factor of F plasmid, rather than a pilus synthesis function, was
involved in curli production during biofilm formation, which promoted
cell-surface interactions. Curli played an important role in the maturation
process. Microarray experiments were performed to identify the genes involved in 
curli biosynthesis and regulation. The results suggested that a natural F plasmid
was more likely an external activator that indirectly promoted curli production
via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the 
RpoS and HN-S global regulators). These data provided new insights into the role 
of a natural F plasmid during the development of E. coli biofilms.

DOI: 10.1128/JB.00823-08 
PMCID: PMC2576669
PMID: 18790864  [Indexed for MEDLINE]


707. Mol Microbiol. 2008 Nov;70(3):608-22. doi: 10.1111/j.1365-2958.2008.06423.x. Epub
2008 Aug 29.

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits
replication fork progression and is lethal.

Uchida K(1), Furukohri A, Shinozaki Y, Mori T, Ogawara D, Kanaya S, Nohmi T, Maki
H, Akiyama M.

Author information: 
(1)Department of Molecular Biology, Graduate School of Biological Sciences, Nara 
Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which
is induced as part of the SOS stress-response system and functions in translesion
synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA
lesions. As the number of DinB molecules, even in unstressed cells, is greater
than that required to accomplish TLS, it is thought that dinB plays some
additional physiological role. Here, we overexpressed dinB under the tightly
regulable arabinose promoter and looked for a distinct phenotype. Upon induction 
of dinB expression, progression of the replication fork was immediately inhibited
at random genomic positions, and the colony-forming ability of the cells was
reduced. Overexpression of mutated dinB alleles revealed that the structural
requirements for these two inhibitory effects and for TLS were distinct. The
extent of in vivo inhibition displayed by a mutant DinB matched the extent of its
in vitro impedance, at near-physiological concentration, of a moving Pol III. We 
suggest that DinB targets Pol III, thereby acting as a brake on replication fork 
progression. Because the brake operates when cells have excess DinB, as they do
under stress conditions, it may serve as a checkpoint that modulates replication 
to safeguard genome stability.

DOI: 10.1111/j.1365-2958.2008.06423.x 
PMID: 18761688  [Indexed for MEDLINE]


708. PLoS Comput Biol. 2008 Nov;4(11):e1000237. doi: 10.1371/journal.pcbi.1000237.
Epub 2008 Nov 28.

Dynamic changes in protein functional linkage networks revealed by integration
with gene expression data.

Hegde SR(1), Manimaran P, Mande SC.

Author information: 
(1)Centre for DNA Fingerprinting and Diagnostics, Nacharam, Hyderabad, India.

Response of cells to changing environmental conditions is governed by the
dynamics of intricate biomolecular interactions. It may be reasonable to assume, 
proteins being the dominant macromolecules that carry out routine cellular
functions, that understanding the dynamics of protein:protein interactions might 
yield useful insights into the cellular responses. The large-scale protein
interaction data sets are, however, unable to capture the changes in the profile 
of protein:protein interactions. In order to understand how these interactions
change dynamically, we have constructed conditional protein linkages for
Escherichia coli by integrating functional linkages and gene expression
information. As a case study, we have chosen to analyze UV exposure in wild-type 
and SOS deficient E. coli at 20 minutes post irradiation. The conditional
networks exhibit similar topological properties. Although the global topological 
properties of the networks are similar, many subtle local changes are observed,
which are suggestive of the cellular response to the perturbations. Some such
changes correspond to differences in the path lengths among the nodes of
carbohydrate metabolism correlating with its loss in efficiency in the UV treated
cells. Similarly, expression of hubs under unique conditions reflects the
importance of these genes. Various centrality measures applied to the networks
indicate increased importance for replication, repair, and other stress proteins 
for the cells under UV treatment, as anticipated. We thus propose a novel
approach for studying an organism at the systems level by integrating genome-wide
functional linkages and the gene expression data.

DOI: 10.1371/journal.pcbi.1000237 
PMCID: PMC2580820
PMID: 19043542  [Indexed for MEDLINE]


709. Vaccine. 2008 Oct 23;26(45):5722-7. doi: 10.1016/j.vaccine.2008.08.018. Epub 2008
Aug 30.

Genetic stability of the live attenuated Bordetella pertussis vaccine candidate
BPZE1.

Feunou PF(1), Ismaili J, Debrie AS, Huot L, Hot D, Raze D, Lemoine Y, Locht C.

Author information: 
(1)Inserm U629, Lille, France.

Despite the extensive use of efficacious pertussis vaccines, Bordetella pertussis
infections are still among the main causes for childhood morbidity and mortality.
Severe pertussis occurs mostly in very young children, often too young to be
sufficiently protected by current vaccines, which require several administrations
in regimens that vary between countries. Since natural infection with B.
pertussis is able to induce protection, we have developed the live attenuated B. 
pertussis vaccine strain BPZE1 that protects mice upon a single intranasal
administration. This strain was obtained by genetically inactivating pertussis
toxin via two point mutations in the ptx gene, by deleting dnt encoding
dermonecrotic toxin, and by replacing the B. pertussis ampG gene by Escherichia
coli ampG, resulting in the removal of tracheal cytotoxin. Here, we assessed the 
genetic stability of BPZE1 after 20 and 27 weeks of continuous passaging in vitro
and in vivo, respectively. BPZE1 was passaged 20 times in vitro and 9 times in
vivo in Balb/C mice. After these passages, 8 hemolytic colonies were analyzed by 
PCR for the absence of dnt and B. pertussis ampG and the presence of E. coli
ampG, by DNA sequencing for the presence of the two ptx point mutations and by
DNA microarrays for the global genomic stability. In addition, the protective
capacity of BPZE1 was evaluated after the passages. No genetic or protective
difference was detected between the passaged bacteria and non-passaged BPZE1,
indicating that stability of the vaccine strain is not a concern for BPZE1 to be 
considered as an attenuated live vaccine against whooping cough.

DOI: 10.1016/j.vaccine.2008.08.018 
PMID: 18762220  [Indexed for MEDLINE]


710. Anal Chem. 2008 Oct 15;80(20):7786-91. doi: 10.1021/ac8012048. Epub 2008 Aug 27.

Bacterial DNA sample preparation from whole blood using surface-modified Si
pillar arrays.

Hwang KY(1), Lim HK, Jung SY, Namkoong K, Kim JH, Huh N, Ko C, Park JC.

Author information: 
(1)Bio & Health Lab., Samsung Advanced Institute of Technology (SAIT), Mt. 14-1, 
Nongseo-Dong, Giheung-Gu, Yongin-Si, Gyeonggi-Do, South Korea, 449-712.

A novel bacterial DNA sample preparation device for molecular diagnostics has
been developed. On the basis of optimized conditions for bacterial adhesion,
surface-modified silicon pillar arrays for bacterial cell capture were
fabricated, and their ability to capture bacterial cells was demonstrated. The
capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus
epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow
rate of 400 microL/min. Moreover, the proposed method captured E. coli cells
present in 50% whole blood effectively. The captured cells from whole blood were 
then in- situ lyzed on the surface of the microchip, and the eluted DNA was
successfully amplified by qPCR. These results demonstrate that the full process
of pathogen capture to DNA isolation from whole blood could be automated in a
single microchip.

DOI: 10.1021/ac8012048 
PMID: 18729477  [Indexed for MEDLINE]


711. BMC Genomics. 2008 Oct 14;9:483. doi: 10.1186/1471-2164-9-483.

The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 
controls a subset of genes of the McbR regulon in response to the availability of
sulphide acceptor molecules.

Rückert C(1), Milse J, Albersmeier A, Koch DJ, Pühler A, Kalinowski J.

Author information: 
(1)Institut für Systembiologie & Genomforschung, Universität Bielefeld,
Universitätsstr. 25, D-33615 Bielefeld, Germany.
christian.rueckert@cebitec.uni-bielefeld.de

BACKGROUND: Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC
13032 has been studied intensively in the last few years, due to its industrial
as well as scientific importance. Previously, the gene cg0156 was shown to belong
to the regulon of McbR, a global transcriptional repressor of sulphur metabolism 
in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of
the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting
candidate for study to further the understanding of the regulation of sulphur
metabolism in C. glutamicum.
RESULTS: Deletion of cg0156, now designated cysR, results in the inability of the
mutant to utilise sulphate and aliphatic sulphonates. DNA microarray
hybridisations revealed 49 genes with significantly increased and 48 with
decreased transcript levels in presence of the native CysR compared to a cysR
deletion mutant. Among the genes positively controlled by CysR were the gene
cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel
retardation experiments demonstrated that binding of CysR to DNA depends in vitro
on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of 
the transcription start points of five transcription units helped to identify a
10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests
proved this motif to be necessary for CysR-dependent transcriptional regulation.
CONCLUSION: CysR acts as the functional analogue of the unrelated LysR-type
regulator CysB from Escherichia coli, controlling sulphide production in response
to acceptor availability. In both bacteria, gene duplication events seem to have 
taken place which resulted in the evolution of dedicated regulators for the
control of sulphonate utilisation. The striking convergent evolution of network
topology indicates the strong selective pressure to control the metabolism of the
essential but often toxic sulphur-containing (bio-)molecules.

DOI: 10.1186/1471-2164-9-483 
PMCID: PMC2580772
PMID: 18854009  [Indexed for MEDLINE]


712. BMC Syst Biol. 2008 Oct 1;2:84. doi: 10.1186/1752-0509-2-84.

Network evaluation from the consistency of the graph structure with the measured 
data.

Saito S(1), Aburatani S, Horimoto K.

Author information: 
(1)Biological Network Team, Computational Biology Research Center (CBRC),
National Institute of Advanced Industrial Science and Technology (AIST), Tokyo
135-0064, Japan. sh.saito@infocom.co.jp

BACKGROUND: A knowledge-based network, which is constructed by extracting as many
relationships identified by experimental studies as possible and then
superimposing them, is one of the promising approaches to investigate the
associations between biological molecules. However, the molecular relationships
change dynamically, depending on the conditions in a living cell, which suggests 
implicitly that all of the relationships in the knowledge-based network do not
always exist. Here, we propose a novel method to estimate the consistency of a
given network with the measured data: i) the network is quantified into a
log-likelihood from the measured data, based on the Gaussian network, and ii) the
probability of the likelihood corresponding to the measured data, named the graph
consistency probability (GCP), is estimated based on the generalized extreme
value distribution.
RESULTS: The plausibility and the performance of the present procedure are
illustrated by various graphs with simulated data, and with two types of actual
gene regulatory networks in Escherichia coli: the SOS DNA repair system with the 
corresponding data measured by fluorescence, and a set of 29 networks with data
measured under anaerobic conditions by microarray. In the simulation study, the
procedure for estimating GCP is illustrated by a simple network, and the
robustness of the method is scrutinized in terms of various aspects: dimensions
of sampling data, parameters in the simulation study, magnitudes of data noise,
and variations of network structures. In the actual networks, the former example 
revealed that our method operates well for an actual network with a size similar 
to those of the simulated networks, and the latter example illustrated that our
method can select the activated network candidates consistent with the actual
data measured under specific conditions, among the many network candidates.
CONCLUSION: The present method shows the possibility of bridging between the
static network from the literature and the corresponding measurements, and thus
will shed light on the network structure variations in terms of the changes in
molecular interaction mechanisms that occur in response to the environment in a
living cell.

DOI: 10.1186/1752-0509-2-84 
PMCID: PMC2566979
PMID: 18828895  [Indexed for MEDLINE]


713. J Microbiol Methods. 2008 Oct;75(2):365-8. doi: 10.1016/j.mimet.2008.06.020. Epub
2008 Jul 2.

Simultaneous detection of six human diarrheal pathogens by using DNA microarray
combined with tyramide signal amplification.

Jin D(1), Qi H, Chen S, Zeng T, Liu Q, Wang S.

Author information: 
(1)Beijing Institute of Radiation Medicine, No. 27 Taiping road, Beijing, China. 
dazhijin_y@hotmail.com

Multiplex PCR and DNA microarray were combined with tyramide signal amplification
(TSA) to develop a reliable method suitable for simultaneous detection of six
species of human diarrheal pathogens (Yersinia enterocolitica, Shigella spp,
Salmonella typhi, Brucella spp, Vibrio cholera and Escherichia coli O157:H7).
Meanwhile, our method could distinguish V. cholera serotype O1 from O139, and
O157:H7 from O157: non-H7. This assay conferred a specificity of 100% for target 
pathogens. The limit of detection was 103 degrees CFU/mL approximately. The
results of 98.6% (357/362) clinical specimens and 100% (5/5) mocked double-blind 
samples were the same to that from conventional assay. Consequently this assay is
sensitive and a specific tool suitable for diagnostic detection and surveillance 
of multiple human pathogens.

DOI: 10.1016/j.mimet.2008.06.020 
PMID: 18655810  [Indexed for MEDLINE]


714. Mol Microbiol. 2008 Oct;70(1):183-96. doi: 10.1111/j.1365-2958.2008.06400.x. Epub
2008 Aug 18.

mRNA processing by RNases J1 and J2 affects Bacillus subtilis gene expression on 
a global scale.

Mäder U(1), Zig L, Kretschmer J, Homuth G, Putzer H.

Author information: 
(1)Interfaculty Institute for Genetics and Functional Genomics, Department for
Functional Genomics, Ernst-Moritz-Arndt-University Greifswald,
Walther-Rathenau-Str. 49A, D-17489 Greifswald, Germany.

Ribonucleases J1 and J2 of Bacillus subtilis are evolutionarily conserved enzymes
combining an endoribonucleolytic and a 5'-3' exoribonucleolytic activity in a
single polypeptide. Their endoribonucleolytic cleavage specificity resembles that
of RNase E, a key player in the processing and degradation of RNA in Escherichia 
coli. The biological significance of the paralogous RNases J1 and J2 in Bacillus 
subtilis is still unknown. Based on the premise that cleavage of an mRNA might
alter its stability and hence its abundance, we have analysed the transcriptomes 
and proteomes of single and double mutant strains. The absence or decrease of
both RNases J1 and J2 together profoundly alters the expression level of hundreds
of genes. By contrast, the effect on global gene expression is minimal in single 
mutant strains, suggesting that the two nucleases have largely overlapping
substrate specificities. Half-life measurements of individual mRNAs show that
RNases J1/J2 can alter gene expression by modulating transcript stability. The
absence/decrease of RNases J1 and J2 results in similar numbers of transcripts
whose abundance is either increased or decreased, suggesting a complex role of
these ribonucleases in both degradative and regulatory processing events that
have an important impact on gene expression.

DOI: 10.1111/j.1365-2958.2008.06400.x 
PMID: 18713320  [Indexed for MEDLINE]


715. Mol Microbiol. 2008 Oct;70(1):236-57. doi: 10.1111/j.1365-2958.2008.06411.x. Epub
2008 Aug 18.

The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly
regulating the expression of GGDEF proteins.

Jonas K(1), Edwards AN, Simm R, Romeo T, Römling U, Melefors O.

Author information: 
(1)Swedish Institute for Infectious Disease Control, SE-17182, Solna; and
Department of Microbiology, Tumor and Cell Biology, Karolinska Institute,
SE-17177 Stockholm, Sweden.

The carbon storage regulator CsrA is an RNA binding protein that controls carbon 
metabolism, biofilm formation and motility in various eubacteria. Nevertheless,
in Escherichia coli only five target mRNAs have been shown to be directly
regulated by CsrA at the post-transcriptional level. Here we identified two new
direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF 
domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more
than 10-fold. RNA mobility shift assays confirmed the direct and specific binding
of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH
resulted in a more than 20-fold increase in the cellular concentration of the
second messenger cyclic di-GMP (c-di-GMP), implying that both proteins possess
diguanylate cyclase activity. Phenotypic characterization revealed that both
proteins are involved in the regulation of motility in a c-di-GMP-dependent
manner. CsrA was also found to regulate the expression of five additional
GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels 
of c-di-GMP. All together, these data demonstrate a global role for CsrA in the
regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins 
at the post-transcriptional level.

DOI: 10.1111/j.1365-2958.2008.06411.x 
PMCID: PMC2735045
PMID: 18713317  [Indexed for MEDLINE]


716. Mol Microbiol. 2008 Oct;70(1):60-75. doi: 10.1111/j.1365-2958.2008.06388.x. Epub 
2008 Aug 11.

Global gene expression mediated by Thermus thermophilus SdrP, a CRP/FNR family
transcriptional regulator.

Agari Y(1), Kashihara A, Yokoyama S, Kuramitsu S, Shinkai A.

Author information: 
(1)RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148,
Japan.

Erratum in
    Mol Microbiol. 2014 Jul;93(2):390.

Thermus thermophilus SdrP is one of four cyclic AMP receptor protein
(CRP)/fumarate and nitrate reduction regulator (FNR) family proteins from the
extremely thermophilic bacterium T. thermophilus HB8. Expression of sdrP mRNA
increased in the stationary phase during cultivation at 70 degrees C. Although
the sdrP gene was non-essential, an sdrP-deficient strain showed growth defects, 
particularly when grown in a synthetic medium, and increased sensitivity to
disulphide stress. The expression of several genes was altered in the sdrP
disruptant. Among them, we found eight SdrP-dependent promoters using in vitro
transcription assays. A predicted SdrP binding site similar to that recognized by
Escherichia coli CRP was found upstream of each SdrP-dependent promoter. In the
wild-type strain, expression of these eight genes tended to increase upon entry
into the stationary phase. Transcriptional activation in vitro was independent of
any added effector molecule. The hypothesis that apo-SdrP is the active form of
the protein was supported by the observation that the three-dimensional structure
of apo-SdrP is similar to that of the DNA-binding form of E. coli CRP. Based on
the properties of the SdrP-regulated genes found in this study, it is speculated 
that SdrP is involved in nutrient and energy supply, redox control, and
polyadenylation of mRNA.

DOI: 10.1111/j.1365-2958.2008.06388.x 
PMID: 18699868  [Indexed for MEDLINE]


717. PLoS One. 2008 Sep 24;3(9):e3265. doi: 10.1371/journal.pone.0003265.

Protein microarray on-demand: a novel protein microarray system.

Chatterjee DK(1), Sitaraman K, Baptista C, Hartley J, Hill TM, Munroe DJ.

Author information: 
(1)Protein Expression Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick,
Maryland, USA. chatterjee@mail.ncifcrf.gov

We describe a novel, simple and low-cost protein microarray strategy wherein the 
microarrays are generated by printing expression ready plasmid DNAs onto slides
that can be converted into protein arrays on-demand. The printed expression
plasmids serve dual purposes as they not only direct the synthesis of the protein
of interest; they also serve to capture the newly synthesized proteins through a 
high affinity DNA-protein interaction. To accomplish this we have exploited the
high-affinity binding (approximately 3-7 x 10 (-13) M) of E. coli Tus protein to 
Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. 
In our system, each protein of interest is synthesized as a Tus fusion protein
and each expression construct directing the protein synthesis contains embedded
Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for
the newly synthesized Tus fusion protein. This "all DNA" microarray can be
converted to a protein microarray on-demand without need for any additional
capture reagent.

DOI: 10.1371/journal.pone.0003265 
PMCID: PMC2533396
PMID: 18813342  [Indexed for MEDLINE]


718. J Biol Chem. 2008 Sep 12;283(37):25238-46. doi: 10.1074/jbc.M804122200. Epub 2008
Jun 30.

Molecular identification of pseudouridine-metabolizing enzymes.

Preumont A(1), Snoussi K, Stroobant V, Collet JF, Van Schaftingen E.

Author information: 
(1)de Duve Institute, Université Catholique de Louvain, Avenue Hippocrate 75,
Brussels, Belgium.

Pseudouridine, a non-classical nucleoside present in human urine as a degradation
product of RNAs, is one of the few molecules that has a glycosidic C-C bond.
Through a data base mining approach involving transcriptomic data, we have
molecularly identified two enzymes that are involved in the metabolism of
pseudouridine in uropathogenic Escherichia coli, the principal agent of urinary
tract infections in humans. The first enzyme, coded by the gene yeiC,
specifically phosphorylates pseudouridine to pseudouridine 5'-phosphate.
Accordingly, yeiC(-) mutants are unable to metabolize pseudouridine, in contrast 
to wild-type E. coli UTI89. The second enzyme, encoded by the gene yeiN belonging
to the same operon as yeiC, catalyzes the conversion of pseudouridine
5'-phosphate to uracil and ribose 5-phosphate in a divalent cation-dependent
manner. Remarkably, the glycosidic C-C bond of pseudouridine is cleaved in the
course of this reaction, indicating that YeiN is the first molecularly identified
enzyme able to hydrolyze a glycosidic C-C bond. Though this reaction is easily
reversible, the association of YeiN with pseudouridine kinase indicates that it
serves physiologically to metabolize pseudouridine 5'-phosphate rather than to
form it. YeiN is homologous to Thermotoga maritima IndA, a protein with a new
fold, which we now show to act also as a pseudouridine-5'-phosphate glycosidase. 
Data base mining indicates that most eukaryotes possess homologues of
pseudouridine kinase and pseudouridine-5'-phosphate glycosidase and that these
are most often associated in a single bifunctional protein. The gene encoding
this bifunctional protein is absent from the genomes of man and other mammals,
indicating that the capacity for metabolizing pseudouridine has been lost late in
evolution.

DOI: 10.1074/jbc.M804122200 
PMID: 18591240  [Indexed for MEDLINE]


719. Arch Microbiol. 2008 Sep;190(3):341-53. doi: 10.1007/s00203-008-0379-1. Epub 2008
May 21.

Characterization of a Haloferax volcanii member of the enolase superfamily:
deletion mutant construction, expression analysis, and transcriptome comparison.

Dambeck M(1), Soppa J.

Author information: 
(1)Biocentre, Institute for Molecular Biosciences, Goethe-University,
Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. m.dambeck@bio.uni-frankfurt.de

The enolase superfamily (COG4948) contains proteins with very different
biological functions including regulators like the Escherichia coli RspA and
metabolic enzymes like enolase. To unravel the biological function of an archaeal
family member, an in frame deletion mutant of a gene encoding a COG4948 protein
of Haloferax volcanii was generated. The mutant had a lag phase of 3 days after
transition from a richer to a poorer medium, in contrast to the wild-type, and
the gene was therefore named "important for transition" (iftA). After inoculation
of fresh casamino acids or complex medium with stationary phase wild-type cells, 
the transcript level of iftA was transiently induced at the onset of growth. In
contrast, in minimal (or "poor") glucose medium, both transcript and protein were
present throughout growth, even in late stationary phase. A comparison of the
transcriptomes of deletion mutant and wild-type revealed that transcript levels
of a very restricted set of genes were differentially regulated, including genes 
encoding proteins involved in phosphate metabolism, regulators and stress
response proteins. Taken together, the results indicate that IftA might have a
dual function, i.e., transiently after transition to fresh medium and permanently
during growth in glucose medium.

DOI: 10.1007/s00203-008-0379-1 
PMID: 18493744  [Indexed for MEDLINE]


720. Arch Microbiol. 2008 Sep;190(3):319-32. doi: 10.1007/s00203-008-0369-3. Epub 2008
Apr 16.

Insights into the NrpR regulon in Methanosarcina mazei Gö1.

Weidenbach K(1), Ehlers C, Kock J, Ehrenreich A, Schmitz RA.

Author information: 
(1)Institut für Allgemeine Mikrobiologie, Christian-Albrechts Universität zu
Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.

The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues
of NrpR, the transcriptional repressor of nitrogen assimilation genes recently
discovered and characterized in Methanococcus maripaludis. Insertion of a
puromycin-resistance conferring cassette into MM1085 encoding a single NrpR
domain with an N-terminal helix-turn-helix domain (NrpRI) lead to a significant
reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen
limitation. Consistent with this finding, loss of NrpRI resulted in significantly
increased transcript levels of genes involved in nitrogen fixation or nitrogen
assimilation though growing under nitrogen sufficiency as demonstrated by
quantitative reverse transcriptional PCR analysis. Genome-wide analysis using
DNA-microarrays confirmed that transcript levels of 27 ORFs were significantly
elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including
genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA(1),
glnK(1)), and 17 additional genes involved in metabolism (4), encoding a flagella
related protein (1) and genes encoding hypothetical proteins (12). Using cell
extracts of Escherichia coli expressing MM1085 fused to the maltose binding
protein (MBP-NrpRI) and employing promoter binding studies by DNA-affinity
chromatography demonstrated that MBP-NrpRI binds specifically to the
nifH-promoter. Deletion of various bases in the promoter region of nifH confirmed
that the regulatory element ACC-N(7)-GGT is required for specific binding of
NrpRI to the promoter.

DOI: 10.1007/s00203-008-0369-3 
PMID: 18415079  [Indexed for MEDLINE]


721. Biochim Biophys Acta. 2008 Sep;1778(9):1698-713. Epub 2007 Aug 11.

Proteome of the Escherichia coli envelope and technological challenges in
membrane proteome analysis.

Weiner JH(1), Li L.

Author information: 
(1)Membrane Protein Research Group and The Institute for Biomolecular Design,
University of Alberta, Canada. joel.weiner@ualberta.ca

The envelope of Escherichia coli is a complex organelle composed of the outer
membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each
compartment has a unique complement of proteins, the proteome. Determining the
proteome of the envelope is essential for developing an in silico bacterial
model, for determining cellular responses to environmental alterations, for
determining the function of proteins encoded by genes of unknown function and for
development and testing of new experimental technologies such as mass
spectrometric methods for identifying and quantifying hydrophobic proteins. The
availability of complete genomic information has led several groups to develop
computer algorithms to predict the proteome of each part of the envelope by
searching the genome for leader sequences, beta-sheet motifs and stretches of
alpha-helical hydrophobic amino acids. In addition, published experimental data
has been mined directly and by machine learning approaches. In this review we
examine the somewhat confusing available literature and relate published
experimental data to the most recent gene annotation of E. coli to describe the
predicted and experimental proteome of each compartment. The problem of
characterizing integral versus membrane-associated proteins is discussed. The E. 
coli envelope proteome provides an excellent test bed for developing mass
spectrometric techniques for identifying hydrophobic proteins that have generally
been refractory to analysis. We describe the gel based and solution based
proteome analysis approaches along with protein cleavage and proteolysis methods 
that investigators are taking to tackle this difficult problem.

DOI: 10.1016/j.bbamem.2007.07.020 
PMID: 17904518  [Indexed for MEDLINE]


722. Int J Antimicrob Agents. 2008 Sep;32(3):272-7. doi:
10.1016/j.ijantimicag.2008.04.020. Epub 2008 Jul 18.

Genotyping DNA chip for the simultaneous assessment of antibiotic resistance and 
pathogenic potential of extraintestinal pathogenic Escherichia coli.

Barl T(1), Dobrindt U, Yu X, Katcoff DJ, Sompolinsky D, Bonacorsi S, Hacker J,
Bachmann TT.

Author information: 
(1)Division of Pathway Medicine, University of Edinburgh, Chancellor's Building, 
49 Little France Crescent, Edinburgh EH16 4SB, UK.

Urinary tract infections (UTIs) are among the most frequently occurring
infections and are mostly caused by extraintestinal pathogenic Escherichia coli. 
DNA microarrays are potent molecular diagnostic tools for rapid diagnosis of
bacterial infections with high relevance for UTIs. In this study, we present the 
integration and application of two DNA chip modules for the simultaneous
detection of single nucleotide polymorphisms in gyrA (quinolone resistance) and
fimH (increased adhesion to urinary tract epithelium). The performance of the
combined diagnostic chip was assessed by genotyping 140 E. coli strains.
Resistance-causing mutations could only be identified in UTI isolates. A complete
genotyping assay could be performed in <4h after DNA extraction. Together with
the excellent genotyping results, this constitutes a competitive alternative as a
standard tool for routine clinical diagnostics.

DOI: 10.1016/j.ijantimicag.2008.04.020 
PMID: 18640014  [Indexed for MEDLINE]


723. J Bacteriol. 2008 Sep;190(17):5890-7. doi: 10.1128/JB.00459-08. Epub 2008 Jun 20.

The uncharacterized transcription factor YdhM is the regulator of the nemA gene, 
encoding N-ethylmaleimide reductase.

Umezawa Y(1), Shimada T, Kori A, Yamada K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, 
Japan.

N-ethylmaleimide (NEM) has been used as a specific reagent of Cys modification in
proteins and thus is toxic for cell growth. On the Escherichia coli genome, the
nemA gene coding for NEM reductase is located downstream of the gene encoding an 
as-yet-uncharacterized transcription factor, YdhM. Disruption of the ydhM gene
results in reduction of nemA expression even in the induced state, indicating
that the two genes form a single operon. After in vitro genomic SELEX screening, 
one of the target recognition sequences for YdhM was identified within the
promoter region for this ydhM-nemA operon. Both YdhM binding in vitro to the ydhM
promoter region and transcription repression in vivo of the ydhM-nemA operon by
YdhM were markedly reduced by the addition of NEM. Taken together, we propose
that YdhM is the repressor for the nemA gene, thus hereafter designated NemR. The
repressor function of NemR was inactivated by the addition of not only NEM but
also other Cys modification reagents, implying that Cys modification of NemR
renders it inactive. This is an addition to the mode of controlling activity of
transcription factors by alkylation with chemical agents.

DOI: 10.1128/JB.00459-08 
PMCID: PMC2519536
PMID: 18567656  [Indexed for MEDLINE]


724. J Food Prot. 2008 Sep;71(9):1774-84.

Detection of virulence-associated genes in Escherichia coli O157 and non-O157
isolates from beef cattle, humans, and chickens.

Lefebvre B(1), Diarra MS, Moisan H, Malouin F.

Author information: 
(1)Centre d'Etude et de Valorisation de la Diversité Microbienne, Départment de
biologie, Faculté des Sciences, Université de Sherbrooke, 2500 Boulevard
Université, Sherbrooke, Quebec, Canada J1K 2R1.

Food-producing animals can be reservoirs of pathogenic Escherichia coli strains
that can induce diseases in animals or humans. Contamination of food by E. coli
O157:H7 raises immediate concerns about public health, although it is not clear
whether all E. coli O157 isolates of animal origin are equally harmful to humans.
Inversely, the pathogenic potential of atypical E. coli O157 isolates and several
non-O157 serotypes often is ignored. We used a DNA microarray capable of
detecting a subset of 346 genes to compare the virulence-associated genes present
in eight E. coli O157 isolates from human cases, 14 antibiotic-resistant and/or
hypermutable E. coli O157 isolates from beef cattle, and four
antibiotic-resistant, sorbitol-negative, non-O157 E. coli isolates from healthy
broiler chickens. Hybridization on arrays (HOA) revealed that O157 isolates from 
beef cattle and humans were genetically distinct, although they possessed most of
the same subset of virulence genes. HOA allowed discrimination between
hypermutable and antibiotic-resistant O157 isolates from beef cattle based on
hybridization results for the stx2 and ycgG genes (hypermutable) or ymfL, stx1,
stx2, and hlyE(avian) genes (resistant). However, the absence of hybridization to
gene yfdR characterized human isolates. HOA also revealed that an atypical
sorbitol-fermenting bovine O157 isolate lacked some genes of the type 3 secretion
system, plasmid pO157, and the stx1 and stx2 genes. This isolate had a particular
pathotype (eaeA(beta) tir(alpha) espA(alpha) espB(alpha) espD(alpha)) not found
in typical E. coli O157:H7. HOA revealed that some non-O157 E. coli isolates from
healthy chickens carried genes responsible for salmochelin- and
yersiniabactin-mediated iron uptake generally associated with pathogenic strains.


PMID: 18810861  [Indexed for MEDLINE]


725. Nat Methods. 2008 Sep;5(9):789-95. doi: 10.1038/nmeth.1239.

eSGA: E. coli synthetic genetic array analysis.

Butland G(1), Babu M, Díaz-Mejía JJ, Bohdana F, Phanse S, Gold B, Yang W, Li J,
Gagarinova AG, Pogoutse O, Mori H, Wanner BL, Lo H, Wasniewski J, Christopolous
C, Ali M, Venn P, Safavi-Naini A, Sourour N, Caron S, Choi JY, Laigle L,
Nazarians-Armavil A, Deshpande A, Joe S, Datsenko KA, Yamamoto N, Andrews BJ,
Boone C, Ding H, Sheikh B, Moreno-Hagelseib G, Greenblatt JF, Emili A.

Author information: 
(1)Banting and Best Department of Medical Research, Terrence Donnelly Centre for 
Cellular and Biomolecular Research, University of Toronto, 160 College Street,
Toronto M5S 3E1, Canada.

Comment in
    Nat Rev Genet. 2008 Sep;9(9):652.

Physical and functional interactions define the molecular organization of the
cell. Genetic interactions, or epistasis, tend to occur between gene products
involved in parallel pathways or interlinked biological processes.
High-throughput experimental systems to examine genetic interactions on a
genome-wide scale have been devised for Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster,
but have not been reported previously for prokaryotes. Here we describe the
development of a quantitative screening procedure for monitoring bacterial
genetic interactions based on conjugation of Escherichia coli deletion or
hypomorphic strains to create double mutants on a genome-wide scale. The patterns
of synthetic sickness and synthetic lethality (aggravating genetic interactions) 
we observed for certain double mutant combinations provided information about
functional relationships and redundancy between pathways and enabled us to group 
bacterial gene products into functional modules.

DOI: 10.1038/nmeth.1239 
PMID: 18677321  [Indexed for MEDLINE]


726. PLoS One. 2008 Aug 20;3(8):e2981. doi: 10.1371/journal.pone.0002981.

Origin of co-expression patterns in E. coli and S. cerevisiae emerging from
reverse engineering algorithms.

Zampieri M(1), Soranzo N, Bianchini D, Altafini C.

Author information: 
(1)SISSA-ISAS, International School for Advanced Studies, Trieste, Italy.

BACKGROUND: The concept of reverse engineering a gene network, i.e., of inferring
a genome-wide graph of putative gene-gene interactions from compendia of high
throughput microarray data has been extensively used in the last few years to
deduce/integrate/validate various types of "physical" networks of interactions
among genes or gene products.
RESULTS: This paper gives a comprehensive overview of which of these networks
emerge significantly when reverse engineering large collections of gene
expression data for two model organisms, E. coli and S. cerevisiae, without any
prior information. For the first organism the pattern of co-expression is shown
to reflect in fine detail both the operonal structure of the DNA and the
regulatory effects exerted by the gene products when co-participating in a
protein complex. For the second organism we find that direct transcriptional
control (e.g., transcription factor-binding site interactions) has little
statistical significance in comparison to the other regulatory mechanisms (such
as co-sharing a protein complex, co-localization on a metabolic pathway or
compartment), which are however resolved at a lower level of detail than in E.
coli.
CONCLUSION: The gene co-expression patterns deduced from compendia of profiling
experiments tend to unveil functional categories that are mainly associated to
stable bindings rather than transient interactions. The inference power of this
systematic analysis is substantially reduced when passing from E. coli to S.
cerevisiae. This extensive analysis provides a way to describe the different
complexity between the two organisms and discusses the critical limitations
affecting this type of methodologies.

DOI: 10.1371/journal.pone.0002981 
PMCID: PMC2500178
PMID: 18714358  [Indexed for MEDLINE]


727. PLoS One. 2008 Aug 20;3(8):e2984. doi: 10.1371/journal.pone.0002984.

DnaC inactivation in Escherichia coli K-12 induces the SOS response and
expression of nucleotide biosynthesis genes.

Løbner-Olesen A(1), Slominska-Wojewodzka M, Hansen FG, Marinus MG.

Author information: 
(1)Department of Science, Systems and Models, Roskilde University, Roskilde,
Denmark.

BACKGROUND: Initiation of chromosome replication in E. coli requires the DnaA and
DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to
synchronize cell populations.
METHODOLOGY/PRINCIPAL FINDINGS: DNA microarrays were used to measure mRNA
steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at
permissive and non-permissive temperatures and their expression profiles were
compared to MG1655 wildtype cells. For both mutants there was altered expression 
of genes involved in nucleotide biosynthesis at the non-permissive temperature.
Transcription of the dnaA and dnaC genes was increased at the non-permissive
temperature in the respective mutant strains indicating auto-regulation of both
genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C
and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at
non-permissive temperature had completed the early stages of chromosome
replication initiation.
CONCLUSION/SIGNIFICANCE: We suggest that in dnaC2 cells the SOS response is
triggered by persistent open-complex formation at oriC and/or by arrested forks
that require DnaC for replication restart.

DOI: 10.1371/journal.pone.0002984 
PMCID: PMC2500167
PMID: 18714349  [Indexed for MEDLINE]


728. Foodborne Pathog Dis. 2008 Aug;5(4):437-47. doi: 10.1089/fpd.2008.0100.

Examination of stress and virulence gene expression in Escherichia coli O157:H7
using targeted microarray analysis.

Allen KJ(1), Lepp D, McKellar RC, Griffiths MW.

Author information: 
(1)Department of Food Science and Canadian Research Institute for Food Safety,
University of Guelph, Guelph, Ontario, Canada.

Escherichia coli O157:H7 poses a threat to humans through food- and water-borne
transmission. To investigate how environmental stresses affect the Escherichia
coli O157:H7 transcriptome, we designed a targeted microarray consisting of
stress response and virulence genes (n = 125) to analyze the impact of acidified 
(pH 3.5), cold (7.5 degrees C), and fresh tryptic soy broth (TSB) (37 degrees C) 
on E. coli O157:H7 stress response and virulence gene expression. Nutrient
replenishment with fresh TSB resulted in 72 differentially expressed genes (> or 
= 1.5-fold change; p < 0.05), with 65 induced. All queried global and specific
stress regulators were affected, as were 12 virulence genes. Cold-shocked cells
displayed 17 differentially expressed genes, with 10 being induced. Induction of 
rpoS, members of the sigma(H) regulon (clpB, dnaK, ftsH), and acid resistance
(AR) genes (gadA, gadX) was observed. Porin transcript (ompC, ompF) and gapA and 
tufA ancillary genes were repressed. Acid shock resulted in 24 differentially
expressed genes, with 21 induced. No induction of any stationary phase AR system 
was observed, though acid-coping mechanisms were recruited, including mar and
phoB induction, and repression of ompC and ompF. Stress regulators were induced, 
including relA, soxS, rpoE, and rpoH. The microarray data were validated by
quantitative real-time polymerase chain reaction. Exposure to sublethal stress
events led to the induction of diverse stress response networks. In the food
chain, sublethal events may render cells increasingly resistant to future
stresses, potentially leading to increased survival.

DOI: 10.1089/fpd.2008.0100 
PMID: 18713062  [Indexed for MEDLINE]


729. Foodborne Pathog Dis. 2008 Aug;5(4):517-29. doi: 10.1089/fpd.2008.0112.

Transcriptome analysis of organisms with food safety relevance.

Puttamreddy S(1), Carruthers MD, Madsen ML, Minion FC.

Author information: 
(1)Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, Iowa 50011, USA.

Transcriptome analysis using microarrays has become a powerful tool to better
understand the process of disease and other complex biological processes such as 
food spoilage and biofilm formation. This review is divided into two basic
sections: 1) a short history and description of microarrays and 2) a discussion
of studies involving bacterial food safety pathogens that focused on whole genome
transcript analysis. Not included are the many studies using microarrays to
identify, diagnose, or genetically characterize these organisms. This review
focuses on studies involving Escherichia coli O157:H7, Salmonella spp.,
Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. Many
of the studies involve altering the growth environment to simulate stress
conditions and the use of host-pathogen model systems to explore virulence
mechanisms. Few studies use conditions that might be considered unique to the
food industry. Exceptions are studies of biofilm-specific transcriptome changes
and analysis following pressure treatment. This review should not be considered
as a comprehensive review, and where appropriate, species-specific reviews are
cited that are more complete.

DOI: 10.1089/fpd.2008.0112 
PMID: 18673071  [Indexed for MEDLINE]


730. Infect Immun. 2008 Aug;76(8):3679-89. doi: 10.1128/IAI.01581-07. Epub 2008 Jun 9.

Shiga toxin 2 causes apoptosis in human brain microvascular endothelial cells via
C/EBP homologous protein.

Fujii J(1), Wood K, Matsuda F, Carneiro-Filho BA, Schlegel KH, Yutsudo T,
Binnington-Boyd B, Lingwood CA, Obata F, Kim KS, Yoshida S, Obrig T.

Author information: 
(1)Department of Bacteriology, Graduate School of Medical Sciences, Kyushu
University, Fukuoka, 812-8582, Japan. junfujii@bact.med.kyushu-u.ac.jp.

Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be
cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing 
apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced 
DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with
cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to
search for apoptotic potential signals in response to Stx2 revealed that Stx2
treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth
arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression 
was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the
activation of caspase-3 and prevented apoptotic cell death. These results suggest
that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation 
of both the intrinsic and extrinsic pathways of apoptosis.

DOI: 10.1128/IAI.01581-07 
PMCID: PMC2493216
PMID: 18541659  [Indexed for MEDLINE]


731. J Bacteriol. 2008 Aug;190(15):5439-54. doi: 10.1128/JB.00272-08. Epub 2008 Jun 6.

Cadmium toxicity in glutathione mutants of Escherichia coli.

Helbig K(1), Grosse C, Nies DH.

Author information: 
(1)Institute for Biology, Life Science Faculty, Martin-Luther-University
Halle-Wittenberg, Kurt-Mothes-Str 3, 06099 Halle, Germany.

The higher affinity of Cd(2+) for sulfur compounds than for nitrogen and oxygen
led to the theoretical consideration that cadmium toxicity should result mainly
from the binding of Cd(2+) to sulfide, thiol groups, and sulfur-rich complex
compounds rather than from Cd(2+) replacement of transition-metal cations from
nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by
using Escherichia coli for a global transcriptome analysis of cells synthesizing 
glutathione (GSH; wild type), gamma-glutamylcysteine (DeltagshB mutant), or
neither of the two cellular thiols (DeltagshA mutant). The resulting data, some
of which were validated by quantitative reverse transcription-PCR, were sorted
using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which 
groups genes hierarchically with respect to the cellular functions of their
respective products. The main difference among the three strains concerned
tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium
shock and strongly up-regulated in DeltagshA cells but repressed in DeltagshB
cells containing gamma-glutamylcysteine instead of GSH. Overall, however, all
three E. coli strains responded to cadmium shock similarly, with the
up-regulation of genes involved in protein, disulfide bond, and oxidative damage 
repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins
containing sensitive iron-sulfur clusters; the storage of iron; and the
detoxification of Cd(2+) by efflux. General energy conservation pathways and iron
uptake were down-regulated. These findings indicated that the toxic action of
Cd(2+) indeed results from the binding of the metal cation to sulfur, lending
support to the hypothesis tested.

DOI: 10.1128/JB.00272-08 
PMCID: PMC2493267
PMID: 18539742  [Indexed for MEDLINE]


732. J Bacteriol. 2008 Aug;190(15):5256-64. doi: 10.1128/JB.01536-07. Epub 2008 May
30.

Modulation of hexa-acyl pyrophosphate lipid A population under Escherichia coli
phosphate (Pho) regulon activation.

Lamarche MG(1), Kim SH, Crépin S, Mourez M, Bertrand N, Bishop RE, Dubreuil JD,
Harel J.

Author information: 
(1)Groupe de Recherche sur les Maladies Infectieuses du Porc, Université de
Montréal, Faculté de Médecine Vétérinaire, CP 5000, Saint-Hyacinthe, Québec,
Canada.

Environmental phosphate is an important signal for microorganism gene regulation,
and it has recently been shown to trigger some key bacterial virulence
mechanisms. In many bacteria, the Pho regulon is the major circuit involved in
adaptation to phosphate limitation. The Pho regulon is controlled jointly by the 
two-component regulatory system PhoR/PhoB and by the phosphate-specific transport
(Pst) system, which both belong to the Pho regulon. We showed that a pst mutation
results in virulence attenuation in extraintestinal pathogenic Escherichia coli
(ExPEC) strains. Our results indicate that the bacterial cell surface of the pst 
mutants is altered. In this study, we show that pst mutants of ExPEC strains
display an increased sensitivity to different cationic antimicrobial peptides and
vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is
significantly less abundant in pst mutants. Among differentially expressed genes 
in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to
lipid A, forming the 1-diphosphate species, was found to be downregulated. Our
results strongly suggest that the Pho regulon is involved in lipid A
modifications, which could contribute to bacterial surface perturbations. Since
the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A
modification mechanism could be widely distributed among gram-negative bacterial 
species.

DOI: 10.1128/JB.01536-07 
PMCID: PMC2493271
PMID: 18515419  [Indexed for MEDLINE]


733. J Food Prot. 2008 Aug;71(8):1673-8.

Comparison of Shiga-like toxin II expression between two genetically diverse
lineages of Escherichia coli O157:H7.

Dowd SE(1), Williams JB.

Author information: 
(1)U.S. Department of Agriculture, Agricultural Research Service Livestock Issues
Research Unit, 1604 West FM 1294, Lubbock, Texas 79403, USA.
sdowd@lbk.ars.usda.gov

The existence of two separate lineages of Escherichia coli O157:H7 has previously
been reported, and research indicates that one of these lineages (lineage I)
might be more pathogenic toward human hosts. We postulated that the lineage more 
pathogenic expresses higher levels of Shiga toxin 2 (Stx2) than do the
nonpathogenic lineage II. A comprehensive set of methodologies were used to
investigate the difference in Stx2 protein and mRNA expression between the two
lineages. An initial Stx2-specific enzyme-linked immunosorbent assay was
conducted, and lineage I overall demonstrated significantly more toxin proteins
expressed (P < 0.01). Gene expression analyses all showed significantly higher
stx2 gene expression in lineage I (P = 0.02). PCR mapping revealed a possible
explanation for decreased amounts of stx2 transcripts in the potentially
nonpathogenic lineage II isolates, suggesting that genomic changes have modified 
the toxin-encoding region of the phage. This study provides additional data to
support the existence of two diverse lineages of E. coli O157:H7, one of which
may have lower pathogenic potential in relation to human hosts. The PCR described
also provides a possible screening tool for E. coli O157 populations to
differentiate these lineages. This study provides useful information on the
ecology of E. coli O157, with broad implications within the clinical, scientific,
and livestock industries.


PMID: 18724763  [Indexed for MEDLINE]


734. BMB Rep. 2008 Jul 31;41(7):511-5.

Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a
cell-based in vivo assay for protein-RNA interaction.

Jeong YY(1), Kim SH, Jang SI, You JC.

Author information: 
(1)National Research Laboratory for Molecular Virology, Department of Pathology, 
College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu,
Seoul 137-701, Korea.

The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key
role in viral genomic packaging by specifically recognizing the Psi(Psi) RNA
sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was
developed to probe the specific interactions in vivo between the NC and Psi-RNA
using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the
extendibility of this cell-based assay to RNAs other than Psi-RNA, this study
tested the RNA aptamers isolated in vitro using the SELEX method, but whose
specific binding ability to NC in a living cellular environment has not been
established. The results demonstrate for the first time that each of those
aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent
manner within the cell. This confirms that the cell-based assay developed for
NC-Psi interaction can be further extended and applied to NC-binding RNAs other
than Psi-RNA.


PMID: 18682034  [Indexed for MEDLINE]


735. BMC Mol Biol. 2008 Jul 23;9:66. doi: 10.1186/1471-2199-9-66.

Statistical modelling of transcript profiles of differentially regulated genes.

Eastwood DC(1), Mead A, Sergeant MJ, Burton KS.

Author information: 
(1)Warwick HRI, University of Warwick, Wellesbourne, Warwickshire, CV35 9EF, UK. 
daniel.eastwood@warwick.ac.uk

BACKGROUND: The vast quantities of gene expression profiling data produced in
microarray studies, and the more precise quantitative PCR, are often not
statistically analysed to their full potential. Previous studies have summarised 
gene expression profiles using simple descriptive statistics, basic analysis of
variance (ANOVA) and the clustering of genes based on simple models fitted to
their expression profiles over time. We report the novel application of
statistical non-linear regression modelling techniques to describe the shapes of 
expression profiles for the fungus Agaricus bisporus, quantified by PCR, and for 
E. coli and Rattus norvegicus, using microarray technology. The use of parametric
non-linear regression models provides a more precise description of expression
profiles, reducing the "noise" of the raw data to produce a clear "signal" given 
by the fitted curve, and describing each profile with a small number of
biologically interpretable parameters. This approach then allows the direct
comparison and clustering of the shapes of response patterns between genes and
potentially enables a greater exploration and interpretation of the biological
processes driving gene expression.
RESULTS: Quantitative reverse transcriptase PCR-derived time-course data of genes
were modelled. "Split-line" or "broken-stick" regression identified the initial
time of gene up-regulation, enabling the classification of genes into those with 
primary and secondary responses. Five-day profiles were modelled using the
biologically-oriented, critical exponential curve, y(t) = A + (B + Ct)Rt +
epsilon. This non-linear regression approach allowed the expression patterns for 
different genes to be compared in terms of curve shape, time of maximal
transcript level and the decline and asymptotic response levels. Three distinct
regulatory patterns were identified for the five genes studied. Applying the
regression modelling approach to microarray-derived time course data allowed 11% 
of the Escherichia coli features to be fitted by an exponential function, and 25%
of the Rattus norvegicus features could be described by the critical exponential 
model, all with statistical significance of p < 0.05.
CONCLUSION: The statistical non-linear regression approaches presented in this
study provide detailed biologically oriented descriptions of individual gene
expression profiles, using biologically variable data to generate a set of
defining parameters. These approaches have application to the modelling and
greater interpretation of profiles obtained across a wide range of platforms,
such as microarrays. Through careful choice of appropriate model forms, such
statistical regression approaches allow an improved comparison of gene expression
profiles, and may provide an approach for the greater understanding of common
regulatory mechanisms between genes.

DOI: 10.1186/1471-2199-9-66 
PMCID: PMC2525656
PMID: 18651954  [Indexed for MEDLINE]


736. Sci Total Environ. 2008 Jul 15;398(1-3):203-11. doi:
10.1016/j.scitotenv.2008.03.004.

Development and application of an oligonucleotide microarray and real-time
quantitative PCR for detection of wastewater bacterial pathogens.

Lee DY(1), Lauder H, Cruwys H, Falletta P, Beaudette LA.

Author information: 
(1)National Water Research Institute, Environment Canada, Burlington, ON, Canada.
daeyoung.lee@ec.gc.ca

Conventional microbial water quality test methods are well known for their
technical limitations, such as lack of direct pathogen detection capacity and low
throughput capability. The microarray assay has recently emerged as a promising
alternative for environmental pathogen monitoring. In this study, bacterial
pathogens were detected in municipal wastewater using a microarray equipped with 
short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes
have been designed against 38 species, 4 genera, and 1 family of pathogens. The
detection sensitivity of the microarray for a waterborne pathogen Aeromonas
hydrophila was determined to be approximately 1.0% of the total DNA, or
approximately 10(3)A. hydrophila cells per sample. The efficacy of the DNA
microarray was verified in a parallel study where pathogen genes and E. coli
cells were enumerated using real-time quantitative PCR (qPCR) and standard
membrane filter techniques, respectively. The microarray and qPCR successfully
detected multiple wastewater pathogen species at different stages of the
disinfection process (i.e. secondary effluents vs. disinfected final effluents)
and at two treatment plants employing different disinfection methods (i.e.
chlorination vs. UV irradiation). This result demonstrates the effectiveness of
the DNA microarray as a semi-quantitative, high throughput pathogen monitoring
tool for municipal wastewater.

DOI: 10.1016/j.scitotenv.2008.03.004 
PMID: 18423816  [Indexed for MEDLINE]


737. J Biol Chem. 2008 Jul 11;283(28):19201-10. doi: 10.1074/jbc.M710366200. Epub 2008
May 16.

Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis
through up-regulation of serum- and glucocorticoid-inducible kinase 1.

Dehner M(1), Hadjihannas M, Weiske J, Huber O, Behrens J.

Author information: 
(1)Department of Experimental Medicine II, Nikolaus-Fiebiger-Center for Molecular
Medicine, University of Erlangen, Glueckstrasse 6, 91054 Erlangen, Germany.

In human cancers, mutations in components of the Wnt signaling pathway lead to
beta-catenin stabilization and result in augmented gene transcription. HCT116
colon cancer cells carry stabilizing mutations in beta-catenin and exhibit an
elevated activation of Wnt signaling. To clarify the role of an overactive Wnt
signaling, we used DNA microarray analysis to search for genes whose expression
is up-regulated after knockdown of the wild type adenomatous polyposis coli (APC)
tumor suppressor in HCT116 cells, which further enhances Wnt signaling
activation. Serum and glucocorticoid-inducible kinase 1 (SGK1) was among the most
up-regulated genes following APC knockdown through small interfering RNA.
Up-regulation of SGK1 in response to small interfering RNA against APC was
inhibited by concomitant knockdown of beta-catenin. Quantitative real time
reverse transcription-PCR, Western blot, and chromatin immunoprecipitation
analyses confirmed that SGK1 is a direct beta-catenin target gene. SGK1
negatively regulates the pro-apoptotic transcription factor Forkhead box O3a
(FoxO3a) via phosphorylation and exclusion from the nucleus. We show that Wnt
signaling activation results in FoxO3a exclusion from the nucleus and inhibits
expression of FoxO3a target genes. Importantly, FoxO3a mutants that fail to be
phosphorylated and therefore are regulated by SGK1 are not influenced by
activation of Wnt signaling. In line, knockdown of SGK1 relieves the effects of
Wnt signaling on FoxO3a localization and FoxO3a-dependent transcription. Finally,
we show that induction of Wnt signaling inhibits FoxO3a-induced apoptosis.
Collectively our results indicate that evasion of apoptosis is another feature
employed by an overactive Wnt signaling.

DOI: 10.1074/jbc.M710366200 
PMID: 18487207  [Indexed for MEDLINE]


738. Environ Microbiol. 2008 Jul;10(7):1877-87. doi: 10.1111/j.1462-2920.2008.01610.x.
Epub 2008 Apr 8.

The hyperthermophilic anaerobe Thermotoga Maritima is able to cope with limited
amount of oxygen: insights into its defence strategies.

Le Fourn C(1), Fardeau ML, Ollivier B, Lojou E, Dolla A.

Author information: 
(1)Unité Interactions et Modulateurs de Réponse, IBSM - CNRS, Marseille, France.

Thermotoga maritima, an anaerobic hyperthermophilic bacterium, was found able to 
grow in the presence of low concentrations of oxygen of up to 0.5% (v/v).
Differential proteomics and transcripts analysis by qRT-PCR were used to identify
the defence strategies used by T. maritima to protect itself against oxygen. A
flavoprotein, homologous to rubredoxin oxygen oxidoreductase was found to be
overproduced when cells were cultured in oxidative conditions. The recombinant
protein, produced in Escherichia coli, exhibited an oxygen reductase activity,
which could account for the observed decrease in oxygen concentration during
growth. The gene encoding this oxygen reductase belongs to a multicistronic unit 
that includes genes encoding proteins involved in exopolysaccharide biosynthesis,
which may be related to a biofilm formation induced by the presence of oxygen.
Enzymes involved in reactive oxygen species detoxification, iron-sulfur centre
synthesis/repair and the cysteine biosynthesis pathway were also overproduced.
All these enzymatic systems together contribute to the defence strategy of T.
maritima against oxygen. Because of the position of T. maritima in deep branches 
of the phylogenetic tree, we suggest that these strategies can be considered as
ancestral mechanisms first developed by anaerobic microorganisms on the early
Earth to protect themselves against primary abiotic or biotic oxygen production.

DOI: 10.1111/j.1462-2920.2008.01610.x 
PMID: 18397308  [Indexed for MEDLINE]


739. Nucleic Acids Res. 2008 Jul;36(12):3950-5. doi: 10.1093/nar/gkn339. Epub 2008 May
30.

The Escherichia coli RutR transcription factor binds at targets within genes as
well as intergenic regions.

Shimada T(1), Ishihama A, Busby SJ, Grainger DC.

Author information: 
(1)Department of Frontier Bioscience and Micro-Nano Technology Research Centre,
Hosei University, Koganei, Tokyo 184-8584, Nippon Institute for Biological
Science, Ome, Tokyo 198-0024, Japan.

The Escherichia coli RutR protein is the master regulator of genes involved in
pyrimidine catabolism. Here we have used chromatin immunoprecipitation in
combination with DNA microarrays to measure the binding of RutR across the
chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets
were identified and analysis of these targets generated a DNA consensus logo for 
RutR binding. Complementary in vitro binding assays showed high-affinity RutR
binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking 
predicted key binding determinants. Surprisingly, most of the DNA targets for
RutR are located within coding segments of the genome and appear to have little
or no effect on transcript levels in the conditions tested. This contrasts
sharply with other E. coli transcription factors whose binding sites are
primarily located in intergenic regions. We suggest that either RutR has yet
undiscovered function or that evolution has been slow to eliminate non-functional
DNA sites for RutR because they do not have an adverse effect on cell fitness.

DOI: 10.1093/nar/gkn339 
PMCID: PMC2475637
PMID: 18515344  [Indexed for MEDLINE]


740. Pediatr Nephrol. 2008 Jul;23(7):1059-71. doi: 10.1007/s00467-007-0717-6. Epub
2008 Jan 23.

Global gene expression profiling of renal scarring in a rat model of
pyelonephritis.

Ichino M(1), Mori T, Kusaka M, Kuroyanagi Y, Ishikawa K, Shiroki R, Kowa H,
Kurahashi H, Hoshinaga K.

Author information: 
(1)Department of Urology, Fujita Health University School of Medicine, 1-98
Dengakugakubo, Kutsukake-cho, Toyoake, Aichi, Japan. michino@fujita-hu.ac.jp

Renal scarring is a serious complication of chronic pyelonephritis that occurs
due to vesicoureteral reflux. In our study, we performed global expression
profiling of the kidney during renal scarring formation in a rat pyelonephritis
model. An inoculum of Escherichia coli was injected directly into the renal
cortex. Histologically, renal scarring developed during the 3-to-4 week period
after injection. The time-course expression profile of 18,442 genes was then
analyzed using microarrays, followed by validation with real-time reverse
transcriptase-polymerase chain reaction (RT-PCR). Most of the genes found to be
up-regulated during renal scarring are associated with immune and defense
responses, including cytokines, chemokines and their receptors, complement
factors, adhesion molecules and extracellular matrix proteins. These genes were
up-regulated as early as 1 week after injection, when no fibrotic changes were
yet evident, peaked at 2 weeks, and gradually decreased thereafter. However, a
subset of cytokine genes was found to be persistently activated even at 6 weeks
after injection, including interleukin (IL)-1beta, transforming growth factor
(TGF)-beta, and IL-3. Further statistical analysis indicated that the pathways
mediated by these cytokines are activated concomitantly with renal scarring
formation. The products of these genes may thus potentially be novel non-invasive
diagnostic or prognostic biomarkers of renal scarring.

DOI: 10.1007/s00467-007-0717-6 
PMID: 18214547  [Indexed for MEDLINE]


741. Sheng Wu Gong Cheng Xue Bao. 2008 Jul;24(7):1300-5.

[Application of microarrays in screening the antibacterial peptide associated
genes of Musca domestica].

[Article in Chinese]

Liu L(1), Jin X, Zhu J, Xiao P, Li Y, Gong J.

Author information: 
(1)4 Department of Clinical Laboratory, Fourth Hospital of Changsha, Changsha
410006, China.

To screen the candidate genes associated with Musca domestica antibacterial
peptides using DNA microarray technique, the hybrid probes were designed from the
conservative domains of the encoded area of the insect antibacterial peptide
genes in GenBank with biology software Designer 2.0, and were synthesized by a
chemical process, with the assistance of the automated Gen III Microarray
Spotter, those oligo probes were printed on a special ready-made glass, and a
cDNA microarray was constructed. The total RNA was extracted from the fat body of
Musca domestic third-instar larve induced after 24 hours by Escherichia coli and 
Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3
using the method of reverse transcription PCR, after prehybridization,
hybridization and washing procedure, the results of hybridization were scanned
using computer system, and the data were analyzed using the software of MIDAS,
fifteen valid hybridization signals were detected through two times of
hybridization and scanning (the positive samples as a control were excluded). DNA
microarray technique can be successfully applied screen the candidate genes
associated with Musca domestica antibacterial peptides, and further provide
significant evidence to discover its antibacterial peptide new genes.


PMID: 18837412  [Indexed for MEDLINE]


742. Toxicol Appl Pharmacol. 2008 Jul 1;230(1):1-8. doi: 10.1016/j.taap.2008.01.033.
Epub 2008 Feb 12.

Variation in the ovine cortisol response to systemic bacterial endotoxin
challenge is predominantly determined by signalling within the
hypothalamic-pituitary-adrenal axis.

You Q(1), Karrow NA, Cao H, Rodriguez A, Mallard BA, Boermans HJ.

Author information: 
(1)Centre for Genetic Improvement of Livestock, Department of Animal and Poultry 
Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

Bi-directional communication between the neuroendocrine and immune systems is
designed, in part, to maintain or restore homeostasis during physiological
stress. Exposure to endotoxin during Gram-negative bacterial infection for
example, elicits the release of pro-inflammatory cytokines that activate the
hypothalamic-pituitary-adrenal axis (HPAA). The secretion of adrenal
glucocorticoids subsequently down regulates the host inflammatory response,
minimizing potential tissue damage. Sequence and epigenetic variants in genes
involved in regulating the neuroendocrine and immune systems are likely to
contribute to individual differences in the HPAA response, and this may influence
the host anti-inflammatory response to toxin exposure and susceptibility to
inflammatory disease. In this study, high (HCR) and low (LCR) cortisol responders
were selected from a normal population of 110 female sheep challenged iv with
Escherichia coli endotoxin (400 ng/kg) to identify potential determinants that
contribute to variation in the cortisol response phenotype. This phenotype was
stable over several years in the HCR and LCR animals, and did not appear to be
attributed to differences in expression of hepatic immune-related genes or
systemic pro-inflammatory cytokine concentrations. Mechanistic studies using
corticotrophin-releasing factor (0.5 microg/kg body weight), arginine vasopressin
(0.5 microg/kg), and adrenocorticotropic hormone (0.5 microg/kg) administered iv 
demonstrated that variation in this phenotype is largely determined by signalling
within the HPAA. Future studies will use this ovine HCR/LCR model to investigate 
potential genetic and epigenetic variants that may contribute to variation in
cortisol responsiveness to bacterial endotoxin.

DOI: 10.1016/j.taap.2008.01.033 
PMID: 18339413  [Indexed for MEDLINE]


743. Zhonghua Yi Xue Za Zhi. 2008 Jul 1;88(25):1778-82.

[Expression and characterizations of a novel full-length gene PKIbeta related to 
human glioma].

[Article in Chinese]

Qi ZY(1), Hui GZ, Li Y, Gu SH, Xie Y.

Author information: 
(1)Department of Neurosurgery, First Affiliated Hospital of Suzhou University,
Suzhou 215006, China.

OBJECTIVE: To obtain differentially expressed genes related to human glioma using
cDNA microarray and to characterization of one novel full-length gene.
METHOD: Four samples of human glioma samples, 1 fetal brain tissue sample, and 2 
normal brain tissue samples were used to extract the total RNA, and the mRNA was 
used to make probes. After hybridization and washing procedure, the products of
hybridization were scanned using computer system. One gene, named 436F11 clone,
was subsequently analyzed by Northern blotting, bioinformatics, and protein
expression.
RESULT: Fifteen differentially expressed new genes related to human glioma were
obtained through four times of hybridization and scanning. Northern blotting
confirmed that over-expression of 436F11 gene in the normal human brain tissue
and low-expression in the human glioma tissues. The analysis of BLASTn and BLASTx
showed that the clone of 436F11 was a novel full-length gene coding 78 amino
acids of protein with a theoretical relative molecular weight of 8648 and an
isoelectric point of 4.69 and that it was 60% identical to mouse PKIbeta amino
acid, so it was called human PKIbeta gene. After it was transfected into
Escherichia coli, higher-expressed protein of PKIbeta was obtained which yielded 
a major clear band on an SDS-PAGE gel after purification. The products obtained
from amino acid sequencing and molecular weight detection were exactly the same
as the products performed by bioinformatic analysis.
CONCLUSION: cDNA microarray technology can be successfully applied to identify
differentially expressed genes. PKIbeta may be a novel full-length gene related
to human glioma and may provide a new way to gene therapy of glioma.


PMID: 19035091  [Indexed for MEDLINE]


744. BMC Bioinformatics. 2008 Jun 25;9:294. doi: 10.1186/1471-2105-9-294.

Relating gene expression data on two-component systems to functional annotations 
in Escherichia coli.

Denton AM(1), Wu J, Townsend MK, Sule P, Prüss BM.

Author information: 
(1)Department of Computer Science and Operations Research, North Dakota State
University, Fargo, ND 58105, USA. anne.denton@ndsu.edu

BACKGROUND: Obtaining physiological insights from microarray experiments requires
computational techniques that relate gene expression data to functional
information. Traditionally, this has been done in two consecutive steps. The
first step identifies important genes through clustering or statistical
techniques, while the second step assigns biological functions to the identified 
groups. Recently, techniques have been developed that identify such relationships
in a single step.
RESULTS: We have developed an algorithm that relates patterns of gene expression 
in a set of microarray experiments to functional groups in one step. Our only
assumption is that patterns co-occur frequently. The effectiveness of the
algorithm is demonstrated as part of a study of regulation by two-component
systems in Escherichia coli. The significance of the relationships between
expression data and functional annotations is evaluated based on density
histograms that are constructed using product similarity among expression
vectors. We present a biological analysis of three of the resulting functional
groups of proteins, develop hypotheses for further biological studies, and test
one of these hypotheses experimentally. A comparison with other algorithms and a 
different data set is presented.
CONCLUSION: Our new algorithm is able to find interesting and biologically
meaningful relationships, not found by other algorithms, in previously analyzed
data sets. Scaling of the algorithm to large data sets can be achieved based on a
theoretical model.

DOI: 10.1186/1471-2105-9-294 
PMCID: PMC2478693
PMID: 18578884  [Indexed for MEDLINE]


745. BMC Microbiol. 2008 Jun 19;8:101. doi: 10.1186/1471-2180-8-101.

Predicting transcriptional regulatory interactions with artificial neural
networks applied to E. coli multidrug resistance efflux pumps.

Veiga DF(1), Vicente FF, Nicolás MF, Vasconcelos AT.

Author information: 
(1)Laboratório Nacional de Computação Científica, Laboratório de Bioinformática, 
Av, Getúlio Vargas, 333 Petrópolis, Rio de Janeiro, Brasil. diogofv@lncc.br

BACKGROUND: Little is known about bacterial transcriptional regulatory networks
(TRNs). In Escherichia coli, which is the organism with the largest wet-lab
validated TRN, its set of interactions involves only approximately 50% of the
repertoire of transcription factors currently known, and ~25% of its genes. Of
those, only a small proportion describes the regulation of processes that are
clinically relevant, such as drug resistance mechanisms.
RESULTS: We designed feed-forward (FF) and bi-fan (BF) motif predictors for E.
coli using multi-layer perceptron artificial neural networks (ANNs). The motif
predictors were trained using a large dataset of gene expression data; the
collection of motifs was extracted from the E. coli TRN. Each network motif was
mapped to a vector of correlations which were computed using the gene expression 
profile of the elements in the motif. Thus, by combining network structural
information with transcriptome data, FF and BF predictors were able to classify
with a high precision of 83% and 96%, respectively, and with a high recall of 86%
and 97%, respectively. These results were found when motifs were represented
using different types of correlations together, i.e., Pearson, Spearman, Kendall,
and partial correlation. We then applied the best predictors to hypothesize new
regulations for 16 operons involved with multidrug resistance (MDR) efflux pumps,
which are considered as a major bacterial mechanism to fight antimicrobial
agents. As a result, the motif predictors assigned new transcription factors for 
these MDR proteins, turning them into high-quality candidates to be
experimentally tested.
CONCLUSION: The motif predictors presented herein can be used to identify novel
regulatory interactions by using microarray data. The presentation of an example 
motif to predictors will make them categorize whether or not the example motif is
a BF, or whether or not it is an FF. This approach is useful to find new "pieces"
of the TRN, when inspecting the regulation of a small set of operons.
Furthermore, it shows that correlations of expression data can be used to
discriminate between elements that are arranged in structural motifs and those in
random sets of transcripts.

DOI: 10.1186/1471-2180-8-101 
PMCID: PMC2453137
PMID: 18565227  [Indexed for MEDLINE]


746. PLoS One. 2008 Jun 18;3(6):e2456. doi: 10.1371/journal.pone.0002456.

Critical dynamics in genetic regulatory networks: examples from four kingdoms.

Balleza E(1), Alvarez-Buylla ER, Chaos A, Kauffman S, Shmulevich I, Aldana M.

Author information: 
(1)Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México,
Cuernavaca, Morelos, México.

The coordinated expression of the different genes in an organism is essential to 
sustain functionality under the random external perturbations to which the
organism might be subjected. To cope with such external variability, the global
dynamics of the genetic network must possess two central properties. (a) It must 
be robust enough as to guarantee stability under a broad range of external
conditions, and (b) it must be flexible enough to recognize and integrate
specific external signals that may help the organism to change and adapt to
different environments. This compromise between robustness and adaptability has
been observed in dynamical systems operating at the brink of a phase transition
between order and chaos. Such systems are termed critical. Thus, criticality, a
precise, measurable, and well characterized property of dynamical systems, makes 
it possible for robustness and adaptability to coexist in living organisms. In
this work we investigate the dynamical properties of the gene transcription
networks reported for S. cerevisiae, E. coli, and B. subtilis, as well as the
network of segment polarity genes of D. melanogaster, and the network of flower
development of A. thaliana. We use hundreds of microarray experiments to infer
the nature of the regulatory interactions among genes, and implement these data
into the Boolean models of the genetic networks. Our results show that, to the
best of the current experimental data available, the five networks under study
indeed operate close to criticality. The generality of this result suggests that 
criticality at the genetic level might constitute a fundamental evolutionary
mechanism that generates the great diversity of dynamically robust living forms
that we observe around us.

DOI: 10.1371/journal.pone.0002456 
PMCID: PMC2423472
PMID: 18560561  [Indexed for MEDLINE]


747. Science. 2008 Jun 6;320(5881):1313-7. doi: 10.1126/science.1154456. Epub 2008 May
8.

Predictive behavior within microbial genetic networks.

Tagkopoulos I(1), Liu YC, Tavazoie S.

Author information: 
(1)Department of Electrical Engineering, Princeton University, Princeton, NJ
08544, USA.

Comment in
    Science. 2008 Jun 6;320(5881):1297-8.

The homeostatic framework has dominated our understanding of cellular physiology.
We question whether homeostasis alone adequately explains microbial responses to 
environmental stimuli, and explore the capacity of intracellular networks for
predictive behavior in a fashion similar to metazoan nervous systems. We show
that in silico biochemical networks, evolving randomly under precisely defined
complex habitats, capture the dynamical, multidimensional structure of diverse
environments by forming internal representations that allow prediction of
environmental change. We provide evidence for such anticipatory behavior by
revealing striking correlations of Escherichia coli transcriptional responses to 
temperature and oxygen perturbations-precisely mirroring the covariation of these
parameters upon transitions between the outside world and the mammalian
gastrointestinal tract. We further show that these internal correlations reflect 
a true associative learning paradigm, because they show rapid decoupling upon
exposure to novel environments.

DOI: 10.1126/science.1154456 
PMCID: PMC2931280
PMID: 18467556  [Indexed for MEDLINE]


748. Arch Toxicol. 2008 Jun;82(6):355-62. Epub 2007 Nov 20.

Gene expression profile in monocyte during in vitro mineral fiber degradation.

Dika Nguea H(1), de Reydellet A, Lehuédé P, De Meringo A, Le Faou A, Marcocci L, 
Rihn BH.

Author information: 
(1)Laboratoire de Bactériologie Virologie, Faculté de Médecine, Nancy-University,
9 Avenue de la Forêt de Haye BP 184, 54505, Vandoeuvre-lès-Nancy Cedex, France.

A human monocytes cell line, U-937, incubated in the presence of filtered medium 
from Escherichia coli culture (FS) has been previously reported to degrade man
made mineral fiber and it has been indicated as a good paradigm of in vivo fiber 
biopersistence evaluation (manuscript accepted for publication). In the present
paper, a study is reported aimed to define the molecular modification occurring
in the U-937 monocytes during in vitro fiber degradation. The induction of gene
expression was investigated in U-937 exposed to rock wool fibers (HDN) in the
presence of FS by transcriptome analysis using 20 K DNA microarrays and
quantitative RT-PCR. The over-expression of genes related to mobility and
cellular adhesion, oxidative stress, immune system stimulation, enzymes, and ions
transport protein systems were identified. Among them NCF1 gene, the gene
encoding a subunit of NADPH oxidase, over-expression was detected. As the product
of this gene allows the formation of superoxide anion that could lead to
oxidative stress, HDN fibers were exposed to hydrogen peroxide. Fiber degradation
similar to those observed upon incubation with U-937 in the presence of FS was
obtained thus suggesting that reactive oxygen species production may be
responsible for fiber degradation by U-937 monocytes.

DOI: 10.1007/s00204-007-0258-6 
PMID: 18026935  [Indexed for MEDLINE]


749. Infect Immun. 2008 Jun;76(6):2411-9. doi: 10.1128/IAI.01730-07. Epub 2008 Mar 24.

Identification of a novel prophage-like gene cluster actively expressed in both
virulent and avirulent strains of Leptospira interrogans serovar Lai.

Qin JH(1), Zhang Q, Zhang ZM, Zhong Y, Yang Y, Hu BY, Zhao GP, Guo XK.

Author information: 
(1)Department of Microbiology and Parasitology, Institutes of Medical Sciences,
Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, China.

DNA microarray analysis was used to compare the differential gene expression
profiles between Leptospira interrogans serovar Lai type strain 56601 and its
corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster
of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains
but concomitantly upregulated in strain 56601 in contrast to that of IPAV.
Reverse transcription-PCR assays proved that the gene cluster comprised five
transcripts. Gene annotation of this cluster revealed characteristics of a
putative prophage-like remnant with at least 8 of 34 sequences encoding
prophage-like proteins, of which the LA0195 protein is probably a putative
prophage CI-like regulator. The transcription initiation activities of putative
promoter-regulatory sequences of transcripts I, II, and III, all proximal to the 
LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector 
pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT)
activities. The strong promoter activities of both transcripts I and II indicated
by the E. coli CAT assay were well correlated with the in vitro sequence-specific
binding of the recombinant LA0195 protein to the corresponding promoter probes
detected by the electrophoresis mobility shift assay. On the other hand, the
promoter activity of transcript III was very low in E. coli and failed to show
active binding to the LA0195 protein in vitro. These results suggested that the
LA0195 protein is likely involved in the transcription of transcripts I and II.
However, the identical complete DNA sequences of this prophage remnant from these
two strains strongly suggests that possible regulatory factors or signal
transduction systems residing outside of this region within the genome may be
responsible for the differential expression profiling in these two strains.

DOI: 10.1128/IAI.01730-07 
PMCID: PMC2423081
PMID: 18362131  [Indexed for MEDLINE]


750. J Med Virol. 2008 Jun;80(6):1042-50. doi: 10.1002/jmv.21131.

DNA microarray technology for simultaneous detection and species identification
of seven human herpes viruses.

Zheng ZB(1), Wu YD, Yu XL, Shang SQ.

Author information: 
(1)Laboratory Center, Children's Hospital, Zhejiang University School of
Medicine, Hangzhou, China.

The aim of the study was to develop a multiplex PCR-based DNA microarray
technology for simultaneous detection and species identification of seven human
herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2),
varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV),
and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to
accurate diagnosis of herpesvirus-associated diseases. Primers and
oligonucleotide probes were designed and synthesized based on the highly
conserved regions of the DNA polymerase gene in human herpes viruses. DNA
microarrays were made by printing the oligonucleotide probes onto special glass
slides. After amplification and labeling with CY5, the PCR products were
hybridized with the DNA microarrays and species identified. Sixty-one
cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this
technique, and the results were compared with those of TaqMan PCR. Several
specimens were sequenced further after cloning. The PCR products of the seven
human herpes viruses ranged from 224 to 252 bp, and could be species identified
with DNA microarrays. The detection limits were 10(1) copies/microl for each
virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. 
coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human
genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human
herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and
specificity of the DNA microarray technology was 96.2% and 99.3%, respectively.
This multiplex PCR-based DNA microarray technology, which is rapid, specific and 
sensitive, serves as an effective technique for simultaneous detection and
species identification of seven human herpes viruses.

DOI: 10.1002/jmv.21131 
PMID: 18428126  [Indexed for MEDLINE]


751. J Proteome Res. 2008 Jun;7(6):2234-45. doi: 10.1021/pr800055r. Epub 2008 May 9.

Protein disorder is positively correlated with gene expression in Escherichia
coli.

Paliy O(1), Gargac SM, Cheng Y, Uversky VN, Dunker AK.

Author information: 
(1)Department of Biochemistry and Molecular Biology, Wright State University,
Dayton, Ohio 45435, USA. oleg.paliy@wright.edu

We considered, on a global scale, the relationship between the predicted fraction
of protein disorder and the RNA and protein expression in Escherichia coli.
Fraction of protein disorder correlated positively with both measured RNA
expression levels of E. coli genes in three different growth media and with
predicted abundance levels of E. coli proteins. Though weak, the correlation was 
highly significant. Correlation of protein disorder with RNA expression did not
depend on the growth rate of E. coli cultures and was not caused by a small
subset of genes showing exceptionally high concordance in their disorder and
expression levels. Global analysis was complemented by detailed consideration of 
several groups of proteins.

DOI: 10.1021/pr800055r 
PMCID: PMC2754758
PMID: 18465893  [Indexed for MEDLINE]


752. Science. 2008 May 16;320(5878):935-8. doi: 10.1126/science.1152763.

Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. 
coli.

Cardinale CJ(1), Washburn RS, Tadigotla VR, Brown LM, Gottesman ME, Nudler E.

Author information: 
(1)Department of Biochemistry, New York University School of Medicine, New York, 
NY 10016, USA.

Transcription of the bacterial genome by the RNA polymerase must terminate at
specific points. Transcription can be terminated by Rho factor, an essential
protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits
Rho, to assess its role on a genome-wide scale. Rho is revealed as a global
regulator of gene expression that matches Escherichia coli transcription to
translational needs. We also found that genes in E. coli that are most repressed 
by Rho are prophages and other horizontally acquired portions of the genome.
Elimination of these foreign DNA elements increases resistance to bicyclomycin.
Although rho remains essential, such reduced-genome bacteria no longer require
Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E.
coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho
termination, supported by NusA and NusG, is required to suppress the toxic
activity of foreign genes.

DOI: 10.1126/science.1152763 
PMCID: PMC4059013
PMID: 18487194  [Indexed for MEDLINE]


753. Cancer Res. 2008 May 15;68(10):3655-61. doi: 10.1158/0008-5472.CAN-07-2940.

Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice
attenuates intestinal tumorigenesis.

Zeilstra J(1), Joosten SP, Dokter M, Verwiel E, Spaargaren M, Pals ST.

Author information: 
(1)Department of Pathology, Academic Medical Center, University of Amsterdam,
Amsterdam, the Netherlands.

Mutation of the genes encoding the WNT signaling components adenomatous polyposis
coli or beta-catenin plays a critical role in the initiation of colorectal
cancer. These mutations cause constitutively active beta-catenin/TCF-mediated
transcription, driving the transformation of intestinal crypts to colorectal
cancer precursor lesions, called dysplastic aberrant crypt foci. CD44 is a
prominent WNT signaling target in the intestine and is selectively expressed on
the renewing epithelial cells lining the crypts. The expression of CD44 is
dramatically increased in aberrant crypt foci in both humans and
tumor-susceptible Apc(Min/+) mice, suggesting a role for CD44 in intestinal
tumorigenesis. To study this role, we crossed C57BL/6J-Cd44(-/-) mice with
C57BL/6J-Apc(Min/+) mice. Compared with C57BL/6J-Cd44(+/+)/Apc(Min/+) mice,
C57BL/6J-Cd44(-/-)/Apc(Min/+) mice showed an almost 50% reduction in the number
of intestinal adenomas. This reduction was primarily caused by a decrease in the 
formation of aberrant crypts, implying the involvement of CD44 in tumor
initiation. The absence of CD44 in the normal (nonneoplastic) crypts of
Cd44(-/-)/Apc(Min/+) mice did not alter the proliferative capacity and size of
the intestinal stem cell and transit-amplifying compartments. However, compared
with Cd44(+/+)/Apc(Min/+) mice, Cd44(-/-)/Apc(Min/+) showed an increase in the
number of apoptotic epithelial cells at the base of the crypt which correlated
with an increased expression of the proapoptotic genes Bok and Dr6. Our results
show an important role for CD44 in intestinal tumorigenesis and suggest that CD44
does not affect proliferation but is involved in the control of the balance
between survival and apoptosis in the intestinal crypt.

DOI: 10.1158/0008-5472.CAN-07-2940 
PMID: 18483247  [Indexed for MEDLINE]


754. PLoS One. 2008 May 14;3(5):e2163. doi: 10.1371/journal.pone.0002163.

A functional gene array for detection of bacterial virulence elements.

Jaing C(1), Gardner S, McLoughlin K, Mulakken N, Alegria-Hartman M, Banda P,
Williams P, Gu P, Wagner M, Manohar C, Slezak T.

Author information: 
(1)Chemistry, Materials, Earth and Life Sciences, Lawrence Livermore National
Laboratory, Livermore, California, United States of America. jaing2/at/llnl.gov

Emerging known and unknown pathogens create profound threats to public health.
Platforms for rapid detection and characterization of microbial agents are
critically needed to prevent and respond to disease outbreaks. Available
detection technologies cannot provide broad functional information about known or
novel organisms. As a step toward developing such a system, we have produced and 
tested a series of high-density functional gene arrays to detect elements of
virulence and antibiotic resistance mechanisms. Our first generation array
targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis
and Staphylococcus aureus. We determined optimal probe design parameters for gene
family detection and discrimination. When tested with organisms at varying
phylogenetic distances from the four target strains, the array detected orthologs
for the majority of targeted gene families present in bacteria belonging to the
same taxonomic family. In combination with whole-genome amplification, the array 
detects femtogram concentrations of purified DNA, either spiked in to an aerosol 
sample background, or in combinations from one or more of the four target
organisms. This is the first report of a high density NimbleGen microarray system
targeting microbial antibiotic resistance and virulence mechanisms. By targeting 
virulence gene families as well as genes unique to specific biothreat agents,
these arrays will provide important data about the pathogenic potential and drug 
resistance profiles of unknown organisms in environmental samples.

DOI: 10.1371/journal.pone.0002163 
PMCID: PMC2367441
PMID: 18478124  [Indexed for MEDLINE]


755. Am J Obstet Gynecol. 2008 May;198(5):539.e1-8. doi: 10.1016/j.ajog.2007.11.021.

Angiogenesis gene expression in mouse uterus during the common pathway of
parturition.

Haddad R(1), Romero R, Gould BR, Tromp G, Gotsch F, Edwin SS, Zingg HH.

Author information: 
(1)Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy 
Shriver National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD, USA.

OBJECTIVE: The objective of the study was to investigate changes in the
expression of angiogenesis-related genes during the common terminal pathway of
parturition including spontaneous labor at term, as well as preterm labor (PTL), 
induced by either bacteria or ovariectomy.
STUDY DESIGN: Preterm pregnant mice (14.5 days of gestation) were treated with
the following: (1) intrauterine injection of media; (2) intrauterine injection of
heat-inactivated Escherichia coli; (3) ovariectomy; and (4) sham operation.
Tissues from mice at term (19.5 days of gestation) were collected at term not in 
labor, term in labor, and 12 hours postpartum. Angiogenesis-related gene
expression levels were quantitated by the measurement of specific mRNAs in
uterine tissue by RT-qPCR and analyzed by repeated-measures analysis of variance.
RESULTS: The following results were found: (1) microarray analysis of the uterine
transcriptome indicated an enrichment for the gene ontology category of
angiogenesis in bacteria-induced PTL samples (P < or = .093); (2) several genes
related to angiogenesis demonstrated significantly increased expression in
samples in either term spontaneous labor or preterm labor; and (3) qRT-PCR
measurements demonstrated that spontaneous term labor and preterm labor induced
by either bacteria or ovariectomy all substantially increased the expression of
multiple angiogenesis-related genes (P < or = .0003; Angpt2, Ctgf, Cyr61, Dscr1, 
Pgf, Serpine1, Thbs1, and Wisp 1).
CONCLUSION: Spontaneous labor at term, as well as pathologically induced preterm 
labor, all result in greatly increased expression of angiogenesis-related genes
in the uterus.

DOI: 10.1016/j.ajog.2007.11.021 
PMID: 18455529  [Indexed for MEDLINE]


756. Appl Environ Microbiol. 2008 May;74(9):2690-9. doi: 10.1128/AEM.02809-07. Epub
2008 Mar 14.

The R1 conjugative plasmid increases Escherichia coli biofilm formation through
an envelope stress response.

Yang X(1), Ma Q, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A&M University,
College Station, TX 77843-3122, USA.

Differential gene expression in biofilm cells suggests that adding the
derepressed conjugative plasmid R1drd19 increases biofilm formation by affecting 
genes related to envelope stress (rseA and cpxAR), biofilm formation (bssR and
cstA), energy production (glpDFK), acid resistance (gadABCEX and hdeABD), and
cell motility (csgBEFG, yehCD, yadC, and yfcV); genes encoding outer membrane
proteins (ompACF), phage shock proteins (pspABCDE), and cold shock proteins
(cspACDEG); and phage-related genes. To investigate the link between the
identified genes and biofilm formation upon the addition of R1drd19, 40 isogenic 
mutants were classified according to their different biofilm formation
phenotypes. Cells with class I mutations (those in rseA, bssR, cpxA, and ompA)
exhibited no difference from the wild-type strain in biofilm formation and no
increase in biofilm formation upon the addition of R1drd19. Cells with class II
mutations (those in gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA,
slp, cpxP, cpxR, cstA, rseC, ompF, and yqjD) displayed increased biofilm
formation compared to the wild-type strain but decreased biofilm formation upon
the addition of R1drd19. Class III mutants showed increased biofilm formation
compared to the wild-type strain and increased biofilm formation upon the
addition of R1drd19. Cells with class IV mutations displayed increased biofilm
formation compared to the wild-type strain but little difference upon the
addition of R1drd19, and class V mutants exhibited no difference from the
wild-type strain but increased biofilm formation upon the addition of R1drd19.
Therefore, proteins encoded by the genes corresponding to the class I mutant
phenotype are involved in R1drd19-promoted biofilm formation, primarily through
their impact on cell motility. We hypothesize that the pili formed upon the
addition of the conjugative plasmid disrupt the membrane (induce ompA) and
activate the two-component system CpxAR as well as the other envelope stress
response system, RseA-sigma(E), both of which, along with BssR, play a key role
in bacterial biofilm formation.

DOI: 10.1128/AEM.02809-07 
PMCID: PMC2394867
PMID: 18344336  [Indexed for MEDLINE]


757. Int J Antimicrob Agents. 2008 May;31(5):440-51. doi:
10.1016/j.ijantimicag.2007.11.017. Epub 2008 Feb 19.

Development of a miniaturised microarray-based assay for the rapid identification
of antimicrobial resistance genes in Gram-negative bacteria.

Batchelor M(1), Hopkins KL, Liebana E, Slickers P, Ehricht R, Mafura M, Aarestrup
F, Mevius D, Clifton-Hadley FA, Woodward MJ, Davies RH, Threlfall EJ, Anjum MF.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories Agency,
Addlestone, Surrey, UK.

We describe the development of a miniaturised microarray for the detection of
antimicrobial resistance genes in Gram-negative bacteria. Included on the array
are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides,
tetracyclines and beta-lactams, including extended-spectrum beta-lactamases.
Validation of the array with control strains demonstrated a 99% correlation
between polymerase chain reaction and array results. There was also good
correlation between phenotypic and genotypic results for a large panel of
Escherichia coli and Salmonella isolates. Some differences were also seen in the 
number and type of resistance genes harboured by E. coli and Salmonella strains. 
The array provides an effective, fast and simple method for detection of
resistance genes in clinical isolates suitable for use in diagnostic
laboratories, which in future will help to understand the epidemiology of
isolates and to detect gene linkage in bacterial populations.

DOI: 10.1016/j.ijantimicag.2007.11.017 
PMID: 18243668  [Indexed for MEDLINE]


758. Mol Genet Genomics. 2008 May;279(5):523-34. doi: 10.1007/s00438-008-0330-9.

Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia
coli strain 83972.

Hancock V(1), Seshasayee AS, Ussery DW, Luscombe NM, Klemm P.

Author information: 
(1)Microbial Adhesion Group, Risø DTU, Technical University of Denmark, Building 
301, 2800 Kgs Lyngby, Denmark.

Escherichia coli strains are the major cause of urinary tract infections in
humans. Such strains can be divided into virulent, UPEC strains causing
symptomatic infections, and asymptomatic, commensal-like strains causing
asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain 83972.
Global gene expression profiling of strain 83972 has been carried out under seven
different sets of environmental conditions ranging from laboratory minimal medium
to human bladders. The data reveal highly specific gene expression responses to
different conditions. A number of potential fitness factors for the human urinary
tract could be identified. Also, presence/ absence data of the gene expression
was used as an adaptive genomics tool to model the gene pool of 83972 using
primarily UPEC strain CFT073 as a scaffold. In our analysis, 96% of the
transcripts filtered present in strain 83972 can be found in CFT073, and genes on
six of the seven pathogenicity islands were expressed in 83972. Despite the very 
different patient symptom profiles, the two strains seem to be very similar.
Genes expressed in CFT073 but not in 83972 were identified and can be considered 
as virulence factor candidates. Strain 83972 is a deconstructed pathogen rather
than a commensal strain that has acquired fitness properties.

DOI: 10.1007/s00438-008-0330-9 
PMCID: PMC2329726
PMID: 18317809  [Indexed for MEDLINE]


759. Nature. 2008 May 1;453(7191):115-9. doi: 10.1038/nature06888.

Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions.

Mili S(1), Moissoglu K, Macara IG.

Author information: 
(1)Department of Microbiology, Center for Cell Signaling, University of Virginia,
HSC, Charlottesville, Virginia 22908-0577, USA. sm2ju@virginia.edu

RNA localization is important for the establishment and maintenance of polarity
in multiple cell types. Localized RNAs are usually transported along microtubules
or actin filaments and become anchored at their destination to some underlying
subcellular structure. Retention commonly involves actin or actin-associated
proteins, although cytokeratin filaments and dynein anchor certain RNAs. RNA
localization is important for diverse processes ranging from cell fate
determination to synaptic plasticity; however, so far there have been few
comprehensive studies of localized RNAs in mammalian cells. Here we have
addressed this issue, focusing on migrating fibroblasts that polarize to form a
leading edge and a tail in a process that involves asymmetric distribution of
RNAs. We used a fractionation scheme combined with microarrays to identify, on a 
genome-wide scale, RNAs that localize in protruding pseudopodia of mouse
fibroblasts in response to migratory stimuli. We find that a diverse group of
RNAs accumulates in such pseudopodial protrusions. Through their 3' untranslated 
regions these transcripts are anchored in granules concentrated at the plus ends 
of detyrosinated microtubules. RNAs in the granules associate with the
adenomatous polyposis coli (APC) tumour suppressor and the fragile X mental
retardation protein (FMRP). APC is required for the accumulation of transcripts
in protrusions. Our results suggest a new type of RNA anchoring mechanism as well
as a new, unanticipated function for APC in localizing RNAs.

DOI: 10.1038/nature06888 
PMCID: PMC2782773
PMID: 18451862  [Indexed for MEDLINE]


760. Nature. 2008 Apr 17;452(7189):840-5. doi: 10.1038/nature06847.

Evolvability and hierarchy in rewired bacterial gene networks.

Isalan M(1), Lemerle C, Michalodimitrakis K, Horn C, Beltrao P, Raineri E,
Garriga-Canut M, Serrano L.

Author information: 
(1)EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG),
UPF, 08003 Barcelona, Spain. isalan@crg.es

Comment in
    Nature. 2008 Apr 17;452(7189):824-5.

Sequencing DNA from several organisms has revealed that duplication and drift of 
existing genes have primarily moulded the contents of a given genome. Though the 
effect of knocking out or overexpressing a particular gene has been studied in
many organisms, no study has systematically explored the effect of adding new
links in a biological network. To explore network evolvability, we constructed
598 recombinations of promoters (including regulatory regions) with different
transcription or sigma-factor genes in Escherichia coli, added over a wild-type
genetic background. Here we show that approximately 95% of new networks are
tolerated by the bacteria, that very few alter growth, and that expression level 
correlates with factor position in the wild-type network hierarchy. Most
importantly, we find that certain networks consistently survive over the wild
type under various selection pressures. Therefore new links in the network are
rarely a barrier for evolution and can even confer a fitness advantage.

DOI: 10.1038/nature06847 
PMCID: PMC2666274
PMID: 18421347  [Indexed for MEDLINE]


761. BMC Microbiol. 2008 Apr 11;8:60. doi: 10.1186/1471-2180-8-60.

Limited functional conservation of a global regulator among related bacterial
genera: Lrp in Escherichia, Proteus and Vibrio.

Lintner RE(1), Mishra PK, Srivastava P, Martinez-Vaz BM, Khodursky AB, Blumenthal
RM.

Author information: 
(1)Department of Medical Microbiology and Immunology, University of Toledo Health
Sciences Center, Toledo, OH 43614-2598, USA. lintner@wi.mit.edu

BACKGROUND: Bacterial genome sequences are being determined rapidly, but few
species are physiologically well characterized. Predicting regulation from genome
sequences usually involves extrapolation from better-studied bacteria, using the 
hypothesis that a conserved regulator, conserved target gene, and predicted
regulator-binding site in the target promoter imply conserved regulation between 
the two species. However many compared organisms are ecologically and
physiologically diverse, and the limits of extrapolation have not been well
tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects
expression of approximately 400 genes. Proteus mirabilis and Vibrio cholerae have
highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The
functional equivalence of Lrp from these related species was assessed.
RESULTS: Heterologous Lrp regulated gltB, livK and lrp transcriptional fusions in
an E. coli background in the same general way as the native Lrp, though with
significant differences in extent. Microarray analysis of these strains revealed 
that the heterologous Lrp proteins significantly influence only about half of the
genes affected by native Lrp. In P. mirabilis, heterologous Lrp restored
swarming, though with some pattern differences. P. mirabilis produced
substantially more Lrp than E. coli or V. cholerae under some conditions. Lrp
regulation of target gene orthologs differed among the three native hosts.
Strikingly, while Lrp negatively regulates its own gene in E. coli, and was shown
to do so even more strongly in P. mirabilis, Lrp appears to activate its own gene
in V. cholerae.
CONCLUSION: The overall similarity of regulatory effects of the Lrp orthologs
supports the use of extrapolation between related strains for general purposes.
However this study also revealed intrinsic differences even between orthologous
regulators sharing >90% overall identity, and 100% identity for the DNA-binding
helix-turn-helix motif, as well as differences in the amounts of those
regulators. These results suggest that predicting regulation of specific target
genes based on genome sequence comparisons alone should be done on a conservative
basis.

DOI: 10.1186/1471-2180-8-60 
PMCID: PMC2374795
PMID: 18405378  [Indexed for MEDLINE]


762. Microbiology. 2008 Apr;154(Pt 4):1117-31. doi: 10.1099/mic.0.2007/012534-0.

The importance of the Rcs phosphorelay in the survival and pathogenesis of the
enteropathogenic yersiniae.

Hinchliffe SJ(1), Howard SL, Huang YH, Clarke DJ, Wren BW.

Author information: 
(1)Department of Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine, London, UK.

The human-pathogenic yersiniae represent an ideal species group to study the
evolution of highly virulent bacteria, with Yersinia pestis having emerged from
the enteropathogen Y. pseudotuberculosis an estimated 20 000 years ago. Sequence 
data reveal that the Y. pestis genome is in the early stages of decay and
contains hundreds of non-functioning pseudogenes, some of which may be important 
in the enteric lifestyle of Y. pseudotuberculosis. Bioinformatic analysis of
pseudogenes from seven Y. pestis genome sequences identified rcsD as a gene
disrupted early in the evolution of this organism. This phosphotransfer protein
is part the of the Rcs phosphorelay, a two-component system present in the
Enterobacteriaceae which has been shown to regulate the expression of capsular
polysaccharide and other virulence determinants in several species including
Escherichia coli and Salmonella. Using microarray analysis, we determined that
the Y. pseudotuberculosis Rcs phosphorelay regulates the expression of 136 coding
sequences, of which 60 % are predicted to affect the cell envelope. Several
putative virulence determinants were identified as being regulated by this
phosphorelay, along with proteins involved in biofilm formation, motility,
mammalian cell adhesion and stress survival. Phenotypic assays on defined mutants
confirmed a role for the phosphorelay in these processes in both Y.
pseudotuberculosis and Y. enterocolitica.

DOI: 10.1099/mic.0.2007/012534-0 
PMID: 18375804  [Indexed for MEDLINE]


763. Cancer. 2008 Mar 15;112(6):1325-36. doi: 10.1002/cncr.23312.

Multiple gene methylation of nonsmall cell lung cancers evaluated with
3-dimensional microarray.

Wang Y(1), Zhang D, Zheng W, Luo J, Bai Y, Lu Z.

Author information: 
(1)State Key Laboratory of Bioelectronics, College of Biological Science and
Medical Engineering, Southeast University, Nanjing, China.

BACKGROUND: Aberrant DNA methylation of the CpG islands for cancer-related genes 
is among the earliest and most frequent alterations in cancer and may be useful
for diagnosing cancer or evaluating recurrent disease.
METHODS: In this study, a 3-dimensional (3-D), polyacrylamide gel-based DNA
microarray coupled with linker-polymerase chain reaction (PCR) was developed to
detect hypermethylation of CpG islands in multiple genes from a large group of
different samples. The authors determined the frequency of aberrant promoter
methylation of 15 genes in 28 resected primary nonsmall cell lung cancers
(NSCLCs) and in 12 corresponding nonmalignant lung tissues.
RESULTS: Methylation frequencies in the tumor samples were detected in 18% of
samples for the breast cancer 1 gene BRCA1, in 43% of samples for the tissue
inhibitor of metalloproteinase 3 gene TIMP-3, in 38% of samples for the
cyclin-dependent kinase inhibitor 4A gene p16INK4a, in 54% of samples for the
cadherin 13 gene CDH13, in 50% of samples for the death-associated protein kinase
gene DAPK, in 11% of samples for the E-cadherin gene ECAD, in 25% of samples for 
the insulin-like growth factor binding protein 7 gene IGFBP7, in 18% of samples
for the Ras association domain family 1 gene RASSF1, in 68% of samples for the
adenomatous polyposis coli gene APC, in 7% of samples for the cyclin-dependent
kinase inhibitor gene p15, in 18% of samples for the CD44 cell adhesion molecule 
gene, in 29% of samples for the human Mut-L homolog gene hMLH, in 32% of samples 
for the human telomerase reverse transcriptase gene hTERT, in 64% of samples for 
the calcitonin gene-related polypeptide alpha gene CALCA, and in 54% of samples
for the estrogen receptor gene ER; however, methylation was not observed in the
majority of corresponding nonmalignant tissues. Six samples in from 28 tumors had
>6 genes methylated, and 1 sample had 13 genes methylated. Methylation of these
genes was correlated with some clinicopathologic patient characteristics.
CONCLUSIONS: This study demonstrated that a 3-D microarray could be used to
detect DNA hypermethylation and provided a high-throughput platform for DNA
hypermethylation analysis.

Copyright (c) 2008 American Cancer Society.

DOI: 10.1002/cncr.23312 
PMID: 18286531  [Indexed for MEDLINE]


764. Physiol Genomics. 2008 Mar 14;33(1):121-32. doi:
10.1152/physiolgenomics.00095.2007. Epub 2008 Jan 29.

Genome-wide identification and characterization of transcripts translationally
regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells.

Kitamura H(1), Ito M, Yuasa T, Kikuguchi C, Hijikata A, Takayama M, Kimura Y,
Yokoyama R, Kaji T, Ohara O.

Author information: 
(1)Laboratories for Immunogenomics, RIKEN Research Center for Allergy and
Immunology, Yokohama, Japan. ktmr@rcai.riken.jp

Although Escherichia coli LPS is known to elicit various proinflammatory
responses in macrophages, its effect on the translational states of transcripts
has not yet been explored on a genome-wide scale. To address this, we
investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein 
particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix
Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total
cellular, polysomal, and free mRNP fractions enabled us to identify transcripts
that were modulated at the translational level by LPS: among 19,791 transcripts, 
115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100
ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based
analysis suggested that translation-dependent downregulated genes frequently
include those encoding proteins in the mitochondrial respiratory chain. In fact, 
the mRNA levels of some transcripts for complexes I, IV, and V in the
mitochondrial respiratory chain were translationally downregulated, eventually
contributing to the decline of their protein levels. Moreover, the amount of
metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased
without any change of its mRNA level in total cellular fraction after LPS
stimulation. Consistently, the total amounts and activities of complexes I and IV
were attenuated by LPS stimulation, and the attenuation was independent of nitric
oxide. These results demonstrated that translational suppression may play a
critical role in the LPS-mediated attenuation of mitochondrial oxidative
phosphorylation in a nitric oxide-independent manner in J774.1 cells.

DOI: 10.1152/physiolgenomics.00095.2007 
PMID: 18230670  [Indexed for MEDLINE]


765. Infect Immun. 2008 Mar;76(3):1143-52. doi: 10.1128/IAI.01386-07. Epub 2008 Jan 7.

Comparison of carbon nutrition for pathogenic and commensal Escherichia coli
strains in the mouse intestine.

Fabich AJ(1), Jones SA, Chowdhury FZ, Cernosek A, Anderson A, Smalley D, McHargue
JW, Hightower GA, Smith JT, Autieri SM, Leatham MP, Lins JJ, Allen RL, Laux DC,
Cohen PS, Conway T.

Author information: 
(1)Advanced Center for Genome Technology, University of Oklahoma, Norman,
Oklahoma 73019, USA.

The carbon sources that support the growth of pathogenic Escherichia coli O157:H7
in the mammalian intestine have not previously been investigated. In vivo, the
pathogenic E. coli EDL933 grows primarily as single cells dispersed within the
mucus layer that overlies the mouse cecal epithelium. We therefore compared the
pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in 
vitro, using a mixture of the sugars known to be present in cecal mucus, and
found that the two strains used the 13 sugars in a similar order and
cometabolized as many as 9 sugars at a time. We conducted systematic mutation
analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways
used for catabolism of 13 mucus-derived sugars and five other compounds for which
the corresponding bacterial gene system was induced in the transcriptome of cells
grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic
backgrounds was fed to streptomycin-treated mice, together with the respective
wild-type parent strain, and their colonization was monitored by fecal plate
counts. None of the mutations corresponding to the five compounds not found in
mucosal polysaccharides resulted in colonization defects. Based on the mutations 
that caused colonization defects, we determined that both E. coli EDL933 and E.
coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In 
addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose,
whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The
colonization defects of six catabolic lesions were found to be additive with E.
coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E.
coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize
the mouse intestine. The results suggest a strategy whereby invading pathogens
gain advantage by simultaneously consuming several sugars that may be available
because they are not consumed by the commensal intestinal microbiota.

DOI: 10.1128/IAI.01386-07 
PMCID: PMC2258830
PMID: 18180286  [Indexed for MEDLINE]


766. J Bacteriol. 2008 Mar;190(6):2065-74. doi: 10.1128/JB.01740-07. Epub 2008 Jan 11.

The Rcs phosphorelay is a cell envelope stress response activated by
peptidoglycan stress and contributes to intrinsic antibiotic resistance.

Laubacher ME(1), Ades SE.

Author information: 
(1)Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, PA 16802, USA.

Gram-negative bacteria possess stress responses to maintain the integrity of the 
cell envelope. Stress sensors monitor outer membrane permeability, envelope
protein folding, and energization of the inner membrane. The systems used by
gram-negative bacteria to sense and combat stress resulting from disruption of
the peptidoglycan layer are not well characterized. The peptidoglycan layer is a 
single molecule that completely surrounds the cell and ensures its structural
integrity. During cell growth, new peptidoglycan subunits are incorporated into
the peptidoglycan layer by a series of enzymes called the penicillin-binding
proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan
stress, global gene expression analysis was used to identify Escherichia coli
stress responses activated following inhibition of specific PBPs by the
beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of
PBPs with different roles in peptidoglycan synthesis has different consequences
for cell morphology and viability, suggesting that not all perturbations to the
peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition 
of different PBPs resulted in both shared and unique stress responses. The
regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition
of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay 
increased survival in the presence of these antibiotics, independently of capsule
synthesis. Both activation of the phosphorelay and survival required signal
transduction via the outer membrane lipoprotein RcsF and the response regulator
RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and
contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.

DOI: 10.1128/JB.01740-07 
PMCID: PMC2258881
PMID: 18192383  [Indexed for MEDLINE]


767. J Proteome Res. 2008 Mar;7(3):1315-25. doi: 10.1021/pr700642g. Epub 2008 Jan 26.

Development of a novel oligonucleotide array-based transcription factor assay
platform for genome-wide active transcription factor profiling in Saccharomyces
cerevisiae.

Zhao Y(1), Shao W, Wei H, Qiao J, Lu Y, Sun Y, Mitchelson K, Cheng J, Zhou Y.

Author information: 
(1)Medical Systems Biology Research Center, Tsinghua University, Beijing 100084, 
China.

Transcription factors (TFs) play a central role in regulating gene expression and
in providing interconnecting regulatory networks between related pathway
elements. Although single TF assays provide some insights into pathway
regulation, a method that allows the parallel investigation of all active TFs is 
highly desired to elucidate the complex inter-regulated cellular mechanisms. We
have developed a novel oligonucleotide array-based transcription factor assay
platform for genome-wide active TF profiling of Saccharomyces cerevisiae, which
can simultaneously analyze the activities of 93 different TFs. The platform has
been validated using 28 purified TFs produced in Escherichia coli, cell extracts 
from yeast strains overexpressing particular TFs, and by detailed control
experiments. We then used the platform to examine the activity changes of all
yeast TFs during diauxic shift, and results showed, in good agreement with
previous studies, that the Sip4 was induced specifically. Other individual TFs
required for growth in synthetic complete medium were also identified.
Genome-wide analysis of TF activity is extremely useful in investigating complex 
gene regulatory networks and for the development of systematic understanding of
the complexity of genomic functions. These results obtained in this report
demonstrate the validity, and for the first time the utility, of this technology 
for genome-wide investigation of TF activities.

DOI: 10.1021/pr700642g 
PMID: 18220337  [Indexed for MEDLINE]


768. Metab Eng. 2008 Mar;10(2):109-20. Epub 2007 Nov 5.

Genome-scale analysis of anti-metabolite directed strain engineering.

Bonomo J(1), Lynch MD, Warnecke T, Price JV, Gill RT.

Author information: 
(1)Department of Chemical and Biological Engineering, University of Colorado, UCB
424 Boulder, CO 80309, USA.

Classic strain engineering methods have previously been limited by the
low-throughput of conventional sequencing technology. Here, we applied a new
genomics technology, scalar analysis of library enrichments (SCALEs), to measure 
>3 million Escherichia coli genomic library clone enrichment patterns resulting
from growth selections employing three aspartic-acid anti-metabolites. Our
objective was to assess the extent to which access to genome-scale enrichment
patterns would provide strain-engineering insights not reasonably accessible
through the use of conventional sequencing. We determined that the SCALEs method 
identified a surprisingly large range of anti-metabolite tolerance regions (423, 
865, or 909 regions for each of the three anti-metabolites) when compared to the 
number of regions (1-3 regions) indicated by conventional sequencing.
Genome-scale methods uniquely enable the calculation of clone fitness values by
providing concentration data for all clones within a genomic library before and
after a period of selection. We observed that clone fitness values differ
substantially from clone concentration values and that this is due to differences
in overall clone fitness distributions for each selection. Finally, we show that 
many of the clones of highest fitness overlapped across all selections,
suggesting that inhibition of aspartate metabolism, as opposed to specific
inhibited enzymes, dominated each selection. Our follow up studies confirmed our 
observed growth phenotypes and showed that intracellular amino-acid levels were
also altered in several of the identified clones. These results demonstrate that 
genome-scale methods, such as SCALEs, can be used to dramatically improve
understanding of classic strain engineering approaches.

DOI: 10.1016/j.ymben.2007.10.002 
PMID: 18093856  [Indexed for MEDLINE]


769. Mol Genet Genomics. 2008 Mar;279(3):267-77. Epub 2007 Dec 20.

RpoS regulation of gene expression during exponential growth of Escherichia coli 
K12.

Dong T(1), Kirchhof MG, Schellhorn HE.

Author information: 
(1)Department of Biology, McMaster University, Life Sciences Building, Rm. 433,
1280 Main Street West, Hamilton, ON, Canada, L8S 4K1. dongt2@mcmaster.ca

RpoS is a major regulator of genes required for adaptation to stationary phase in
E. coli. However, the exponential phase expression of some genes is affected by
rpoS mutation, suggesting RpoS may also have an important physiological role in
growing cells. To test this hypothesis, we examined the regulatory role of RpoS
in exponential phase using both genomic and biochemical approaches. Microarray
expression data revealed that, in the rpoS mutant, the expression of 268 genes
was attenuated while the expression of 24 genes was enhanced. Genes responsible
for carbon source transport (the mal operon for maltose), protein folding (dnaK
and mopAB), and iron acquisition (fepBD, entCBA, fecI, and exbBD) were positively
controlled by RpoS. The importance of RpoS-mediated control of iron acquisition
was confirmed by cellular metal analysis which revealed that the intracellular
iron content of wild type cells was two-fold higher than in rpoS mutant cells.
Surprisingly, many previously identified RpoS stationary-phase dependent genes
were not controlled by RpoS in exponential phase and several genes were
RpoS-regulated only in exponential phase, suggesting the involvement of other
regulators. The expression of RpoS-dependent genes osmY, tnaA and malK was
controlled by Crl, a transcriptional regulator that modulates RpoS activity. In
summary, the identification of a group of exponential phase genes controlled by
RpoS reveals a novel aspect of RpoS function.

DOI: 10.1007/s00438-007-0311-4 
PMID: 18158608  [Indexed for MEDLINE]


770. Mol Microbiol. 2008 Mar;67(6):1242-56. doi: 10.1111/j.1365-2958.2008.06117.x.
Epub 2008 Jan 15.

Promoter specificity for 6S RNA regulation of transcription is determined by core
promoter sequences and competition for region 4.2 of sigma70.

Cavanagh AT(1), Klocko AD, Liu X, Wassarman KM.

Author information: 
(1)Department of Bacteriology, University of Wisconsin, Madison, WI, USA.

6S RNA binds sigma70-RNA polymerase and downregulates transcription at many
sigma70-dependent promoters, but others escape regulation even during stationary 
phase when the majority of the transcription machinery is bound by the RNA. We
report that core promoter elements determine this promoter specificity; a weak
-35 element allows a promoter to be 6S RNA sensitive, and an extended -10 element
similarly determines 6S RNA inhibition except when a consensus -35 element is
present. These two features together predicted that hundreds of mapped
Escherichia coli promoters might be subject to 6S RNA dampening in stationary
phase. Microarray analysis confirmed 6S RNA-dependent downregulation of
expression from 68% of the predicted genes, which corresponds to 49% of the
expressed genes containing mapped E. coli promoters and establishes 6S RNA as a
global regulator in stationary phase. We also demonstrate a critical role for
region 4.2 of sigma70 in RNA polymerase interactions with 6S RNA. Region 4.2
binds the -35 element during transcription initiation; therefore we propose one
mechanism for 6S RNA regulation of transcription is through competition for
binding region 4.2 of sigma70.

DOI: 10.1111/j.1365-2958.2008.06117.x 
PMID: 18208528  [Indexed for MEDLINE]


771. Nan Fang Yi Ke Da Xue Xue Bao. 2008 Mar;28(3):392-5.

[Construction and identification of a eukaryotic expression vector for the small 
interfering RNA targeting nucleostemin gene].

[Article in Chinese]

Zhang GY(1), Zhao GQ, Yin L, Zhang QX.

Author information: 
(1)Department of Histology and Embryology, College of Basic Medical Sciences,
Zhengzhou University, Zhengzhou 450052, China. E-mail: zgy@zzu.edu.cn.

OBJECTIVE: To construct a eukaryotic expression vector for the small interfering 
RNA (siRNA) targeting nucleostemin (NS) gene.
METHODS: The siRNA targeting NS gene was designed according to the sequence of NS
mRNA available in GenBank. Three siRNA sequences were obtained, and the
corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for
constructing the recombinant plasmids, which were transformed into E.coli
DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing,
were transfected into EC9706 cell line via liposome, and the mRNA and protein
expressions of NS gene in the cells were determined by RT-PCR and Western
blotting, respectively.
RESULTS: Three recombinant plasmids were identified by PCR and sequence analysis,
the results of which showed correct insertion of the designed sequences in the
plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and
protein expressions of NS gene in the transfected cells.
CONCLUSION: The recombinant plasmid expressing the siRNA targeting NS gene has
been successfully constructed, which provides the basis for studying RNA
interference of the NS gene.


PMID: 18359698  [Indexed for MEDLINE]


772. Syst Appl Microbiol. 2008 Mar;31(1):50-61. doi: 10.1016/j.syapm.2008.01.001. Epub
2008 Feb 11.

A novel DNA microarray design for accurate and straightforward identification of 
Escherichia coli safety and laboratory strains.

Bauer AP(1), Ludwig W, Schleifer KH.

Author information: 
(1)Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4,
85350 Freising, Germany. andibauer78@gmx.net

Escherichia coli K-12, B, C and W strains and their derivates are declared in
biological safety guidelines as risk group 1 organisms as they are unable to
colonise the human gut. Differentiation and identification of these safety
strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage
sensitivity tests or PCR-based methods. However, these methods are either tedious
and time consuming (phage sensitivity, PFGE) or based on single specific
fragments (PCR) or patterns (PFGE) lacking additional information for further
differentiation of the strains. In the current study, subtractive hybridisation
techniques were applied to detect specific DNA fragments which were used to
design a microarray (chip) for accurate and simple identification of these
organisms, and to differentiate them from other E. coli strains. The chip can be 
used to identify E. coli safety strains and monitor them during ongoing
experiments for changes in their genome and culture purity. The hybridisation
layout of the microarray was arranged in such a way that the respective lineages 
of safety strains could be easily identified as distinct letters (K, B, C or W). 
Differentiation of single strains or subtyping was possible with further probes. 
In addition, a set of probes targeting genes coding for common virulence factors 
has been included, both to differentiate safety strains from pathogenic variants 
and to make sure that no transfer of these genes happens during handling or
storage. The reliability of the approach has been tested on a comprehensive
selection of E. coli laboratory strains and pathogenic representatives.

DOI: 10.1016/j.syapm.2008.01.001 
PMID: 18262744  [Indexed for MEDLINE]


773. Wei Sheng Yan Jiu. 2008 Mar;37(2):245-8.

[Studies on rapid detection of food-borne pathogenic bacteria by nucleic acid
testing and related technology].

[Article in Chinese]

Cao W(1), Wang M, Wang X, Liu X.

Author information: 
(1)Institute of Nutrition and Food Safety, Chinese Center for Disease Control and
Prevention, Beijing 100050, China.

The traditional methods of bacteria isolation, cultivation and identification are
time-consuming, which can't meet the needs of the control and prevention of
food-borne diseases. Recently, various kinds of rapid methods for food-borne
pathogenic bacteria detection have emerged with the prompt development of nucleic
acid testing technology. The application studies on polymerase chain reaction and
the techniques derived from it, nucleic acid isothermal amplification,
oligonucleotide microarray, immunomagnetic separation and DNA biosensing on
food-borne pathogenic bacteria including Salmonella, Staphylococcus aureus and
Enterohemorrhagic Escherchia coli, etc. were reviewed.


PMID: 18589620  [Indexed for MEDLINE]


774. BMC Microbiol. 2008 Feb 26;8:37. doi: 10.1186/1471-2180-8-37.

Rapid acid treatment of Escherichia coli: transcriptomic response and recovery.

Kannan G(1), Wilks JC, Fitzgerald DM, Jones BD, Bondurant SS, Slonczewski JL.

Author information: 
(1)Department of Biology, Kenyon College, Gambier, OH, 43022 USA.
kannang@kenyon.edu

BACKGROUND: Many E. coli genes show pH-dependent expression during logarithmic
growth in acid (pH 5-6) or in base (pH 8-9). The effect of rapid pH change,
however, has rarely been tested. Rapid acid treatment could distinguish between
genes responding to external pH, and genes responding to cytoplasmic
acidification, which occurs transiently following rapid external acidification.
It could reveal previously unknown acid-stress genes whose effects are transient,
as well as show which acid-stress genes have a delayed response.
RESULTS: Microarray hybridization was employed to observe the global gene
expression of E. coli K-12 W3110 following rapid acidification of the external
medium, from pH 7.6 to pH 5.5. Fluorimetric observation of pH-dependent tetR-YFP 
showed that rapid external acidification led to a half-unit drop in cytoplasmic
pH (from pH 7.6 to pH 6.4) which began to recover within 20 s. Following acid
treatment, 630 genes were up-regulated and 586 genes were down-regulated.
Up-regulated genes included amino-acid decarboxylases (cadA, adiY, gadA),
succinate dehydrogenase (sdhABCD), biofilm-associated genes (bdm, gatAB, and
ymgABC), and the Gad, Fur and Rcs regulons. Genes with response patterns
consistent with cytoplasmic acid stress were revealed by addition of benzoate, a 
membrane-permeant acid that permanently depresses cytoplasmic pH without
affecting external pH. Several genes (yagU, ygiN, yjeI, and yneI) were
up-regulated specifically by external acidification, while other genes (fimB,
ygaC, yhcN, yhjX, ymgABC, yodA) presented a benzoate response consistent with
cytoplasmic pH stress. Other genes (the nuo operon for NADH dehydrogenase I, and 
the HslUV protease) showed delayed up-regulation by acid, with expression rising 
by 10 min following the acid shift.
CONCLUSION: Transcriptomic profiling of E. coli K-12 distinguished three
different classes of change in gene expression following rapid acid treatment:
up-regulation with or without recovery, and delayed response to acid. For eight
genes showing acid response and recovery (fimB, ygaC, yhcN, yhjX, ymgABC, yodA), 
responses to the permeant acid benzoate revealed expression patterns consistent
with sensing of cytoplasmic pH. The delayed acid response of nuo genes shows that
NADH dehydrogenase I is probably induced as a secondary result of acid-associated
metabolism, not as a direct response to cytoplasmic acidification.

DOI: 10.1186/1471-2180-8-37 
PMCID: PMC2270276
PMID: 18302792  [Indexed for MEDLINE]


775. BMC Syst Biol. 2008 Feb 19;2:18. doi: 10.1186/1752-0509-2-18.

Dissecting the logical types of network control in gene expression profiles.

Marr C(1), Geertz M, Hütt MT, Muskhelishvili G.

Author information: 
(1)Computational Systems Biology Group, Jacobs University, Campus Ring 1, 28759
Bremen, Germany. c.marr@jacobs-university.de

BACKGROUND: In the bacterium Escherichia coli the transcriptional regulation of
gene expression involves both dedicated regulators binding specific DNA sites
with high affinity and also global regulators - abundant DNA architectural
proteins of the bacterial nucleoid binding multiple sites with a wide range of
affinities and thus modulating the superhelical density of DNA. The first form of
transcriptional regulation is predominantly pairwise and specific, representing
digitial control, while the second form is (in strength and distribution)
continuous, representing analog control.
RESULTS: Here we look at the properties of effective networks derived from
significant gene expression changes under variation of the two forms of control
and find that upon limitations of one type of control (caused e.g. by mutation of
a global DNA architectural factor) the other type can compensate for compromised 
regulation. Mutations of global regulators significantly enhance the digital
control, whereas in the presence of global DNA architectural proteins regulation 
is mostly of the analog type, coupling spatially neighboring genomic loci. Taken 
together our data suggest that two logically distinct - digital and analog -
types of control are balancing each other.
CONCLUSION: By revealing two distinct logical types of control, our approach
provides basic insights into both the organizational principles of
transcriptional regulation and the mechanisms buffering genetic flexibility. We
anticipate that the general concept of distinguishing logical types of control
will apply to many complex biological networks.

DOI: 10.1186/1752-0509-2-18 
PMCID: PMC2263018
PMID: 18284674  [Indexed for MEDLINE]


776. BMC Evol Biol. 2008 Feb 18;8:52. doi: 10.1186/1471-2148-8-52.

Epigenetic inheritance based evolution of antibiotic resistance in bacteria.

Adam M(1), Murali B, Glenn NO, Potter SS.

Author information: 
(1)Division of Developmental Biology, Children's Hospital Research Foundation,
3333 Burnet Ave, Cincinnati, OH 45229, USA. mik1jg@cchmc.org

BACKGROUND: The evolution of antibiotic resistance in bacteria is a topic of
major medical importance. Evolution is the result of natural selection acting on 
variant phenotypes. Both the rigid base sequence of DNA and the more plastic
expression patterns of the genes present define phenotype.
RESULTS: We investigated the evolution of resistant E. coli when exposed to low
concentrations of antibiotic. We show that within an isogenic population there
are heritable variations in gene expression patterns, providing phenotypic
diversity for antibiotic selection to act on. We studied resistance to three
different antibiotics, ampicillin, tetracycline and nalidixic acid, which act by 
inhibiting cell wall synthesis, protein synthesis and DNA synthesis,
respectively. In each case survival rates were too high to be accounted for by
spontaneous DNA mutation. In addition, resistance levels could be ramped higher
by successive exposures to increasing antibiotic concentrations. Furthermore,
reversion rates to antibiotic sensitivity were extremely high, generally over
50%, consistent with an epigenetic inheritance mode of resistance. The gene
expression patterns of the antibiotic resistant E. coli were characterized with
microarrays. Candidate genes, whose altered expression might confer survival,
were tested by driving constitutive overexpression and determining antibiotic
resistance. Three categories of resistance genes were identified. The endogenous 
beta-lactamase gene represented a cryptic gene, normally inactive, but when by
chance expressed capable of providing potent ampicillin resistance. The glutamate
decarboxylase gene, in contrast, is normally expressed, but when overexpressed
has the incidental capacity to give an increase in ampicillin resistance. And the
DAM methylase gene is capable of regulating the expression of other genes,
including multidrug efflux pumps.
CONCLUSION: In this report we describe the evolution of antibiotic resistance in 
bacteria mediated by the epigenetic inheritance of variant gene expression
patterns. This provides proof in principle that epigenetic inheritance, as well
as DNA mutation, can drive evolution.

DOI: 10.1186/1471-2148-8-52 
PMCID: PMC2262874
PMID: 18282299  [Indexed for MEDLINE]


777. Genetics. 2008 Feb;178(2):1049-60. doi: 10.1534/genetics.107.082040. Epub 2008
Feb 1.

Metabolic changes associated with adaptive diversification in Escherichia coli.

Le Gac M(1), Brazas MD, Bertrand M, Tyerman JG, Spencer CC, Hancock RE, Doebeli
M.

Author information: 
(1)Department of Zoology, University of British Columbia, Vancouver, British
Columbia V6T 1Z4, Canada. legac@zoology.ubc.ca

During a 1000-generation evolution experiment, two types of morphologically and
kinetically distinct bacteria repeatedly diverged from a common ancestor in a
fully sympatric seasonal environment containing glucose and acetate. To
investigate the metabolic modifications associated with this adaptive
diversification, we compared transcription profiles of the two derived types and 
the common ancestor. Both derived types share a suite of common metabolic changes
that may represent adaptation to the environment preceding the diversification
event. These include improved translation efficiency, glucose uptake capacity via
the mal/lamB genes, upregulation of various transporters during stationary phase,
and likely the disruption of the rbs operon. The diversification event is
associated with the overexpression of genes involved in the TCA cycle, glyoxylate
shunt, acetate consumption, and anaerobic respiration in one type and in acetate 
excretion in the other. These results reveal that competition for both carbon and
oxygen have likely played an important role in the adaptation of Escherichia coli
during this adaptive diversification event, where one derived type mainly
consumes glucose at a fast rate when oxygen is not limiting, and the other
derived type consumes glucose and acetate at a slower rate, even when oxygen is
limiting.

DOI: 10.1534/genetics.107.082040 
PMCID: PMC2248342
PMID: 18245349  [Indexed for MEDLINE]


778. Infect Immun. 2008 Feb;76(2):845-56. Epub 2007 Dec 10.

Genetic diversity among Escherichia coli O157:H7 isolates and identification of
genes linked to human infections.

Wu G(1), Carter B, Mafura M, Liebana E, Woodward MJ, Anjum MF.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories Agency
-Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT13 3NB, United Kingdom.
g.wu@vla.defra.gsi.gov.uk

An Escherichia coli oligonucleotide microarray based on three sequenced genomes
was validated for comparative genomic microarray hybridization and used to study 
the diversity of E. coli O157 isolates from human infections and food and animal 
sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and
-negative strains) were found to be related to the two sequenced E. coli O157:H7 
strains, EDL933 and Sakai. However, these strains showed much greater genetic
diversity than those reported previously, and most of them could not be
categorized as either lineage I or II. Some genes were found more often in
isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976,
associated with stx2AB and stx1AB, were in all isolates from human sources but in
only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or
-dominant genes were also more frequently associated with isolates from human.
The results suggested that it might be more effective to concentrate our efforts 
on finding markers that are directly related to infection rather than those
specific to certain lineages. In addition, two Stx-negative O157 cattle isolates 
(one confirmed to be H7) were significantly different from other Stx-positive and
-negative E. coli O157:H7 strains and were more similar to MG1655 in their gene
content. This work demonstrates that not all E. coli O157:H7 strains belong to
the same clonal group, and those that were similar to E. coli K-12 might be less 
virulent.

DOI: 10.1128/IAI.00956-07 
PMCID: PMC2223464
PMID: 18070900  [Indexed for MEDLINE]


779. Infect Immun. 2008 Feb;76(2):695-703. Epub 2007 Nov 26.

Molecular basis of commensalism in the urinary tract: low virulence or virulence 
attenuation?

Zdziarski J(1), Svanborg C, Wullt B, Hacker J, Dobrindt U.

Author information: 
(1)Institut für Molekulare Infektionsbiologie, Julius-Maximilians-Universität
Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

In some patients, Escherichia coli strains establish significant bacteriuria
without causing symptoms of urinary tract infection (UTI). These
asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence
factors than the uropathogenic E. coli (UPEC) strains that cause severe,
symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence
genes, and the molecular basis of their low virulence therefore remains unclear. 
This study examined whether ABU strains might evolve from UPEC by genome loss and
virulence gene attenuation. The presence of conserved E. coli K-12 genes was
examined using an E. coli K-12 strain MG1655-specific DNA array and the
distribution of UPEC virulence-related genes was examined with the E. coli
pathoarray. Two groups of strains could be distinguished. Several ABU strains
were shown by multilocus sequence typing and by comparative genomic analyses to
be related to UPEC but to have smaller genome sizes. There were significant
alterations in essential virulence genes, including reductive evolution by point 
mutations, DNA rearrangements, and deletions. Other strains were unrelated to
UPEC and lacked most of the virulence-associated genes. The results suggest that 
some ABU strains arise from virulent strains by attenuation of virulence genes
while others are nonvirulent and resemble commensal strains. We propose that
virulence attenuation might constitute a general mechanism for mucosal pathogens 
to evolve toward commensalism.

DOI: 10.1128/IAI.01215-07 
PMCID: PMC2223460
PMID: 18039831  [Indexed for MEDLINE]


780. Insect Mol Biol. 2008 Feb;17(1):39-51. doi: 10.1111/j.1365-2583.2008.00778.x.

The Gram-negative bacteria-binding protein gene family: its role in the innate
immune system of anopheles gambiae and in anti-Plasmodium defence.

Warr E(1), Das S, Dong Y, Dimopoulos G.

Author information: 
(1)W. Harry Feinstone Department of Molecular Microbiology and Immunology,
Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street,
Baltimore, MD 21205-2179, USA.

Gram-negative bacteria-binding proteins (GNBPs) are pattern recognition receptors
which contribute to the defensive response against Plasmodium infection in
Anopheles. We have characterized the GNBP gene family in Anopheles gambiae at the
molecular level, and show that they are functionally diverse components of the A.
gambiae innate immune system. GNBPB4 is a major factor in the defence against a
broad range of pathogens, while the other GNBPs have narrower defence
specificities. GNBPB4 is associated with the regulation of immune signalling
pathways and was found to interact with the Gram-negative Escherichia coli and
weakly co-localized with Plasmodium berghei ookinetes in the mosquito midgut
epithelium.

DOI: 10.1111/j.1365-2583.2008.00778.x 
PMID: 18237283  [Indexed for MEDLINE]


781. Microbiology. 2008 Feb;154(Pt 2):608-18. doi: 10.1099/mic.0.2007/012146-0.

Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by
FNR, NarL and NarP.

Partridge JD(1), Browning DF, Xu M, Newnham LJ, Scott C, Roberts RE, Poole RK,
Green J.

Author information: 
(1)Department of Molecular Biology and Biotechnology, The University of
Sheffield, Sheffield, UK.

In Escherichia coli K-12 the expression of many genes is controlled by the
oxygen-responsive transcription factor FNR and the nitrate- and
nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is 
shown to be the first gene of a six-gene operon (ydhYVWXUT) that encodes proteins
predicted to be components of an oxidoreductase. Mapping the ydhY-T transcript
start and site-directed mutagenesis confirmed that the ydhY-T genes are
transcribed from an FNR-dependent class II promoter and showed that the FNR site 
is centred at -42.5. In the presence of nitrate or nitrite, NarXL and NarPQ
repressed ydhY-T expression. Analysis of the DNA sequence of the ydhY promoter
region (PydhY) revealed the presence of four heptameric sequences similar to
NarL/P binding sites centred at -42, -16, +6 and +15. The latter heptamers are
arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP.
Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation 
of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression,
whereas mutation of heptamer -42 and heptamer -16 did not affect the response to 
nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to
NarP, the NarL footprint extended further upstream to encompass the -16 heptamer.
The extended NarL footprint was consistent with the presence of multiple
NarL-PydhY complexes in gel retardation assays. Mutation of heptamer -42, which
is located within the FNR binding site, or heptamer +6 (but not heptamers -16 or 
+15) impaired nitrate-mediated repression. Thus, although the region of the
ydhY-T promoter containing the -16 and +15 heptamers was recognized by NarL in
vitro, mutation of these heptamers did not affect NarL-mediated repression in
vivo.

DOI: 10.1099/mic.0.2007/012146-0 
PMID: 18227264  [Indexed for MEDLINE]


782. Microbiology. 2008 Feb;154(Pt 2):559-70. doi: 10.1099/mic.0.2007/013334-0.

Genomic comparison of the O-antigen biosynthesis gene clusters of Escherichia
coli O55 strains belonging to three distinct lineages.

Iguchi A(1), Ooka T, Ogura Y, Asadulghani, Nakayama K, Frankel G, Hayashi T.

Author information: 
(1)Division of Bioenvironmental Science, Frontier Science Research Center,
University of Miyazaki, Miyazaki, Japan.

Typical enteropathogenic Escherichia coli (EPEC) O55 : H7 is regarded as the
closest relative of enterohaemorrhagic E. coli (EHEC) O157 : H7. Both serotypes
usually express the gamma1 intimin subclass and trigger actin polymerization by
the Tir-TccP pathway. However, atypical O55 : H7 strains capable of triggering
actin polymerization via the Tir-Nck pathway have recently been identified. In
this study, we investigated the genotypic differences and phylogenetic
relationships between typical and atypical O55 : H7 strains. We show that the
atypical O55 : H7 strains, which express the theta intimin subclass and lack both
tccP and tccP2, belong to an E. coli lineage distinct from the typical O55 : H7
and from the EPEC O55 : H6, which also uses the Tir-Nck actin polymerization
pathway. We conducted genomic comparisons of the chromosomal regions covering the
O-antigen gene cluster and its flanking regions between the three O55 lineages by
RFLP analysis of PCR products and DNA sequencing analysis of about 65 kb
chromosomal regions. This unexpectedly revealed that horizontal transfer of large
fragments (> or =40 kb) encoding the O55-antigen gene cluster and part of the
neighbouring colanic acid gene cluster was involved in the emergence of the three
O55 E. coli lineages. The data provide new insights into the mechanisms involved 
in the generation of a wide variety of O-serotypes in Gram-negative bacteria.

DOI: 10.1099/mic.0.2007/013334-0 
PMID: 18227260  [Indexed for MEDLINE]


783. Mol Microbiol. 2008 Feb;67(3):516-27. Epub 2007 Dec 9.

Activation of the Cpx regulon destabilizes the F plasmid transfer activator,
TraJ, via the HslVU protease in Escherichia coli.

Lau-Wong IC(1), Locke T, Ellison MJ, Raivio TL, Frost LS.

Author information: 
(1)Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada T6G 2E9.

The Escherichia coli CpxAR two-component signal transduction system senses and
responds to extracytoplasmic stress. The cpxA101* allele was previously found to 
reduce F plasmid conjugation by post-transcriptional inactivation of the positive
activator TraJ. Microarray analysis revealed upregulation of the
protease-chaperone pair, HslVU, which was shown to degrade TraJ in an E. coli
C600 cpxA101* background. Double mutants of cpxA101* and hslV or hslU restored
TraJ and F conjugation to wild-type levels. The constitutive overexpression of
nlpE, an outer membrane lipoprotein that induces the Cpx stress response, also
led to HslVU-mediated degradation of TraJ and repression of F transfer. However, 
Cpx-mediated TraJ degradation appears to be growth phase-dependent, as induction 
of nlpE in mid-log phase cells did not appreciably alter TraJ levels. Further,
His6-TraJ was sensitive to HslVU degradation in vitro only when it was purified
from cells overexpressing nlpE. Thus, TraJ appears to become resistant to HslVU
during normal growth, with this resistance mapping to the F transfer region.
Extracytoplasmic stress prevents this modification of TraJ, leaving it
susceptible to HslVU. Thus, the CpxAR stress response indirectly controls the
synthesis of the F mating apparatus, a complex transenvelope type IV secretion
system, by degrading TraJ.

DOI: 10.1111/j.1365-2958.2007.06055.x 
PMID: 18069965  [Indexed for MEDLINE]


784. PLoS Genet. 2008 Feb;4(2):e35. doi: 10.1371/journal.pgen.0040035.

Expression profiles reveal parallel evolution of epistatic interactions involving
the CRP regulon in Escherichia coli.

Cooper TF(1), Remold SK, Lenski RE, Schneider D.

Author information: 
(1)Department of Biology and Biochemistry, University of Houston, Houston, Texas,
United States of America. tcooper@central.uh.edu

The extent and nature of epistatic interactions between mutations are issues of
fundamental importance in evolutionary biology. However, they are difficult to
study and their influence on adaptation remains poorly understood. Here, we use a
systems-level approach to examine epistatic interactions that arose during the
evolution of Escherichia coli in a defined environment. We used expression arrays
to compare the effect on global patterns of gene expression of deleting a central
regulatory gene, crp. Effects were measured in two lineages that had
independently evolved for 20,000 generations and in their common ancestor. We
found that deleting crp had a much more dramatic effect on the expression profile
of the two evolved lines than on the ancestor. Because the sequence of the crp
gene was unchanged during evolution, these differences indicate epistatic
interactions between crp and mutations at other loci that accumulated during
evolution. Moreover, a striking degree of parallelism was observed between the
two independently evolved lines; 115 genes that were not crp-dependent in the
ancestor became dependent on crp in both evolved lines. An analysis of changes in
crp dependence of well-characterized regulons identified a number of regulatory
genes as candidates for harboring beneficial mutations that could account for
these parallel expression changes. Mutations within three of these genes have
previously been found and shown to contribute to fitness. Overall, these findings
indicate that epistasis has been important in the adaptive evolution of these
lines, and they provide new insight into the types of genetic changes through
which epistasis can evolve. More generally, we demonstrate that expression
profiles can be profitably used to investigate epistatic interactions.

DOI: 10.1371/journal.pgen.0040035 
PMCID: PMC2242816
PMID: 18282111  [Indexed for MEDLINE]

Conflict of interest statement: Competing interests. The authors have declared
that no competing interests exist.


785. BMC Genomics. 2008 Jan 29;9:53. doi: 10.1186/1471-2164-9-53.

A process for analysis of microarray comparative genomics hybridisation studies
for bacterial genomes.

Carter B(1), Wu G, Woodward MJ, Anjum MF.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories
Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK.
b.carter@vla.defra.gsi.gov.uk

BACKGROUND: Microarray based comparative genomic hybridisation (CGH) experiments 
have been used to study numerous biological problems including understanding
genome plasticity in pathogenic bacteria. Typically such experiments produce
large data sets that are difficult for biologists to handle. Although there are
some programmes available for interpretation of bacterial transcriptomics data
and CGH microarray data for looking at genetic stability in oncogenes, there are 
none specifically to understand the mosaic nature of bacterial genomes.
Consequently a bottle neck still persists in accurate processing and mathematical
analysis of these data. To address this shortfall we have produced a simple and
robust CGH microarray data analysis process that may be automated in the future
to understand bacterial genomic diversity.
RESULTS: The process involves five steps: cleaning, normalisation, estimating
gene presence and absence or divergence, validation, and analysis of data from
test against three reference strains simultaneously. Each stage of the process is
described and we have compared a number of methods available for characterising
bacterial genomic diversity, for calculating the cut-off between gene presence
and absence or divergence, and shown that a simple dynamic approach using a
kernel density estimator performed better than both established, as well as a
more sophisticated mixture modelling technique. We have also shown that current
methods commonly used for CGH microarray analysis in tumour and cancer cell lines
are not appropriate for analysing our data.
CONCLUSION: After carrying out the analysis and validation for three sequenced
Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic
test strains were used to demonstrate the benefits of applying this simple and
robust process to CGH microarray studies using bacterial genomes.

DOI: 10.1186/1471-2164-9-53 
PMCID: PMC2262894
PMID: 18230148  [Indexed for MEDLINE]


786. BMC Microbiol. 2008 Jan 23;8:12. doi: 10.1186/1471-2180-8-12.

Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced
lethality in a chicken embryo model.

Townsend MK(1), Carr NJ, Iyer JG, Horne SM, Gibbs PS, Prüss BM.

Author information: 
(1)Department of Veterinary and Microbiological Sciences, North Dakota State
University, Fargo, USA. megan.townsend@ndsu.edu

BACKGROUND: The Yersinia enterocolitica flagellar master regulator FlhD/FlhC
affects the expression levels of non-flagellar genes, including 21 genes that are
involved in central metabolism. The sigma factor of the flagellar system, FliA,
has a negative effect on the expression levels of seven plasmid-encoded virulence
genes in addition to its positive effect on the expression levels of eight of the
flagellar operons. This study investigates the phenotypes of flhD and fliA
mutants that result from the complex gene regulation.
RESULTS: Phenotypes relating to central metabolism were investigated with
Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA
mutants exhibited increased growth on purines and reduced growth on
N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both
mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. 
Several intermediates of the tricarboxylic acid and the urea cycle, as well as
several dipeptides, provided differential growth conditions for the two mutants. 
Gene expression was determined for selected genes and correlated with the
observed phenotypes. Phenotypes relating to virulence were determined with the
chicken embryo lethality assay. The assay that was previously established for
Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant
caused reduced chicken embryo lethality when compared to wild-type bacteria. In
contrast, the fliA mutant caused wild-type lethality. This indicates that the
virulence phenotype of the flhD mutant might be due to genes that are regulated
by FlhD/FlhC but not FliA, such as those that encode the flagellar type III
secretion system.
CONCLUSION: Phenotypes of flhD and fliA mutants are related to central metabolism
and virulence and correlate with gene regulation.

DOI: 10.1186/1471-2180-8-12 
PMCID: PMC2262085
PMID: 18215272  [Indexed for MEDLINE]


787. Adv Appl Microbiol. 2008;65:93-113. doi: 10.1016/S0065-2164(08)00604-7.

Global regulators of transcription in Escherichia coli: mechanisms of action and 
methods for study.

Grainger DC(1), Busby SJ.

Author information: 
(1)Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,
UK.

DOI: 10.1016/S0065-2164(08)00604-7 
PMID: 19026863  [Indexed for MEDLINE]


788. Appl Environ Microbiol. 2008 Jan;74(2):535-9. Epub 2007 Nov 26.

The BaeSR two-component regulatory system mediates resistance to condensed
tannins in Escherichia coli.

Zoetendal EG(1), Smith AH, Sundset MA, Mackie RI.

Author information: 
(1)Department of Animal Sciences and Division of Nutritional Sciences, Institute 
for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana,
Illinois, USA.

The gene expression profiles of Escherichia coli strains grown anaerobically with
or without Acacia mearnsii (black wattle) extract were compared to identify
tannin resistance strategies. The cell envelope stress protein gene spy and the
multidrug transporter-encoding operon mdtABCD, both under the control of the
BaeSR two-component regulatory system, were significantly up-regulated in the
presence of tannins. BaeSR mutants were more tannin sensitive than their
wild-type counterparts.

DOI: 10.1128/AEM.02271-07 
PMCID: PMC2223256
PMID: 18039828  [Indexed for MEDLINE]


789. Dev Biol (Basel). 2008;132:153-9.

Unique co-expression of immune cell-related genes in IBDV resistant chickens
indicates the activation of specific cellular host-response mechanisms.

Koren E(1), Zhou H, Cahaner A, Heller ED, Pitcovski J, Lamont SJ.

Author information: 
(1)MIGAL, Kiryat Shmona, Israel.

Infectious bursal disease virus (IBDV) causes highly contagious,
immunosuppressive disease that leads to high mortality in young chickens. The
purpose of this study was to look for the genetic regulation of the immune acute 
immune response to IBDV in our selected lines. Chicks of a F2 generation of two
lines divergently selected for early high (HH) or low (LL) antibody (Ab) response
to Escherichia coLi vaccination were challenged with virulent IBDV. Viral load in
infected bursae was used to determine resistant (R) and susceptible (S) birds. By
using a 13K chicken cDNA microarray, and pooled spleen mRNA of R, S and
non-challenged, control (C) chicks, several genes were identified with
differential expression associated with host resistance to IBDV. These genes were
also subjected to RT-PCR on individual samples to verify the results obtained
from microarrays. The major finding was the co-upregulation of seven genes--ETS2,
H963, RGS1, ABIN-2, CREM/ICER, DUSP1 and CXCR4- in several R, but not S or C
individuals, and characterized by a high correlation of expression levels.
Resistance also generally coincided with reduced transcript levels of acute-phase
serum amyloid A (A-SAA) and increased levels of IL-8. Based on reported functions
of these genes, these findings suggest that resistance was mediated by the
activation of specific cellular mechanisms, indicated by increased activity of
splenic macrophages and T-lymphocytes 3 days post-challenge.

DOI: 10.1159/000317155 
PMID: 18817297  [Indexed for MEDLINE]


790. Folia Microbiol (Praha). 2008;53(4):295-302. doi: 10.1007/s12223-008-0046-6. Epub
2008 Aug 31.

In vitro antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs
complexes.

Bruno JG(1), Carrillo MP, Phillips T.

Author information: 
(1)Operational Technologies Corporation, San Antonio, TX 78229, USA.
john.bruno@otcorp.com

DNA aptamers were developed against lipopolysaccharide (LPS) from E. coli O111:B4
and shown to bind both LPS and E. coli by a colorimetric enzyme-based microplate 
assay. The polyclonal aptamers were coupled to human C1qrs protein either
directly using a bifunctional linker or indirectly using biotinylated aptamers
and a streptavidin-C1qrs complex. Both systems significantly reduced colony
counts when applied to E. coli O111:B4 and K12 strains across a series of 10x
dilutions of the bacteria in the presence of human serum; it was diluted 1: 10(3)
in order to avoid significant bacterial lysis by the competing alternate pathway 
of complement activation. A number of candidate DNA aptamer sequences were cloned
and sequenced from the anti-LPS aptamer library for future screening of
antibacterial or "antibiotic" potential and to aid in eventual development of an 
alternative therapy for antibiotic-resistant bacterial infections.

DOI: 10.1007/s12223-008-0046-6 
PMID: 18759112  [Indexed for MEDLINE]


791. Genome Inform. 2008;21:42-52.

Phenotype profiling of single gene deletion mutants of E. coli using Biolog
technology.

Tohsato Y(1), Mori H.

Author information: 
(1)Department of Bioscience and Bioinformatics, Ritsumeikan University, Kusatsu, 
Shiga, Japan. yukako@sk.ritsumei.ac.jp

Phenotype MicroArray (PM) technology is high-throughput phenotyping system and is
directly applicable to assay the effects of genetic changes in cells. In this
study, we performed comprehensive PM analysis using single gene deletion mutants 
of central metabolic pathway and related genes. To elucidate the structure of
central metabolic networks in Escherichia coli K-12, we focused 288 different PM 
conditions of carbon and nitrogen sources and performed bioinformatic analysis.
For data processing, we employed noise reduction procedures. The distance between
each of the mutants was defined by Manhattan distance and agglomerative Ward's
hierarchical method was applied for clustering analysis. As a result, five
clusters were revealed which represented to activate or repress cellular
respiratory activities. Furthermore, the results might suggest that
Glyceraldehyde-3P plays a key role as a molecular switch of central metabolic
network.


PMID: 19425146  [Indexed for MEDLINE]


792. J Bacteriol. 2008 Jan;190(1):438-41. Epub 2007 Nov 2.

Dam methyltransferase is required for stable lysogeny of the Shiga toxin
(Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7.

Murphy KC(1), Ritchie JM, Waldor MK, Løbner-Olesen A, Marinus MG.

Author information: 
(1)University of Massachusetts Medical School, 364 Plantation Street, Worcester, 
MA 01605, USA.

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic
Escherichia coli, is encoded by 933W, a lambda-like prophage. 933W prophage
induction contributes to Stx2 production, and here, we provide evidence that Dam 
methyltransferase is essential for maintenance of 933W lysogeny. Our findings are
consistent with the idea that the 933W prophage has a relatively low threshold
for induction, which may promote Stx2 production during infection.

DOI: 10.1128/JB.01373-07 
PMCID: PMC2223730
PMID: 17981979  [Indexed for MEDLINE]


793. Mediators Inflamm. 2008;2008:725854. doi: 10.1155/2008/725854.

Prolonged classical NF-kappaB activation prevents autophagy upon E. coli
stimulation in vitro: a potential resolving mechanism of inflammation.

Schlottmann S(1), Buback F, Stahl B, Meierhenrich R, Walter P, Georgieff M,
Senftleben U.

Author information: 
(1)Department of Anesthesiology and Intensive Care, University of Ulm, 89075 Ulm,
Germany.

Activation of NF-kappaB is known to prevent apoptosis but may also act as
proapoptotic factor in order to eliminate inflammatory cells. Here, we show that 
classical NF-kappaB activation in RAW 264.7 and bone marrow-derived macrophages
upon short E. coli coculture is necessary to promote cell death at late time
points. At 48 hours subsequent to short-term, E. coli challenge increased
survival of NF-kappaB-suppressed macrophages was associated with pattern of
autophagy whereas macrophages with normal NF-kappaB signalling die. Cell death of
normal macrophages was indicated by preceding downregulation of autophagy
associated genes atg5 and beclin1. Restimulation of macrophages with LPS at 48
hours after E. coli treatment results in augmented proinflammatory cytokine
production in NF-kappaB-suppressed macrophages compared to control cells. We thus
demonstrate that classical NF-kappaB activation inhibits autophagy and promotes
delayed programmed cell death. This mechanism is likely to prevent the recovery
of inflammatory cells and thus contributes to the resolution of inflammation.

DOI: 10.1155/2008/725854 
PMCID: PMC2430012
PMID: 18566685  [Indexed for MEDLINE]


794. Methods Enzymol. 2008;447:47-64. doi: 10.1016/S0076-6879(08)02203-9.

Genomic analysis of mRNA decay in E. coli with DNA microarrays.

Lin PH(1), Singh D, Bernstein JA, Lin-Chao S.

Author information: 
(1)Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.

The decay of mRNA plays an important role in the regulation of gene expression.
Although relatively ignored for many years and regarded as a simple
ribonucleotide salvage pathway, mRNA decay has been established in recent years
as a well-defined cellular process that plays an integral role in determining
gene expression. The recent application of microarray methods to the study of
diverse organisms will help us to better understand these gene regulatory
circuits and the influence of transcript stability on gene expression. DNA
microarray technology is the method of choice to study individual mRNA half-lives
on a global scale. It is important to standardize these methods to generate
reproducible and reliable results. In this chapter, we describe experimental
designs for the analysis of mRNA decay on a genome-wide scale and provide
detailed protocols for each experimental step. We also present an analysis of the
decay of chromosomally encoded mRNAs in E. coli.

DOI: 10.1016/S0076-6879(08)02203-9 
PMID: 19161837  [Indexed for MEDLINE]


795. Methods Enzymol. 2008;437:499-519. doi: 10.1016/S0076-6879(07)37025-0.

Microbial responses to nitric oxide and nitrosative stress: growth, "omic," and
physiological methods.

Pullan ST(1), Monk CE, Lee L, Poole RK.

Author information: 
(1)Department of Molecular Biology and Biotechnology, University of Sheffield,
Sheffield, United Kingdom.

The study of bacterial responses to nitric oxide (NO), nitrosating agents, and
other agents of nitrosative stress has a short history but has rapidly produced
important insights into the interactions of these agents with model microbial
systems as well as pathogenic species. Several methodological problems arise in
attempting to define the global responses to these agents, whether in simply
measuring growth or performing "omic" experiments in which the objective is to
determine the genome-wide (transcriptomic) or proteome-wide responses. The first 
problem is the relatively long timescale over which the experiments are
conducted--minutes, hours, or days in the case of slow-growing cultures. The
second problem is not unique to NO and its congeners but concerns the
difficulties encountered when sensitive and comprehensive analytical techniques
(such as transcriptomics) are applied to cultures whose growth and physiology are
perturbed by an inhibitor. In essence, the problem is "seeing the wood for the
trees." This chapter reviews briefly the state of knowledge of NO responses and
mechanisms in bacteria, particularly Escherichia coli and Campylobacter jejuni.
Continuous culture has several advantages for investigating the consequences of
NO exposure, and this approach is outlined with examples of recent results and
conclusions. The major advantage of the chemostat is establishment of a
reproducible quasi-steady state in growth, in which the growth rate can be
controlled and maintained. Contrary to common belief, neither the concept nor the
apparatus is difficult. Commercially available and homemade systems are described
with practical advice. Establishing continuous cultures paves the way for other
"omic" approaches, particularly proteomics and metabolomics, which are not
covered here, as their application to the field of NO biology is in its infancy. 
A key to the literature describing methods suitable for assessing toxicity to
microbes of NO and reactive nitrogen species is given.

DOI: 10.1016/S0076-6879(07)37025-0 
PMID: 18433644  [Indexed for MEDLINE]


796. Methods Enzymol. 2008;437:211-33. doi: 10.1016/S0076-6879(07)37012-2.

Genome-wide identification of binding sites for the nitric oxide-sensitive
transcriptional regulator NsrR.

Efromovich S(1), Grainger D, Bodenmiller D, Spiro S.

Author information: 
(1)Department of Mathematical Sciences, University of Texas at Dallas,
Richardson, Texas, USA.

NsrR is a nitric oxide-sensitive regulator of transcription. In Escherichia coli,
NsrR is a repressor of the hmp gene encoding the flavohemoglobin that detoxifies 
nitric oxide. Three other transcription units (ytfE, ygbA, and hcp-hcr) are known
to be subject to regulation by NsrR. This chapter describes experimental and
statistical protocols used to identify NsrR-binding sites in the E. coli
chromosome using chromatin immunoprecipitation and microarray analysis. The
methods are applicable, with suitable modifications, to any regulatory protein
and any organism.

DOI: 10.1016/S0076-6879(07)37012-2 
PMID: 18433631  [Indexed for MEDLINE]


797. Microbiology. 2008 Jan;154(Pt 1):167-75. doi: 10.1099/mic.0.2007/011981-0.

The ferric yersiniabactin uptake receptor FyuA is required for efficient biofilm 
formation by urinary tract infectious Escherichia coli in human urine.

Hancock V(1), Ferrières L, Klemm P.

Author information: 
(1)Microbial Adhesion Group, BioCentrum-DTU, Technical University of Denmark,
DK-2800 Lyngby, Denmark.

Urinary tract infection (UTI) is the most common infection in patients with
indwelling urinary catheters, and bacterial biofilm formation is a major problem 
in this type of infection. Escherichia coli is responsible for the large majority
of UTIs. Free iron is strictly limited in the human urinary tract and there is
fierce competition between the host and infectious bacteria for this essential
metal. Urinary tract infectious E. coli have highly efficient mechanisms of iron 
acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding
the yersiniabactin receptor, is one of the most upregulated genes in biofilm; it 
was upregulated 63-fold in the E. coli UTI strain VR50. FyuA was found to be
highly important for biofilm formation in iron-poor environments such as human
urine. Mutants in fyuA show aberrant biofilm formation and the cells become
filamentous; a VR50fyuA mutant showed a 92 % reduction in biofilm formation in
urine flow-cell chambers compared with the wild-type. The FyuA/yersiniabactin
system is known to be important for virulence. Here we demonstrate a direct link 
between FyuA and biofilm formation in iron-poor environments. We also show that
the availability of iron greatly influences UTI strains' ability to form biofilm.

DOI: 10.1099/mic.0.2007/011981-0 
PMID: 18174135  [Indexed for MEDLINE]


798. Microbiology. 2008 Jan;154(Pt 1):148-66. doi: 10.1099/mic.0.2007/012021-0.

Low temperature (23 degrees C) increases expression of biofilm-, cold-shock- and 
RpoS-dependent genes in Escherichia coli K-12.

White-Ziegler CA(1), Um S, Pérez NM, Berns AL, Malhowski AJ, Young S.

Author information: 
(1)Department of Biological Sciences and Program in Biochemistry, Smith College, 
Northampton, MA 01063, USA. cwhitezi@smith.edu

Temperature serves as a cue to regulate gene expression in Escherichia coli and
other bacteria. Using DNA microarrays, we identified 297 genes whose expression
is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these
genes, 122 are RpoS-controlled, confirming genome-wide the model that low
temperature serves as a primary cue to trigger the general stress response.
Several genes expressed at 23 degrees C overlap with the cold-shock response,
suggesting that strategies used to adapt to sudden shifts in temperature also
mediate long-term growth at 23 degrees C. Another category of genes more highly
expressed at 23 degrees C are associated with biofilm development, implicating
temperature as an important cue influencing this developmental pathway. In a
candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA,
yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and
DsrA-dependent for their transcription at 23 degrees C. In contrast,
transcription of three genes (ycgZ, dps and ymgB) was either partially or fully
independent of these regulators, signifying there is an alternative
thermoregulatory mechanism(s) that increases gene expression at 23 degrees C.
Increased expression at 23 degrees C compared to 37 degrees C is retained in
various media tested for most of the genes, supporting the relative importance of
this cue in adaptation to changing environments. Both the RpoS-dependent gene
otsA and the RpoS-independent gene ymgB demonstrated increased expression levels 
within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to 
this environmental cue. Despite changes in gene expression for many
RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and
viability at 4 degrees C did not demonstrate significant impairment in rpoS : :
Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain.
Biofilm formation was favoured at low temperature and is moderately impaired in
both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes
controlled by these regulators play a role necessary for optimal biofilm
formation at 23 degrees C. Taken together, our data demonstrate that a large
number of genes are increased in expression at 23 degrees C to globally respond
to this environmental change and that at least two thermoregulatory pathways are 
involved in co-ordinating this response - the RpoS/DsrA pathway and an
alternative thermoregulatory pathway, independent of these regulators.

DOI: 10.1099/mic.0.2007/012021-0 
PMID: 18174134  [Indexed for MEDLINE]


799. Mol Microbiol. 2008 Jan;67(2):336-49. Epub 2007 Nov 25.

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a
DsbD-independent manner.

Vertommen D(1), Depuydt M, Pan J, Leverrier P, Knoops L, Szikora JP, Messens J,
Bardwell JC, Collet JF.

Author information: 
(1)de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins.
DsbA is recycled by DsbB, which generates disulphides from quinone reduction.
DsbA is not known to have any proofreading activity and can form incorrect
disulphides in proteins with multiple cysteines. These incorrect disulphides are 
thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in
the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway
is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC
and DsbA pathways are more intimately connected than previously thought.
dsbA(-)dsbC(-) mutants have a number of phenotypes not exhibited by either
dsbA(-), dsbC(-) or dsbA(-)dsbD(-) mutations: they exhibit an increased
permeability of the outer membrane, are resistant to the lambdoid phage Phi80,
and are unable to assemble the maltoporin LamB. Using differential
two-dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry
analysis, we estimated the abundance of about 130 secreted proteins in various
dsb(-) strains. dsbA(-)dsbC(-) mutants exhibit unique changes at the protein
level that are not exhibited by dsbA(-)dsbD(-) mutants. Our data indicate that
DsbC can assist DsbA in a DsbD-independent manner to oxidatively fold envelope
proteins. The view that DsbC's function is limited to the disulphide
isomerization pathway should therefore be reinterpreted.

DOI: 10.1111/j.1365-2958.2007.06030.x 
PMCID: PMC2614554
PMID: 18036138  [Indexed for MEDLINE]


800. Mol Syst Biol. 2008;4:181. doi: 10.1038/msb.2008.20. Epub 2008 Apr 15.

Synthetic gene brushes: a structure-function relationship.

Buxboim A(1), Daube SS, Bar-Ziv R.

Author information: 
(1)Department of Materials and Interfaces, The Weizmann Institute of Science,
Rehovot, Israel.

We present the assembly of gene brushes by means of a photolithographic approach 
that allows us to control the density of end-immobilized linear double-stranded
DNA polymers coding for entire genes. For 2 kbp DNAs, the mean distance varies
from 300 nm, where DNAs are dilute and assume relaxed conformations, down to 30
nm, where steric repulsion at dense packing forces stretching out. We
investigated the gene-to-protein relationship of firefly luciferase under the
T7/E.Coli-extract expression system, as well as transcription-only reactions with
T7 RNA polymerase, and found both systems to be highly sensitive to brush
density, conformation, and orientation. A 'structure-function' picture emerges in
which extension of genes induced by moderate packing exposes coding sequences and
improves their interaction with the transcription/translation machinery. However,
tighter packing impairs the penetration of the machinery into the brush. The
response of expression to two-dimensional gene crowding at the nanoscale
identifies gene brushes as basic controllable units en route to multicomponent
synthetic systems. In turn, these brushes could deepen our understanding of
biochemical reactions taking place under confinement and molecular crowding in
living cells.

DOI: 10.1038/msb.2008.20 
PMCID: PMC2387232
PMID: 18414482  [Indexed for MEDLINE]


801. Nucleic Acids Res. 2008 Jan;36(Database issue):D519-23. Epub 2007 Nov 13.

Enteropathogen Resource Integration Center (ERIC): bioinformatics support for
research on biodefense-relevant enterobacteria.

Glasner JD(1), Plunkett G 3rd, Anderson BD, Baumler DJ, Biehl BS, Burland V,
Cabot EL, Darling AE, Mau B, Neeno-Eckwall EC, Pot D, Qiu Y, Rissman AI, Worzella
S, Zaremba S, Fedorko J, Hampton T, Liss P, Rusch M, Shaker M, Shaull L, Shetty
P, Thotakura S, Whitmore J, Blattner FR, Greene JM, Perna NT.

Author information: 
(1)Genome Center, University of Wisconsin, 425G Henry Mall, Madison, Madison, WI 
53703, USA. jglasner@wisc.edu

ERIC, the Enteropathogen Resource Integration Center (www.ericbrc.org), is a new 
web portal serving as a rich source of information about enterobacteria on the
NIAID established list of Select Agents related to biodefense-diarrheagenic
Escherichia coli, Shigella spp., Salmonella spp., Yersinia enterocolitica and
Yersinia pestis. More than 30 genomes have been completely sequenced, many more
exist in draft form and additional projects are underway. These organisms are
increasingly the focus of studies using high-throughput experimental technologies
and computational approaches. This wealth of data provides unprecedented
opportunities for understanding the workings of basic biological systems and
discovery of novel targets for development of vaccines, diagnostics and
therapeutics. ERIC brings information together from disparate sources and
supports data comparison across different organisms, analysis of varying data
types and visualization of analyses in human and computer-readable formats.

DOI: 10.1093/nar/gkm973 
PMCID: PMC2238966
PMID: 17999997  [Indexed for MEDLINE]


802. Nucleic Acids Res. 2008 Jan;36(Database issue):D866-70. Epub 2007 Oct 11.

Many Microbe Microarrays Database: uniformly normalized Affymetrix compendia with
structured experimental metadata.

Faith JJ(1), Driscoll ME, Fusaro VA, Cosgrove EJ, Hayete B, Juhn FS, Schneider
SJ, Gardner TS.

Author information: 
(1)Program in Bioinformatics, Boston University, 24 Cummington St. and Department
of Biomedical Engineering, Boston University, 44 Cummington St., Boston,
Massachusetts, 02215, USA.

Many Microbe Microarrays Database (M3D) is designed to facilitate the analysis
and visualization of expression data in compendia compiled from multiple
laboratories. M3D contains over a thousand Affymetrix microarrays for Escherichia
coli, Saccharomyces cerevisiae and Shewanella oneidensis. The expression data is 
uniformly normalized to make the data generated by different laboratories and
researchers more comparable. To facilitate computational analyses, M3D provides
raw data (CEL file) and normalized data downloads of each compendium. In
addition, web-based construction, visualization and download of custom datasets
are provided to facilitate efficient interrogation of the compendium for more
focused analyses. The experimental condition metadata in M3D is human curated
with each chemical and growth attribute stored as a structured and computable set
of experimental features with consistent naming conventions and units. All
versions of the normalized compendia constructed for each species are maintained 
and accessible in perpetuity to facilitate the future interpretation and
comparison of results published on M3D data. M3D is accessible at
http://m3d.bu.edu/.

DOI: 10.1093/nar/gkm815 
PMCID: PMC2238822
PMID: 17932051  [Indexed for MEDLINE]


803. Oncology. 2008;75(1-2):81-91. doi: 10.1159/000155210. Epub 2008 Sep 11.

Molecular profiling of isolated histological components of wilms tumor implicates
a common role for the Wnt signaling pathway in kidney and tumor development.

Maschietto M(1), de Camargo B, Brentani H, Grundy P, Sredni ST, Torres C, Mota
LD, Cunha IW, Patrão DF, Costa CM, Soares FA, Brentani RR, Carraro DM.

Author information: 
(1)Laboratories of Genomics and Molecular Biology, Hospital A.C. Camargo, São
Paulo, Brazil.

Wilms tumor (WT), a tumor composed of three histological components - blastema
(BL), epithelia and stroma - is considered an appropriate model system to study
the biological relationship between differentiation and tumorigenesis. To
investigate molecular associations between nephrogenesis and WT, the gene
expression pattern of individual cellular components was analyzed, using a
customized platform containing 4,608 genes. WT gene expression patterns were
compared to genes regulated during kidney differentiation. BL had a closer gene
expression pattern to the earliest stage of normal renal development. The BL gene
expression pattern was compared to that of fetal kidney (FK) and also between FK 
and mature kidney, identifying 25 common deregulated genes supposedly involved in
the earliest events of WT onset. Quantitative RT-PCR was performed, confirming
the difference in expression levels for 13 of 16 genes (81.2%) in the initial set
and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of
genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 
and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed
in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed
positive nuclear immunostaining for an independent set of WT samples, similarly
to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK
until week 18. Taken together, these results provided molecular evidence of the
recapitulation of the embryonic kidney by WT as well as involvement of the Wnt
pathway in the earliest events of WT onset.

Copyright 2008 S. Karger AG, Basel.

DOI: 10.1159/000155210 
PMID: 18784435  [Indexed for MEDLINE]


804. RNA Biol. 2008 Jan-Mar;5(1):30-40. Epub 2008 Feb 4.

Isolation of small RNA-binding proteins from E. coli: evidence for frequent
interaction of RNAs with RNA polymerase.

Windbichler N(1), von Pelchrzim F, Mayer O, Csaszar E, Schroeder R.

Author information: 
(1)Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

Bacterial small RNAs (sRNAs) are non-coding RNAs that regulate gene expression
enabling cells to adapt to various growth conditions. Assuming that most RNAs
require proteins to exert their activities, we purified and identified
sRNA-binding factors via affinity chromatography and mass spectrometry. We
consistently obtained RNA polymerase betasubunit, host factor Hfq and ribosomal
protein S1 as sRNA-binding proteins in addition to several other factors. Most
importantly, we observed that RNA polymerase not only binds several sRNAs but
also reacts with them, both cleaving and extending the RNAs at their 3' ends. The
fact that the RNA polymerase reacts with sRNAs maps their interaction site to the
active centre cleft of the enzyme and shows that it takes RNAs as template to
perform RNA-dependent RNA polymerase activity. We further performed genomic SELEX
to isolate RNA polymerase-binding RNAs and obtained a large number of E. coli
sequences binding with high affinity to this enzyme. In vivo binding of some of
the RNAs to the RNA polymerase was confirmed via co-immunoprecipitation in cell
extracts prepared from different growth conditions. Our observations show that
RNA polymerase is able to bind and react with many different RNAs and we suggest 
that RNAs are involved in transcriptional regulation more frequently than
anticipated.


PMID: 18388495  [Indexed for MEDLINE]


805. Wei Sheng Wu Xue Bao. 2008 Jan;48(1):103-11.

[Differential expression of virulence and potential virulence genes of avian
pathogenic Escherichia coli in vitro with DNA microarray analysis].

[Article in Chinese]

Huan H(1), Zhou Q, Zhao L, Gao S, Liu X.

Author information: 
(1)Animal Infectious Disease Laboratory, Ministry of Agriculture, Yangzhou
University, Yangzhou, Jiangsu 225009, China. coocean@yzu.edu.cn

We constructed avian pathogenic Escherichia coli (APEC) specific DNA microarray
to analyze the transcriptomes of APEC highly pathogenic strain E058 and low
pathogenic strain E526 (both belonging to O2 serotype). For cultures in
Luria-Bertani (LB) broth medium, 16 distinctly expressed genes were observed in
strain E526, and all of them were down-regulated. For cultures in the serum of
chickens, 15 distinctly expressed genes were screened in strain E526, and all of 
them were also down-regulated. The results suggest that DNA microarray could be
used to screen distinctly expressed genes among virulence- and potential
virulence-associated genes of APEC. The expression of 11 common virulence- or
potential virulence-associated genes were down-regulated for strain E526 compared
to those of strain E058 cultured both in LB broth and chicken serum. Meanwhile, 4
potential virulence-associated genes, aes-3, aes-10, aes-13 and aes-15 were
down-regulated for strain E526 but not for strain E058, when they grew in the
chicken serum. Besides known virulence factors, we observed 10 new potential
virulence-related genes including aes-1, aes-2, aes-3, aes-4, aes-6, aes-8,
aes-10, aes-13, aes-15 and aes-31.


PMID: 18338585  [Indexed for MEDLINE]


806. World J Gastroenterol. 2007 Dec 21;13(47):6370-8.

Probiotic bacteria change Escherichia coli-induced gene expression in cultured
colonocytes: Implications in intestinal pathophysiology.

Panigrahi P(1), Braileanu GT, Chen H, Stine OC.

Author information: 
(1)University of Maryland School of Medicine, Department of Pediatrics, 22 South 
Greene Street, N5W68, Baltimore, MD 21201, United States. ppanigrahi@pol.net

AIM: To investigate the change in eukaryotic gene expression profile in Caco-2
cells after infection with strains of Escherichia coli and commensal probiotic
bacteria.
METHODS: A 19,200 gene/expressed sequence tag gene chip was used to examine
expression of genes after infection of Caco-2 cells with strains of normal flora 
E. coli, Lactobacillus plantarum, and a combination of the two.
RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of
177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes.
During mixed infection, 27 genes were up-regulated and 59 were down-regulated,
with nullification of stimulatory/inhibitory effects on most of the genes.
Expression of several new genes was noted in this group.
CONCLUSION: The commensal bacterial strains used in this study induced the
expression of a large number of genes in colonocyte-like cultured cells and
changed the expression of several genes involved in important cellular processes 
such as regulation of transcription, protein biosynthesis, metabolism, cell
adhesion, ubiquitination, and apoptosis. Such changes induced by the presence of 
probiotic bacteria may shape the physiologic and pathologic responses they
trigger in the host.


PMCID: PMC4205456
PMID: 18081226  [Indexed for MEDLINE]


807. BMC Genomics. 2007 Dec 19;8:467.

Identification of chromosomal alpha-proteobacterial small RNAs by comparative
genome analysis and detection in Sinorhizobium meliloti strain 1021.

Ulvé VM(1), Sevin EW, Chéron A, Barloy-Hubler F.

Author information: 
(1)CNRS UMR6061 Génétique et Développement, Groupe Modèles Génétiques, Université
de Rennes 1, IFR140 GFAS, Faculté de médecine, 2 avenue du Professeur Léon
Bernard, CS 34317, 35043 Rennes Cedex, France. vincent.ulve@rennes.inra.fr

BACKGROUND: Small untranslated RNAs (sRNAs) seem to be far more abundant than
previously believed. The number of sRNAs confirmed in E. coli through various
approaches is above 70, with several hundred more sRNA candidate genes under
biological validation. Although the total number of sRNAs in any one species is
still unclear, their importance in cellular processes has been established.
However, unlike protein genes, no simple feature enables the prediction of the
location of the corresponding sequences in genomes. Several approaches, of
variable usefulness, to identify genomic sequences encoding sRNA have been
described in recent years.
RESULTS: We used a combination of in silico comparative genomics and
microarray-based transcriptional profiling. This approach to screening identified
~60 intergenic regions conserved between Sinorhizobium meliloti and related
members of the alpha-proteobacteria sub-group 2. Of these, 14 appear to
correspond to novel non-coding sRNAs and three are putative peptide-coding or 5' 
UTR RNAs (ORF smaller than 100 aa). The expression of each of these new small RNA
genes was confirmed by Northern blot hybridization.
CONCLUSION: Small non coding RNA (sra) genes can be found in the intergenic
regions of alpha-proteobacteria genomes. Some of these sra genes are only present
in S. meliloti, sometimes in genomic islands; homologues of others are present in
related genomes including those of the pathogens Brucella and Agrobacterium.

DOI: 10.1186/1471-2164-8-467 
PMCID: PMC2245857
PMID: 18093320  [Indexed for MEDLINE]


808. Bioorg Med Chem. 2007 Dec 15;15(24):7688-95. Epub 2007 Aug 31.

Elucidation of the RNA target of linezolid by using a linezolid-neomycin B
heteroconjugate and genomic SELEX.

Kim HJ(1), Kwon M, Yu J.

Author information: 
(1)Department of Chemistry and Education, Seoul National University, Seoul
151-742, Republic of Korea.

A covalently modified heteroconjugate between linezolid and neomycin B leads to
an enhanced and more specific binding affinity to hairpin RNA targets in
comparison to neomycin B itself. This heteroconjugate was used as a lure to
select linezolid-specific hairpin RNA from an Escherichia coli genome RNA. The
selected RNA obtained after eight cycles not only has typical stem-loop
structures but also includes known sequences of the linezolid binding site. The
results of RNA footprinting show that the binding site of the heteroconjugate
encompasses both stem and loop regions, suggesting that the possible binding site
for linezolid is in the terminal loop. In addition, findings from application of 
a surface plasmon resonance assay clearly demonstrate that linezolid binds to
selected hairpin RNA in a highly specific manner with a low millimolar affinity. 
The results suggest that heteroconjugates might represent a generally useful
approach in studies aimed at uncovering loop-specific RNA binding ligands that
would be otherwise difficult to identify owing to their weak affinities.

DOI: 10.1016/j.bmc.2007.08.053 
PMID: 17869523  [Indexed for MEDLINE]


809. Appl Environ Microbiol. 2007 Dec;73(23):7657-63. Epub 2007 Oct 5.

Improvement of NADPH-dependent bioconversion by transcriptome-based molecular
breeding.

Hibi M(1), Yukitomo H, Ito M, Mori H.

Author information: 
(1)Biofrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahimachi,
Machida, Tokyo 194-8533, Japan.

Transcriptome data for a xylitol-producing recombinant Escherichia coli were
obtained and used to tune up its productivity. Structural genes of
NADPH-dependent D-xylose reductase and D-xylose permease were inserted into an
Escherichia coli chromosome to construct a recombinant strain producing xylitol
from D-xylose for use as a model system for NADPH-dependent bioconversion.
Transcriptome analysis of xylitol-producing and nonproducing conditions for the
recombinant revealed that xylitol production down-regulated 56 genes. These genes
were then selected as candidate factors for suppression of the NADPH supply and
were disrupted to validate their functions. Of the gene disruptants, that
resulting from the deletion of yhbC showed the best bioconversion rate. Also, the
deletion accelerated cell growth during log phase. The features of the mutant
could be maintained in jar fermenter-scale production of xylitol. Thus, our novel
molecular host strain breeding method using transcriptome analysis was fully
effective and could be applied to improving various industrial strains.

DOI: 10.1128/AEM.01754-07 
PMCID: PMC2168071
PMID: 17921263  [Indexed for MEDLINE]


810. Appl Environ Microbiol. 2007 Dec;73(23):7757-8. Epub 2007 Sep 21.

Microarray analysis of Escherichia coli strains from interstitial beach waters of
Lake Huron (Canada).

Kon T(1), Weir SC, Trevors JT, Lee H, Champagne J, Meunier L, Brousseau R, Masson
L.

Author information: 
(1)Department of Environmental Biology, University of Guelph, Guelph, Ontario,
Canada N1G 2W1.

DNA microarray analyses revealed that clusters of repetitive extragenic
palindromic PCR-related Escherichia coli isolates were isogenic only within
interstitial Lake Huron beach water samples and not in surrounding waters. This
suggested that adaptation and growth occurred within the interstitial water sites
tested. All isolates were nonpathogenic, and three lake isolates possessed
tetracycline resistance genes.

DOI: 10.1128/AEM.01333-07 
PMCID: PMC2168082
PMID: 17890330  [Indexed for MEDLINE]


811. Insect Biochem Mol Biol. 2007 Dec;37(12):1338-47. Epub 2007 Sep 6.

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on
antibacterial peptide gene expression in Bombyx mori.

Ha Lee J(1), Hee Lee I, Noda H, Mita K, Taniai K.

Author information: 
(1)Invertebrate Gene Function Research Unit, National Institute of Agrobiological
Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B
gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of
CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella
minnesota were completely eliminated after digestion of the LPSs with muramidase.
The results clearly indicate that a polymer form of PGN in the LPSs elicited
CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes
on the array were activated by LPS in the cells. In contrast, E. coli PGN
strongly elicited five antibacterial peptide genes and numerous other genes, and 
PGN from Micrococcus luteus activated only several genes. Semi-quantitative
RT-PCR revealed that all antibacterial genes activated by both PGNs, but the
extents were 10-100 times higher with E. coli PGN. Similarly, higher elicitor
activity of E. coli than M. luteus was indicated using peptidoglycan recognition 
protein gene, which is involved in pro-phenol oxidase cascade.

DOI: 10.1016/j.ibmb.2007.08.007 
PMID: 17967352  [Indexed for MEDLINE]


812. Insect Mol Biol. 2007 Dec;16(6):761-76.

Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.

Bartholomay LC(1), Mayhew GF, Fuchs JF, Rocheleau TA, Erickson SM, Aliota MT,
Christensen BM.

Author information: 
(1)Department of Pathobiological Sciences, University of Wisconsin-Madison,
Madison, WI, USA.

Pathogens that infect and/or are transmitted by mosquitoes typically are exposed 
to the body cavity, and to haemocytes circulating therein, during development or 
dissemination. Aedes aegypti haemocytes produce a range of immune
response-related gene products, and an endpoint response of phagocytosis and/or
melanization that is temporally and structurally distinct for the invading
pathogen. Expressed sequence tags were generated from haemocyte libraries and
then used to design oligonucleotide microarrays. Arrays were screened with
haemocyte material collected 1-, 8- and 24-h post-inoculation with Escherichia
coli or Micrococcus luteus bacteria. Data from these studies support the
discovery of novel immune response-activated genes, provide an expanded
understanding of antimicrobial peptide biology and highlight the coordination of 
immune factors that leads to an endpoint response.

DOI: 10.1111/j.1365-2583.2007.00773.x 
PMID: 18093005  [Indexed for MEDLINE]


813. J Bacteriol. 2007 Dec;189(24):8855-62. Epub 2007 Oct 12.

Cohabitation of two different lexA regulons in Pseudomonas putida.

Abella M(1), Campoy S, Erill I, Rojo F, Barbé J.

Author information: 
(1)Departament de Genètica i Microbiologia, Ed. Cn, Universitat Autònoma de
Barcelona, 08193 Bellaterra, Spain.

In contrast to the vast majority of the members of the domain Bacteria, several
Pseudomonas and Xanthomonas species have two lexA genes, whose products have been
shown to recognize different LexA binding motifs, making them an interesting
target for studying the interplay between cohabiting LexA regulons in a single
species. Here we report an analysis of the genetic composition of the two LexA
regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The
data obtained indicate that one of the two LexA proteins (LexA1) seems to be in
control of the conventional Escherichia coli-like SOS response, while the other
LexA protein (LexA2) regulates only its own transcriptional unit, which includes 
the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida
prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA
damage-mediated induction of several P. putida resident prophage genes. In silico
searches suggested that this marked asymmetry in regulon contents also occurs in 
other Pseudomonas species with two lexA genes, and the implications of this
asymmetry in the evolution of the SOS network are discussed.

DOI: 10.1128/JB.01213-07 
PMCID: PMC2168638
PMID: 17933893  [Indexed for MEDLINE]


814. J Bacteriol. 2007 Dec;189(24):8944-52. Epub 2007 Oct 5.

Response of Desulfovibrio vulgaris to alkaline stress.

Stolyar S(1), He Q, Joachimiak MP, He Z, Yang ZK, Borglin SE, Joyner DC, Huang K,
Alm E, Hazen TC, Zhou J, Wall JD, Arkin AP, Stahl DA.

Author information: 
(1)University of Washington, Department of Civil and Environmental Engineering,
616 NE North Lake Place, Box 355014, Seattle, WA 98195-2700, USA.
sstolyar@washington.edu

The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 
10 stress was studied using oligonucleotide microarrays and a study set of
mutants with genes suggested by microarray data to be involved in the alkaline
stress response deleted. The data showed that the response of D. vulgaris to
increased pH is generally similar to that of Escherichia coli but is apparently
controlled by unique regulatory circuits since the alternative sigma factors
(sigma S and sigma E) contributing to this stress response in E. coli appear to
be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli
were up-regulated in D. vulgaris; these genes included three ATPase genes and a
tryptophan synthase gene. Transcription of chaperone and protease genes (encoding
ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As
in E. coli, genes involved in flagellum synthesis were down-regulated. The
transcriptional data also identified regulators, distinct from sigma S and sigma 
E, that are likely part of a D. vulgaris Hildenborough-specific stress response
system. Characterization of a study set of mutants with genes implicated in
alkaline stress response deleted confirmed that there was protective involvement 
of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative
regulators/histidine kinases (DVU0331 and DVU2580).

DOI: 10.1128/JB.00284-07 
PMCID: PMC2168612
PMID: 17921288  [Indexed for MEDLINE]


815. J Bacteriol. 2007 Dec;189(24):8772-85. Epub 2007 Aug 31.

Analysis of promoter targets for Escherichia coli transcription elongation factor
GreA in vivo and in vitro.

Stepanova E(1), Lee J, Ozerova M, Semenova E, Datsenko K, Wanner BL, Severinov K,
Borukhov S.

Author information: 
(1)Department of Cell Biology, UMDNJ-SOM at Stratford, 2 Medical Center Drive,
Stratford, NJ 08084-1489, USA.

Comment in
    J Bacteriol. 2007 Dec;189(24):8769-71.

Transcription elongation factor GreA induces nucleolytic activity of bacterial
RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to
transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating 
RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is 
unclear which of these functions is (are) most relevant in vivo. By comparing
global gene expression profiles of Escherichia coli strains lacking Gre factors
and strains expressing either the wild type (wt) or a functionally inactive GreA 
mutant, we identified genes that are potential targets of GreA action. Data
analysis revealed that in the presence of chromosomally expressed GreA, 19 genes 
are upregulated; an additional 105 genes are activated upon overexpression of the
wt but not the mutant GreA. Primer extension reactions with selected
transcription units confirmed the gene array data. The most prominent stimulatory
effect (threefold to about sixfold) of GreA was observed for genes of ribosomal
protein operons and the tna operon, suggesting that transcript cleavage by GreA
contributes to optimal expression levels of these genes in vivo. In vitro
transcription assays indicated that the stimulatory effect of GreA upon the
transcription of these genes is mostly due to increased RNAP recycling due to
facilitated promoter escape. We propose that transcript cleavage during early
stages of initiation is thus the main in vivo function of GreA. Surprisingly, the
presence of the wt GreA also led to the decreased transcription of many genes.
The mechanism of this effect is unknown and may be indirect.

DOI: 10.1128/JB.00911-07 
PMCID: PMC2168603
PMID: 17766423  [Indexed for MEDLINE]


816. J Biomed Inform. 2007 Dec;40(6):698-706. Epub 2007 Feb 22.

Mining pathway signatures from microarray data and relevant biological knowledge.

Panteris E(1), Swift S, Payne A, Liu X.

Author information: 
(1)School of Information Systems, Computing and Mathematics, Brunel University,
Uxbridge, Middlesex UB8 3PH, UK. eleftherios.panteris@brunel.ac.uk

High-throughput technologies such as DNA microarray are in the process of
revolutionizing the way modern biological research is being done. Bioinformatics 
tools are becoming increasingly important to assist biomedical scientists in
their quest in understanding complex biological processes. Gene expression
analysis has attracted a large amount of attention over the last few years mostly
in the form of algorithms, exploring cluster and regulatory relationships among
genes of interest, and programs that try to display the multidimensional
microarray data in appropriate formats so that they make biological sense. To
reduce the dimensionality of microarray data and make the corresponding analysis 
more biologically relevant, in this paper we propose a biologically-led approach 
to biochemical pathway analysis using microarray data and relevant biological
knowledge. The method selects a subset of genes for each pathway that describes
the behaviour of the pathway at a given experimental condition, and transforms
them into pathway signatures. The metabolic pathways of Escherichia coli are used
as a case study.

DOI: 10.1016/j.jbi.2007.01.004 
PMID: 17395545  [Indexed for MEDLINE]


817. FEMS Microbiol Lett. 2007 Nov;276(2):202-6.

Global gene expression profiling of wild type and lysC knockout Escherichia coli 
W3110.

Liu DY(1), Liu CH, Lai MT, Lin HK, Hseu TH.

Author information: 
(1)Department of Life Science, Institute of Biotechnology, National Tsing Hua
University, Hsinchu, Taiwan.

Aspartokinase III, encoded by lysC, is responsible for the first step of lysine
biosynthesis in Escherichia coli. In this study, a lysC knockout E. coli W3110
strain was generated to study the differential gene expression profiles of wild
type and lysC knockout strains. Several significant changes were observed,
including biosynthesis of lysine, oxaloacetate, alpha-ketoglutarate and glutamate
genes. Genes related to transporters and heat shock proteins were also affected
by lysC knockout. The results indicated that the lysC knockout strain exhibited
some phenomena similar to lysine starvation. The data generated by this study
further clarify the systematic role of lysC in lysine biosynthesis.

DOI: 10.1111/j.1574-6968.2007.00932.x 
PMID: 17956427  [Indexed for MEDLINE]


818. Genet Sel Evol. 2007 Nov-Dec;39(6):651-68. Epub 2007 Dec 6.

Analysis of the real EADGENE data set: multivariate approaches and post analysis 
(open access publication).

Sørensen P(1), Bonnet A, Buitenhuis B, Closset R, Déjean S, Delmas C, Duval M,
Glass L, Hedegaard J, Hornshøj H, Hulsegge I, Jaffrézic F, Jensen K, Jiang L, de 
Koning DJ, Lê Cao KA, Nie H, Petzl W, Pool MH, Robert-Granié C, San Cristobal M, 
Lund MS, van Schothorst EM, Schuberth HJ, Seyfert HM, Tosser-Klopp G, Waddington 
D, Watson M, Yang W, Zerbe H.

Author information: 
(1)University of Aarhus, Faculty of Agricultural Sciences, Dept. of Genetics and 
Biotechnology, PO Box 50 DK-8830 Tjele, Denmark. pso@agrsci.dk

The aim of this paper was to describe, and when possible compare, the
multivariate methods used by the participants in the EADGENE WP1.4 workshop. The 
first approach was for class discovery and class prediction using evidence from
the data at hand. Several teams used hierarchical clustering (HC) or principal
component analysis (PCA) to identify groups of differentially expressed genes
with a similar expression pattern over time points and infective agent (E. coli
or S. aureus). The main result from these analyses was that HC and PCA were able 
to separate tissue samples taken at 24 h following E. coli infection from the
other samples. The second approach identified groups of differentially
co-expressed genes, by identifying clusters of genes highly correlated when
animals were infected with E. coli but not correlated more than expected by
chance when the infective pathogen was S. aureus. The third approach looked at
differential expression of predefined gene sets. Gene sets were defined based on 
information retrieved from biological databases such as Gene Ontology. Based on
these annotation sources the teams used either the GlobalTest or the Fisher exact
test to identify differentially expressed gene sets. The main result from these
analyses was that gene sets involved in immune defence responses were
differentially expressed.

DOI: 10.1051/gse:2007030 
PMCID: PMC2682812
PMID: 18053574  [Indexed for MEDLINE]


819. Genet Sel Evol. 2007 Nov-Dec;39(6):633-50. Epub 2007 Dec 6.

Analysis of the real EADGENE data set: comparison of methods and guidelines for
data normalisation and selection of differentially expressed genes (open access
publication).

Jaffrézic F(1), de Koning DJ, Boettcher PJ, Bonnet A, Buitenhuis B, Closset R,
Déjean S, Delmas C, Detilleux JC, Dovc P, Duval M, Foulley JL, Hedegaard J,
Hornshøj H, Hulsegge I, Janss L, Jensen K, Jiang L, Lavric M, Lê Cao KA, Lund MS,
Malinverni R, Marot G, Nie H, Petzl W, Pool MH, Robert-Granié C, San Cristobal M,
van Schothorst EM, Schuberth HJ, Sørensen P, Stella A, Tosser-Klopp G, Waddington
D, Watson M, Yang W, Zerbe H, Seyfert HM.

Author information: 
(1)INRA, UR337, Jouy-en-Josas, France (INRA_J). florence.jaffrezic@jouy.inra.fr

A large variety of methods has been proposed in the literature for microarray
data analysis. The aim of this paper was to present techniques used by the
EADGENE (European Animal Disease Genomics Network of Excellence) WP1.4
participants for data quality control, normalisation and statistical methods for 
the detection of differentially expressed genes in order to provide some more
general data analysis guidelines. All the workshop participants were given a real
data set obtained in an EADGENE funded microarray study looking at the gene
expression changes following artificial infection with two different mastitis
causing bacteria: Escherichia coli and Staphylococcus aureus. It was reassuring
to see that most of the teams found the same main biological results. In fact,
most of the differentially expressed genes were found for infection by E. coli
between uninfected and 24 h challenged udder quarters. Very little
transcriptional variation was observed for the bacteria S. aureus. Lists of
differentially expressed genes found by the different research teams were,
however, quite dependent on the method used, especially concerning the data
quality control step. These analyses also emphasised a biological problem of
cross-talk between infected and uninfected quarters which will have to be dealt
with for further microarray studies.

DOI: 10.1186/1297-9686-39-6-633 
PMCID: PMC2682811
PMID: 18053573  [Indexed for MEDLINE]


820. Int J Med Microbiol. 2007 Nov;297(7-8):625-39. Epub 2007 Apr 25.

Aspects of genome plasticity in pathogenic Escherichia coli.

Bielaszewska M(1), Dobrindt U, Gärtner J, Gallitz I, Hacker J, Karch H, Müller D,
Schubert S, Alexander Schmidt M, Sorsa LJ, Zdziarski J.

Author information: 
(1)Institut für Hygiene, Universitätsklinikum Münster, Robert-Koch-Str. 41,
D-48149 Münster, Germany.

The species Escherichia coli comprises not only non-pathogenic or commensal
variants that belong to the normal intestinal flora of most mammals, but also
various pathogenic strains causing diverse intestinal and extraintestinal
infections in man and animals. Virulence factors and mechanisms involved in
pathogenesis have been successfully analyzed for many years resulting in a wealth
of knowledge about many E. coli pathotypes. However, our knowledge on the genome 
content, diversity and variability between pathogenic and also non-pathogenic
subtypes is only slowly accumulating. Pathotypes have been largely defined by the
presence or absence of particular DNA segments that in most cases appear to have 
been acquired via horizontal gene transfer events. As these regions are
frequently subjected to excisions, rearrangements, and transfers they contribute 
to the previously unexpected and underestimated rapid evolution of E. coli
variants resulting in the development of novel strains and even pathotypes. In
these studies various novel aspects of genome diversity and plasticity in
extraintestinal and intestinal pathogenic E. coli pathotypes have been addressed 
and the results have been directly applied for the improvement of diagnostic
methods.

DOI: 10.1016/j.ijmm.2007.03.001 
PMID: 17462951  [Indexed for MEDLINE]


821. J Antimicrob Chemother. 2007 Nov;60(5):937-46. Epub 2007 Sep 25.

Oligonucleotide microarray for molecular characterization and genotyping of
Salmonella spp. strains.

Majtan T(1), Majtanova L, Timko J, Majtan V.

Author information: 
(1)Department of Genomics and Biotechnology, Institute of Molecular Biology SAS, 
Dubravska cesta 21, 84551 Bratislava 45, Slovakia. tomas.majtan@uchsc.edu

OBJECTIVES: To characterize and subtype multidrug-resistant Salmonella isolates
by determining the virulence factors, prophage sequences and antimicrobial
resistance genes using a novel Salmonella-specific oligonucleotide microarray.
METHODS: Preliminary screening of 24 Salmonella clinical isolates was carried out
by using susceptibility testing, plasmid profiling and class 1 integron PCR.
Subsequently, oligonucleotide microarray was involved in genotypic
characterization and localization of monitored genetic markers. The presence of
antimicrobial resistance genes was also detected and confirmed by PCR and
subsequent sequencing. The potential spread of emerging bla(SHV-2) was
investigated by bacterial conjugation.
RESULTS: All Salmonella strains revealed resistance to two or more (up to nine)
antibiotics. Nineteen of them carried class 1 integrons including dfrA1, dfrA12, 
aadA1, aadA2, bla(PSE-1) and bla(TEM-1) gene cassettes, respectively.
Twenty-three out of 24 Salmonella isolates possessed one or more plasmids.
Oligonucleotide microarray characterization and typing revealed the conserved
character of Salmonella pathogenicity island virulence factors among three
Salmonella enterica serovars, significant variability in prophage sequences and
many different antimicrobial resistance gene patterns. Differential labelling of 
genomic and plasmid DNA, respectively, and hybridization to the microarray made
it possible to localize important resistance determinants. Microarray results
were successfully confirmed and verified by using PCR. The emerging bla(SHV-2)
gene from Salmonella Kentucky SK10944 conferring resistance to ceftriaxone and
cefotaxime was transferred via bacterial conjugation to Escherichia coli K-12
3110.
CONCLUSIONS: Salmonella isolates were quickly and thoroughly characterized by a
novel oligonucleotide microarray, which could become a useful tool for detection 
of virulence and resistance genes and monitoring of their dissemination among
salmonellae and closely related bacteria.

DOI: 10.1093/jac/dkm326 
PMID: 17897936  [Indexed for MEDLINE]


822. J Bacteriol. 2007 Nov;189(21):7539-48. Epub 2007 Aug 24.

Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator 
NarL is independent of oxygen- responsive regulator Fnr in Escherichia coli K-12.

Lin HY(1), Bledsoe PJ, Stewart V.

Author information: 
(1)Food Science Graduate Group, Section of Microbiology, University of
California, One Shields Avenue, Davis, CA 95616-8665, USA.

The facultative aerobe Escherichia coli K-12 can use respiratory nitrate
ammonification to generate energy during anaerobic growth. The toxic compound
nitric oxide is a by-product of this metabolism. Previous transcript microarray
studies identified the yeaR-yoaG operon, encoding proteins of unknown function,
among genes whose transcription is induced in response to nitrate, nitrite, or
nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression 
through the NarX-NarL and NarQ-NarP two-component systems. All known
Nar-activated genes also require the oxygen-responsive Fnr transcription
activator. However, previous studies indicated that yeaR-yoaG operon
transcription does not require Fnr activation. Here, we report results from
mutational analyses demonstrating that yeaR-yoaG operon transcription is
activated by phospho-NarL protein independent of the Fnr protein. The
phospho-NarL protein binding site is centered at position -43.5 with respect to
the transcription initiation site. Expression from the Shewanella oneidensis MR-1
nnrS gene promoter, cloned into E. coli, similarly was activated by phospho-NarL 
protein independent of the Fnr protein. Recently, yeaR-yoaG operon transcription 
was shown to be regulated by the nitric oxide-responsive NsrR repressor (N.
Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses
reveal the individual contributions of the Nar and NsrR regulators to overall
yeaR-yoaG operon expression and document the NsrR operator centered at position
-32. Thus, control of yeaR-yoaG operon transcription provides an example of
overlapping regulation by nitrate and nitrite, acting through the Nar regulatory 
system, and nitric oxide, acting through the NsrR repressor.

DOI: 10.1128/JB.00953-07 
PMCID: PMC2168752
PMID: 17720788  [Indexed for MEDLINE]


823. Jpn J Infect Dis. 2007 Nov;60(6):355-61.

Comparison of whole genome amplification methods for detecting pathogenic
bacterial genomic DNA using microarray.

Uda A(1), Tanabayashi K, Fujita O, Hotta A, Yamamoto Y, Yamada A.

Author information: 
(1)Department of Veterinary Science, National Institute of Infectious Diseases,
Tokyo 162-8640, Japan. auda@nih.go.jp

The genetic diagnosis of pathogenic agents using microarrays has the advantage of
high-throughput detection, but a relatively large amount of DNA sample is
required. To obtain a sufficient amount of DNA for molecular diagnoses, several
whole genome amplification (WGA) methods have been proposed. In this study, using
Francisella tularensis and Escherichia coli as models, we compared four WGA
methods in terms of their efficiency of amplification of whole genomic DNA in
order to identify the most suitable method for preparing DNA to be used for
microarray analysis. It was possible to obtain more than 1.5 microg of products
from 10 ng of F. tularensis and E. coli genomic DNA using four methods, but
biases in the amplification of bacterial genes were least prominent in the
multiple displacement amplification (MDA) or OmniPlex WGA. When the amplified
DNAs were applied to microarray slides consisting of 32 different genes probes,
DNAs amplified by Phi29 v2 of MDA and OmniPlex WGA showed high signal intensity
as well as a high signal-to-noise ratio for all 32 genes. These results indicate 
that Phi29 v2 and OmniPlex WGA are useful methods for obtaining sufficient DNA
from a limited amount of samples for the detection of microbes using microarrays.


PMID: 18032834  [Indexed for MEDLINE]


824. Mol Microbiol. 2007 Nov;66(3):744-57. Epub 2007 Oct 5.

RutR is the uracil/thymine-sensing master regulator of a set of genes for
synthesis and degradation of pyrimidines.

Shimada T(1), Hirao K, Kori A, Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience and Micro-Nano Technology Research Centre,
Hosei University, Koganei, Tokyo 184-8584, Japan.

Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been 
identified, which formed complexes with transcription factor RutR. The RutR
regulon was found to include a large number of genes encoding components for not 
only degradation of pyrimidines but also transport of glutamate, synthesis of
glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of
purines. DNase I footprinting indicated that RutR recognizes a palindromic
sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding
carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps
with the PepA (CarP) repressor binding site, implying competition between RutR
and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the 
carAB P1 promoter. Accordingly, in the rutR-deletion mutant or in the presence of
uracil, the activation in vivo of carAB P1 promoter was markedly reduced.
Northern blot analysis of the RutR target genes indicated that RutR represses the
Gad system genes involved in glutamate-dependent acid resistance and allantoin
degradation. Altogether we propose that RutR is the pyrimidine sensor and the
master regulator for a large set of the genes involved in the synthesis and
degradation of pyrimidines.

DOI: 10.1111/j.1365-2958.2007.05954.x 
PMID: 17919280  [Indexed for MEDLINE]


825. BMC Microbiol. 2007 Oct 29;7:97.

Global transcriptional response of Escherichia coli O157:H7 to growth transitions
in glucose minimal medium.

Bergholz TM(1), Wick LM, Qi W, Riordan JT, Ouellette LM, Whittam TS.

Author information: 
(1)Microbial Evolution Laboratory, National Food Safety and Toxicology Center,
Michigan State University, East Lansing, Michigan 48824, USA. largeter@msu.edu

BACKGROUND: Global patterns of gene expression of Escherichia coli K-12 during
growth transitions have been deeply investigated, however, comparable studies of 
E. coli O157:H7 have not been explored, particularly with respect to factors
regulating virulence genes and genomic islands specific to this pathogen. To
examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 
Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 
10 time points from early exponential to full stationary phase, and relative gene
expression was measured by co-hybridization on high-density DNA microarrays.
Expression levels of 14 genes, including those encoding Shiga toxins and other
virulence factors associated with the locus of enterocyte effacement (LEE), were 
confirmed by Q-PCR.
RESULTS: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239
O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in
expression significantly over time. QT cluster analysis placed 2468 of the 2552
significant ORFs into 12 groups; each group representing a distinct expression
pattern. ORFs from the largest cluster (n = 1078) decreased in expression from
late exponential to early stationary phase: most of these ORFs are involved in
functions associated with steady state growth. Also represented in this cluster
are ORFs of the TAI island, encoding tellurite resistance and urease activity,
which decreased approximately 4-fold. Most ORFs of the LEE pathogenicity island
also decreased approximately 2-fold by early stationary phase. The ORFs encoding 
proteins secreted via the LEE encoded type III secretion system, such as tccP and
espJ, also decreased in expression from exponential to stationary phase. Three of
the clusters (n = 154) comprised genes that are transiently upregulated at the
transition into stationary phase and included genes involved in nutrient
scavenging. Upregulated genes with an increase in mRNA levels from late
exponential to early stationary phase belonged to one cluster (n = 923) which
includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These
transcript levels remained relatively high for > 3 h in stationary phase. The
Shiga toxin genes (stx1AB and stx2B) were significantly induced after transition 
into stationary phase.
CONCLUSION: Expression of more than 300 O157-specific ORFs, many implicated in
virulence of the O157 pathogen, was modulated in a growth dependent manner. These
results provide a baseline transcriptional profile that can be compared to
patterns of gene expression of this important foodborne pathogen under adverse
environmental conditions.

DOI: 10.1186/1471-2180-7-97 
PMCID: PMC2241611
PMID: 17967175  [Indexed for MEDLINE]


826. J Mol Biol. 2007 Oct 12;373(1):11-26. Epub 2007 Aug 2.

Structure and function of the Escherichia coli protein YmgB: a protein critical
for biofilm formation and acid-resistance.

Lee J(1), Page R, García-Contreras R, Palermino JM, Zhang XS, Doshi O, Wood TK,
Peti W.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A & M University,
College Station, TX 77843-3122, USA.

The Escherichia coli gene cluster ymgABC was identified in transcriptome studies 
to have a role in biofilm development and stability. In this study, we showed
that YmgB represses biofilm formation in rich medium containing glucose,
decreases cellular motility, and protects the cell from acid indicating that YmgB
has a major role in acid-resistance in E. coli. Our data show that these
phenotypes are potentially mediated through interactions with the important cell 
signal indole. In addition, gel mobility-shift assays suggest that YmgB may be a 
non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we
showed that YmgB binds, either directly or indirectly, via a probable ligand,
genes important for biofilm formation. To advance our understanding of the
function of YmgB, we used X-ray crystallography to solve the structure of the
protein to 1.8 A resolution. YmgB is a biological dimer that is structurally
homologous to the E. coli gene regulatory protein Hha, despite having only 5%
sequence identity. This supports our DNA microarray data showing that YmgB is a
gene regulatory protein. Therefore, this protein, which clearly has a critical
role in acid-resistance in E. coli, has been renamed as AriR for regulator of
acid resistance influenced by indole.

DOI: 10.1016/j.jmb.2007.07.037 
PMCID: PMC2185545
PMID: 17765265  [Indexed for MEDLINE]


827. J Theor Biol. 2007 Oct 7;248(3):512-21. Epub 2007 Jun 11.

Randomized probe selection algorithm for microarray design.

Gasieniec L(1), Li CY, Sant P, Wong PW.

Author information: 
(1)Department of Computer Science, The University of Liverpool, Ashton Building, 
Ashton Street, Liverpool, L69 3BX, UK. leszek@csc.liv.ac.uk

DNA microarray technology, originally developed to measure the level of gene
expression, has become one of the most widely used tools in genomic study. The
crux of microarray design lies in how to select a unique probe that distinguishes
a given genomic sequence from other sequences. Due to its significance, probe
selection attracts a lot of attention. Various probe selection algorithms have
been developed in recent years. Good probe selection algorithms should produce a 
small number of candidate probes. Efficiency is also crucial because the data
involved are usually huge. Most existing algorithms are usually not sufficiently 
selective and quite a large number of probes are returned. We propose a new
direction to tackle the problem and give an efficient algorithm based on
randomization to select a small set of probes and demonstrate that such a small
set of probes is sufficient to distinguish each sequence from all the other
sequences. Based on the algorithm, we have developed probe selection software
RandPS, which runs efficiently in practice. The software is available on our
website (http://www.csc.liv.ac.uk/ approximately cindy/RandPS/RandPS.htm). We
test our algorithm via experiments on different genomes (Escherichia coli,
Saccharamyces cerevisiae, etc.) and our algorithm is able to output unique probes
for most of the genes efficiently. The other genes can be identified by a
combination of at most two probes.

DOI: 10.1016/j.jtbi.2007.05.036 
PMID: 17628606  [Indexed for MEDLINE]


828. Genet Mol Res. 2007 Oct 5;6(4):730-42.

Genome-wide partial correlation analysis of Escherichia coli microarray data.

Veiga DF(1), Vicente FF, Grivet M, de la Fuente A, Vasconcelos AT.

Author information: 
(1)Laboratório Nacional de Computação Científica, Laboratório de Bioinformática, 
Petrópolis, RJ, Brasil. atrv@lncc.br

Transcriptional control is an essential regulatory mechanism employed by
bacteria. Much about transcriptional regulation remains to be discovered, even
for the most widely studied bacterium, Escherichia coli. In the present study, we
made a genome-wide low-order partial correlation analysis of E. coli microarray
data with the purpose of recovering regulatory interactions from transcriptome
data. As a result, we produced whole genome transcription factor regulation and
co-regulation graphs using the predicted interactions, and we demonstrated how
they can be used to investigate regulation and biological function. We concluded 
that partial correlation analysis can be employed as a method to predict putative
regulatory interactions from expression data, as a complementary approach to
transcription factor binding site tools and other tools designed to detect
co-regulated genes.


PMID: 18058701  [Indexed for MEDLINE]


829. Appl Environ Microbiol. 2007 Oct;73(20):6566-76. Epub 2007 Sep 7.

Impact of feed supplementation with antimicrobial agents on growth performance of
broiler chickens, Clostridium perfringens and enterococcus counts, and antibiotic
resistance phenotypes and distribution of antimicrobial resistance determinants
in Escherichia coli isolates.

Diarra MS(1), Silversides FG, Diarrassouba F, Pritchard J, Masson L, Brousseau R,
Bonnet C, Delaquis P, Bach S, Skura BJ, Topp E.

Author information: 
(1)Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, 
British Columbia, Canada. diarram@agr.gc.ca

The effects of feed supplementation with the approved antimicrobial agents
bambermycin, penicillin, salinomycin, and bacitracin or a combination of
salinomycin plus bacitracin were evaluated for the incidence and distribution of 
antibiotic resistance in 197 commensal Escherichia coli isolates from broiler
chickens over 35 days. All isolates showed some degree of multiple antibiotic
resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur
(51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The
levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 
35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7
to day 35 (P < 0.001). Comparing treatments, the levels of resistance to
ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin
treatment) were significantly higher in isolates from chickens receiving feed
supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA 
microarray analysis capable of detecting commonly found antimicrobial resistance 
genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to
28-day-old chickens fed different growth promoters. Results showed a decrease in 
the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1 
integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant
E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of
isolates positive for sulI, aadA, and integron class 1 were significantly higher 
in salinomycin-treated chickens than in the control or other treatment groups (P 
< 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates
can be found in broiler chickens regardless of the antimicrobial growth promoters
used. However, the phenotype and the distribution of resistance determinants in
E. coli can be modulated by feed supplementation with some of the antimicrobial
agents used in broiler chicken production.

DOI: 10.1128/AEM.01086-07 
PMCID: PMC2075079
PMID: 17827305  [Indexed for MEDLINE]


830. Artif Intell Med. 2007 Oct;41(2):151-9. Epub 2007 Sep 14.

A multi-approaches-guided genetic algorithm with application to operon
prediction.

Wang S(1), Wang Y, Du W, Sun F, Wang X, Zhou C, Liang Y.

Author information: 
(1)College of Computer Science and Technology, Jilin University, Key Laboratory
of Symbol Computation and Knowledge Engineering of the Ministry of Education,
Changchun 130012, China.

OBJECTIVE: The prediction of operons is critical to the reconstruction of
regulatory networks at the whole genome level. Multiple genome features have been
used for predicting operons. However, multiple genome features are usually dealt 
with using only single method in the literatures. The aim of this paper is to
develop a combined method for operon prediction by using different methods to
preprocess different genome features in order for exerting their unique
characteristics.
METHODS: A novel multi-approach-guided genetic algorithm for operon prediction is
presented. We exploit different methods for intergenic distance, cluster of
orthologous groups (COG) gene functions, metabolic pathway and microarray
expression data. A novel local-entropy-minimization method is proposed to
partition intergenic distance. Our program can be used for other newly sequenced 
genomes by transferring the knowledge that has been obtained from Escherichia
coli data. We calculate the log-likelihood for COG gene functions and Pearson
correlation coefficient for microarray expression data. The genetic algorithm is 
used for integrating the four types of data.
RESULTS: The proposed method is examined on E. coli K12 genome, Bacillus subtilis
genome, and Pseudomonas aeruginosa PAO1 genome. The accuracies of prediction for 
these three genomes are 85.9987%, 88.296%, and 81.2384%, respectively.
CONCLUSION: Simulated experimental results demonstrate that in the genetic
algorithm the preprocessing for genome data using multiple approaches ensures the
effective utilization of different biological characteristics. Experimental
results also show that the proposed method is applicable for predicting operons
in prokaryote.

DOI: 10.1016/j.artmed.2007.07.010 
PMID: 17869072  [Indexed for MEDLINE]


831. Diagn Microbiol Infect Dis. 2007 Oct;59(2):149-56. Epub 2007 Jul 26.

Development of a base stacking hybridization-based microarray method for rapid
identification of clinical isolates.

Zhu LX(1), Wang D, Zhang GB, Jiang D, Zhang ZW, Zhang Q, Mitchelson K, Cheng J.

Author information: 
(1)Medical Systems Biology Research Center, School of Medicine Tsinghua
University, Beijing 100084, China.

A base stacking hybridization-based microarray method was developed for rapid
identification of clinical isolates within 2 h. The oligonucleotide probe
sequences for species or genus-level identification were targeted against
ribosomal RNA. Isolates were lysed and directly hybridized to the
microarray-bound capture probes without conventional DNA or RNA isolation and
prior polymerase chain reaction amplification. Five bacterial species encountered
frequently in the clinical setting, Pseudomonas aeruginosa, Staphylococcus
aureus, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, and one
genus Enterococcus, could be discriminated by the microarray-based assay.
Identification by this method matched biochemical identification for 150 of 152
clinical strains. This base-stacking hybridization microarray offers a simple,
fast (</=2 h), and accurate identification of bacterial cultures and is a
potential tool for routine clinical diagnosis.

DOI: 10.1016/j.diagmicrobio.2007.05.001 
PMID: 17662562  [Indexed for MEDLINE]


832. Infect Immun. 2007 Oct;75(10):4875-84. Epub 2007 Jul 16.

Global effects of the cell-to-cell signaling molecules autoinducer-2,
autoinducer-3, and epinephrine in a luxS mutant of enterohemorrhagic Escherichia 
coli.

Kendall MM(1), Rasko DA, Sperandio V.

Author information: 
(1)University of Texas Southwestern Medical Center at Dallas, Dallas, TX
75390-9048, USA.

Erratum in
    Infect Immun. 2008 Mar;76(3):1319.

Intrakingdom cell-to-cell communication and interkingdom cell-to-cell
communication play essential roles in the virulence of enterohemorrhagic
Escherichia coli (EHEC). Four signals, autoinducer 2 (AI-2), AI-3, and the human 
hormones epinephrine and norepinephrine, are important in this communication. The
effect of these signaling compounds on the transcriptome of EHEC was examined in 
this study. We demonstrated that the luxS mutation affects primarily central
metabolic genes in both pathogenic and nonpathogenic strains of E. coli and that 
addition of exogenous AI-2 does not fully restore the expression profile in a
luxS-deficient strain lacking the ability to synthesize AI-2. Addition of AI-3 or
epinephrine increased expression of the locus of enterocyte effacement regulon,
which is known to play a pivotal role in EHEC virulence. Moreover, when
epinephrine was added to the culture medium, the greatest number of gene
alterations was observed. These alterations included a greater proportion of
alterations in EHEC genes than in MG1655 genes, suggesting that epinephrine may
be a global virulence signal. Detailed examination with real-time reverse
transcriptase PCR (RT-PCR) confirmed the increases in virulence gene expression
with addition of AI-3 and epinephrine. Additional studies with real-time RT-PCR
examining the EHEC secreted effectors and putative fimbrial gene expression
showed a variable expression profile, indicating that there is differential
regulation of the secreted molecules. This study began to examine the global
signaling networks in EHEC and revealed expression profiles that are signal and
pathogen specific.

DOI: 10.1128/IAI.00550-07 
PMCID: PMC2044543
PMID: 17635870  [Indexed for MEDLINE]


833. Infect Immun. 2007 Oct;75(10):4831-7. Epub 2007 Jul 2.

Bartonella quintana lipopolysaccharide is a natural antagonist of Toll-like
receptor 4.

Popa C(1), Abdollahi-Roodsaz S, Joosten LA, Takahashi N, Sprong T, Matera G,
Liberto MC, Foca A, van Deuren M, Kullberg BJ, van den Berg WB, van der Meer JW, 
Netea MG.

Author information: 
(1)Department of General Internal Medicine, Radboud University Nijmegen Medical
Centre, The Netherlands.

Bartonella quintana is a gram-negative microorganism that causes trench fever and
chronic bacteremia. B. quintana lipopolysaccharide (LPS) was unable to induce the
production of proinflammatory cytokines in human monocytes. Interestingly, B.
quintana LPS is a potent antagonist of Toll-like receptor 4 (TLR4), as it
inhibited both mRNA transcription and the release of tumor necrosis factor alpha,
interleukin 1beta (IL-1beta), and IL-6 by Escherichia coli LPS in human
monocytes, at ratios ranging from 1,000:1 to 10:1 (B. quintana LPS to E. coli
LPS). Likewise, B. quintana LPS blocked the interaction of E. coli LPS with TLR4 
in transfected cell lines. The extent of the inhibitory effect of B. quintana LPS
was demonstrated in microarray studies, which showed downregulation of
practically all genes induced by LPS in monocytes. Because of the role of TLR4 in
inflammation, B. quintana LPS may prove useful as a potent anti-TLR4 agent with
therapeutic potential in both infections and autoimmune inflammation.

DOI: 10.1128/IAI.00237-07 
PMCID: PMC2044526
PMID: 17606598  [Indexed for MEDLINE]


834. Int J Med Microbiol. 2007 Oct;297(6):417-29. Epub 2007 May 7.

Rapid and sensitive detection of fluoroquinolone-resistant Escherichia coli from 
urine samples using a genotyping DNA microarray.

Yu X(1), Susa M, Weile J, Knabbe C, Schmid RD, Bachmann TT.

Author information: 
(1)Institute of Technical Biochemistry, University of Stuttgart, Germany.

Urinary tract infections (UTI) are among the most common bacterial infections in 
humans, with Escherichia coli being the major cause of infection. Fluoroquinolone
resistance of uropathogenic E. coli has increased significantly over the last
decade. In this study a microarray-based assay was developed and applied, which
provides a rapid, sensitive and specific detection of fluoroquinolone-resistant
E. coli in urine. The capture probes were designed against previously identified 
and described hotspots for quinolone resistance (codons 83 and 87 of gyrA). The
key goals of this development were to reduce assay time while increasing the
sensitivity and specificity as compared with a pilot version of a gyrA genotyping
DNA microarray. The performance of the assay was demonstrated with pure cultures 
of 30 E. coli isolates as well as with urine samples spiked with 6 E. coli
isolates. The microarray results were confirmed by standard DNA sequencing and
were in full agreement with the phenotypic antimicrobial susceptibility testing
using standard methods. The DNA microarray test displayed an assay time of 3.5h, 
a sensitivity of 100CFU/ml, and the ability to detect fluoroquinolone-resistant
E. coli in the presence of a 10-fold excess of fluoroquinolone-susceptible E.
coli cells. As a consequence, we believe that this microarray-based determination
of antibiotics resistance has a true potential for the application in clinical
routine laboratories in the future.

DOI: 10.1016/j.ijmm.2007.03.018 
PMID: 17482874  [Indexed for MEDLINE]


835. J Bacteriol. 2007 Oct;189(19):6936-44. Epub 2007 Jul 20.

Global analysis of mRNA decay in Halobacterium salinarum NRC-1 at single-gene
resolution using DNA microarrays.

Hundt S(1), Zaigler A, Lange C, Soppa J, Klug G.

Author information: 
(1)Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität,
Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany.

RNA degradation is an important factor in the regulation of gene expression. It
allows organisms to quickly respond to changing environmental conditions by
adapting the expression of individual genes. The stability of individual mRNAs
within an organism varies considerably, contributing to differential amounts of
proteins expressed. In this study we used DNA microarrays to analyze mRNA
degradation in exponentially growing cultures of the extremely halophilic
euryarchaeon Halobacterium salinarum NRC-1 on a global level. We determined mRNA 
half-lives for 1,717 open reading frames, 620 of which are part of known or
predicted operons. Under the tested conditions transcript stabilities ranged from
5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives
between 8 and 12 min. The overall mean half-life was 10 min, which is
considerably longer than the ones found in the other prokaryotes investigated
thus far. As previously observed in Escherichia coli and Saccharomyces
cerevisiae, we could not detect a significant correlation between transcript
length and transcript stability, but there was a relationship between gene
function and transcript stability. Genes that are known or predicted to be
transcribed in operons exhibited similar mRNA half-lives. These results provide
initial insights into mRNA turnover in a euryarchaeon. Moreover, our model
organism, H. salinarum NRC-1, is one of just two archaea sequenced to date that
are missing the core subunits of the archaeal exosome. This complex orthologous
to the RNA degrading exosome of eukarya is found in all other archaeal genomes
sequenced thus far.

DOI: 10.1128/JB.00559-07 
PMCID: PMC2045193
PMID: 17644597  [Indexed for MEDLINE]


836. Mol Microbiol. 2007 Oct;66(2):315-28. Epub 2007 Sep 10.

Microarray analysis of Mu transposition in Salmonella enterica, serovar
Typhimurium: transposon exclusion by high-density DNA binding proteins.

Manna D(1), Porwollik S, McClelland M, Tan R, Higgins NP.

Author information: 
(1)Department of Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, Birmingham, AL-35294, USA.

All organisms contain transposons with the potential to disrupt and rearrange
genes. Despite the presence of these destabilizing sequences, some genomes show
remarkable stability over evolutionary time. Do bacteria defend the genome
against disruption by transposons? Phage Mu replicates by transposition and
virtually all genes are potential insertion targets. To test whether bacteria
limit Mu transposition to specific parts of the chromosome, DNA arrays of
Salmonella enterica were used to quantitatively measure target site preference
and compare the data with Escherichia coli. Essential genes were as susceptible
to transposon disruption as non-essential ones in both organisms, but the
correlation of transposition hot spots among homologous genes was poor. Genes in 
highly transcribed operons were insulated from transposon mutagenesis in both
organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which
the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid
cold spot, and an ectopic parS site placed in the Salmonella chromosome created a
new cold spot in the presence of ParB. Our data show that competition between
cellular proteins and transposition proteins on plasmids and the chromosome is a 
dominant factor controlling the genetic footprint of transposons in living cells.

DOI: 10.1111/j.1365-2958.2007.05915.x 
PMID: 17850262  [Indexed for MEDLINE]


837. BMC Bioinformatics. 2007 Sep 19;8:350.

Selection of long oligonucleotides for gene expression microarrays using weighted
rank-sum strategy.

Hu G(1), Llinás M, Li J, Preiser PR, Bozdech Z.

Author information: 
(1)School of Biological Sciences, Nanyang Technological University, No, 60
Nanyang Drive, 637551, Singapore. hu0002an@ntu.edu.sg

BACKGROUND: The design of long oligonucleotides for spotted DNA microarrays
requires detailed attention to ensure their optimal performance in the
hybridization process. The main challenge is to select an optimal oligonucleotide
element that represents each genetic locus/gene in the genome and is unique,
devoid of internal structures and repetitive sequences and its Tm is uniform with
all other elements on the microarray. Currently, all of the publicly available
programs for DNA long oligonucleotide microarray selection utilize various
combinations of cutoffs in which each parameter (uniqueness, Tm, and secondary
structure) is evaluated and filtered individually. The use of the cutoffs can,
however, lead to information loss and to selection of suboptimal
oligonucleotides, especially for genomes with extreme distribution of the GC
content, a large proportion of repetitive sequences or the presence of large gene
families with highly homologous members.
RESULTS: Here we present the program OligoRankPick which is using a weighted
rank-based strategy to select microarray oligonucleotide elements via an integer 
weighted linear function. This approach optimizes the selection criteria (weight 
score) for each gene individually, accommodating variable properties of the DNA
sequence along the genome. The designed algorithm was tested using three
microbial genomes Escherichia coli, Saccharomyces cerevisiae and the human
malaria parasite species Plasmodium falciparum. In comparison to other published 
algorithms OligoRankPick provides significant improvements in oligonucleotide
design for all three genomes with the most significant improvements observed in
the microarray design for P. falciparum whose genome is characterized by large
fluctuations of GC content, and abundant gene duplications.
CONCLUSION: OligoRankPick is an efficient tool for the design of long
oligonucleotide DNA microarrays which does not rely on direct oligonucleotide
exclusion by parameter cutoffs but instead optimizes all parameters in context of
each other. The weighted rank-sum strategy utilized by this algorithm provides
high flexibility of oligonucleotide selection which accommodates extreme
variability of DNA sequence properties along genomes of many organisms.

DOI: 10.1186/1471-2105-8-350 
PMCID: PMC2099447
PMID: 17880708  [Indexed for MEDLINE]


838. Appl Environ Microbiol. 2007 Sep;73(17):5692-7. Epub 2007 Jul 13.

Pathotyping Escherichia coli by using miniaturized DNA microarrays.

Anjum MF(1), Mafura M, Slickers P, Ballmer K, Kuhnert P, Woodward MJ, Ehricht R.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories
Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
m.anjum@vla.defra.gsi.gov.uk

The detection of virulence determinants harbored by pathogenic Escherichia coli
is important for establishing the pathotype responsible for infection. A
sensitive and specific miniaturized virulence microarray containing 60
oligonucleotide probes was developed. It detected six E. coli pathotypes and will
be suitable in the future for high-throughput use.

DOI: 10.1128/AEM.00419-07 
PMCID: PMC2042074
PMID: 17630299  [Indexed for MEDLINE]


839. IET Syst Biol. 2007 Sep;1(5):306-12.

Inference of gene networks from temporal gene expression profiles.

Bansal M(1), di Bernardo D.

Author information: 
(1)Telethon Institute of Genetics and Medicine, Via P. Castellino 111, Naples
80131, Italy. bansal@tigem.it

Genes interact with each other in complex networks that enable the processing of 
information and the metabolism of nutrients inside the cell. A novel inference
algorithm based on linear ordinary differential equations is proposed. The
algorithm can infer the local network of gene-gene interactions surrounding a
gene of interest from time-series gene expression profiles. The performance of
the algorithm has been tested on in silico simulated gene expression data and on 
a nine gene subnetwork part of the DNA-damage response pathway (SOS pathway) in
the bacteria Escherichia coli. This approach can infer regulatory interactions
even when only a small number of measurements is available.


PMID: 17907680  [Indexed for MEDLINE]


840. Infect Immun. 2007 Sep;75(9):4597-607. Epub 2007 Jun 25.

Differential effects of epinephrine, norepinephrine, and indole on Escherichia
coli O157:H7 chemotaxis, colonization, and gene expression.

Bansal T(1), Englert D, Lee J, Hegde M, Wood TK, Jayaraman A.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A&M University, 200
Jack E. Brown Engineering, 3122 TAMU, College Station, TX 77843-3122, USA.

During infection in the gastrointestinal tract, enterohemorrhagic Escherichia
coli (EHEC) O157:H7 is exposed to a wide range of signaling molecules, including 
the eukaryotic hormones epinephrine and norepinephrine, and bacterial signal
molecules such as indole. Since these signaling molecules have been shown to be
involved in the regulation of phenotypes such as motility and virulence that are 
crucial for EHEC infections, we hypothesized that these molecules also govern the
initial recognition of the large intestine environment and attachment to the host
cell surface. Here, we report that, compared to indole, epinephrine and
norepinephrine exert divergent effects on EHEC chemotaxis, motility, biofilm
formation, gene expression, and colonization of HeLa cells. Using a novel
two-fluorophore chemotaxis assay, it was found that EHEC is attracted to
epinephrine and norepinephrine while it is repelled by indole. In addition,
epinephrine and norepinephrine also increased EHEC motility and biofilm formation
while indole attenuated these phenotypes. DNA microarray analysis of
surface-associated EHEC indicated that epinephrine/norepinephrine up-regulated
the expression of genes involved in surface colonization and virulence while
exposure to indole decreased their expression. The gene expression data also
suggested that autoinducer 2 uptake was repressed upon exposure to
epinephrine/norepinephrine but not indole. In vitro adherence experiments
confirmed that epinephrine and norepinephrine increased attachment to epithelial 
cells while indole decreased adherence. Taken together, these results suggest
that epinephrine and norepinephrine increase EHEC infection while indole
attenuates the process.

DOI: 10.1128/IAI.00630-07 
PMCID: PMC1951185
PMID: 17591798  [Indexed for MEDLINE]


841. Int J Antimicrob Agents. 2007 Sep;30(3):259-63. Epub 2007 Jul 13.

Partial characterisation of the acrAB locus in two Citrobacter freundii clinical 
isolates.

Sánchez-Céspedes J(1), Vila J.

Author information: 
(1)Servei de Microbiologia, Centre de Diagnòstic Biomèdic, Hospital Clínic,
IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Villarroel 170, 08036
Barcelona, Spain.

We studied the mechanisms of resistance to fluoroquinolones in two Citrobacter
freundii strains (1.44 and 1.38) isolated from the same patient and belonging to 
the same clone by pulsed-field gel electrophoresis. This study allowed partial
characterisation of the acrA and acrB genes of this microorganism. As previously 
reported, the two strains showed the same substitutions in the GyrA and ParC
proteins (Thr-83-->Ile and Asp-87-->Tyr in GyrA and Ser-83-->Ile in ParC).
However, differences were observed in the amount of ciprofloxacin accumulated,
with strain 1.38 showing less accumulation. Expression of genes in both strains
was analysed using DNA microarrays for Escherichia coli. Ten genes were
overexpressed in strain 1.38 compared with strain 1.44, including genes acrA and 
acrB. Nucleotide similarity between the partially sequenced acrA and acrB genes
of C. freundii and E. coli was 80.7% and 85%, respectively. The acrA and acrB
genes of C. freundii are similar to those described in E. coli and their
overexpression may play an important role in modulating the final minimum
inhibitory concentration of fluoroquinolones in collaboration with mutations in
the gyrA and parC genes.

DOI: 10.1016/j.ijantimicag.2007.05.010 
PMID: 17631985  [Indexed for MEDLINE]


842. Microbiology. 2007 Sep;153(Pt 9):2922-40.

Effects of Fis on Escherichia coli gene expression during different growth
stages.

Bradley MD(1), Beach MB, de Koning AP, Pratt TS, Osuna R.

Author information: 
(1)Department of Biological Science, University at Albany, 1400 Washington
Avenue, Albany, NY 12222, USA.

Fis is a nucleoid-associated protein in Escherichia coli that is abundant during 
early exponential growth in rich medium but is in short supply during stationary 
phase. Its role as a transcriptional regulator has been demonstrated for an
increasing number of genes. In order to gain insight into the global effects of
Fis on E. coli gene expression during different stages of growth in rich medium, 
DNA microarray analyses were conducted in fis and wild-type strains during early,
mid-, late-exponential and stationary growth phases. The results uncovered 231
significantly regulated genes that were distributed over 15 functional
categories. Regulatory effects were observed at all growth stages examined.
Coordinate upregulation was observed for a number of genes involved in
translation, flagellar biosynthesis and motility, nutrient transport, carbon
compound metabolism, and energy metabolism at different growth stages. Coordinate
down-regulation was also observed for genes involved in stress response, amino
acid and nucleotide biosynthesis, energy and intermediary metabolism, and
nutrient transport. As cells transitioned from the early to the late-exponential 
growth phase, different functional categories of genes were regulated, and a
gradual shift occurred towards mostly down-regulation. The results demonstrate
that the growth phase-dependent Fis expression triggers coordinate regulation of 
15 categories of functionally related genes during specific stages of growth of
an E. coli culture.

DOI: 10.1099/mic.0.2007/008565-0 
PMID: 17768236  [Indexed for MEDLINE]


843. Nat Med. 2007 Sep;13(9):1042-9. Epub 2007 Sep 2.

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting
lethal, endotoxin-induced shock.

Vogl T(1), Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van Zoelen MA, Nacken W, 
Foell D, van der Poll T, Sorg C, Roth J.

Author information: 
(1)Institute of Experimental Dermatology, University of Münster, D-48129 Münster,
Germany.

To identify new components that regulate the inflammatory cascade during sepsis, 
we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and
myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of 
phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are
protected from endotoxin-induced lethal shock and Escherichia coli-induced
abdominal sepsis. Both proteins are released during activation of phagocytes, and
Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of
phagocytes. Mrp8 is the active component that induces intracellular translocation
of myeloid differentiation primary response protein 88 and activation of
interleukin-1 receptor-associated kinase-1 and nuclear factor-kappaB, resulting
in elevated expression of tumor necrosis factor-alpha (TNF-alpha). Using
phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells
transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in 
vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex,
thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes
are new inflammatory components that amplify phagocyte activation during sepsis
upstream of TNFalpha-dependent effects.

DOI: 10.1038/nm1638 
PMID: 17767165  [Indexed for MEDLINE]


844. Pak J Biol Sci. 2007 Aug 15;10(16):2612-21.

Variable Number Of Tandem Repeats (VNTR) and its application in bacterial
epidemiology.

Ramazanzadeh R(1), McNerney R.

Author information: 
(1)Department of Microbiology, Faculty of Medicine, Kurdistan University of
Medical Science, Sanandaj, Iran.

Molecular epidemiology is the using of molecular techniques to study bacterial
distribution in human populations. Recently molecular epidemiologist benefit from
several techniques such as Variable Number Tandem Repeat (VNTR) typing method to 
typing bacterial strains. Variable Number Tandem Repeat (VNTR) typing is a tool
for genotyping and provides data in a simple and numeric format based on the
number of repetitive sequences. VNTR for first time identified in M. tuberculosis
as Mycobacterial Interspersed Repeat Units (MIRUs). General terms of VNTR have
now been reported in Bacillus anthracis, Legionella pneumophila, Pseudomonas
aeruginosa, Salmonella enterica and Escherichia coli O157.


PMID: 19070072  [Indexed for MEDLINE]


845. BMC Bioinformatics. 2007 Aug 14;8:299.

Constructing gene co-expression networks and predicting functions of unknown
genes by random matrix theory.

Luo F(1), Yang Y, Zhong J, Gao H, Khan L, Thompson DK, Zhou J.

Author information: 
(1)Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge,
Tennessee 37831, USA. luofeng@cs.clemson.edu

BACKGROUND: Large-scale sequencing of entire genomes has ushered in a new age in 
biology. One of the next grand challenges is to dissect the cellular networks
consisting of many individual functional modules. Defining co-expression networks
without ambiguity based on genome-wide microarray data is difficult and current
methods are not robust and consistent with different data sets. This is
particularly problematic for little understood organisms since not much existing 
biological knowledge can be exploited for determining the threshold to
differentiate true correlation from random noise. Random matrix theory (RMT),
which has been widely and successfully used in physics, is a powerful approach to
distinguish system-specific, non-random properties embedded in complex systems
from random noise. Here, we have hypothesized that the universal predictions of
RMT are also applicable to biological systems and the correlation threshold can
be determined by characterizing the correlation matrix of microarray profiles
using random matrix theory.
RESULTS: Application of random matrix theory to microarray data of S. oneidensis,
E. coli, yeast, A. thaliana, Drosophila, mouse and human indicates that there is 
a sharp transition of nearest neighbour spacing distribution (NNSD) of
correlation matrix after gradually removing certain elements insider the matrix. 
Testing on an in silico modular model has demonstrated that this transition can
be used to determine the correlation threshold for revealing modular
co-expression networks. The co-expression network derived from yeast cell cycling
microarray data is supported by gene annotation. The topological properties of
the resulting co-expression network agree well with the general properties of
biological networks. Computational evaluations have showed that RMT approach is
sensitive and robust. Furthermore, evaluation on sampled expression data of an in
silico modular gene system has showed that under-sampled expressions do not
affect the recovery of gene co-expression network. Moreover, the cellular roles
of 215 functionally unknown genes from yeast, E. coli and S. oneidensis are
predicted by the gene co-expression networks using guilt-by-association
principle, many of which are supported by existing information or our
experimental verification, further demonstrating the reliability of this approach
for gene function prediction.
CONCLUSION: Our rigorous analysis of gene expression microarray profiles using
RMT has showed that the transition of NNSD of correlation matrix of microarray
profile provides a profound theoretical criterion to determine the correlation
threshold for identifying gene co-expression networks.

DOI: 10.1186/1471-2105-8-299 
PMCID: PMC2212665
PMID: 17697349  [Indexed for MEDLINE]


846. BMC Microbiol. 2007 Aug 14;7:78.

Identification of human pathogens isolated from blood using microarray
hybridisation and signal pattern recognition.

Wiesinger-Mayr H(1), Vierlinger K, Pichler R, Kriegner A, Hirschl AM, Presterl E,
Bodrossy L, Noehammer C.

Author information: 
(1)Molecular Diagnostics, Austrian Research Centers GmbH - ARC, Mendelstrasse 1, 
A-2444 Seibersdorf, Austria. herbert.wiesinger@arcs.ac.at

BACKGROUND: Pathogen identification in clinical routine is based on the
cultivation of microbes with subsequent morphological and physiological
characterisation lasting at least 24 hours. However, early and accurate
identification is a crucial requisite for fast and optimally targeted
antimicrobial treatment. Molecular biology based techniques allow fast
identification, however discrimination of very closely related species remains
still difficult.
RESULTS: A molecular approach is presented for the rapid identification of
pathogens combining PCR amplification with microarray detection. The DNA chip
comprises oligonucleotide capture probes for 25 different pathogens including
Gram positive cocci, the most frequently encountered genera of
Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The
observed detection limits varied from 10 cells (e.g. E. coli) to 10(5) cells (S. 
aureus) per mL artificially spiked blood. Thus the current low sensitivity for
some species still represents a barrier for clinical application. Successful
discrimination of closely related species was achieved by a signal pattern
recognition approach based on the k-nearest-neighbour method. A prototype
software providing this statistical evaluation was developed, allowing correct
identification in 100 % of the cases at the genus and in 96.7 % at the species
level (n = 241).
CONCLUSION: The newly developed molecular assay can be carried out within 6 hours
in a research laboratory from pathogen isolation to species identification. From 
our results we conclude that DNA microarrays can be a useful tool for rapid
identification of closely related pathogens particularly when the protocols are
adapted to the special clinical scenarios.

DOI: 10.1186/1471-2180-7-78 
PMCID: PMC1994958
PMID: 17697354  [Indexed for MEDLINE]


847. FEMS Microbiol Lett. 2007 Aug;273(2):214-23. Epub 2007 Jun 15.

Escherichia coli O157:H7 gene expression in the presence of catecholamine
norepinephrine.

Dowd SE(1).

Author information: 
(1)United States Department of Agriculture, Agriculture Research Service,
Livestock Issues Research Unit, Lubbock, TX 79403, USA. sdowd@lbk.ars.usda.gov

Various forms of host stresses (e.g. physiological, psychological) are thought to
influence susceptibility to pathogenic microorganisms. Catecholamines such as
norepinephrine are released into the GI environment during acute stress and may
influence the infective process of bacterial pathogens associated with the GI
tract. To examine the effects of norepinephrine on expression of virulence
factors in Escherichia coli O157:H7, the clinical-type isolate EDL933 (ATCC
43895) was grown in serum-Standard American Petroleum Institute media in the
presence or absence of norepinephrine. After 5 h of exposure to norepinephrine,
treatment and control cultures (not exposed to norepinephrine) were harvested,
their RNA isolated, and gene expression evaluated. There was a dramatic increase 
in the expression of virulence factor transcripts including stx1, stx2, and eae. 
Also induced were transcripts involved in iron metabolism. Conversely, there was 
comparative repression of iron acquisition and phage shock protein-related
transcripts in the presence of norepinephrine. Novel observations from these data
suggested that exposure to norepinephrine induced glutamate decarboxylase acid
resistance as well as an SOS response in E. coli O157:H7. The results corroborate
many of the previous findings detailed in the literature and provide new
observations that could expand the scope of microbial endocrinology.

DOI: 10.1111/j.1574-6968.2007.00800.x 
PMID: 17573936  [Indexed for MEDLINE]


848. J Bacteriol. 2007 Aug;189(16):6080-4. Epub 2007 Jun 8.

folA, a new member of the TyrR regulon in Escherichia coli K-12.

Yang J(1), Ogawa Y, Camakaris H, Shimada T, Ishihama A, Pittard AJ.

Author information: 
(1)Department of Microbiology and Immunology, The University of Melbourne,
Victoria 3010, Australia.

The folA gene was identified as a new member of the TyrR regulon by genomic
SELEX. Binding of TyrR to two sites in folA activated its transcription.
Mutations in the N-terminal or central domain of TyrR, the alpha subunit of RNA
polymerase, or integration host factor all abolished activation of the folA
promoter.

DOI: 10.1128/JB.00482-07 
PMCID: PMC1952039
PMID: 17557822  [Indexed for MEDLINE]


849. J Bacteriol. 2007 Aug;189(15):5429-40. Epub 2007 May 25.

Human body temperature (37degrees C) increases the expression of iron,
carbohydrate, and amino acid utilization genes in Escherichia coli K-12.

White-Ziegler CA(1), Malhowski AJ, Young S.

Author information: 
(1)Department of Biological Sciences, Smith College, Northampton, MA 01063, USA. 
cwhitezi@smith.edu

Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose
expression is increased at human body temperature (37 degrees C) compared to
growth at 23 degrees C. Genes involved in the uptake and utilization of amino
acids, carbohydrates, and iron dominated the list, supporting a model in which
temperature serves as a host cue to increase expression of bacterial genes needed
for growth. Using quantitative real-time PCR, we investigated the
thermoregulatory response for representative genes in each of these three
categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene 
papB. Increased expression at 37 degrees C compared to 23 degrees C was retained 
in both exponential and stationary phases for all of the genes and in most of the
various media tested, supporting the relative importance of this cue in adapting 
to changing environments. Because iron acquisition is important for both growth
and virulence, we analyzed the regulation of the iron utilization genes cirA and 
fes and found that growth in iron-depleted medium abrogated the thermoregulatory 
effect, with high-level expression at both temperatures, contrasting with papB
thermoregulation, which was not greatly altered by limiting iron levels. A
positive role for the environmental regulator H-NS was found for fes, cirA, hisJ,
and srlE transcription, whereas it had a primarily negative effect on cysP and
garP expression. Together, these studies indicate that temperature is a broadly
used cue for regulating gene expression in E. coli and that H-NS regulates iron, 
carbohydrate, and amino acid utilization gene expression.

DOI: 10.1128/JB.01929-06 
PMCID: PMC1951813
PMID: 17526711  [Indexed for MEDLINE]


850. J Bacteriol. 2007 Aug;189(15):5472-81. Epub 2007 May 25.

CsrA inhibits translation initiation of Escherichia coli hfq by binding to a
single site overlapping the Shine-Dalgarno sequence.

Baker CS(1), Eöry LA, Yakhnin H, Mercante J, Romeo T, Babitzke P.

Author information: 
(1)Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, PA 16802, USA.

Csr (carbon storage regulation) of Escherichia coli is a global regulatory system
that consists of CsrA, a homodimeric RNA binding protein, two noncoding small
RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering
this protein, and CsrD, a specificity factor that targets CsrB and CsrC for
degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and
pgaA by binding to their leader transcripts and preventing ribosome binding.
Translation inhibition is thought to contribute to the observed mRNA
destabilization. Each of the previously known target transcripts contains
multiple CsrA binding sites. A position-specific weight matrix search program was
developed using known CsrA binding sites in mRNA. This search tool identified a
potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a
gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This
putative CsrA binding site matched the SELEX-derived binding site consensus
sequence in 8 out of 12 positions. Results from gel mobility shift and footprint 
assays demonstrated that CsrA binds specifically to this site in the hfq leader
transcript. Toeprint and cell-free translation results indicated that bound CsrA 
inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of 
csrA caused elevated expression of an hfq'-'lacZ translational fusion, while
overexpression of csrA inhibited expression of this fusion. We also found that
hfq mRNA is stabilized upon entry into stationary-phase growth by a
CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first
example of a CsrA-regulated gene that contains only one CsrA binding site.

DOI: 10.1128/JB.00529-07 
PMCID: PMC1951803
PMID: 17526692  [Indexed for MEDLINE]


851. J Bacteriol. 2007 Aug;189(15):5534-41. Epub 2007 May 18.

PdhR (pyruvate dehydrogenase complex regulator) controls the respiratory electron
transport system in Escherichia coli.

Ogasawara H(1), Ishida Y, Yamada K, Yamamoto K, Ishihama A.

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Kajino-cho 3-7-2,
Koganei, Tokyo 184-8584, Japan.

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the
metabolic interconnection between glycolysis and the citric acid cycle.
Transcription of the Escherichia coli genes for all three components of the PDH
complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, 
a GntR family transcription regulator, and derepressed by pyruvate. After a
systematic search for the regulation targets of PdhR using genomic systematic
evolution of ligands by exponential enrichment (SELEX), we have identified two
novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the
cytochrome bo-type oxidase, both together forming the pathway of respiratory
electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh
and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift
and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined,
which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in 
vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter
assays in vivo using a two-fluorescent-protein vector also indicated that the
newly identified operons are repressed by PdhR and derepressed by the addition of
pyruvate. Taken together, we propose that PdhR is a master regulator for
controlling the formation of not only the PDH complex but also the respiratory
electron transport system.

DOI: 10.1128/JB.00229-07 
PMCID: PMC1951801
PMID: 17513468  [Indexed for MEDLINE]


852. Mol Microbiol. 2007 Aug;65(3):714-27. Epub 2007 Jul 3.

Global analysis of tolerance to secretin-induced stress in Yersinia
enterocolitica suggests that the phage-shock-protein system may be a remarkably
self-contained stress response.

Seo J(1), Savitzky DC, Ford E, Darwin AJ.

Author information: 
(1)Department of Microbiology, New York University School of Medicine, New York, 
NY 10016, USA.

The phage-shock-protein (Psp) system is essential for Yersinia enterocolitica
virulence. Mislocalized secretins induce psp gene expression, and kill psp null
strains. We used transposon mutagenesis to investigate whether other genes are
required to tolerate secretin-induced stress. Our motivation included the
possibility of identifying signal transducers required to activate psp gene
expression. Besides Psp, only defects in the RpoE system and the TrkA potassium
transporter caused secretin sensitivity. These mutations did not cause the same
specific/severe sensitivity as defects in the Psp system, nor did they affect psp
gene expression. The Escherichia coli Psp system was reported to be induced via
the ArcB redox sensor and to activate anaerobic metabolism. Our screen did not
identify arcB, or any genes involved in anaerobic metabolism/regulation.
Therefore, we investigated the role of ArcB in Y. enterocolitica and E. coli.
ArcB was not required for secretin-dependent induction of psp gene expression.
Furthermore, microarray analysis uncovered a restricted transcriptional response 
to prolonged secretin stress in Y. enterocolitica. Taken together, these data do 
not support the proposal that the Psp system is induced via ArcB and activates
anaerobic metabolism. Rather, they suggest that Psp proteins may sense an
inducing trigger and mediate their physiological output(s) directly.

DOI: 10.1111/j.1365-2958.2007.05821.x 
PMID: 17608794  [Indexed for MEDLINE]


853. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12663-8. Epub 2007 Jul 24.

Intracellular crowding defines the mode and sequence of substrate uptake by
Escherichia coli and constrains its metabolic activity.

Beg QK(1), Vazquez A, Ernst J, de Menezes MA, Bar-Joseph Z, Barabási AL, Oltvai
ZN.

Author information: 
(1)Department of Pathology, University of Pittsburgh, 3550 Terrace Street,
Pittsburgh, PA 15261, USA.

The influence of the high intracellular concentration of macromolecules on cell
physiology is increasingly appreciated, but its impact on system-level cellular
functions remains poorly quantified. To assess its potential effect, here we
develop a flux balance model of Escherichia coli cell metabolism that takes into 
account a systems-level constraint for the concentration of enzymes catalyzing
the various metabolic reactions in the crowded cytoplasm. We demonstrate that the
model's predictions for the relative maximum growth rate of wild-type and mutant 
E. coli cells in single substrate-limited media, and the sequence and mode of
substrate uptake and utilization from a complex medium are in good agreement with
subsequent experimental observations. These results suggest that molecular
crowding represents a bound on the achievable functional states of a metabolic
network, and they indicate that models incorporating this constraint can
systematically identify alterations in cellular metabolism activated in response 
to environmental change.

DOI: 10.1073/pnas.0609845104 
PMCID: PMC1937523
PMID: 17652176  [Indexed for MEDLINE]


854. PLoS One. 2007 Jul 25;2(7):e663.

Printing multistrain bacterial patterns with a piezoelectric inkjet printer.

Merrin J(1), Leibler S, Chuang JS.

Author information: 
(1)Laboratory of Living Matter and Center for Physics and Biology, Rockefeller
University, New York, New York, United States of America.

Many studies involving interacting microorganisms would benefit from simple
devices able to deposit cells in precisely defined patterns. We describe an
inexpensive bacterial piezoelectric inkjet printer (adapted from the design of
the POSaM oligonucleotide microarrayer) that can be used to "print out" different
strains of bacteria or chemicals in small droplets onto a flat surface at high
resolution. The capabilities of this device are demonstrated by printing ordered 
arrays comprising two bacterial strains labeled with different fluorescent
proteins. We also characterized several properties of this piezoelectric printer,
such as the droplet volume (of the order of tens of pl), the distribution of
number of cells in each droplet, and the dependence of droplet volume on printing
frequency. We established the limits of the printing resolution, and determined
that the printed viability of Escherichia coli exceeded 98.5%.

DOI: 10.1371/journal.pone.0000663 
PMCID: PMC1914381
PMID: 17653283  [Indexed for MEDLINE]


855. Chem Biol Interact. 2007 Jul 20;168(3):171-83. Epub 2007 Apr 13.

Transcriptional response of genes involved in cell defense system in human cells 
stressed by H2O2 and pre-treated with (Tunisian) Rhamnus alaternus extracts:
combination with polyphenolic compounds and classic in vitro assays.

Ammar RB(1), Bouhlel I, Valenti K, Sghaier MB, Kilani S, Mariotte AM,
Dijoux-Franca MG, Laporte F, Ghedira K, Chekir-Ghedira L.

Author information: 
(1)Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de
Pharmacie/Médecine Dentaire de Monastir, Rue Avicenne 5000 Monastir, Tunisie.

The ability of three Rhamnus alaternus leaves extracts on antigenotoxic and gene 
expression level effects was respectively investigated in a bacterial assay
system, i.e. the SOS chromotest with Escherichia coli PQ37 and in human K562
lymphoblast cell line. Total oligomers flavonoids (TOF) enriched, methanol and
ethyl acetate extracts were prepared from powdered R. alaternus leaves and
characterized quantitatively for the presence of polyphenolic compounds. We
explored the response to oxidative stress using the transcriptional profile of
genes in K562 cells stressed with H2O2 after incubation with plant extracts. For 
this purpose, we used a cDNA microarrays containing 82 genes related to cell
defense, essentially represented by antioxidant and DNA repair genes. Analysis
revealed that SOD1, AOE 372, TXN genes involved in the antioxidant defense system
and XPC, LIG4, POLD2, PCNA genes implied in the DNA repair system were among the 
most expressed ones in the presence of the tested extracts. These results were in
accordance with those obtained when we tested the antigenotoxic and antioxidant
effects of the same extracts with, respectively the SOS chromotest and the
xanthine/xanthine oxidase enzymatic assay system. The effect of the tested
extracts on SOS response induced by both Aflatoxin B1 (AFB1: 10 microg/assay) and
nifuroxazide (20 microg/assay) showed that the TOF extract exhibited the highest 
antimutagenic level towards the indirect mutagen AFB1. Whereas ethyl acetate
extract showed the highest antimutagenic effect towards the direct mutagen,
nifuroxazide. None of the tested extracts induced mutagenic activity. However all
the tested extracts exhibited xanthine oxidase inhibiting and superoxide anions
scavenging effects. R. alaternus extracts contain compounds with significant
antioxidant and antigenotoxic activities. These compounds modulate gene
expression as detected by using cDNA arrays.

DOI: 10.1016/j.cbi.2007.04.002 
PMID: 17512922  [Indexed for MEDLINE]


856. Bioinformatics. 2007 Jul 1;23(13):1640-7. Epub 2007 May 7.

Comparing association network algorithms for reverse engineering of large-scale
gene regulatory networks: synthetic versus real data.

Soranzo N(1), Bianconi G, Altafini C.

Author information: 
(1)SISSA-ISAS, International School for Advanced Studies, via Beirut 2-4, 34014
Trieste, Italy.

MOTIVATION: Inferring a gene regulatory network exclusively from microarray
expression profiles is a difficult but important task. The aim of this work is to
compare the predictive power of some of the most popular algorithms in different 
conditions (like data taken at equilibrium or time courses) and on both synthetic
and real microarray data. We are in particular interested in comparing similarity
measures both of linear type (like correlations and partial correlations) and of 
non-linear type (mutual information and conditional mutual information), and in
investigating the underdetermined case (less samples than genes).
RESULTS: In our simulations we see that all network inference algorithms obtain
better performances from data produced with 'structural' perturbations, like gene
knockouts at steady state, than with any dynamical perturbation. The predictive
power of all algorithms is confirmed on a reverse engineering problem from
Escherichia coli gene profiling data: the edges of the 'physical' network of
transcription factor-binding sites are significantly overrepresented among the
highest weighting edges of the graph that we infer directly from the data without
any structure supervision. Comparing synthetic and in vivo data on the same
network graph allows us to give an indication of how much more complex a real
transcriptional regulation program is with respect to an artificial model.
AVAILABILITY: Software is freely available at the URL
http://people.sissa.it/~altafini/papers/SoBiAl07/.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online.

DOI: 10.1093/bioinformatics/btm163 
PMID: 17485431  [Indexed for MEDLINE]


857. Clin Microbiol Rev. 2007 Jul;20(3):511-32, table of contents.

Laboratory diagnostic techniques for Entamoeba species.

Fotedar R(1), Stark D, Beebe N, Marriott D, Ellis J, Harkness J.

Author information: 
(1)St. Vincent's Hospital, Department of Microbiology, Sydney, Darlinghurst, NSW 
2010, Australia.

The genus Entamoeba contains many species, six of which (Entamoeba histolytica,
Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and
Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica 
is the causative agent of amebiasis and is considered a leading parasitic cause
of death worldwide in humans. Although recent studies highlight the recovery of
E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there 
is still no convincing evidence of a causal link between the presence of these
two species and the symptoms of the host. New approaches to the identification of
E. histolytica are based on detection of E. histolytica-specific antigen and DNA 
in stool and other clinical samples. Several molecular diagnostic tests,
including conventional and real-time PCR, have been developed for the detection
and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical 
samples. The purpose of this review is to discuss different methods that exist
for the identification of E. histolytica, E. dispar, and E. moshkovskii which are
available to the clinical diagnostic laboratory. To address the need for a
specific diagnostic test for amebiasis, a substantial amount of work has been
carried out over the last decade in different parts of the world. The molecular
diagnostic tests are increasingly being used for both clinical and research
purposes. In order to minimize undue treatment of individuals infected with other
species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made
for specific diagnosis of E. histolytica infection and not to treat based simply 
on the microscopic examination of Entamoeba species in the stool. The
incorporation of many new technologies into the diagnostic laboratory will lead
to a better understanding of the public health problem and measures to control
the disease.

DOI: 10.1128/CMR.00004-07 
PMCID: PMC1932757
PMID: 17630338  [Indexed for MEDLINE]


858. J Bacteriol. 2007 Jul;189(13):4791-9. Epub 2007 Apr 27.

Genomic SELEX search for target promoters under the control of the PhoQP-RstBA
signal relay cascade.

Ogasawara H(1), Hasegawa A, Kanda E, Miki T, Yamamoto K, Ishihama A.

Author information: 
(1)Hosei University, Department of Frontier Bioscience, Kajino-cho 3-7-2,
Koganei, Tokyo 184-8584, Japan.

RstBA, a two-component regulatory system of Escherichia coli with an unidentified
regulatory function, is under the control of a Mg(2+)-sensing PhoQP two-component
system. In order to identify the network of transcription regulation downstream
of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by 
using the genomic SELEX system. A gel mobility shift assay indicated the binding 
of RstA to two SELEX DNA fragments, one including the promoter region of asr
(acid shock RNA) and another including the promoter for csgD (a regulator of the 
curli operon). Using a DNase I footprinting assay, we determined the RstA-binding
sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of
the asr gene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in
low-phosphate minimal medium as detected by promoter assay. The acid-induced in
vivo transcription of asr was reduced after the deletion of rstA. In vivo
transcription of the asr promoter was observed only in the presence of RstA. In
agreement with the PhoQP-RstBA network, the addition of Mg(2+) led to a severe
reduction of the asr promoter activity, and the disruption of phoP also reduced
the asr promoter activity, albeit to a lesser extent. These observations
altogether indicate that RstA is an activator of asr transcription. In contrast, 
transcription of csgD was repressed by overexpression of RstA, indicating that
RstA is a repressor for csgD. With these data taken together, we conclude that
the expression of both asr and csgD is under the direct control of the
PhoQP-RstBA signal relay cascade.

DOI: 10.1128/JB.00319-07 
PMCID: PMC1913430
PMID: 17468243  [Indexed for MEDLINE]


859. Plant Mol Biol. 2007 Jul;64(5):481-97. Epub 2007 Apr 21.

Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast
development.

Motohashi R(1), Yamazaki T, Myouga F, Ito T, Ito K, Satou M, Kobayashi M, Nagata 
N, Yoshida S, Nagashima A, Tanaka K, Takahashi S, Shinozaki K.

Author information: 
(1)Faculty of Agriculture, University of Shizuoka, 836 Ohya, Suruga-ku, Shizuoka,
422-8529, Japan. motohasi@agr.shizuoka.ac.jp

To study the functions of nuclear genes involved in chloroplast development, we
systematically analyzed albino and pale green Arabidopsis thaliana mutants by use
of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study,
we focused on one of these albino mutants, designated apg3-1 (for a lbino or p
ale g reen mutant 3). A gene encoding a ribosome release factor 1 (RF1) homologue
was disrupted by the insertion of a Ds transposon into the APG3 gene; a T-DNA
insertion into the same gene caused a similar phenotype (apg3-2). The APG3 gene
(At3g62910) has 15 exons and encodes a protein (422-aa) with a transit peptide
that functions in targeting the protein to chloroplasts. The amino acid sequence 
of APG3 showed 40.6% homology with an RF1 of Escherichia coli, and
complementation analysis using the E. coli rf1 mutant revealed that APG3
functions as an RF1 in E. coli, although complementation was not successful in
the RF2-deficient (rf2) mutants of E. coli. These results indicate that the APG3 
protein is an orthologue of E. coli RF1, and is essential for chloroplast
translation machinery; it was accordingly named AtcpRF1. Since the chloroplasts
of apg3-1 plants contained few internal thylakoid membranes, and chloroplast
proteins related to photosynthesis were not detected by immunoblot analysis,
AtcpRF1 is thought to be essential for chloroplast development.

DOI: 10.1007/s11103-007-9166-7 
PMID: 17450416  [Indexed for MEDLINE]


860. Plant Mol Biol. 2007 Jul;64(5):499-518. Epub 2007 Apr 17.

A functional genomics approach to (iso)flavonoid glycosylation in the model
legume Medicago truncatula.

Modolo LV(1), Blount JW, Achnine L, Naoumkina MA, Wang X, Dixon RA.

Author information: 
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401,
USA.

Analysis of over 200,000 expressed sequence tags from a range of Medicago
truncatula cDNA libraries resulted in the identification of over 150 different
family 1 glycosyltransferase (UGT) genes. Of these, 63 were represented by full
length clones in an EST library collection. Among these, 19 gave soluble proteins
when expressed in E. coli, and these were screened for catalytic activity against
a range of flavonoid and isoflavonoid substrates using a high-throughput HPLC
assay method. Eight UGTs were identified with activity against isoflavones,
flavones, flavonols or anthocyanidins, and several showed high catalytic
specificity for more than one class of (iso)flavonoid substrate. All tested UGTs 
preferred UDP-glucose as sugar donor. Phylogenetic analysis indicated that the
Medicago (iso)flavonoid glycosyltransferase gene sequences fell into a number of 
different clades, and several clustered with UGTs annotated as glycosylating
non-flavonoid substrates. Quantitative RT-PCR and DNA microarray analysis
revealed unique transcript expression patterns for each of the eight UGTs in
Medicago organs and cell suspension cultures, and comparison of these patterns
with known phytochemical profiles suggested in vivo functions for several of the 
enzymes.

DOI: 10.1007/s11103-007-9167-6 
PMID: 17437063  [Indexed for MEDLINE]


861. BMC Bioinformatics. 2007 Jun 14;8:203.

Using expression arrays for copy number detection: an example from E. coli.

Skvortsov D(1), Abdueva D, Stitzer ME, Finkel SE, Tavaré S.

Author information: 
(1)Molecular and Computational Biology Program, Department of Biological
Sciences, University of Southern California, Los Angeles, CA 90089-2910, USA.
dskvortsov@mednet.ucla.edu <dskvortsov@mednet.ucla.edu>

BACKGROUND: The sequencing of many genomes and tiling arrays consisting of
millions of DNA segments spanning entire genomes have made high-resolution copy
number analysis possible. Microarray-based comparative genomic hybridization
(array CGH) has enabled the high-resolution detection of DNA copy number
aberrations. While many of the methods and algorithms developed for the analysis 
microarrays have focused on expression analysis, the same technology can be used 
to detect genetic alterations, using for example standard commercial Affymetrix
arrays. Due to the nature of the resultant data, standard techniques for
processing GeneChip expression experiments are inapplicable.
RESULTS: We have developed a robust and flexible methodology for high-resolution 
analysis of DNA copy number of whole genomes, using Affymetrix high-density
expression oligonucleotide microarrays. Copy number is obtained from fluorescence
signals after processing with novel normalization, spatial artifact correction,
data transformation and deletion/duplication detection. We applied our approach
to identify deleted and amplified regions in E. coli mutants obtained after
prolonged starvation.
CONCLUSION: The availability of Affymetrix expression chips for a wide variety of
organisms makes the proposed array CGH methodology useful more generally.

DOI: 10.1186/1471-2105-8-203 
PMCID: PMC1914360
PMID: 17570850  [Indexed for MEDLINE]


862. BMC Microbiol. 2007 Jun 8;7:53.

Identification of regulatory network topological units coordinating the
genome-wide transcriptional response to glucose in Escherichia coli.

Gutierrez-Ríos RM(1), Freyre-Gonzalez JA, Resendis O, Collado-Vides J, Saier M,
Gosset G.

Author information: 
(1)Departamentos de Microbiología Molecular, Instituto de Biotecnología,
Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Morelos,
México. rmaria@ibt.unam.mx

BACKGROUND: Glucose is the preferred carbon and energy source for Escherichia
coli. A complex regulatory network coordinates gene expression, transport and
enzyme activities in response to the presence of this sugar. To determine the
extent of the cellular response to glucose, we applied an approach combining
global transcriptome and regulatory network analyses.
RESULTS: Transcriptome data from isogenic wild type and crp- strains grown in
Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify 
differentially transcribed genes. We detected 180 and 200 genes displaying
increased and reduced relative transcript levels in the presence of glucose,
respectively. The observed expression pattern in LB was consistent with a
gluconeogenic metabolic state including active transport and interconversion of
small molecules and macromolecules, induction of protease-encoding genes and a
partial heat shock response. In LB+G, catabolic repression was detected for
transport and metabolic interconversion activities. We also detected an increased
capacity for de novo synthesis of nucleotides, amino acids and proteins. Cluster 
analysis of a subset of genes revealed that CRP mediates catabolite repression
for most of the genes displaying reduced transcript levels in LB+G, whereas Fis
participates in the upregulation of genes under this condition. An analysis of
the regulatory network, in terms of topological functional units, revealed 8
interconnected modules which again exposed the importance of Fis and CRP as
directly responsible for the coordinated response of the cell. This effect was
also seen with other not extensively connected transcription factors such as FruR
and PdhR, which showed a consistent response considering media composition.
CONCLUSION: This work allowed the identification of eight interconnected
regulatory network modules that includes CRP, Fis and other transcriptional
factors that respond directly or indirectly to the presence of glucose. In most
cases, each of these modules includes genes encoding physiologically related
functions, thus indicating a connection between regulatory network topology and
related cellular functions involved in nutrient sensing and metabolism.

DOI: 10.1186/1471-2180-7-53 
PMCID: PMC1905917
PMID: 17559662  [Indexed for MEDLINE]


863. BMC Genomics. 2007 Jun 4;8:143.

Conservation of the links between gene transcription and chromosomal organization
in the highly reduced genome of Buchnera aphidicola.

Viñuelas J(1), Calevro F, Remond D, Bernillon J, Rahbé Y, Febvay G, Fayard JM,
Charles H.

Author information: 
(1)UMR 203 Biologie Fonctionnelle Insectes et Interactions, IFR41, INRA,
INSA-Lyon, Villeurbanne, France. jose.vinuelas@insa-lyon.fr

BACKGROUND: Genomic studies on bacteria have clearly shown the existence of
chromosomal organization as regards, for example, to gene localization, order and
orientation. Moreover, transcriptomic analyses have demonstrated that, in
free-living bacteria, gene transcription levels and chromosomal organization are 
mutually influenced. We have explored the possible conservation of relationships 
between mRNA abundances and chromosomal organization in the highly reduced genome
of Buchnera aphidicola, the primary endosymbiont of the aphids, and a close
relative to Escherichia coli.
RESULTS: Using an oligonucleotide-based microarray, we normalized the
transcriptomic data by genomic DNA signals in order to have access to inter-gene 
comparison data. Our analysis showed that mRNA abundances, gene organization
(operon) and gene essentiality are correlated in Buchnera (i.e., the most
expressed genes are essential genes organized in operons) whereas no link between
mRNA abundances and gene strand bias was found. The effect of Buchnera genome
evolution on gene expression levels has also been analysed in order to assess the
constraints imposed by the obligate symbiosis with aphids, underlining the
importance of some gene sets for the survival of the two partners. Finally, our
results show the existence of spatial periodic transcriptional patterns in the
genome of Buchnera.
CONCLUSION: Despite an important reduction in its genome size and an apparent
decay of its capacity for regulating transcription, this work reveals a
significant correlation between mRNA abundances and chromosomal organization of
the aphid-symbiont Buchnera.

DOI: 10.1186/1471-2164-8-143 
PMCID: PMC1899503
PMID: 17547756  [Indexed for MEDLINE]


864. Appl Environ Microbiol. 2007 Jun;73(12):4082-8. Epub 2007 Apr 20.

DNA microarray-based identification of serogroups and virulence gene patterns of 
Escherichia coli isolates associated with porcine postweaning diarrhea and edema 
disease.

Han W(1), Liu B, Cao B, Beutin L, Krüger U, Liu H, Li Y, Liu Y, Feng L, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, 23
Hongda Street, TEDA, Tianjin 300457, China.

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease
(ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139,
O141, O147, O149, and O157 being the most commonly reported worldwide. In this
study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli
serogroups, as well as 11 genes encoding adhesion factors and exotoxins
associated with PWD and ED, was developed for the identification of related
serogroups and virulence gene patterns. The microarray method was tested against 
186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference
strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of
other bacterial species and shown to be highly specific with reproducible
results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3
g of porcine feces in mock samples. Seventeen porcine feces samples from local
hoggeries were examined using the microarray, and the result for one sample was
verified by the conventional serotyping methods. This microarray can be readily
used to screen for the presence of PWD- and ED-associated E. coli in porcine
feces samples.

DOI: 10.1128/AEM.01820-06 
PMCID: PMC1932722
PMID: 17449692  [Indexed for MEDLINE]


865. Appl Environ Microbiol. 2007 Jun;73(11):3645-55. Epub 2007 Apr 6.

Detection and genotyping of Arcobacter and Campylobacter isolates from retail
chicken samples by use of DNA oligonucleotide arrays.

Quiñones B(1), Parker CT, Janda JM Jr, Miller WG, Mandrell RE.

Author information: 
(1)U.S. Department of Agriculture, Agricultural Research Service, Western
Regional Research Center, Produce Safety and Microbiology Research Unit, Albany, 
CA 94710, USA.

To explore the use of DNA microarrays for pathogen detection in food, we produced
DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter
and the presence of Campylobacter in retail chicken samples. Probes were selected
that target housekeeping and virulence-associated genes in both Arcobacter
butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These
microarrays showed a high level of probe specificity; the signal intensities
detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold
higher than the background levels. Specific identification of A. butzleri, C.
coli, and C. jejuni was achieved without the need for a PCR amplification step.
By adapting an isolation method that employed membrane filtration and selective
media, C. jejuni isolates were recovered from package liquid from whole chicken
carcasses prior to enrichment. Increasing the time of enrichment resulted in the 
isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni
isolates were further classified by using an additional subset of probes
targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results
demonstrated that most of the C. jejuni isolates likely possess class B, C, or H 
LOS. Validation experiments demonstrated that the DNA microarray had a detection 
sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the
use of C. jejuni sequence-specific primers to label genomic DNA improved the
sensitivity of this DNA microarray for detection of C. jejuni in whole chicken
carcass samples. C. jejuni was efficiently detected directly both in package
liquid from whole chicken carcasses and in enrichment broths.

DOI: 10.1128/AEM.02984-06 
PMCID: PMC1932690
PMID: 17416693  [Indexed for MEDLINE]


866. Curr Opin Microbiol. 2007 Jun;10(3):262-70. Epub 2007 Jun 15.

Target identification of small noncoding RNAs in bacteria.

Vogel J(1), Wagner EG.

Author information: 
(1)Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin,
Germany. vogel@mpiib-berlin.mpg.de

Small noncoding RNAs have been discovered at a staggering rate in Escherichia
coli and many other bacteria. Most of the sRNAs of known function regulate gene
expression by binding to specific mRNAs or proteins. Given the scores of sRNAs of
unknown function, the identification of their cellular targets has become urgent.
Here, we review the diverse strategies that have been used to identify and
validate bacterial sRNA targets. These include the pulse-expression of sRNAs
followed by global transcriptome analysis (microarrays), new biocomputational
prediction algorithms, and novel gfp reporter gene fusions to validate candidate 
target gene regulation.

DOI: 10.1016/j.mib.2007.06.001 
PMID: 17574901  [Indexed for MEDLINE]


867. Curr Opin Microbiol. 2007 Jun;10(3):257-61. Epub 2007 Jun 5.

Identification of bacterial small non-coding RNAs: experimental approaches.

Altuvia S(1).

Author information: 
(1)Department of Molecular Genetics and Biotechnology, The Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel. shoshy@cc.huji.ac.il

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs)
have been discovered in the past six years. The majority of these sRNAs were
discovered in Escherichia coli, and a smaller subset was characterized in other
bacteria, many of which were pathogenic. Many of these genes were identified as a
result of systematic screens using computational prediction of sRNAs and
experimental-based approaches, including microarray and shotgun cloning. A
smaller number of sRNAs were discovered by direct labeling or by functional
genetic screens. Many of the discovered genes, ranging in size from 50 to 500
nucleotides, are conserved and located in intergenic regions, in-between open
reading frames. The expression of many of these genes is growth phase dependent
or stress related. As each search employed specific parameters, this led to the
identification of genes with distinct characteristics. Consequently, unique sRNAs
such as those that are species-specific, sRNA genes that are transcribed under
unique conditions or genes located on the antisense strand of protein-encoding
genes, were probably missed.

DOI: 10.1016/j.mib.2007.05.003 
PMID: 17553733  [Indexed for MEDLINE]


868. Eukaryot Cell. 2007 Jun;6(6):919-30. Epub 2007 Apr 13.

Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii.

Ledford HK(1), Chin BL, Niyogi KK.

Author information: 
(1)Department of Plant and Microbial Biology, 111 Koshland Hall, University of
California-Berkeley, Berkeley, CA 94720-3102, USA.

In an aerobic environment, responding to oxidative cues is critical for
physiological adaptation (acclimation) to changing environmental conditions. The 
unicellular alga Chlamydomonas reinhardtii was tested for the ability to
acclimate to specific forms of oxidative stress. Acclimation was defined as the
ability of a sublethal pretreatment with a reactive oxygen species to activate
defense responses that subsequently enhance survival of that stress. C.
reinhardtii exhibited a strong acclimation response to rose bengal, a
photosensitizing dye that produces singlet oxygen. This acclimation was dependent
upon photosensitization and occurred only when pretreatment was administered in
the light. Shifting cells from low light to high light also enhanced resistance
to singlet oxygen, suggesting an overlap in high-light and singlet oxygen
response pathways. Microarray analysis of RNA levels indicated that a relatively 
small number of genes respond to sublethal levels of singlet oxygen. Constitutive
overexpression of either of two such genes, a glutathione peroxidase gene and a
glutathione S-transferase gene, was sufficient to enhance singlet oxygen
resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined
responses to reactive oxygen but did not acclimate to singlet oxygen, possibly
reflecting the relative importance of singlet oxygen stress for photosynthetic
organisms.

DOI: 10.1128/EC.00207-06 
PMCID: PMC1951523
PMID: 17435007  [Indexed for MEDLINE]


869. Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2676-84.

Unique gene expression profiles of donor-matched human retinal and choroidal
vascular endothelial cells.

Smith JR(1), Choi D, Chipps TJ, Pan Y, Zamora DO, Davies MH, Babra B, Powers MR, 
Planck SR, Rosenbaum JT.

Author information: 
(1)Casey Eye Institute, Oregon Health and Science University, Portland, Oregon
97239, USA. smithjus@ohsu.edu

PURPOSE: Consistent with clinical observations that posterior uveitis frequently 
involves the retinal vasculature and recent recognition of vascular
heterogeneity, the hypothesis for this study was that retinal vascular
endothelium was a cell population of unique molecular phenotype.
METHODS: Donor-matched cultures of primary retinal and choroidal endothelial
cells from six human cadavers were incubated with either Toxoplasma gondii
tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide
(100 ng/mL); control cultures were simultaneously incubated with medium. Gene
expression profiling of endothelial cells was performed using oligonucleotide
arrays containing probes designed to detect 8746 human transcripts. After
normalization, differential gene expression was assessed by the significance
analysis of microarrays, with the false-discovery rate set at 5%. For selected
genes, differences in the level of expression between retinal and choroidal cells
were evaluated by real-time RT-PCR.
RESULTS: Graphic descriptive analysis demonstrated a strong correlation between
gene expression of unstimulated retinal and choroidal endothelial cells, but also
highlighted distinctly different patterns of expression that were greater than
differences noted between donors or between unstimulated and stimulated cells.
Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note,
the 330 transcripts that were present at higher levels in retinal cells included 
a larger percentage of transcripts encoding molecules involved in the immune
response. Differential gene expression was confirmed for 12 transcripts by
RT-PCR.
CONCLUSIONS: Retinal and choroidal vascular endothelial cells display distinctive
gene expression profiles. The findings suggest the possibility of treating
posterior uveitis by targeting specific interactions between the retinal
endothelial cell and an infiltrating leukocyte.

DOI: 10.1167/iovs.06-0598 
PMID: 17525199  [Indexed for MEDLINE]


870. J Bacteriol. 2007 Jun;189(12):4410-7. Epub 2007 Apr 20.

The NsrR regulon of Escherichia coli K-12 includes genes encoding the hybrid
cluster protein and the periplasmic, respiratory nitrite reductase.

Filenko N(1), Spiro S, Browning DF, Squire D, Overton TW, Cole J, Constantinidou 
C.

Author information: 
(1)School of Biosciences, University of Birmingham, Birmingham B15 2TT, United
Kingdom.

Successful pathogens must be able to protect themselves against reactive nitrogen
species generated either as part of host defense mechanisms or as products of
their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of
transcription factors) plays key roles in this stress response. Microarray
analysis revealed that NsrR represses nine operons encoding 20 genes in
Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were
previously shown to be regulated by NsrR. Novel NsrR targets revealed by this
study include hcp-hcr (which were predicted in a recent bioinformatic study to be
NsrR regulated) and the well-studied nrfA promoter that directs the expression of
the periplasmic respiratory nitrite reductase. Conversely, transcription from the
ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR
is consistent with the ability of the periplasmic nitrite reductase to reduce
nitric oxide and hence protect against reactive nitrogen species. Gel retardation
assays were used to show that both FNR and NarL bind to the hcp promoter. The
expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As
hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair
iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr,
hmpA, and ytfE are the three transcripts most tightly regulated by NsrR
highlights the possibility that the hybrid cluster protein, HCP, might also be
part of a defense mechanism against reactive nitrogen stress.

DOI: 10.1128/JB.00080-07 
PMCID: PMC1913375
PMID: 17449618  [Indexed for MEDLINE]


871. Protein Expr Purif. 2007 Jun;53(2):356-62. Epub 2006 Dec 24.

Purification of recombinant human interferon-epsilon and oligonucleotide
microarray analysis of interferon-epsilon-regulated genes.

Peng FW(1), Duan ZJ, Zheng LS, Xie ZP, Gao HC, Zhang H, Li WP, Hou YD.

Author information: 
(1)Institute for Viral Disease Control and Prevention, Chinese Center for Disease
Control and Prevention, 100 Ying-Xin Street, Xuan Wu District, Beijing 100052,
China.

Recently identified interferon-epsilon (IFN-epsilon) belongs to type I
interferons. IFN-epsilon is highly and constitutively expressed in the brain, but
its biochemical and biological characteristics are poorly understood. In this
study, full-length IFN-epsilon cDNA was cloned from human peripheral blood
lymphocyte by RT-PCR, and was expressed in Escherichia coli (E. coli). Reverse
phase high pressure liquid chromatography was used to purify recombinant human
IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About
0.8mg of highly purified rhIFN-epsilon protein was obtained from 100ml of E. coli
culture. Functional study of rhIFN-epsilon demonstrated that the antiviral
activity of rhIFN-epsilon was 6+/-0.5x10(5)IU/mg, which was lower than that of
rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis
virus) assay system. As for the activity to promote NK cytotoxicity and
antiproliferation activities, rhIFN-epsilon was about 60 times less potent than
rhIFN-alpha-2b. However, oligonucleotide microarray analyses revealed dramatic
differences in gene expression profiles of cultured human cells treated with
IFN-epsilon and IFN-alpha-2b. Particularly, differential regulation of genes
related to central nervous system by rhIFN-epsilon suggests a role for
IFN-epsilon in maintenance of the structure and function of brain.

DOI: 10.1016/j.pep.2006.12.013 
PMID: 17287131  [Indexed for MEDLINE]


872. Forensic Sci Int. 2007 May 24;168(2-3):183-99. Epub 2006 Aug 28.

Interrogating genomic diversity of E. coli O157:H7 using DNA tiling arrays.

Jackson SA(1), Mammel MK, Patel IR, Mays T, Albert TJ, LeClerc JE, Cebula TA.

Author information: 
(1)Division of Molecular Biology, Office of Applied Research and Safety
Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug
Administration, Laurel, MD 20708, USA.

Here, we describe a novel microarray-based approach for investigating the genomic
diversity of Escherichia coli O157:H7 in a semi-high throughput manner using a
high density, oligonucleotide-based microarray. This microarray, designed to
detect polymorphisms at each of 60,000 base-pair (bp) positions within an E. coli
genome, is composed of overlapping 29-mer oligonucleotides specific for 60
equally spaced, 1000-bp loci of the E. coli O157:H7 strain EDL933 chromosome. By 
use of a novel 12-well microarray that permitted the simultaneous investigation
of 12 strains, the genomes of 44 individual isolates of E. coli O157:H7 were
interrogated. These analyses revealed more than 150 single nucleotide
polymorphisms (SNPs) and several deletions and amplifications in the test
strains. Pyrosequencing was used to confirm the usefulness of the novel SNPs by
determining their allelic frequency among a collection of diverse isolates of E. 
coli O157:H7. The tiling DNA microarray system would be useful for the tracking
and identification of individual strains of E. coli O157:H7 needed for forensic
investigations.

DOI: 10.1016/j.forsciint.2006.06.079 
PMID: 16934953  [Indexed for MEDLINE]


873. BMC Microbiol. 2007 May 18;7:42.

Indole is an inter-species biofilm signal mediated by SdiA.

Lee J(1), Jayaraman A, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A & M University,
College Station, TX 77843-3122, USA. Jintae.Lee@chemail.tamu.edu
<Jintae.Lee@chemail.tamu.edu>

BACKGROUND: As a stationary phase signal, indole is secreted in large quantities 
into rich medium by Escherichia coli and has been shown to control several genes 
(e.g., astD, tnaB, gabT), multi-drug exporters, and the pathogenicity island of
E. coli; however, its impact on biofilm formation has not been well-studied.
RESULTS: Through a series of global transcriptome analyses, confocal microscopy, 
isogenic mutants, and dual-species biofilms, we show here that indole is a
non-toxic signal that controls E. coli biofilms by repressing motility, inducing 
the sensor of the quorum sensing signal autoinducer-1 (SdiA), and influencing
acid resistance (e.g., hdeABD, gadABCEX). Isogenic mutants showed these
associated proteins are directly related to biofilm formation (e.g., the sdiA
mutation increased biofilm formation 50-fold), and SdiA-mediated transcription
was shown to be influenced by indole. The reduction in motility due to indole
addition results in the biofilm architecture changing from scattered towers to
flat colonies. Additionally, there are 12-fold more E. coli cells in dual-species
biofilms grown in the presence of Pseudomonas cells engineered to express toluene
o-monooxygenase (TOM, which converts indole to an insoluble indigoid) than in
biofilms with pseudomonads that do not express TOM due to a 22-fold reduction in 
extracellular indole. Also, indole stimulates biofilm formation in pseudomonads. 
Further evidence that the indole effects are mediated by SdiA and homoserine
lactone quorum sensing is that the addition of N-butyryl-, N-hexanoyl-, and
N-octanoyl-L-homoserine lactones repress E. coli biofilm formation in the
wild-type strain but not with the sdiA mutant.
CONCLUSION: Indole is an interspecies signal that decreases E. coli biofilms
through SdiA and increases those of pseudomonads. Indole may be manipulated to
control biofilm formation by oxygenases of bacteria that do not synthesize it in 
a dual-species biofilm. Furthermore, E. coli changes its biofilm in response to
signals it cannot synthesize (homoserine lactones), and pseudomonads respond to
signals they do not synthesize (indole).

DOI: 10.1186/1471-2180-7-42 
PMCID: PMC1899176
PMID: 17511876  [Indexed for MEDLINE]


874. BMC Genomics. 2007 May 16;8:121.

Genome evolution in major Escherichia coli O157:H7 lineages.

Zhang Y(1), Laing C, Steele M, Ziebell K, Johnson R, Benson AK, Taboada E, Gannon
VP.

Author information: 
(1)Laboratory for Foodborne Zoonoses, Health Canada, Lethbridge, AB, Canada.
yzhang@inspection.gc.ca <yzhang@inspection.gc.ca>

BACKGROUND: Genetic analysis of Escherichia coli O157:H7 strains has shown
divergence into two distinct lineages, lineages I and II, that appear to have
distinct ecological characteristics, with lineage I strains more commonly
associated with human disease. In this study, microarray-based comparative
genomic hybridization (CGH) was used to identify genomic differences among 31 E. 
coli O157:H7 strains that belong to various phage types (PTs) and different
lineage-specific polymorphism assay (LSPA) types.
RESULTS: A total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 
strains and 1,751 were variably present or absent. Based on this data, E. coli
O157:H7 strains were divided into three distinct clusters, which consisted of 15 
lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA 
type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221),
respectively. Eleven different genomic regions that were dominant in lineage I
strains (present in > or =80% of lineage I and absent from > or = 92% of lineage 
II strains) spanned segments containing as few as two and up to 25 ORFs each.
These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 
83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic
backbone region. All four lineage I/II strains were of PT 2 and possessed eight
of these 11 lineage I-dominant loci. Several differences in virulence-associated 
loci were noted between lineage I and lineage II strains, including divergence
within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded
effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II
strains.
CONCLUSION: CGH data suggest the existence of two dominant lineages as well as
LSPA type and PT-related subgroups within E. coli O157:H7. The genomic
composition of these subgroups supports the phylogeny that has been inferred from
other methods and further suggests that genomic divergence from an ancestral form
and lateral gene transfer have contributed to their evolution. The genomic
features identified in this study may contribute to apparent differences in the
epidemiology and ecology of strains of different E. coli O157:H7 lineages.

DOI: 10.1186/1471-2164-8-121 
PMCID: PMC1890555
PMID: 17506902  [Indexed for MEDLINE]


875. Expert Opin Pharmacother. 2007 May;8(7):895-900.

Microarray analysis to reveal genes involved in colon carcinogenesis.

Joyce T(1), Pintzas A.

Author information: 
(1)Laboratory of Signal Mediated Gene Expression, Institute of Biological
Research and Biotechnology, National Hellenic Research Foundation, Athens,
Greece.

Sporadic colon cancer is a major cause of death throughout the world. Multistage 
development of the disease has been associated with remarkable genetic events,
mainly at the level of oncogenes and oncosuppressor genes, most notably APC
(adenomatous polyposis coli), ras and p53. Despite all of these efforts, the
development of a sensitive and convenient diagnostic system for detecting
colorectal cancers at the early stage is still in progress. In recent years, cDNA
and oligonucleotide microarray technologies have made the analysis of gene
expression profiles of colorectal tumours at the genomic level possible and have 
identified signatures of gene expression associated with pre-cancerous
phenotypes, cancers of the early stage and/or metastatic cancer. The contribution
of this powerful technology in identification of novel important genes for
prognosis, diagnosis and therapy of sporadic colorectal will be discussed.

DOI: 10.1517/14656566.8.7.895	 
PMID: 17472535  [Indexed for MEDLINE]


876. J Bacteriol. 2007 May;189(9):3532-46. Epub 2007 Mar 9.

Defining genomic islands and uropathogen-specific genes in uropathogenic
Escherichia coli.

Lloyd AL(1), Rasko DA, Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, 5641 Medical Science Bldg. II, 1150 West Medical Center Dr., Ann Arbor,
MI 48109-0620, USA.

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of
uncomplicated urinary tract infections, which can present clinically as cystitis 
or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with 
acute pyelonephritis, was most cytotoxic and most virulent in mice among our
strain collection. Based on the genome sequence of CFT073, microarrays were
utilized in comparative genomic hybridization (CGH) analysis of a panel of
uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC
(three pyelonephritis and four cystitis) isolates and three fecal/commensal
strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The
CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of
these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific
genes were found by CGH to be present in all UPEC strains but in none of the
fecal/commensal strains. When the sequences of three additional sequenced UPEC
strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the
analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains
were identified. Seven previously unrecognized genomic islands (>30 kb) were
delineated by CGH in addition to the three known pathogenicity islands. These
genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the
importance of horizontal transfer for UPEC and the mosaic structure of the
genome. UPEC strains contain a greater number of iron acquisition systems than do
fecal/commensal strains, which is reflective of the adaptation to the
iron-limiting urinary tract environment. Each strain displayed distinct
differences in the number and type of known virulence factors. The large number
of hypothetical genes in the CFT073 genome, especially those shown to be UPEC
specific, strongly suggests that many urovirulence factors remain
uncharacterized.

DOI: 10.1128/JB.01744-06 
PMCID: PMC1855899
PMID: 17351047  [Indexed for MEDLINE]


877. J Bacteriol. 2007 May;189(9):3489-95. Epub 2007 Mar 9.

The Escherichia coli regulator of sigma 70 protein, Rsd, can up-regulate some
stress-dependent promoters by sequestering sigma 70.

Mitchell JE(1), Oshima T, Piper SE, Webster CL, Westblade LF, Karimova G, Ladant 
D, Kolb A, Hobman JL, Busby SJ, Lee DJ.

Author information: 
(1)School of Biosciences, University of Birmingham, Birmingham B15 2TT, United
Kingdom.

The Escherichia coli Rsd protein forms complexes with the RNA polymerase
sigma(70) factor, but its biological role is not understood. Transcriptome
analysis shows that overexpression of Rsd causes increased expression from some
promoters whose expression depends on the alternative sigma(38) factor, and this 
was confirmed by experiments with lac fusions at selected promoters. The LP18
substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac
fusions. Analysis with a bacterial two-hybrid system shows that the LP18
substitution in Rsd increases its interaction with sigma(70). Our experiments
support a model in which the role of Rsd is primarily to sequester sigma(70),
thereby increasing the levels of RNA polymerase containing the alternative
sigma(38) factor.

DOI: 10.1128/JB.00019-07 
PMCID: PMC1855875
PMID: 17351046  [Indexed for MEDLINE]


878. J Biotechnol. 2007 May 1;129(3):461-71. Epub 2007 Jan 25.

Globin-expression postpones onset of stationary phase specific gene expression in
Escherichia coli.

Frey AD(1), Andersson CI, Schmid VH, Bülow L, Kallio PT.

Author information: 
(1)Institute of Microbiology, ETH-Zürich, CH-8093 Zürich, Switzerland.
alexander.frey@micro.biol.ethz.ch

We have analyzed gene expression of Escherichia coli MG1655 expressing native and
engineered bacterial globin proteins, in order to identify the molecular
mechanisms leading to the improved phenotypical traits relative to control cells 
under oxygen-limited conditions. Regulated expression of hemoglobin and
flavohemoglobin proteins postponed the onset of rpoS expression relative to
plasmid bearing control cells. This change in expression pattern coincided with
the expression pattern of stationary-phase specific genes including
sigma(S)-dependent and sigma(S)-independent genes. Furthermore, several genes
known to affect rpoS transcription, rpoS mRNA stability and sigma(S) turnover
were regulated in such a manner as to ultimately lower the cellular level of
sigma(S) in all globin-expressing strains. In a strain harboring an rpoS-lacZ
fusion, lacZ expression correlated with acetate accumulation, a metabolite that
is known to activate rpoS transcription, but not with growth. Therefore, we
hypothesize that reduced excretion of acetate in globin expressing cells prevents
induction of stationary phase specific genes. Additionally, several genes
responding to carbon starvation (e.g. csrAB, cstA, sspA) were expressed at lower 
levels in globin-expressing cells. These findings are in good agreement with
previous reports showing a more efficient energy household, i.e. also reduced
glucose consumption, in hemoglobin- and flavohemoglobin-expressing cells relative
to controls.

DOI: 10.1016/j.jbiotec.2007.01.012 
PMID: 17320232  [Indexed for MEDLINE]


879. Microbiology. 2007 May;153(Pt 5):1464-73.

Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia 
pseudotuberculosis reveals a tight link between VagH and the type III secretion
system.

Garbom S(1), Olofsson M, Björnfot AC, Srivastava MK, Robinson VL, Oyston PC,
Titball RW, Wolf-Watz H.

Author information: 
(1)Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.

Recently, a number of attenuated mutants of Yersinia pseudotuberculosis have been
identified using a bioinformatics approach. One of the target genes identified in
that study was vagH, which the authors now characterized further. VagH shows
homology to HemK of Escherichia coli, possessing methyltransferase activity
similar to that of HemK, and targeting release factors 1 and 2. Microarray
studies comparing the wild-type and the vagH mutant revealed that the mRNA levels
of only a few genes were altered in the mutant. By proteome analysis, expression 
of the virulence determinant YopD was found to be increased, indicating a
possible connection between VagH and the virulence plasmid-encoded type III
secretion system (T3SS). Further analysis showed that Yop expression and
secretion were repressed in a vagH mutant. This phenotype could be suppressed by 
trans-complementation with the wild-type vagH gene or by deletion of the negative
regulator yopD. Also, in a similar manner to a T3SS-negative mutant, the
avirulent vagH mutant was rapidly cleared from Peyer's patches and could not
reach the spleen after oral infection of mice. In a manner analogous to that of
T3SS mutants, the vagH mutant could not block phagocytosis by macrophages.
However, a vagH mutant showed no defects in the T3SS-independent ability to
proliferate intracellularly and replicated to levels similar to those of the
wild-type in macrophages. In conclusion, the vagH mutant exhibits a virulence
phenotype similar to that of a T3SS-negative mutant, indicating a tight link
between VagH and type III secretion in Y. pseudotuberculosis.

DOI: 10.1099/mic.0.2006/000323-0 
PMID: 17464060  [Indexed for MEDLINE]


880. Microbiology. 2007 May;153(Pt 5):1350-60.

Analysis of the expression, regulation and export of NleA-E in Escherichia coli
O157 : H7.

Roe AJ(1), Tysall L, Dransfield T, Wang D, Fraser-Pitt D, Mahajan A, Constandinou
C, Inglis N, Downing A, Talbot R, Smith DG, Gally DL.

Author information: 
(1)Zoonotic and Animal Pathogens Research Laboratory, Centre for Infectious
Disease, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16
4SB, UK.

Previous work has shown that locus of enterocyte effacement (LEE)-encoded
effector proteins such as Tir and Map can be exported via the type III secretion 
system (T3SS) of Escherichia coli O157 : H7. Additionally, a family of
non-LEE-encoded (Nle) effector proteins has been shown to be secreted from
Citrobacter rodentium, homologues of which are located on the E. coli O157
chromosome. While NleA has been shown to be secreted from pathogenic E. coli, the
secretion of other Nle effector proteins has only been detected under induced
conditions, or using a mutated T3SS. This study aimed to determine: (1) which nle
genes are expressed in E. coli O157 : H7 under secretion-permissive conditions;
(2) if Nle proteins are secreted from wild-type E. coli O157 : H7 under
secretion-permissive conditions; and (3) if nle gene expression is regulated
co-ordinately with other LEE-encoded effectors. Using data generated from a
combination of transcriptome arrays, reporter fusions and proteomics, it was
demonstrated that only nleA is expressed co-ordinately with the LEE. Secretion
and expression of NleA were regulated directly or indirectly by ler, a key
activator of the LEE. MS confirmed the secretion of NleA into the culture
supernatant, while NleB-F were not detected.

DOI: 10.1099/mic.0.2006/003707-0 
PMID: 17464049  [Indexed for MEDLINE]


881. Plant Cell Physiol. 2007 May;48(5):700-14. Epub 2007 Mar 22.

Induction of a novel XIP-type xylanase inhibitor by external ascorbic acid
treatment and differential expression of XIP-family genes in rice.

Tokunaga T(1), Esaka M.

Author information: 
(1)Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama,
Higashi-Hiroshima, 739-8528 Japan.

Rice microarray analysis showed that a number of stress-related genes are induced
by external addition of L-ascorbic acid (AsA). The gene designated as AK073843
which is homologous to class capital SHA, Cyrillic chitinase was found to exhibit
the highest induction among these genes. However, its crucial residues within the
chitinase active site are substituted with other residues, suggesting that the
protein has no chitinase activity. The recombinant protein which is encoded by
the AK073843 gene produced in Escherichia coli has xylanase inhibitor activity,
indicating that the gene encodes a novel rice XIP-type xylanase inhibitor protein
(OsXIP). The expression of OsXIP was enhanced not only by exogenous AsA treatment
but also by various stresses such as citrate and sodium chloride treatments, and 
wounding; however, it was not influenced by increasing endogenous AsA content.
External AsA treatment caused a significant increase in electrolyte leakage from 
rice root. These results suggested that OsXIP was induced by stress which is
caused by external AsA treatment. Rice XIP-family genes, OsXIP, riceXIP and RIXI,
showed differential organ-specific expression. Also, these genes were
differentially induced by stress and stress-related phytohormones. The
transcripts of OsXIP and riceXIP were undetectable under normal conditions, and
were drastically induced by wounding and methyl jasmonate (MeJA) treatment in the
root. RIXI was constitutively expressed in the shoot but not induced by wounding 
and stress-related phytohormones. Thus, XIP-type xylanase inhibitors were
suggested to be specialized in their function and involved in defense mechanisms 
in rice.

DOI: 10.1093/pcp/pcm038 
PMID: 17379695  [Indexed for MEDLINE]


882. Yi Chuan. 2007 May;29(5):629-36.

[Study of hrpN(CSDS001) and the gene expression profile of Arabidopsis thaliana
induced by Harpin(CSDS001)].

[Article in Chinese]

Zhang J(1), Cao ML, Huang YB, Wu BJ.

Author information: 
(1)Maize Research Insititute of Sichuan Agricultural University, Ya'an 625014,
China. zjthebest@gmail.com

Erwinia carotovora subsp. carotovora CSDS001 elicits hypersensitive reaction (HR)
in tobacco. From the genomic libraries of Erwinia carotovora subsp. carotovora
CSDS001, the hrpNCSDS001 gene (GenBank number AY939927), was isolated. The
hrpNCSDS001 fusion protein was produced in Escherichia coli, and was used to
induce HR by injecting into tobacco. We further examined the global regulation of
Arabidopsis thaliana genes in response to HarpinCSDS001 at a concentration of 30 
miccrog/mL. We indicated that 912, 1787, 2393, 1833 and 1,755 genes that were
regulated significantly (log ratio <or=-1 or >or=1) at 3 h, 12 h, 24 h, 36 h and 
48 h respectively after the treatment. Analysis of some transcription factors
(TF) showed that 13 TF families responded to HarpinCSDS001 including ZIM, BES1,
TCP, C2C2, AP2/EREBP, WRKY, bHLH, bZIP, GARP, MYB, NAC, HB, C2H2. These families 
mainly function in biological processes of plant defense,
pho-tosynthesisdevelopment and flowering.


PMID: 17548335  [Indexed for MEDLINE]


883. Science. 2007 Apr 27;316(5824):593-7. Epub 2007 Mar 22.

Multiple high-throughput analyses monitor the response of E. coli to
perturbations.

Ishii N(1), Nakahigashi K, Baba T, Robert M, Soga T, Kanai A, Hirasawa T, Naba M,
Hirai K, Hoque A, Ho PY, Kakazu Y, Sugawara K, Igarashi S, Harada S, Masuda T,
Sugiyama N, Togashi T, Hasegawa M, Takai Y, Yugi K, Arakawa K, Iwata N, Toya Y,
Nakayama Y, Nishioka T, Shimizu K, Mori H, Tomita M.

Author information: 
(1)Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata
997-0017, Japan.

Comment in
    Science. 2007 Apr 27;316(5824):550-1.

Analysis of cellular components at multiple levels of biological information can 
provide valuable functional insights. We performed multiple high-throughput
measurements to study the response of Escherichia coli cells to genetic and
environmental perturbations. Analysis of metabolic enzyme gene disruptants
revealed unexpectedly small changes in messenger RNA and proteins for most
disruptants. Overall, metabolite levels were also stable, reflecting the
rerouting of fluxes in the metabolic network. In contrast, E. coli actively
regulated enzyme levels to maintain a stable metabolic state in response to
changes in growth rate. E. coli thus seems to use complementary strategies that
result in a metabolic network robust against perturbations.

DOI: 10.1126/science.1132067 
PMID: 17379776  [Indexed for MEDLINE]


884. Biosens Bioelectron. 2007 Apr 15;22(9-10):1853-60. Epub 2006 Aug 7.

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays
employing electrochemical detection.

Ghindilis AL(1), Smith MW, Schwarzkopf KR, Roth KM, Peyvan K, Munro SB, Lodes MJ,
Stöver AG, Bernards K, Dill K, McShea A.

Author information: 
(1)CombiMatrix Corporation, 6500 Harbour Heights Pkwy, 301, Mukilteo, WA 98275,
USA. aghindilis@combimatrix.com

Electrochemical detection has been developed and assay performances studied for
the CombiMatrix oligonucleotide microarray platform that contains 12,544
individually addressable microelectrodes (features) in a semiconductor matrix.
The approach is based on the detection of redox active chemistries (such as
horseradish peroxidase (HRP) and the associated substrate TMB) proximal to
specific microarray electrodes. First, microarray probes are hybridized to
biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin,
and enzymatic oxidation of the electron donor substrate then occurs. The
detection current is generated due to electro-reduction of the HRP reaction
product, and it is measured with the CombiMatrix ElectraSense Reader. Performance
of the ElectraSense platform has been characterized using gene expression and
genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay 
resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array
reproducibility and data correlation. The ElectraSense platform was also compared
to the standard fluorescent detection, and good consistency was observed between 
these two different detection techniques. A lower detection limit of 0.75 pM was 
obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained
for fluorescent detection. Thus, the ElectraSense platform has been used to
develop nucleic acid assays for highly accurate genotyping of a variety of
pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia
pestis, and other microorganisms including Escherichia coli, Bacillus subtilis,
etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

DOI: 10.1016/j.bios.2006.06.024 
PMID: 16891109  [Indexed for MEDLINE]


885. Appl Environ Microbiol. 2007 Apr;73(7):2380-5. Epub 2007 Feb 9.

Expression profiles of bovine genes in the rectoanal junction mucosa during
colonization with Escherichia coli O157:H7.

Li J(1), Hovde CJ.

Author information: 
(1)Department of Microbiology, Molecular Biology, and Biochemistry, University of
Idaho, Moscow, Idaho 83844-3052, USA.

A bovine-specific cDNA microarray was used to characterize gene expression in the
bovine rectoanal junction mucosa in response to Escherichia coli O157:H7
colonization, and results were confirmed using quantitative real-time PCR. The
results showed involvement of cell processes including immune response, cell
structure/dynamics, signal transduction, intercellular communication, and
metabolism.

DOI: 10.1128/AEM.02262-06 
PMCID: PMC1855659
PMID: 17293527  [Indexed for MEDLINE]


886. Appl Microbiol Biotechnol. 2007 Apr;74(6):1276-83. Epub 2007 Jan 11.

Characterization of superoxide-stress sensing recombinant Escherichia coli
constructed using promoters for genes zwf and fpr fused to lux operon.

Niazi JH(1), Kim BC, Gu MB.

Author information: 
(1)College of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong,
Sungbuk-gu, Seoul 136-701, Republic of Korea.

To measure the toxicity experienced by superoxide-generating compounds, two
plasmids were constructed in which the superoxide-inducible fpr and zwf promoters
from Escherichia coli were fused to promoterless Vibrio fischeri luxCDABE operon 
present in plasmid pUCD615. The bioluminescent response of E. coli harboring
these constructs was studied as a function of the toxicity and was shown to be
specific for superoxide generating chemicals. The two promoters employed, fpr and
zwf, responded differentially to the redox-chemicals tested. Furthermore, a
DeltamarA strain bearing the fpr::luxCDABE fusion had a weaker response to
paraquat (methyl viologen) than its isogenic parent strain, whereas zwf induction
was not inhibited in DeltamarA or Deltarob strains. The fpr and zwf promoters
were also induced by alkylating agents but were unresponsive in DeltamarA or
Deltarob strains. Using optimized assay conditions, the abilities of these
strains to differentially respond to superoxide stress and alkylating agents that
may be present in contaminants proves them to be good biosensor candidates for
monitoring toxicity.

DOI: 10.1007/s00253-006-0758-y 
PMID: 17216460  [Indexed for MEDLINE]


887. Cell Microbiol. 2007 Apr;9(4):1028-38. Epub 2006 Dec 12.

Hierarchical gene expression profiles of HUVEC stimulated by different lipid A
structures obtained from Porphyromonas gingivalis and Escherichia coli.

Chen C(1), Coats SR, Bumgarner RE, Darveau RP.

Author information: 
(1)Department of Periodontics and Oral Biology, University of Washington,
Seattle, WA 98195, USA. ccchen@usc.edu

The ability of lipid A structural variants to elicit unique endothelial cell gene
expression was examined by measuring global gene expression profiles in human
umbilical cord vein endothelial cells (HUVEC) using Affymetrix full genome chips.
Two lipid A structural variants obtained from Porphyromonas gingivalis designated
PgLPS(1435/1449) and PgLPS(1690) as well as LPS obtained from Escherichia coli
wild type and an E. coli msbB mutant (missing myristic acid in the lipid A) were 
examined. Each of these lipid A structures has been shown to interact with TLR4; 
however, PgLPS(1435/1449) and E. coli msbB LPS have been shown to be TLR4
antagonists while PgLPS(1690) and wild-type E. coli LPS are TLR4 agonists. It was
found that PgLPS(1435/1449) and PgLPS(1690) as well as E. coli msbB LPS activated
a subset of those genes significantly transcribed in response to E. coli
wild-type LPS. Furthermore, the subset of genes expressed in response to the
different lipid A structural forms were those most significantly activated by
wild-type E. coli LPS demonstrating a hierarchy in TLR4-dependent endothelial
cell gene activation. A unique gene expression profile for the weak TLR4 agonist 
PgLPS(1690) was observed and represents a TLR4 hierarchy in endothelial cell gene
activation.

DOI: 10.1111/j.1462-5822.2006.00849.x 
PMID: 17166236  [Indexed for MEDLINE]


888. Environ Toxicol Chem. 2007 Apr;26(4):634-46.

Hazard characterization and identification of a former ammunition site using
microarrays, bioassays, and chemical analysis.

Eisentraeger A(1), Reifferscheid G, Dardenne F, Blust R, Schofer A.

Author information: 
(1)Aachen University of Technology, Medical Faculty, Institute of Hygiene and
Environmental Medicine, Pauwelsstrasse 30, 52074 Aachen, Germany.
adolf.eisentraeger@post.rwth-aachen.de

More than 100,000 tons of 2,4,6-trinitrotoluene were produced at the former
ammunition site Werk Tanne in Clausthal-Zellerfeld, Germany. The production of
explosives and consequent detonation in approximately 1944 by the Allies caused
great pollution in this area. Four soil samples and three water samples were
taken from this site and characterized by applying chemical-analytical methods
and several bioassays. Ecotoxicological test systems, such as the algal growth
inhibition assay with Desmodesmus subspicatus, and genotoxicity tests, such as
the umu and NM2009 tests, were performed. Also applied were the Ames test,
according to International Organization for Standardization 16240, and an Ames
fluctuation test. The toxic mode of action was examined using bacterial gene
profiling assays with a battery of Escherichia coli strains and with the human
liver cell line hepG2 using the PIQOR Toxicology cDNA microarray. Additionally,
the molecular mechanism of 2,4,6-trinitrotoluene in hepG2 cells was analyzed. The
present assessment indicates a danger of pollutant leaching for the
soil-groundwater path. A possible impact for human health is discussed, because
the groundwater in this area serves as drinking water.


PMID: 17447547  [Indexed for MEDLINE]


889. Int J Med Microbiol. 2007 Apr;297(2):83-95. Epub 2007 Feb 5.

Characterization of four novel genomic regions of uropathogenic Escherichia coli 
highly associated with the extraintestinal virulent phenotype: a jigsaw puzzle of
genetic modules.

Sorsa LJ(1), Feldmann F, Hildinger K, Dufke S, Schubert S.

Author information: 
(1)Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie,
Marchioninistr. 17, D-81377 München, Germany.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a major cause of urinary 
tract infections, sepsis, and neonatal meningitis. A variety of virulence factors
in these strains is encoded by mobile genetic elements, such as transposons or
pathogenicity islands (PAIs). Using subtractive cloning of ExPEC genomes, we
recently detected short DNA fragments, which were significantly associated with
the extraintestinal virulent phenotype. In this study, we identified four novel
genomic DNA regions of the highly virulent uropathogenic E. coli strain JS299
carrying these previously identified DNA fragments. Characterization of the
partial sequences of the genomic DNA regions revealed complex DNA arrangements
with variable genetic compositions regarding the G+C contents and codon usage
patterns. The prevalence of 15 previously uncharacterized genes was determined in
a collection of clinical ExPECs and commensal E. coli strains by means of DNA
microarray analyses. From this, 13 novel DNA sequences were demonstrated to be
significantly associated with extraintestinal virulent strains, and thus may
represent new virulence traits. Beside genes predicted to play a role in
metabolic functions, such as sucrose utilization (scr), we identified DNA
sequences shared by both ExPEC and enteropathogenic E. coli (EPEC). These
sequences were significantly more prevalent among ExPECs when compared to
commensal E. coli isolates. Our results support the idea of a considerable
genetic variability among ExPEC strains and suggest that the novel genomic
determinants described in this study may contribute to the ExPEC virulence.

DOI: 10.1016/j.ijmm.2006.11.007 
PMID: 17280868  [Indexed for MEDLINE]


890. J Virol. 2007 Apr;81(8):3740-8. Epub 2007 Jan 24.

Association of gamma interferon and interleukin-17 production in intestinal CD4+ 
T cells with protection against rotavirus shedding in mice intranasally immunized
with VP6 and the adjuvant LT(R192G).

Smiley KL(1), McNeal MM, Basu M, Choi AH, Clements JD, Ward RL.

Author information: 
(1)Division of Infectious Diseases, Cincinnati Children's Hospital Medical
Center, 3333 Burnet Ave., Cincinnati, OH 45229, and Tulane University Medical
Center, New Orleans, LA 70112, USA.

Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the
protein that comprises the intermediate capsid layer of the rotavirus particle,
together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant,
reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus
challenge, and the only lymphocytes required for protection are CD4+ T cells.
Because these cells produce cytokines with antiviral properties, the cytokines
whose expression is upregulated in intestinal memory CD4+ T cells immediately
after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or
indirectly responsible for the rapid suppression of rotavirus shedding. This
study was designed to identify which cytokines are significantly upregulated in
intestinal effector sites and secondary lymphoid tissues of intranasally
immunized BALB/c mice after challenge with murine rotavirus strain EDIM.
Initially, this was done by using microarray analysis to quantify mRNAs for 96
murine common cytokines. With this procedure, the synthesis of mRNAs for gamma
interferon (IFN-gamma) and interleukin-17 (IL-17) was found to be temporarily
upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h 
after rotavirus challenge. These cytokines were also produced in CD4+ T cells
obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in 
vitro exposure to VP6 as determined by intracellular cytokine staining and
secretion of cytokines. Although genetically modified mice that lack receptors
for either IFN-gamma or IL-17 remained protected after immunization, these
results provide suggestive evidence that these cytokines may play direct or
indirect roles in protection against rotavirus after mucosal immunization of mice
with VP6/LT(R192G).

DOI: 10.1128/JVI.01877-06 
PMCID: PMC1866156
PMID: 17251301  [Indexed for MEDLINE]


891. BMC Physiol. 2007 Mar 30;7:3.

Regulatory network of inflammation downstream of proteinase-activated receptors.

Saban R(1), D'Andrea MR, Andrade-Gordon P, Derian CK, Dozmorov I, Ihnat MA, Hurst
RE, Simpson C, Saban MR.

Author information: 
(1)Department of Physiology, The University Oklahoma Health Sciences Center,
Oklahoma City, OK 73104, USA. ricardo-saban@ouhsc.edu

BACKGROUND: Protease-activated receptors (PAR) are present in the urinary
bladder, and their expression is altered in response to inflammation. PARs are a 
unique class of G protein-coupled that carry their own ligands, which remain
cryptic until unmasked by proteolytic cleavage. Although the canonical signal
transduction pathway downstream of PAR activation and coupling with various G
proteins is known and leads to the rapid transcription of genes involved in
inflammation, the effect of PAR activation on the downstream transcriptome is
unknown. We have shown that intravesical administration of PAR-activating
peptides leads to an inflammatory reaction characterized by edema and granulocyte
infiltration. Moreover, the inflammatory response to intravesical instillation of
known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS),
substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser
extent by PAR2-deficiency.
RESULTS: Here, cDNA array experiments determined inflammatory genes whose
expression is dependent on PAR1 activation. For this purpose, we compared the
alteration in gene expression in wild type and PAR1-/- mice induced by classical 
pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered
to be dependent on PAR-1 activation and further annotated in silico by Ingenuity 
Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target 
validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the
following have been implicated in the inflammatory process: b2m, ccl7, cd200,
cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia,
pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a
balanced response to signals of injury requires a transient cellular activation
of a panel of genes together with inhibitory systems that temper the overwhelming
inflammation. In this context, the activation of genes such as dusp1 and nfkbia
seems to counter-balance the inflammatory response to PAR activation by limiting 
prolonged activation of p38 MAPK and increased cytokine production. In contrast, 
transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR
re-sensitization would tend to perpetuate the inflammatory reaction in response
to common pro-inflammatory stimuli.
CONCLUSION: The combination of cDNA array results and genomic networks reveals an
overriding participation of PAR1 in bladder inflammation, provides a working
model for the involvement of downstream signaling, and evokes testable hypotheses
regarding the transcriptome downstream of PAR1 activation. It remains to be
determined whether or not mechanisms targeting PAR1 gene silencing or PAR1
blockade will ameliorate the clinical manifestation of cystitis.

DOI: 10.1186/1472-6793-7-3 
PMCID: PMC1853107
PMID: 17397547  [Indexed for MEDLINE]


892. J Biol Chem. 2007 Mar 23;282(12):8969-77. Epub 2007 Jan 16.

Fungal peptide Destruxin A plays a specific role in suppressing the innate immune
response in Drosophila melanogaster.

Pal S(1), St Leger RJ, Wu LP.

Author information: 
(1)Center for Biosystems Research, University of Maryland Biotechnology
Institute, College Park, Maryland 20742, USA.

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic
depsipeptides that are also studied for their toxicity to cancer cells. They are 
produced by various fungi, and a direct relationship has been established between
Destruxin production and the virulence of the entomopathogen Metarhizium
anisopliae. Aside from opening calcium channels, their in vivo mode of action
during pathogenesis remains largely uncharacterized. To better understand the
effects of a Destruxin, we looked at changes in gene expression following
injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray
results revealed reduced expression of various antimicrobial peptides that play a
major role in the humoral immune response of the fly. Flies co-injected with a
non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria 
Escherichia coli, showed increased mortality and an accompanying increase in
bacterial titers. Mortality due to sepsis was rescued through ectopic activation 
of components in the IMD pathway, one of two signal transduction pathways that
are responsible for antimicrobial peptide induction. These results demonstrate a 
novel role for Destruxin A in specific suppression of the humoral immune response
in insects.

DOI: 10.1074/jbc.M605927200 
PMID: 17227774  [Indexed for MEDLINE]


893. J Biotechnol. 2007 Mar 10;128(4):747-61. Epub 2007 Jan 14.

The plasticity of global proteome and genome expression analyzed in closely
related W3110 and MG1655 strains of a well-studied model organism, Escherichia
coli-K12.

Vijayendran C(1), Polen T, Wendisch VF, Friehs K, Niehaus K, Flaschel E.

Author information: 
(1)International NRW Graduate School in Bioinformatics and Genome Research,
University of Bielefeld, D-33594 Bielefeld, Germany.
cvijayen@cebitec.uni-bielefeld.de

The use of Escherichia coli as a model organism has provided a great deal of
basic information in biomolecular sciences. Examining trait differences among
closely related strains of the same species addresses a fundamental biological
question: how much diversity is there at the single species level? The main aim
of our research was to identify significant differences in the activities of
groups of genes between two laboratory strains of an organism closely related in 
genome structure. We demonstrate that despite strict and controlled growth
conditions, there is high plasticity in the global proteome and genome expression
in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ
insignificantly in genome structure. The growth patterns of these two sub-strains
were very similar in a well-equipped bioreactor, and their genome structures were
shown to be almost identical by DNA microarray. However, detailed profiling of
protein and gene expression by 2-dimensional gel electrophoresis and microarray
analysis showed many differentially expressed genes and proteins, combinations of
which were highly correlated. The differentially regulated genes and proteins
belonged to the following functional categories: genes regulated by sigma subunit
of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in
central metabolism. Genes involved in central cell metabolism - the glycolysis
pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were
differentially regulated at both the mRNA and proteome levels. The strains differ
significantly in central metabolism and thus in the generation of precursor
metabolites and energy. This high plasticity probably represents a universal
feature of metabolic activities in closely related species, and has the potential
to reveal differences in regulatory networks. We suggest that unless care is
taken in the choice of strains for any validating experiment, the results might
be misleading.

DOI: 10.1016/j.jbiotec.2006.12.026 
PMID: 17331609  [Indexed for MEDLINE]


894. BMC Microbiol. 2007 Mar 7;7:15.

Characterization of Escherichia coli MG1655 grown in a low-shear modeled
microgravity environment.

Tucker DL(1), Ott CM, Huff S, Fofanov Y, Pierson DL, Willson RC, Fox GE.

Author information: 
(1)University of Houston, Department of Biology & Biochemistry, Houston, TX
77204-5001, USA. dtucker84@yahoo.com <dtucker84@yahoo.com>

BACKGROUND: Extra-cellular shear force is an important environmental parameter
that is significant both medically and in the space environment. Escherichia coli
cells grown in a low-shear modeled microgravity (LSMMG) environment produced in a
high aspect rotating vessel (HARV) were subjected to transcriptional and
physiological analysis.
RESULTS: Aerobic LSMMG cultures were grown in rich (LB) and minimal (MOPS +
glucose) medium with a normal gravity vector HARV control. Reproducible changes
in transcription were seen, but no specific LSMMG responsive genes were
identified. Instead, absence of shear and a randomized gravity vector appears to 
cause local extra-cellular environmental changes, which elicit reproducible
cellular responses. In minimal media, the majority of the significantly up- or
down-regulated genes of known function were associated with the cell envelope. In
rich medium, most LSMMG down-regulated genes were involved in translation. No
observable changes in post-culture stress responses and antibiotic sensitivity
were seen in cells immediately after exposure to LSMMG. Comparison with earlier
studies of Salmonella enterica serovar Typhimurium conducted under similar growth
conditions, revealed essentially no similarity in the genes that were
significantly up- or down-regulated.
CONCLUSION: Comparison of these results to previous studies suggests that
different organisms may dramatically differ in their responses to medically
significant low-shear and space environments. Depending on their specific
response, some organisms, such as Salmonella, may become preadapted in a manner
that predisposes them to increased virulence.

DOI: 10.1186/1471-2180-7-15 
PMCID: PMC1852313
PMID: 17343762  [Indexed for MEDLINE]


895. PLoS One. 2007 Mar 7;2(3):e273.

An assessment of the role of DNA adenine methyltransferase on gene expression
regulation in E coli.

Seshasayee AS(1).

Author information: 
(1)Genomics and Regulatory Systems Group, EMBL-European Bioinformatics Institute,
Wellcome Trust Genome Campus, Cambridge, United Kingdom. aswin@ebi.ac.uk

N6-Adenine methylation is an important epigenetic signal, which regulates various
processes, such as DNA replication and repair and transcription. In
gamma-proteobacteria, Dam is a stand-alone enzyme that methylates GATC sites,
which are non-randomly distributed in the genome. Some of these overlap with
transcription factor binding sites. This work describes a global computational
analysis of a published Dam knockout microarray alongside other publicly
available data to throw insights into the extent to which Dam regulates
transcription by interfering with protein binding. The results indicate that DNA 
methylation by DAM may not globally affect gene transcription by physically
blocking access of transcription factors to binding sites. Down-regulation of Dam
during stationary phase correlates with the activity of TFs whose binding sites
are enriched for GATC sites.

DOI: 10.1371/journal.pone.0000273 
PMCID: PMC1804101
PMID: 17342207  [Indexed for MEDLINE]


896. Biotechnol Appl Biochem. 2007 Mar;46(Pt 3):157-67.

Escherichia coli minimum genome factory.

Mizoguchi H(1), Mori H, Fujio T.

Author information: 
(1)Biofrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahimachi,
Machida-shi, Tokyo 194-8533, Japan.

Research programmes for constructing a 'cell factory' have been funded in several
countries. In Japan, the 'Minimum genome factory' (MGF) project was launched in
2001. In this project, several model microbes have been genetically reconstructed
to obtain a cell with fewer genes on a chromosome of reduced size. A microbe with
a 'minimum genome' is expected to exhibit less regulation and therefore to be an 
ideal platform for a cell-factory system. The goal of this project is to
construct such a minimum genome microbe for a cell factory. In this project, the 
4.6 Mbp genome of Escherichia coli K-12 has been successfully reduced to 3.6 Mbp.
The constructed reduced-genome strain, MGF-01, shows better growth and higher
threonine production compared with the wild-type strain. Furthermore functional
analyses of all E. coli genes have also been performed. CGH (comparative genomic 
hybridization) analysis revealed that about 2600 genes were commonly conserved in
the 23 E. coli strains tested. This set of conserved genes was hypothesized as a 
core set for E. coli species. Phenotype array analysis of a nearly complete
collection of single-gene knockout mutants of E. coli provided insights into E.
coli metabolic networks. The data sets from the functional genomics will be used 
to improve design of an E. coli MGF. The present minireview summarizes the
progress of the E. coli MGF project and overviews related research.

DOI: 10.1042/BA20060107 
PMID: 17300222  [Indexed for MEDLINE]


897. Blood. 2007 Mar 1;109(5):2066-77. Epub 2006 Nov 14.

Gene expression patterns in blood leukocytes discriminate patients with acute
infections.

Ramilo O(1), Allman W, Chung W, Mejias A, Ardura M, Glaser C, Wittkowski KM,
Piqueras B, Banchereau J, Palucka AK, Chaussabel D.

Author information: 
(1)Baylor National Institute of Allergy and Infections Diseases (NIAID)
Cooperative Center for Translational Research on Human Immunology and Biodefense 
and Baylor Institute for Immunology Research, Dallas, TX 75204, USA.
octavio.ramilo@utsouthwestern.edu

Each infectious agent represents a unique combination of pathogen-associated
molecular patterns that interact with specific pattern-recognition receptors
expressed on immune cells. Therefore, we surmised that the blood immune cells of 
individuals with different infections might bear discriminative transcriptional
signatures. Gene expression profiles were obtained for 131 peripheral blood
samples from pediatric patients with acute infections caused by influenza A
virus, Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus
and Streptococcus pneumoniae) bacteria. Thirty-five genes were identified that
best discriminate patients with influenza A virus infection from patients with
either E coli or S pneumoniae infection. These genes classified with 95% accuracy
(35 of 37 samples) an independent set of patients with either influenza A, E
coli, or S pneumoniae infection. A different signature discriminated patients
with E coli versus S aureus infections with 85% accuracy (34 of 40). Furthermore,
distinctive gene expression patterns were observed in patients presenting with
respiratory infections of different etiologies. Thus, microarray analyses of
patient peripheral blood leukocytes might assist in the differential diagnosis of
infectious diseases.

DOI: 10.1182/blood-2006-02-002477 
PMCID: PMC1801073
PMID: 17105821  [Indexed for MEDLINE]


898. Cell Microbiol. 2007 Mar;9(3):804-16. Epub 2006 Nov 3.

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 
1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and
epithelial barrier repair.

Zyrek AA(1), Cichon C, Helms S, Enders C, Sonnenborn U, Schmidt MA.

Author information: 
(1)Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung
(ZMBE), Westfälische Wilhelms-Universität/Universitätsklinikum Münster,
von-Esmarch-Str. 56, D-48149 Münster, Germany.

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades
in human medicine in Central Europe for the treatment and prevention of
intestinal disorders and diseases. However, the molecular mechanisms underlying
its beneficial effects are only partially understood. To identify molecular
responses induced by EcN that might contribute to its probiotic properties
polarized T84 cells were investigated employing DNA microarrays, quantitative
RT-PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) 
inhibitors. Polarized T84 epithelial cell monolayers were used as a model to
monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) 
strain E2348/69. Co-incubation of EPEC with EcN or addition of EcN following EPEC
infection abolished barrier disruption and, moreover, restored barrier integrity 
as monitored by transepithelial resistance. DNA-microarray analysis of T84 cells 
incubated with EcN identified 300+ genes exhibiting altered expression. EcN
altered the expression, distribution of zonula occludens-2 (ZO-2) protein and of 
distinct PKC isotypes. ZO-2 expression was enhanced in parallel to its
redistribution towards the cell boundaries. This study provides evidence that EcN
induces an overriding signalling effect leading to restoration of a disrupted
epithelial barrier. This is transmitted via silencing of PKCzeta and the
redistribution of ZO-2. We suggest that these properties contribute to the
reported efficacy in the treatment of inflammatory bowel diseases and in part
rationalize the probiotic nature of EcN.

DOI: 10.1111/j.1462-5822.2006.00836.x 
PMID: 17087734  [Indexed for MEDLINE]


899. J Bacteriol. 2007 Mar;189(6):2262-73. Epub 2007 Jan 12.

FNR is a global regulator of virulence and anaerobic metabolism in Salmonella
enterica serovar Typhimurium (ATCC 14028s).

Fink RC(1), Evans MR, Porwollik S, Vazquez-Torres A, Jones-Carson J, Troxell B,
Libby SJ, McClelland M, Hassan HM.

Author information: 
(1)Department of Microbiology, North Carolina State University, Raleigh, NC
27695-7615, USA.

Salmonella enterica serovar Typhimurium must successfully transition the broad
fluctuations in oxygen concentrations encountered in the host. In Escherichia
coli, FNR is one of the main regulatory proteins involved in O2 sensing. To
assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr
mutant in the virulent wild-type strain (ATCC 14028s) and compared their
transcriptional profiles and pathogenicities in mice. Here, we report that, under
anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or
indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation 
by FNR in serovar Typhimurium is similar to, but distinct from, that in E. coli. 
Thus, genes/operons involved in aerobic metabolism, NO. detoxification, flagellar
biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are
regulated by FNR in a fashion similar to that in E. coli. However, genes/operons 
existing in E. coli but regulated by FNR only in serovar Typhimurium include
those coding for ethanolamine utilization, a universal stress protein, a
ferritin-like protein, and a phosphotransacetylase. Interestingly,
Salmonella-specific genes/operons regulated by FNR include numerous virulence
genes within Salmonella pathogenicity island 1 (SPI-1), newly identified
flagellar genes (mcpAC, cheV), and the virulence operon (srfABC). Furthermore,
the role of FNR as a positive regulator of motility, flagellar biosynthesis, and 
pathogenesis was confirmed by showing that the mutant is nonmotile, lacks
flagella, is attenuated in mice, and does not survive inside macrophages. The
inability of the mutant to survive inside macrophages is likely due to its
sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

DOI: 10.1128/JB.00726-06 
PMCID: PMC1899381
PMID: 17220229  [Indexed for MEDLINE]


900. J Bacteriol. 2007 Mar;189(6):2359-68. Epub 2007 Jan 12.

Enhancement of the synthesis of RpoN, Cra, and H-NS by polyamines at the level of
translation in Escherichia coli cultured with glucose and glutamate.

Terui Y(1), Higashi K, Taniguchi S, Shigemasa A, Nishimura K, Yamamoto K,
Kashiwagi K, Ishihama A, Igarashi K.

Author information: 
(1)Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana,
Chiba 260-8675, Japan.

Proteins whose synthesis is enhanced by polyamines at the level of translation
were identified in a polyamine-requiring mutant cultured in the presence of 0.1% 
glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under
these conditions, enhancement of cell growth by polyamines was almost the same as
that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra,
and H-NS was enhanced by polyamines at the level of translation at the early
logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on
synthesis of RpoN, H-NS, and Cra were due to the existence of unusual
Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon 
(Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new
members of the polyamine modulon. Because most of the polyamine modulon genes
thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI
[sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of
mRNA expressed in cells was performed. At the early logarithmic phase of growth, 
a total of 97 species of mRNAs that were up-regulated by polyamines more than
twofold were under the control of seven polyamine modulon genes mentioned above.

DOI: 10.1128/JB.01562-06 
PMCID: PMC1899374
PMID: 17220219  [Indexed for MEDLINE]


901. J Struct Funct Genomics. 2007 Mar;8(1):27-35. Epub 2007 Nov 9.

The transcriptional response of Escherichia coli to recombinant protein
insolubility.

Smith HE(1).

Author information: 
(1)Center for Advanced Research in Biotechnology, University of Maryland
Biotechnology Institute, Rockville, MD 20850, USA smithh@umbi.umd.edu

Bacterial production of recombinant proteins offers several advantages over
alternative expression methods and remains the system of choice for many
structural genomics projects. However, a large percentage of targets accumulate
as insoluble inclusion bodies rather than soluble protein, creating a significant
bottleneck in the protein production pipeline. Numerous strategies have been
reported that can improve in vivo protein solubility, but most do not scale
easily for high-throughput expression screening. To understand better the host
cell response to the accumulation of insoluble protein, we determined genome-wide
changes in bacterial gene expression upon induction of either soluble or
insoluble target proteins. By comparing transcriptional profiles for multiple
examples from the soluble or insoluble class, we identified a pattern of gene
expression that correlates strongly with protein solubility. Direct targets of
the sigma32 heat shock sigma factor, which includes genes involved in protein
folding and degradation, were highly expressed in response to induction of
insoluble protein. This same group of genes was also upregulated by insoluble
protein accumulation under a different growth regime, indicating that
sigma32-mediated gene expression is a general response to protein insolubility.
This knowledge provides a starting point for the rational design of growth
parameters and host strains with improved protein solubility characteristics.
Summary Problems with protein solubility are frequently encountered when
recombinant proteins are expressed in E. coli. The bacterial host responds to
this problem by increasing expression of the protein folding machinery via the
heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might
provide a general mechanism for improving recombinant protein solubility.

DOI: 10.1007/s10969-007-9030-7 
PMID: 17992580  [Indexed for MEDLINE]


902. Mol Microbiol. 2007 Mar;63(5):1468-81.

Increased adherence and actin pedestal formation by dam-deficient
enterohaemorrhagic Escherichia coli O157:H7.

Campellone KG(1), Roe AJ, Løbner-Olesen A, Murphy KC, Magoun L, Brady MJ,
Donohue-Rolfe A, Tzipori S, Gally DL, Leong JM, Marinus MG.

Author information: 
(1)Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester, MA 01655, USA.

Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens
capable of causing severe diarrhoeal illnesses. As a critical step during their
colonization, EHEC adhere intimately to intestinal epithelial cells and generate 
F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. 
Intimate adhesion and pedestal formation result from delivery of the EHEC type
III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell 
and expression of the bacterial outer membrane adhesin, intimin. To investigate a
role for DNA methylation during the regulation of adhesion and pedestal formation
in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC
O157:H7 and demonstrate that this mutation results in increased interactions with
cultured host cells. EHECDeltadam exhibits dramatically elevated levels of
adherence and pedestal formation when compared with wild-type EHEC, and expresses
significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP 
fusions, Northern blotting, reverse transcription polymerase chain reaction, and 
microarray experiments indicate that the abundance of Tir in the dam mutant is
not due to increased transcription levels, raising the possibility that Dam
methylation can indirectly control protein expression by a post-transcriptional
mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable 
of robust intestinal colonization of experimentally infected animals.

DOI: 10.1111/j.1365-2958.2007.05602.x 
PMID: 17302821  [Indexed for MEDLINE]


903. Plant Cell Physiol. 2007 Mar;48(3):381-90. Epub 2007 Jan 23.

Approaches for extracting practical information from gene co-expression networks 
in plant biology.

Aoki K(1), Ogata Y, Shibata D.

Author information: 
(1)Kazusa DNA Research Institute, Kazusa-Kamatari 2-6-7, Kisarazu, 292-0818,
Japan.

Gene co-expression, in many cases, implies the presence of a functional linkage
between genes. Co-expression analysis has uncovered gene regulatory mechanisms in
model organisms such as Escherichia coli and yeast. Recently, accumulation of
Arabidopsis microarray data has facilitated a genome-wide inspection of gene
co-expression profiles in this model plant. An approach using network analysis
has provided an intuitive way to represent complex co-expression patterns between
many genes. Co-expression network analysis has enabled us to extract modules, or 
groups of tightly co-expressed genes, associated with biological processes.
Furthermore, integrated analysis of gene expression and metabolite accumulation
has allowed us to hypothesize the functions of genes associated with specific
metabolic processes. Co-expression network analysis is a powerful approach for
data-driven hypothesis construction and gene prioritization, and provides novel
insights into the system-level understanding of plant cellular processes.

DOI: 10.1093/pcp/pcm013 
PMID: 17251202  [Indexed for MEDLINE]


904. Biochem Biophys Res Commun. 2007 Feb 23;353(4):1028-33. Epub 2006 Dec 28.

Characterization of the ColE2-like replicon of plasmid pTT8 from Thermus
thermophilus.

Aoki K(1), Itoh T.

Author information: 
(1)Department of Biology, Faculty of Science, Shinshu University, Matsumoto,
Nagano 390-8621, Japan.

We identified the 1.6-kb region of Thermus thermophilus plasmid pTT8 capable of
autonomous replication, which shows a significant sequence similarity to the
replicon regions of the ColE2-related plasmids. We showed the requirement of DNA 
polymerase I for pTT8 replication. The putative rep gene coding for the
replication initiator protein, Rep, similar to those of the ColE2-related
plasmids was cloned into an expression vector. The 6xHis-Rep protein expressed in
Escherichia coli was successfully purified by stepwise denaturing with urea and
refolding in the presence of glycerol on Ni-resin. We identified the nucleotide
sequence recognized by the pTT8 Rep protein by the SELEX experiment using the
purified protein, and proposed the existence of the third origin of pTT8
replication different from those predicted previously.

DOI: 10.1016/j.bbrc.2006.12.150 
PMID: 17207772  [Indexed for MEDLINE]


905. Appl Environ Microbiol. 2007 Feb;73(3):890-6. Epub 2006 Dec 8.

High-throughput and quantitative procedure for determining sources of Escherichia
coli in waterways by using host-specific DNA marker genes.

Yan T(1), Hamilton MJ, Sadowsky MJ.

Author information: 
(1)BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA.

Escherichia coli is currently used as an indicator of fecal pollution and to
assess water quality. While several genotypic techniques have been used to
determine potential sources of fecal bacteria impacting waterways and beaches,
they do not allow for the rapid analysis of a large number of samples in a
relatively short period of time. Here we report that gene probes identified by
Hamilton and colleagues (M. J. Hamilton, T. Yan, and M. J. Sadowsky, Appl.
Environ. Microbiol. 72:4012-4019, 2006) were useful for the development of a
high-throughput and quantitative macroarray hybridization system to determine
numbers of E. coli bacteria originating from geese/ducks. The procedure we
developed, using a QBot robot for picking and arraying of colonies, allowed us to
simultaneously analyze up to 20,736 E. coli colonies from water samples, with
minimal time and human input. Statistically significant results were obtained by 
analyzing 700 E. coli colonies per water sample, allowing for the analysis of
approximately 30 sites per macroarray. Macroarray hybridization studies done on
E. coli collected from water samples obtained from two urban Minnesota lakes and 
one rural South Carolina lake indicated that geese/ducks contributed up to 51% of
the fecal bacteria in the urban lake water samples, and the level was below the
detection limit in the rural lake water sample. This technique, coupled with the 
use of other host source-specific gene probes, holds great promise as a new
quantitative microbial source tracking tool to rapidly determine the origins of
E. coli in waterways and on beaches.

DOI: 10.1128/AEM.01395-06 
PMCID: PMC1800753
PMID: 17158618  [Indexed for MEDLINE]


906. Appl Microbiol Biotechnol. 2007 Feb;74(2):406-21. Epub 2006 Nov 25.

Global gene expression analysis of glucose overflow metabolism in Escherichia
coli and reduction of aerobic acetate formation.

Veit A(1), Polen T, Wendisch VF.

Author information: 
(1)Institute of Biotechnology 1, Research Center Juelich, 52428 Juelich, Germany.

During aerobic growth on glucose, Escherichia coli produces acetate in the
so-called overflow metabolism. DNA microarray analysis was used to determine the 
global gene expression patterns of chemostat cultivations of E. coli MG1655 that 
were characterized by different acetate formation rates during aerobic growth on 
glucose. A correlation analysis identified that expression of ten genes (sdhCDAB,
sucB, sucC, acnB, lpdA, fumC and mdh) encoding the TCA cycle enzymes succinate
dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase,
aconitase, fumarase and malate dehydrogenase, respectively, and of the
acs-yjcH-actP operon for acetate utilization correlated negatively with acetate
formation. Relieving transcriptional control of the sdhCDAB-b0725-sucABCD operon 
by chromosomal promoter exchange mutagenesis yielded a strain with increased
specific activities of the TCA cycle enzymes succinate dehydrogenase,
alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase, which are encoded 
by this operon. The resulting strain produced less acetate and directed more
carbon towards carbon dioxide formation than the parent strain MG1655 while
maintaining high growth and glucose consumption rates.

DOI: 10.1007/s00253-006-0680-3 
PMID: 17273855  [Indexed for MEDLINE]


907. Environ Microbiol. 2007 Feb;9(2):332-46.

Temporal gene-expression in Escherichia coli K-12 biofilms.

Domka J(1), Lee J, Bansal T, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, Texas A&M University,
College Station, TX, USA.

Analysis of the temporal development of Escherichia coli K-12 biofilms in complex
medium indicates the greatest differential gene expression between biofilm and
suspension cells occurred in young biofilms at 4 and 7 h (versus 15 and 24 h).
The main classes of genes differentially expressed (biofilm versus biofilm and
biofilm versus suspension cells) include 42 related to stress response (e.g.
cspABFGI), 66 related to quorum sensing (e.g. ydgG, gadABC, hdeABD), 20 related
to motility (e.g. flgBCEFH, fliLMQR, motB), 13 related to fimbriae (e.g. sfmCHM, 
fimZ, csgC), 24 related to sulfur and tryptophan metabolism (e.g. trpLBA, tnaLA, 
cysDNCJH), 80 related to transport (e.g. gatABC, agaBC, ycjJ, ydfJ, phoU,
phnCJKM), and six related to extracellular matrix (e.g. wcaBDEC). Of the 93
mutants identified and studied, 76 showed altered biofilm formation. Biofilm
architecture changed from thin and dense to globular and dispersed to dense and
smooth. The quorum-sensing signal AI-2 controls gene expression most clearly in
mature biofilms (24 h) when intracellular AI-2 levels are highest. Sulfate
transport and metabolism genes (cysAUWDN) and genes with unknown functions
(ymgABCZ) were repressed in young (4, 7 h) biofilms, induced in developed
biofilms (15 h), and repressed in mature (24 h) biofilms. Genes related to both
motility and fimbriae were induced in biofilms at all sampling time points and
colanic acid genes were induced in mature biofilms (24 h). Genes related to
dihydroxyacetone phosphate synthesis from galactitol and galactosamine (e.g.
gatZABCDR, agaBCY) were highly regulated in biofilms. Genes involved in the
biosynthesis of indole and sulfide (tnaLA) are repressed in biofilms after 7 h
(corroborated by decreasing intracellular indole concentrations in biofilms).
Cold-shock protein transcriptional regulators (cspABFGI) appear to be positive
biofilm regulators, and deletions in respiratory genes (e.g. hyaACD, hyfCG, appC,
narG) increased biofilm formation sevenfold.

DOI: 10.1111/j.1462-2920.2006.01143.x 
PMID: 17222132  [Indexed for MEDLINE]


908. FEMS Microbiol Lett. 2007 Feb;267(1):51-5.

The role of the omega subunit of RNA polymerase in expression of the relA gene in
Escherichia coli.

Chatterji D(1), Ogawa Y, Shimada T, Ishihama A.

Author information: 
(1)Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase
constitutes single operon with the spoT gene, which is responsible for the
maintenance of stringent response under nutrient starvation conditions. To
identify the physiological role of the omega subunit, we compared the gene
expression profile of wild-type Escherichia coli with that of an rpoZ deleted
strain by microarray analysis using an E. coli DNA chip. Here we report on a set 
of genes which show changes in expression profile following the removal of rpoZ. 
We have seen that relA, which is responsible for the synthesis of the stringent
factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA
levels and were therefore further analyzed for their expression using a GFP/RFP
two-fluorescent protein promoter assay vector. In the absence of rpoZ, the
promoter for the relA gene was severely impaired, but the promoters from the
ribosomal protein genes were not affected as much. Taking these results together 
we propose that the omega subunit is involved in regulation of the relA gene, but
induction of the stringently controlled genes in the absence of rpoZ is, at least
in part, attributable to a decrease in ppGpp level.

DOI: 10.1111/j.1574-6968.2006.00532.x 
PMID: 17233676  [Indexed for MEDLINE]


909. Genomics. 2007 Feb;89(2):300-5. Epub 2006 Nov 27.

Evaluating microarrays using a semiparametric approach: application to the
central carbon metabolism of Escherichia coli BL21 and JM109.

Phue JN(1), Kedem B, Jaluria P, Shiloach J.

Author information: 
(1)Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Building 14A, Room 170, 9000 Rockville
Pike, Bethesda, MD 20892, USA.

Escherichia coli K (JM109) and E. coli B (BL21) are strains used routinely for
recombinant protein production. These two strains grow and respond differently to
environmental factors such as glucose and oxygen concentration. The differences
have been attributed to differential expression of individual genes that
constitute certain metabolic pathways that are part of the central carbon
metabolism. By implementing a semiparametric algorithm, which is based on a
density ratio model, it was possible to compare and quantify the expression
patterns of groups of genes involved in several central carbon metabolic
pathways. The groups comprising the glyoxylate shunt, TCA cycle, fatty acid, and 
gluconeogenesis and anaplerotic pathways were expressed differently between the
two strains, whereas no differences were apparent for the groups comprising
either glycolysis or the pentose phosphate pathway. These results further
characterized differences between the two E. coli strains and illustrated the
potency of the semiparametric algorithm.

DOI: 10.1016/j.ygeno.2006.10.004 
PMCID: PMC1945183
PMID: 17125967  [Indexed for MEDLINE]


910. Infect Immun. 2007 Feb;75(2):966-76. Epub 2006 Dec 4.

Global gene expression profiling of asymptomatic bacteriuria Escherichia coli
during biofilm growth in human urine.

Hancock V(1), Klemm P.

Author information: 
(1)Microbial Adhesion Group, Center for Biomedical Microbiology, BioCentrum-DTU, 
Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.

Urinary tract infection (UTI) is an important health problem worldwide, with many
millions of cases each year, and Escherichia coli is the most common organism
causing UTI in humans. Also, E. coli is responsible for most infections in
patients with chronic indwelling bladder catheter. The two asymptomatic
bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm
formers in their natural growth medium, human urine, than the two uropathogenic
E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the
expression profile during biofilm growth in urine of the two ABU strains 83972
and VR50. Significant differences in expression levels were seen between the
biofilm expression profiles of the two strains with the corresponding planktonic 
expression profiles in morpholinepropanesulfonic acid minimal laboratory medium
and human urine; 417 and 355 genes were up- and down-regulated, respectively,
during biofilm growth in urine of 83972 and VR50. Many genes involved in
transcription and stress were up-regulated in biofilm-grown cells. The role in
biofilm formation of four of the up-regulated genes, i.e., yceP, yqgA, ygiD, and 
aaeX, was investigated by creating single-knockout mutant versions of 83972 and
VR50; all mutants showed reduced biofilm formation in urine by 18 to 43% compared
with the wild type (P < 0.05). Also, the expression profile of strain 83972 in
the human urinary tract partially overlaps with the biofilm expression profile.

DOI: 10.1128/IAI.01748-06 
PMCID: PMC1828481
PMID: 17145952  [Indexed for MEDLINE]


911. J Bacteriol. 2007 Feb;189(3):911-7. Epub 2006 Nov 17.

AccD6, a member of the Fas II locus, is a functional carboxyltransferase subunit 
of the acyl-coenzyme A carboxylase in Mycobacterium tuberculosis.

Daniel J(1), Oh TJ, Lee CM, Kolattukudy PE.

Author information: 
(1)Burnett College of Biomedical Sciences, University of Central Florida, BMS
136, 4000 Central Florida Blvd., Orlando, FL 32816-2364, USA.

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the
building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS
I) and for the elongation of FAS I end products by the FAS II complex to produce 
meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase
subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with
accD6 located in a genetic locus that contains members of the FAS II complex. We 
found by quantitative real-time PCR analysis that the transcripts of accA3,
accD4, accD5, and accD6 are expressed at high levels during the exponential
growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis
transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and
accE were repressed during later growth stages. AccD4 and AccD5 have been
previously studied, but there are no reports on the function of AccD6. We
expressed AccA3 (alpha3) and AccD6 (beta6) in E. coli and purified them by
affinity chromatography. We report here that reconstitution of the alpha3-beta6
complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this 
carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1
nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the
alpha3-beta6 complex was inhibited by the epsilon subunit. The alpha3-beta6
carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop,
cerulenin, and 1,2-cyclohexanedione. Our results suggest that the beta6 subunit
could play an important role in mycolic acid biosynthesis by providing
malonyl-CoA to the FAS II complex.

DOI: 10.1128/JB.01019-06 
PMCID: PMC1797314
PMID: 17114269  [Indexed for MEDLINE]


912. J Clin Microbiol. 2007 Feb;45(2):370-9. Epub 2006 Nov 15.

Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray.

Ballmer K(1), Korczak BM, Kuhnert P, Slickers P, Ehricht R, Hächler H.

Author information: 
(1)Institute of Veterinary Bacteriology, NENT, Vetsuisse-Faculty, University of
Berne, Berne, Switzerland.

Classical antibody-based serotyping of Escherichia coli is an important method in
diagnostic microbiology for epidemiological purposes, as well as for a rough
virulence assessment. However, serotyping is so tedious that its use is
restricted to a few reference laboratories. To improve this situation we
developed and validated a genetic approach for serotyping based on the microarray
technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen
polymerase (wzy) were selected as target sequences for the O antigen, whereas
fliC and related genes, which code for the flagellar monomer, were chosen as
representatives for the H phenotype. Starting with a detailed bioinformatic
analysis and oligonucleotide design, an ArrayTube-based assay was established: a 
fast and robust DNA extraction method was coupled with a site-specific, linear
multiplex labeling procedure and hybridization analysis of the biotinylated
amplicons. The microarray contained oligonucleotide DNA probes, each in
duplicate, representing 24 of the epidemiologically most relevant of the over 180
known O antigens (O antigens 4, 6 to 9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103,
104, 111, 113, 114, 121, 128, 145, 157, and 172) as well as 47 of the 53
different H antigens (H antigens 1 to 12, 14 to 16, 18 to 21, 23 to 34, 37 to 43,
45, 46, 48, 49, 51 to 54, and 56). Evaluation of the microarray with a set of
defined strains representing all O and H serotypes covered revealed that it has a
high sensitivity and a high specificity. All of the conventionally typed 24 O
groups and all of the 47 H serotypes were correctly identified. Moreover, strains
which were nonmotile or nontypeable by previous serotyping assays yielded
unequivocal results with the novel ArrayTube assay, which proved to be a valuable
alternative to classical serotyping, allowing processing of single colonies
within a single working day.

DOI: 10.1128/JCM.01361-06 
PMCID: PMC1829071
PMID: 17108073  [Indexed for MEDLINE]


913. Mol Cell Probes. 2007 Feb;21(1):56-65. Epub 2006 Sep 17.

Molecular characterisation of Salmonella strains by an oligonucleotide multiprobe
microarray.

Malorny B(1), Bunge C, Guerra B, Prietz S, Helmuth R.

Author information: 
(1)National Salmonella Reference Laboratory, Federal Institute for Risk
Assessment, Diedersdorfer Weg 1, D-12277 Berlin, Germany.

A DNA microarray has been developed for the simultaneous characterisation and
typing of Salmonella enterica subsp. enterica isolates. One-hundred and nine
35-40 mer oligonucleotides probes detect flagellar and somatic antigen encoding
genes (serogroup or serotype specific), important virulence genes located within 
or outside the pathogenicity islands, phage-associated genes and antibiotic
resistance determinants. The probes were printed on glass slides and whole
genomic Cy5-labelled Salmonella DNA was hybridised to the substrate. A set of 19 
different Salmonella strains and one Escherichia coli strain has been selected as
positive and negative controls for each probe. The validity of the results is
confirmed by gene-specific PCRs or phenotypic methods (serotyping, MIC
determination for various antimicrobial agents). Of 2071 data points generated,
an agreement of 97.4% has been obtained between microarray and PCR/phenotypic
results. Twenty-six data points (1.3%) were classified as uncertain and,
similarly, 1.3% showed a discordant result. The microarray described here is a
new tool to study the epidemiology of Salmonella strains on the genotypic level
and might become a powerful method in risk assessment studies.

DOI: 10.1016/j.mcp.2006.08.005 
PMID: 17029709  [Indexed for MEDLINE]


914. BMC Bioinformatics. 2007 Jan 23;8:20.

Transcriptional regulatory network refinement and quantification through kinetic 
modeling, gene expression microarray data and information theory.

Sayyed-Ahmad A(1), Tuncay K, Ortoleva PJ.

Author information: 
(1)Center for Cell and Virus Theory, Department of Chemistry, Indiana University,
Bloomington, IN 47405, USA. asayyeda@cems.umn.edu <asayyeda@cems.umn.edu>

BACKGROUND: Gene expression microarray and other multiplex data hold promise for 
addressing the challenges of cellular complexity, refined diagnoses and the
discovery of well-targeted treatments. A new approach to the construction and
quantification of transcriptional regulatory networks (TRNs) is presented that
integrates gene expression microarray data and cell modeling through information 
theory. Given a partial TRN and time series data, a probability density is
constructed that is a functional of the time course of transcription factor (TF) 
thermodynamic activities at the site of gene control, and is a function of mRNA
degradation and transcription rate coefficients, and equilibrium constants for
TF/gene binding.
RESULTS: Our approach yields more physicochemical information that compliments
the results of network structure delineation methods, and thereby can serve as an
element of a comprehensive TRN discovery/quantification system. The most probable
TF time courses and values of the aforementioned parameters are obtained by
maximizing the probability obtained through entropy maximization. Observed time
delays between mRNA expression and activity are accounted for implicitly since
the time course of the activity of a TF is coupled by probability functional
maximization, and is not assumed to be proportional to expression level of the
mRNA type that translates into the TF. This allows one to investigate
post-translational and TF activation mechanisms of gene regulation. Accuracy and 
robustness of the method are evaluated. A kinetic formulation is used to
facilitate the analysis of phenomena with a strongly dynamical character while a 
physically-motivated regularization of the TF time course is found to overcome
difficulties due to omnipresent noise and data sparsity that plague other methods
of gene expression data analysis. An application to Escherichia coli is
presented.
CONCLUSION: Multiplex time series data can be used for the construction of the
network of cellular processes and the calibration of the associated
physicochemical parameters. We have demonstrated these concepts in the context of
gene regulation understood through the analysis of gene expression microarray
time series data. Casting the approach in a probabilistic framework has allowed
us to address the uncertainties in gene expression microarray data. Our approach 
was found to be robust to error in the gene expression microarray data and
mistakes in a proposed TRN.

DOI: 10.1186/1471-2105-8-20 
PMCID: PMC1790715
PMID: 17244365  [Indexed for MEDLINE]


915. Anal Biochem. 2007 Jan 15;360(2):244-54. Epub 2006 Oct 30.

A microbial diagnostic microarray technique for the sensitive detection and
identification of pathogenic bacteria in a background of nonpathogens.

Kostić T(1), Weilharter A, Rubino S, Delogu G, Uzzau S, Rudi K, Sessitsch A,
Bodrossy L.

Author information: 
(1)Department of Bioresources, ARC Seibersdorf Research GmbH, A-2444 Seibersdorf,
Austria.

A major challenge in microbial diagnostics is the parallel detection and
identification of low-bundance pathogens within a complex microbial community. In
addition, a high specificity providing robust, reliable identification at least
at the species level is required. A microbial diagnostic microarray approach,
using single nucleotide extension labeling with gyrB as the marker gene, was
developed. We present a novel concept applying competitive oligonucleotide probes
to improve the specificity of the assay. Our approach enabled the sensitive and
specific detection of a broad range of pathogenic bacteria. The approach was
tested with a set of 35 oligonucleotide probes targeting Escherichia coli,
Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae,
Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus
mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of
competitive oligonucleotides in the labeling reaction successfully suppressed
cross-reaction by closely related sequences, significantly improving the
performance of the assay. Environmental applicability was tested with
environmental and veterinary samples harboring complex microbial communities.
Detection sensitivity in the range of 0.1% has been demonstrated, far below the
5% detection limit of traditional microbial diagnostic microarrays.

DOI: 10.1016/j.ab.2006.09.026 
PMID: 17123456  [Indexed for MEDLINE]


916. Biosens Bioelectron. 2007 Jan 15;22(6):1041-7. Epub 2006 Jul 11.

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7
that eliminates the risk of a false negative signal.

Kim H(1), Kane MD, Kim S, Dominguez W, Applegate BM, Savikhin S.

Author information: 
(1)Department of Physics, Purdue University, West Lafayette, IN 47907, USA.

A DNA hybridization based optical detection platform for the detection of
foodborne pathogens has been developed with virtually zero probability of the
false negative signal. This portable, low-cost and real-time assaying detection
platform utilizes the color changing molecular beacon as a probe for the optical 
detection of the target sequence. The computer-controlled detection platform
exploits the target hybridization induced change of fluorescence color due to the
Förster (fluorescence) resonance energy transfer (FRET) between a pair of
spectrally shifted fluorophores conjugated to the opposite ends of a beacon
(oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon
design, the presence of two fluorescence molecules allows to actively visualize
both hybridized and unhybridized states of the beacon. This eliminates false
negative signal detection characteristic for the fluorophore-quencher beacon
where bleaching of the fluorophore or washout of a beacon is indistinguishable
from the absence of the target DNA sequence. In perspective, the two-color design
allows also to quantify the concentration of the target DNA in a sample down to <
=1 ng/microl. The new design is suitable for simultaneous reliable detection of
hundreds of DNA target sequences in one test run using a series of beacons
immobilized on a single substrate in a spatial format.

DOI: 10.1016/j.bios.2006.04.032 
PMID: 16815005  [Indexed for MEDLINE]


917. Chem Biol Interact. 2007 Jan 5;165(1):1-13. Epub 2006 Oct 21.

Study of antimutagenic and antioxidant activities of gallic acid and
1,2,3,4,6-pentagalloylglucose from Pistacia lentiscus. Confirmation by microarray
expression profiling.

Abdelwahed A(1), Bouhlel I, Skandrani I, Valenti K, Kadri M, Guiraud P, Steiman
R, Mariotte AM, Ghedira K, Laporte F, Dijoux-Franca MG, Chekir-Ghedira L.

Author information: 
(1)Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de
Pharmacie/Médecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisia.

In vitro antioxidant and antimutagenic activities of two polyphenols isolated
from the fruits of Pistacia lentiscus was assessed. Antioxidant activity was
determined by the ability of each compound to scavenge the free radical
1,1-diphenyl-2-picrylhydrazyl (DPPH*), to inhibit xanthine oxidase and to inhibit
the lipid peroxidation induced by H(2)O(2) in K562 cell line. Antimutagenic
activity was assayed with SOS chromotest using Escherichia coli PQ37 as tester
strain and Comet assay using K562 cell line. 1,2,3,4,6-Pentagalloylglucose was
found to be more effective to scavenge DPPH* radical and protect against lipid
peroxidation. Moreover, these two compounds induced an inhibitory activity
against nifuroxazide and aflatoxin B1 mutagenicity. The protective effect
exhibited by these molecules was also determined by analysis of gene expression
as response to an oxidative stress. For this purpose, we used a cDNA-microarray
containing 82 genes related to cell defense, essentially represented by
antioxidant and DNA repair proteins. We found that 1,2,3,4,6-pentagalloylglucose 
induced a decrease in the expression of 11 transcripts related to antioxidant
enzymes family (GPX1, TXN, AOE372, SHC1 and SEPW1) and DNA repair (POLD1, APEX,
POLD2, MPG, PARP and XRCC5). The use of Gallic acid, induced expression of TXN,
TXNRD1, AOE372, GSS (antioxidant enzymes) and LIG4, POLD2, MPG, GADD45A, PCNA,
RPA2, DDIT3, HMOX2, XPA, TDG, ERCC1 and GTF2H1 (DNA repair) as well as the
repression of GPX1, SEPW1, POLD1 and SHC1 gene expression.

DOI: 10.1016/j.cbi.2006.10.003 
PMID: 17129579  [Indexed for MEDLINE]


918. Appl Environ Microbiol. 2007 Jan;73(2):477-84. Epub 2006 Nov 3.

Occurrence of virulence and antimicrobial resistance genes in Escherichia coli
isolates from different aquatic ecosystems within the St. Clair River and Detroit
River areas.

Hamelin K(1), Bruant G, El-Shaarawi A, Hill S, Edge TA, Fairbrother J, Harel J,
Maynard C, Masson L, Brousseau R.

Author information: 
(1)Biotechnology Research Institute, National Research Council of Canada, 6100
Royalmount Ave., Montreal, Quebec, Canada H4P 2R2.

Although the number of Escherichia coli bacteria in surface waters can differ
greatly between locations, relatively little is known about the distribution of
E. coli pathotypes in surface waters used as sources for drinking or recreation. 
DNA microarray technology is a suitable tool for this type of study due to its
ability to detect high numbers of virulence and antimicrobial resistance genes
simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene 
profiles were determined for 308 E. coli isolates from surface water samples
collected from diverse aquatic ecosystems at six different sites in the St. Clair
River and Detroit River areas. A higher frequency (48%) of E. coli isolates
possessing virulence and antimicrobial resistance genes was observed in an urban 
site located downstream of wastewater effluent outfalls than in the other
examined sites (average of 24%). Most E. coli pathotypes were extraintestinal
pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and 
D. The ExPEC pathotypes were found to occur across all aquatic ecosystems
investigated, including riverine, estuarine, and offshore lake locations. The
results of this environmental study using DNA microarrays highlight the
widespread distribution of E. coli pathotypes in aquatic ecosystems and the
potential public health threat of E. coli pathotypes originating from municipal
wastewater sources.

DOI: 10.1128/AEM.01445-06 
PMCID: PMC1796988
PMID: 17085696  [Indexed for MEDLINE]


919. Appl Environ Microbiol. 2007 Jan;73(1):73-82. Epub 2006 Oct 27.

Effects of target length on the hybridization efficiency and specificity of
rRNA-based oligonucleotide microarrays.

Liu WT(1), Guo H, Wu JH.

Author information: 
(1)Division of Environmental Science and Engineering, National University of
Singapore, Block E2, no. 04-07, 1 Engineering Drive 2, Singapore 117576.
eseliuwt@nus.edu.sg

The effect of target size on microarray hybridization efficiencies and
specificity was investigated using a set of 166 oligonucleotide probes targeting 
the 16S rRNA gene of Escherichia coli. The targets included unfragmented native
rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480
bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp).
Fluorescence intensities of probes hybridized with targets were categorized into 
classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 
60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization
efficiency was defined for those probes conferring intensities in classes I to
IV; those in classes V and VI were regarded as weak and false-negative signals,
respectively. Using unfragmented native rRNA, 13.9% of the probes had
fluorescence intensities in classes I to IV, whereas the majority (57.8%)
exhibited false-negative signals. Similar trends were observed for the 1,480-bp
PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after
hybridization of fragmented rRNA, the percentage of probes in classes I to IV
rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45
bp, this percentage increased approximately 14-fold. Overall, microarray
hybridization efficiencies and specificity were improved with fragmented rRNA (20
to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp).
Such an understanding is important to the application of DNA microarray
technology in microbial community studies.

DOI: 10.1128/AEM.01468-06 
PMCID: PMC1797121
PMID: 17071797  [Indexed for MEDLINE]


920. Bacteriol Virusol Parazitol Epidemiol. 2007 Jan-Jun;52(1-2):5-14.

[Conventional bacteriological and molecular diagnostic for the identification of 
Escherichia coli digestive patho-types].

[Article in Romanian]

Nica M(1).

Author information: 
(1)Spitalul Clinic de Boli Infecţioase şi Tropicale Dr. V. Babeg, Bucureşti,
Laboratorul de microbiologie.


PMID: 18441953  [Indexed for MEDLINE]


921. Biosci Biotechnol Biochem. 2007 Jan;71(1):77-83. Epub 2007 Jan 7.

Effect of the arcA mutation on the expression of flagella genes in Escherichia
coli.

Kato Y(1), Sugiura M, Mizuno T, Aiba H.

Author information: 
(1)Laboratory of Molecular Microbiology, School of Agriculture, Nagoya
University, Japan.

Flagella expression in Escherichia coli is controlled in a hierarchical manner,
in which class-1 gene products, FlhDC, functions as a master regulator to control
class-2 genes that encode motility-related genes. fliA, one of the class 2 genes,
encodes flagellum-specific sigma factor (FliA/Sigma F/Sigma-28), which is
necessary for the expression of class-3 genes. Previously, we carried out
transcriptome analyses of all two-component regulatory systems of E. coli, and
determined that the arcA mutant showed the motility-defective phenotype. In this 
study, we characterized the arcA mutant, and we present evidence that ArcA is
necessary for the expression of FliA, but not for the master regulators, FlhDC.
The phosphorylation site of ArcA is necessary for motility, while a cognate
histidine kinase, ArcB, appears not to be involved in motility. This suggests
that there must be regulatory factors other than ArcB interacting with ArcA to
control flagella genes.


PMID: 17213678  [Indexed for MEDLINE]


922. Chemosphere. 2007 Jan;66(7):1243-8. Epub 2006 Sep 1.

Gene expression analysis and classification of mode of toxicity of polycyclic
aromatic hydrocarbons (PAHs) in Escherichia coli.

Kim YS(1), Min J, Hong HN, Park JH, Park KS, Gu MB.

Author information: 
(1)College of Life Sciences and Biotechnology, Korea University, Anam-dong,
Sungbuk-gu, Seoul 136-701, South Korea.

Escherichia coli is known to respond to certain toxic chemicals through an
increased expression of various stress genes. In this study, therefore, the
expression of recA, katG, fabA and grpE genes was used as a representative for
DNA, oxidative, membrane and protein damage, respectively, after E. coli was
exposed to different polycyclic aromatic hydrocarbons (PAHs), i.e., phenanthrene,
naphthalene and benzo[a]pyrene. To accomplish this, the expression levels of
these four genes were quantified using a real-time RT-PCR analysis when E. coli
cultures were under stressful conditions, such as those caused by an exposure to 
mitomycin C, hydrogen peroxide and phenol. It was found that the primary toxic
effect of each chemical is clearly seen when the expression levels of the
different genes are compared. Tests with the PAHs showed naphthalene and
benzo[a]pyrene to be genotoxic, while phenanthrene had no clear effect on the
expression of any of these genes. Based on these results, the effects due to
these toxic chemicals and the extent of each stress can be evaluated with ease
using the expression levels of different stress responsive genes.

DOI: 10.1016/j.chemosphere.2006.07.040 
PMID: 16949636  [Indexed for MEDLINE]


923. Cytogenet Genome Res. 2007;117(1-4):139-45.

A functional genomics approach to the study of avian innate immunity.

Keeler CL Jr(1), Bliss TW, Lavric M, Maughan MN.

Author information: 
(1)Department of Animal and Food Sciences, College of Agriculture and Natural
Resources, University of Delaware, Newark, DE 19716-2150, USA. ckeeler@udel.edu

A second-generation 4,959 element cDNA microarray has been created and evaluated 
for its potential use in examining the avian innate immune response. The elements
in this array were obtained from EST libraries of stimulated avian PMNC-derived
monocytes/macrophages and supplemented by genes of interest from several specific
innate immune pathways. The elements are spotted in triplicate resulting in
14,877 total spots per slide. The avian innate immunity microarray (AIIM)
contains 25 avian interleukin, chemokine, and cytokine elements. The array also
contains elements for several innate immune pathways, including genes involved in
the Toll-like receptor (TLR) pathway (including six of the currently known avian 
TLR receptors), avian interferon/antiviral response pathway genes, and genes
involved in apoptosis, antigen presentation and the oxidative burst. The AIIM can
be used to evaluate global gene expression patterns in a number of
immunologically relevant tissues and in chickens, turkeys and ducks. The array
has also been evaluated for its ability to monitor the avian immune response to
both bacterial (avian pathogenic Escherichia coli) and viral (avian influenza)
avian pathogens.

Copyright 2007 S. Karger AG, Basel.

DOI: 10.1159/000103174 
PMID: 17675854  [Indexed for MEDLINE]


924. Genome Biol. 2007;8(12):R267.

Characterization of probiotic Escherichia coli isolates with a novel pan-genome
microarray.

Willenbrock H(1), Hallin PF, Wassenaar TM, Ussery DW.

Author information: 
(1)Center for Biological Sequence Analysis, Technical University of Denmark, 2800
Lyngby, Denmark. hanni@cbs.dtu.dk

BACKGROUND: Microarrays have recently emerged as a novel procedure to evaluate
the genetic content of bacterial species. So far, microarrays have mostly covered
single or few strains from the same species. However, with cheaper
high-throughput sequencing techniques emerging, multiple strains of the same
species are rapidly becoming available, allowing for the definition and
characterization of a whole species as a population of genomes--the 'pan-genome'.
RESULTS: Using 32 Escherichia coli and Shigella genome sequences we estimate the 
pan- and core genome of the species. We designed a high-density microarray in
order to provide a tool for characterization of the E. coli pan-genome. Technical
performance of this pan-genome microarray based on control strain samples (E.
coli K-12 and O157:H7) demonstrated a high sensitivity and relatively low false
positive rate. A single-channel analysis approach is robust while allowing the
possibility for deriving presence/absence predictions for any gene included on
our pan-genome microarray. Moreover, the array was highly sufficient to
investigate the gene content of non-pathogenic isolates, despite the strong bias 
towards pathogenic E. coli strains that have been sequenced so far.
CONCLUSION: This high-density microarray provides an excellent tool for
characterizing the genetic makeup of unknown E. coli strains and can also deliver
insights into phylogenetic relationships. Its design poses a considerably larger 
challenge and involves different considerations than the design of single strain 
microarrays. Here, lessons learned and future directions will be discussed in
order to optimize design of microarrays targeting entire pan-genomes.

DOI: 10.1186/gb-2007-8-12-r267 
PMCID: PMC2246269
PMID: 18088402  [Indexed for MEDLINE]


925. Genome Biol. 2007;8(7):R138.

Extensive genomic diversity and selective conservation of virulence-determinants 
in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes.

Ogura Y(1), Ooka T, Asadulghani, Terajima J, Nougayrède JP, Kurokawa K, Tashiro
K, Tobe T, Nakayama K, Kuhara S, Oswald E, Watanabe H, Hayashi T.

Author information: 
(1)Division of Bioenvironmental Science, Frontier Science Research Center,
University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Japan.
y-ogura@med.miyazaki-u.ac.jp <y-ogura@med.miyazaki-u.ac.jp>

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe
food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone 
sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences
encoding many virulence determinants, such as Shiga toxin genes (stx genes) and
the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct
clonal lineages from O157 also cause similar illness in humans. According to the 
'parallel' evolution model, they have independently acquired the major virulence 
determinants, the stx genes and LEE. However, the genomic differences between
O157 and non-O157 EHECs have not yet been systematically analyzed.
RESULTS: Using microarray and whole genome PCR scanning analyses, we performed a 
whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with 
O157. In non-O157 EHEC strains, although genome sizes were similar with or rather
larger than O157 and the backbone regions were well conserved, O157-specific
regions were very poorly conserved. Around only 20% of the O157-specific genes
were fully conserved in each non-O157 serotype. However, the non-O157 EHECs
contained a significant number of virulence genes that are found on prophages and
plasmids in O157, and also multiple prophages similar to, but significantly
divergent from, those in O157.
CONCLUSION: Although O157 and non-O157 EHECs have independently acquired a huge
amount of serotype- or strain-specific genes by lateral gene transfer, they share
an unexpectedly large number of virulence genes. Independent infections of
similar but distinct bacteriophages carrying these virulence determinants are
deeply involved in the evolution of O157 and non-O157 EHECs.

DOI: 10.1186/gb-2007-8-7-r138 
PMCID: PMC2323221
PMID: 17711596  [Indexed for MEDLINE]


926. IET Syst Biol. 2007 Jan;1(1):41-50.

Gene networks reconstruction and time-series prediction from microarray data
using recurrent neural fuzzy networks.

Maraziotis IA(1), Dragomir A, Bezerianos A.

Author information: 
(1)Department of Medical Physics, Medical School, University of Patras, Rio
26500, Greece. imarazi@heart.med.upatras.gr

Reverse engineering problems concerning the reconstruction and identification of 
gene regulatory networks through gene expression data are central issues in
computational molecular biology and have become the focus of much research in the
last few years. An approach has been proposed for inferring the complex causal
relationships among genes from microarray experimental data, which is based on a 
novel neural fuzzy recurrent network. The method derives information on the gene 
interactions in a highly interpretable form (fuzzy rules) and takes into account 
the dynamical aspects of gene regulation through its recurrent structure. To
determine the efficiency of the proposed approach, microarray data from two
experiments relating to Saccharomyces cerevisiae and Escherichia coli have been
used and experiments concerning gene expression time course prediction have been 
conducted. The interactions that have been retrieved among a set of genes known
to be highly regulated during the yeast cell-cycle are validated by previous
biological studies. The method surpasses other computational techniques, which
have attempted genetic network reconstruction, by being able to recover
significantly more biologically valid relationships among genes.


PMID: 17370428  [Indexed for MEDLINE]


927. Infect Immun. 2007 Jan;75(1):278-89. Epub 2006 Oct 30.

In vivo gene expression analysis identifies genes required for enhanced
colonization of the mouse urinary tract by uropathogenic Escherichia coli strain 
CFT073 dsdA.

Haugen BJ(1), Pellett S, Redford P, Hamilton HL, Roesch PL, Welch RA.

Author information: 
(1)Department of Medical Microbiology and Immunology, University of
Wisconsin-Madison, Madison, WI 53706, USA.

Deletional inactivation of the gene encoding d-serine deaminase, dsdA, in
uropathogenic Escherichia coli strain CFT073 results in a hypermotile strain with
a hypercolonization phenotype in the bladder and kidneys of mice in a model of
urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and
CFT073 dsdA were compared by isolating RNA directly from the urine of mice
challenged with each strain individually. Hybridization of cDNAs derived from
these samples to CFT073-specific microarrays allowed identification of genes that
were up- or down-regulated in the dsdA deletion strain during UTI. Up-regulated
genes included the known d-serine-responsive gene dsdX, suggesting in vivo
intracellular accumulation of d-serine by CFT073 dsdA. Genes encoding F1C
fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter
DppA, a heat shock chaperone IbpB, and clusters of open reading frames with
unknown functions were also up-regulated. To determine the role of these genes as
well as motility in the hypercolonization phenotype, mutants were constructed in 
the CFT073 dsdA background and tested in competition against the wild type in the
murine model of UTI. Strains with deletions of one or both of the two P fimbrial 
operons, hlyA, fliC, ibpB, c0468, locus c3566 to c3568, or c2485 to c2490
colonized mouse bladders and kidneys at levels indistinguishable from wild type. 
CFT073 dsdA c2398 and CFT073 dsdA focA maintained a hypercolonization phenotype. 
A CFT073 dsdA dppA mutant was attenuated 10- to 50-fold in its colonization
ability compared to CFT073. Our results support a role for d-serine catabolism
and signaling in global virulence gene regulation of uropathogenic E. coli.

DOI: 10.1128/IAI.01319-06 
PMCID: PMC1828413
PMID: 17074858  [Indexed for MEDLINE]


928. Int J Biochem Cell Biol. 2007;39(10):1886-901. Epub 2007 May 24.

Reversible differentiation of Caco-2 cells reveals galectin-9 as a surface marker
molecule for human follicle-associated epithelia and M cell-like cells.

Pielage JF(1), Cichon C, Greune L, Hirashima M, Kucharzik T, Schmidt MA.

Author information: 
(1)Institute of Infectiology, Center for Molecular Biology of Inflammation
(ZMBE), University of Münster, von-Esmarch-Str. 56, 48149 Münster, Germany.

M cells interspersed in the follicle-associated epithelium of Peyer's patches
represent the major antigen sampling cells of the intestinal mucosa providing
immune surveillance for particulate antigens. Despite their crucial role in
immune defense our knowledge about these elusive cells is still only rudimentary.
A Caco-2 co-culture model for the induction of M cell-like cells and DNA
microarray analysis for differential gene expression profiling were employed to
identify (a) putative suitable surface marker(s). Induction of M cell-like cells 
was demonstrated morphologically by electron microscopy, evaluated by infection
with Yersinia enterocolitica and enteropathogenic Escherichia coli strain
E2348/69 and further monitored by changes in binding of the lectin UEA-1. The
differentiation of Caco-2 cells was found to be reversible, dependent on (a)
lymphocyte-derived soluble factor(s) and accompanied by the up-regulation of the 
glycoprotein lectin galectin-9, which was specifically expressed on these cells
as well as on human follicle-associated epithelial (FAE) cells. Galectin-9
represents a novel surface marker which might be employed for molecular targeting
to the Peyer's patches thereby opening new opportunities for drug and vaccine
development.

DOI: 10.1016/j.biocel.2007.05.009 
PMID: 17596995  [Indexed for MEDLINE]


929. J Bacteriol. 2007 Jan;189(2):531-9. Epub 2006 Nov 3.

Complete and SOS-mediated response of Staphylococcus aureus to the antibiotic
ciprofloxacin.

Cirz RT(1), Jones MB, Gingles NA, Minogue TD, Jarrahi B, Peterson SN, Romesberg
FE.

Author information: 
(1)Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037,
USA.

Staphylococcus aureus infections can be difficult to treat due to both multidrug 
resistance and the organism's remarkable ability to persist in the host.
Persistence and the evolution of resistance may be related to several complex
regulatory networks, such as the SOS response, which modifies transcription in
response to environmental stress. To understand how S. aureus persists during
antibiotic therapy and eventually emerges resistant, we characterized its global 
transcriptional response to ciprofloxacin. We found that ciprofloxacin induces
prophage mobilization as well as significant alterations in metabolism, most
notably the up-regulation of the tricarboxylic acid cycle. In addition, we found 
that ciprofloxacin induces the SOS response, which we show, by comparison of a
wild-type strain and a non-SOS-inducible lexA mutant strain, includes the
derepression of 16 genes. While the SOS response of S. aureus is much more
limited than those of Escherichia coli and Bacillus subtilis, it is similar to
that of Pseudomonas aeruginosa and includes RecA, LexA, several hypothetical
proteins, and a likely error-prone Y family polymerase whose homologs in other
bacteria are required for induced mutation. We also examined induced mutation and
found that either the inability to derepress the SOS response or the lack of the 
LexA-regulated polymerase renders S. aureus unable to evolve antibiotic
resistance in vitro in response to UV damage. The data suggest that up-regulation
of the tricarboxylic acid cycle and induced mutation facilitate S. aureus
persistence and evolution of resistance during antibiotic therapy.

DOI: 10.1128/JB.01464-06 
PMCID: PMC1797410
PMID: 17085555  [Indexed for MEDLINE]


930. J Endotoxin Res. 2007;13(4):227-34.

High glucose up-regulates lipopolysaccharide-stimulated inflammatory cytokine
production via c-jun N-terminal kinase in the monocytic cell line THP-1.

Iwata H(1), Soga Y, Meguro M, Yoshizawa S, Okada Y, Iwamoto Y, Yamashita A,
Takashiba S, Nishimura F.

Author information: 
(1)Department of Pathophysiology - Periodontal Science, Okayama University
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama,
Japan.

Diabetic subjects are susceptible to atherosclerosis. It has been postulated that
inflammation plays a crucial role in atherogenesis. Since previous studies
suggested persistent low-grade infection by Gram-negative bacteria such as
Chlamydia spp. and/or periodontal infection is associated with increased
atherogenesis among diabetic subjects, we hypothesized that macrophages under
hyperglycemia respond to lipopolysaccharide (LPS) challenge in a more exaggerated
manner than under normal glucose conditions. Therefore, we examined cytokine
productivity and associated signal transduction molecules in LPS-stimulated the
monocytic cell line THP-1, under conditions of hyperglycemia. Differentiated
THP-1 cells were cultured under normal and high glucose conditions without fetal 
bovine serum, and were stimulated with Escherichia coli LPS in the presence of
LPS binding protein. Following stimulation, activated signal transduction
molecules were detected by protein microarray and confirmed thereafter. Results
indicated that c-jun N-terminal kinase (JNK) was highly-phosphorylated at high
glucose concentrations, and this was confirmed by Western-immunoblotting. Tumor
necrosis factor-alpha and monocyte chemo-attractant protein-1 production were
significantly enhanced under these conditions. SP600125, a selective inhibitor of
JNK, dose-dependently suppressed the production of these cytokine. Therefore, we 
suggest that this may be one of the mechanisms by which sub-clinical infection by
Gram-negative bacteria promotes atherosclerosis in diabetic subjects.

DOI: 10.1177/0968051907082608 
PMID: 17956941  [Indexed for MEDLINE]


931. J Mol Microbiol Biotechnol. 2007;13(1-3):96-104.

Complete deficiency of 5'-nucleotidase activity in Escherichia coli leads to loss
of growth on purine nucleotides but not of their excretion.

Kakehi M(1), Usuda Y, Tabira Y, Sugimoto S.

Author information: 
(1)Fermentation and Biotechnology Laboratories, Ajinomoto Co., Kawasaki, Japan.

Escherichia coli has many periplasmic phosphatase activities. To test whether it 
can take up and excrete purine nucleotides, we attempted to completely disrupt
periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in
ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they 
were grown with purine nucleotides as the sole carbon source. Using DNA
macroarrays to compare global gene expression in wild-type and ushA knockout
mutant cells cultured with IMP or GMP as the sole carbon source, we identified
two genes that were induced in the ushA knockout mutant cells and encoded signal 
sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase 
activity and was induced by IMP or inosine. An ushA aphA double knockout mutant
was shown to be unable to grow on purine nucleotides as the sole carbon source.
To investigate the excretion of purine nucleotides, we constructed an ushAaphA
double knockout mutant of an inosine-producing strain and found that it
accumulated IMP in the medium. In addition, when the guaBA operon was introduced 
into the ushAaphA double knockout IMP producer, GMP was released into the medium.
These observations imply the existence of efflux activity for purine nucleotides 
in E. coli.

Copyright (c) 2007 S. Karger AG, Basel.

DOI: 10.1159/000103601 
PMID: 17693717  [Indexed for MEDLINE]


932. Nat Methods. 2007 Jan;4(1):87-93. Epub 2006 Nov 12.

SCALEs: multiscale analysis of library enrichment.

Lynch MD(1), Warnecke T, Gill RT.

Author information: 
(1)Department of Chemical and Biological Engineering, University of Colorado,
ECCH 111, Campus Box 424, Boulder, Colorado 80309, USA.

We report a genome-wide, multiscale approach to simultaneously measure the effect
that the increased copy of each gene and/or operon has on a desired trait or
phenotype. The method involves (i) growth selections on a mixture of several
different plasmid-based genomic libraries of defined insert sizes or SCALEs, (ii)
microarray studies of enriched plasmid DNA, and a (iii) mathematical multiscale
analysis that precisely identifies the relevant genetic elements. This approach
allows for identification of all single open reading frames and larger multigene 
fragments within a genomic library that alter the expression of a given
phenotype. We have demonstrated this method in Escherichia coli by monitoring, in
parallel, a population of >10(6) genomic library clones of different insert
sizes, throughout continuous selections over a period of 100 generations.

DOI: 10.1038/nmeth946 
PMID: 17099705  [Indexed for MEDLINE]


933. Nucleic Acids Res. 2007;35(7):e48. Epub 2007 Mar 2.

Improving comparability between microarray probe signals by thermodynamic
intensity correction.

Bruun GM(1), Wernersson R, Juncker AS, Willenbrock H, Nielsen HB.

Author information: 
(1)Niels Bohr Institute, Blegdamsvej 17, Copenhagen, Denmark.

Signals from different oligonucleotide probes against the same target show great 
variation in intensities. However, detection of differences along a sequence e.g.
to reveal intron/exon architecture, transcription boundary as well as simple
absent/present calls depends on comparisons between different probes. It is
therefore of great interest to correct for the variation between probes. Much of 
this variation is sequence dependent. We demonstrate that a thermodynamic model
for hybridization of either DNA or RNA to a DNA microarray, which takes the
sequence-dependent probe affinities into account significantly reduces the signal
fluctuation between probes targeting the same gene transcript. For a test set of 
tightly tiled yeast genes, the model reduces the variance by up to a factor
approximately 1/3. As a consequence of this reduction, the model is shown to
yield a more accurate determination of transcription start sites for a subset of 
yeast genes. In another application, we identify present/absent calls for probes 
hybridized to the sequenced Escherichia coli strain O157:H7 EDL933. The model
improves the correct calls from 85 to 95% relative to raw intensity measures. The
model thus makes applications which depend on comparisons between probes aimed at
different sections of the same target more reliable.

DOI: 10.1093/nar/gkl1098 
PMCID: PMC1874658
PMID: 17337437  [Indexed for MEDLINE]


934. Nucleic Acids Res. 2007;35(2):506-16. Epub 2006 Dec 14.

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis
thaliana can promote the cold adaptation process in Escherichia coli.

Kim JS(1), Park SJ, Kwak KJ, Kim YO, Kim JY, Song J, Jang B, Jung CH, Kang H.

Author information: 
(1)Department of Plant Biotechnology, Agricultural Plant Stress Research Center
and Biotechnology Research Institute, College of Agriculture and Life Sciences,
Chonnam National University, Gwangju, 500-757, Republic of Korea.

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich
RNA-binding proteins (GRPs) have been implicated to play a role during the cold
adaptation process, their importance and function in eukaryotes, including
plants, are largely unknown. To understand the functional role of plant CSDPs and
GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4
and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of
CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli
that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress,
and resulted in better survival rate than control cells during incubation at low 
temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective
evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not
recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1
and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast,
CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity.
Together, these results indicate that CSDPs and GRPs help E.coli grow and survive
better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA
chaperone activity during the cold adaptation process.

DOI: 10.1093/nar/gkl1076 
PMCID: PMC1802614
PMID: 17169986  [Indexed for MEDLINE]


935. Nucleic Acids Res. 2007;35(1):269-78. Epub 2006 Dec 12.

Transcription factor distribution in Escherichia coli: studies with FNR protein.

Grainger DC(1), Aiba H, Hurd D, Browning DF, Busby SJ.

Author information: 
(1)School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15
2TT, UK. d.grainger@bham.ac.uk

Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have 
measured the distribution of the global transcription regulator protein, FNR,
across the entire Escherichia coli chromosome in exponentially growing cells.
Sixty-three binding targets, each located at the 5' end of a gene, were
identified. Some targets are adjacent to poorly transcribed genes where FNR has
little impact on transcription. In stationary phase, the distribution of FNR was 
largely unchanged. Control experiments showed that, like FNR, the distribution of
the nucleoid-associated protein, IHF, is little altered when cells enter
stationary phase, whilst RNA polymerase undergoes a complete redistribution.

DOI: 10.1093/nar/gkl1023 
PMCID: PMC1802558
PMID: 17164287  [Indexed for MEDLINE]


936. PLoS Biol. 2007 Jan;5(1):e8.

Large-scale mapping and validation of Escherichia coli transcriptional regulation
from a compendium of expression profiles.

Faith JJ(1), Hayete B, Thaden JT, Mogno I, Wierzbowski J, Cottarel G, Kasif S,
Collins JJ, Gardner TS.

Author information: 
(1)Bioinformatics Program, Boston University, Boston, Massachusetts, United
States of America.

Machine learning approaches offer the potential to systematically identify
transcriptional regulatory interactions from a compendium of microarray
expression profiles. However, experimental validation of the performance of these
methods at the genome scale has remained elusive. Here we assess the global
performance of four existing classes of inference algorithms using 445
Escherichia coli Affymetrix arrays and 3,216 known E. coli regulatory
interactions from RegulonDB. We also developed and applied the context likelihood
of relatedness (CLR) algorithm, a novel extension of the relevance networks class
of algorithms. CLR demonstrates an average precision gain of 36% relative to the 
next-best performing algorithm. At a 60% true positive rate, CLR identifies 1,079
regulatory interactions, of which 338 were in the previously known network and
741 were novel predictions. We tested the predicted interactions for three
transcription factors with chromatin immunoprecipitation, confirming 21 novel
interactions and verifying our RegulonDB-based performance estimates. CLR also
identified a regulatory link providing central metabolic control of iron
transport, which we confirmed with real-time quantitative PCR. The compendium of 
expression data compiled in this study, coupled with RegulonDB, provides a
valuable model system for further improvement of network inference algorithms
using experimental data.

DOI: 10.1371/journal.pbio.0050008 
PMCID: PMC1764438
PMID: 17214507  [Indexed for MEDLINE]

Conflict of interest statement: Competing interests. A portion of this work was
conducted in collaboration with Cellicon Biotechnologies. JJC and TSG are
founders and shareholders in the company. GC and JW are also shareholders in the 
company. All data, results, and algorithms from this collaboration have been made
publicly available.


937. Water Sci Technol. 2007;55(8-9):413-9.

Using microbial genomics to evaluate the effectiveness of silver to prevent
biofilm formation.

Wu MY(1), Suryanarayanan K, van Ooij WJ, Oerther DB.

Author information: 
(1)Department of Civil and Environment Engineering, University of Cincinnati, 765
Baldwin Hall (MLOO071), Cincinnati, OH 45221-0071, USA. wumi@email.uc.edu

Increasingly, monovalent silver cations are being considered as an alternative
biocide in water distribution systems. The objective of this study was to
understand how bacteria respond when challenged with silver. The approach used
included cultivation of Escherichia coli in planktonic and sessile phases,
exposing biomass to a solution of silver nitrate and evaluating genetic responses
using commercial Affymetrix microarrays. Experimental results showed that lower
pH enhanced silver toxicity in a dose-dependent manner. Sessile biomass
demonstrated resistance to silver, and the microarray results indicated that the 
genetic mechanism for silver resistance was similar to the mechanism for copper
resistance including upregulation of efflux pumps as well as upregulation of
metal oxidoreductases. The gene, copA, a P-type ATPase efflux flux, was
upregulated in response to silver exposure, and the gene of CusCFBA, a Cu(I)
efflux pump, was also upregulated. The gene of CueO, a robust cuprous oxidase,
was also upregulated and may have reduced silver toxicity through oxidation of
silver ions. This study is significant because it provides baseline data to
understand the genetic response of bacteria to silver biocides.


PMID: 17547012  [Indexed for MEDLINE]


938. Gene. 2006 Dec 15;384:73-95. Epub 2006 Jul 20.

Computational prediction of RpoS and RpoD regulatory sites in Geobacter
sulfurreducens using sequence and gene expression information.

Yan B(1), Núñez C, Ueki T, Esteve-Núñez A, Puljic M, Adkins RM, Methé BA, Lovley 
DR, Krushkal J.

Author information: 
(1)Department of Preventive Medicine, University of Tennessee Health Science
Center, Memphis, TN, 38163, USA.

RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and
survival of Geobacter sulfurreducens under conditions typically encountered in
its native subsurface environments. We investigated the conservation of sites
that may be important for RpoS function in G. sulfurreducens. We also employed
sequence information and expression microarray data to predict G. sulfurreducens 
genome sites that may be related to RpoS regulation. Hierarchical clustering
identified three clusters of significantly downregulated genes in the rpoS
deletion mutant. The search for conserved overrepresented motifs in co-regulated 
operons identified likely -35 and -10 promoter elements upstream of a number of
functionally important G. sulfurreducens operons that were downregulated in the
rpoS deletion mutant. Putative -35/-10 promoter elements were also identified in 
the G. sulfurreducens genome using sequence similarity searches to matrices of
-35/-10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due
to a sufficient degree of sequence similarity between -35/-10 promoter elements
for RpoS, RpoD, and other sigma factors, both the sequence similarity searches
and the search for conserved overrepresented motifs using microarray data may
identify promoter elements for both RpoS and other sigma factors.

DOI: 10.1016/j.gene.2006.06.025 
PMID: 17014972  [Indexed for MEDLINE]


939. J Med Chem. 2006 Dec 14;49(25):7331-41.

Isoprenoid biosynthesis as a drug target: bisphosphonate inhibition of
Escherichia coli K12 growth and synergistic effects of fosmidomycin.

Leon A(1), Liu L, Yang Y, Hudock MP, Hall P, Yin F, Studer D, Puan KJ, Morita CT,
Oldfield E.

Author information: 
(1)Center for Biophysics and Computational Biology, University of Illinois at
Urbana - Champaign, 607 South Mathews Avenue, Urbana, Illinois 61801, USA.

We screened a library of 117 bisphosphonates for antibacterial activity against
Escherichia coli. The most potent growth inhibitors where
N-[methyl(4-phenylalkyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonates, known
potent bone resorption inhibitors, and there was a generally good correlation
between cell growth inhibition and E. coli farnesyl diphosphate synthase (FPPS)
inhibition. However, some potent FPPS inhibitors had no activity in cell growth
inhibition, and based on the result of Catalyst pharmacophore modeling, this
could be attributed to the requirement of a large hydrophobic feature for
cellular activity (due most likely to transport). The activity of the most potent
compound, N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate
(13), was strongly potentiated by the drug fosmidomycin. The transcription
profiles for 13 or fosmidomycin alone were different from those found with
carbenicillin or ciprofloxacin alone, but there were many similarities between
the combination (13-fosmidomycin) and carbenicillin or ciprofloxacin, reflecting 
the more potent bactericidal activity of the drug combination on bacterial
growth.

DOI: 10.1021/jm060492b 
PMID: 17149863  [Indexed for MEDLINE]


940. Mol Cell. 2006 Dec 8;24(5):747-57.

The transition between transcriptional initiation and elongation in E. coli is
highly variable and often rate limiting.

Reppas NB(1), Wade JT, Church GM, Struhl K.

Author information: 
(1)Harvard University Biophysics Program, Harvard Medical School, Boston,
Massachusetts 02115, USA.

We perform a genome-wide analysis of the transition between transcriptional
initiation and elongation in Escherichia coli by determining the association of
core RNA polymerase (RNAP) and the promoter-recognition factor sigma70 with
respect to RNA transcripts. We identify 1286 sigma70-associated promoters,
including many internal to known operons, and demonstrate that sigma70 is usually
released very rapidly from elongating RNAP complexes. On average, RNAP density is
higher at the promoter than in the coding sequence, although the ratio is highly 
variable among different transcribed regions. Strikingly, a significant fraction 
of RNAP-bound promoters is not associated with transcriptional activity, perhaps 
due to an intrinsic energetic barrier to promoter escape. Thus, the transition
from transcriptional initiation to elongation is highly variable, often rate
limiting, and in some cases is essentially blocked such that RNAP is effectively 
"poised" to transcribe only under the appropriate environmental conditions. The
genomic pattern of RNAP density in E. coli differs from that in yeast and
mammalian cells.

DOI: 10.1016/j.molcel.2006.10.030 
PMID: 17157257  [Indexed for MEDLINE]


941. Int J Med Microbiol. 2006 Dec;296(8):541-6. Epub 2006 Oct 16.

Specific regions of genome plasticity and genetic diversity of the commensal
Escherichia coli A0 34/86.

Hejnova J(1), Pages D, Rusniok C, Glaser P, Sebo P, Buchrieser C.

Author information: 
(1)Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du
Dr. Roux, F-75724 Paris, France.

Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used 
for prophylactic and therapeutic colonization of the intestine of newborn
infants. To identify traits specific for E. coli A0 34/86, we used a minimal
tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct
restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli
strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC
clones that were sequenced to identify A0 34/86-specific regions. Genes for the
yersiniabactin siderophore system, several proteins homologous to Salmonella
enterica serovar Typhimurium vitamin B12 synthesis proteins, as well as genes
necessary for the degradation of propanediol, the pix fimbriae determinant and
genes coding for a putative phosphoglycerate transport system present also on
pathogenicity island V of E. coli strain 536 were all identified in E. coli A0
34/86. This comparative analysis underlines the important genome heterogeneity
between E. coli strains.

DOI: 10.1016/j.ijmm.2006.06.007 
PMID: 17049458  [Indexed for MEDLINE]


942. J Am Soc Nephrol. 2006 Dec;17(12):3404-14. Epub 2006 Nov 2.

A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal
damage and physiologic response of human disease.

Keepers TR(1), Psotka MA, Gross LK, Obrig TG.

Author information: 
(1)University of Virginia, Department of Internal Medicine, Division of
Nephrology, Box 800133, Charlottesville, VA 22908, USA.

Hemolytic uremic syndrome (HUS), which is caused by Shiga toxin-producing
Escherichia coli infection, is the leading cause of acute renal failure in
children. At present, there is no complete small animal model of this disease.
This study investigated a mouse model using intraperitoneal co-injection of
purified Shiga toxin 2 (Stx2) plus LPS. Through microarray, biochemical, and
histologic analysis, it was found to be a valid model of the human disease.
Biochemical and microarray analysis of mouse kidneys revealed the Stx2 plus LPS
challenge to be distinct from the effects of either agent alone. Microarrays
identified differentially expressed genes that were demonstrated previously to
play a role in this disease. Blood and serum analysis of these mice showed
neutrophilia, thrombocytopenia, red cell hemolysis, and increased serum
creatinine and blood urea nitrogen. In addition, histologic analysis and electron
microscopy of mouse kidneys demonstrated glomerular fibrin deposition, red cell
congestion, microthrombi formation, and glomerular ultrastructural changes. It
was established that this C57BL/6 mouse is a complete model of HUS that includes 
the thrombocytopenia, hemolytic anemia, and renal failure that define the human
disease. In addition, a time course of HUS disease progression that will be
useful for identification of therapeutic targets and development of new
treatments for HUS is described.

DOI: 10.1681/ASN.2006050419 
PMID: 17082244  [Indexed for MEDLINE]


943. J Bacteriol. 2006 Dec;188(23):8160-8. Epub 2006 Sep 22.

DNA adenine methylation regulates virulence gene expression in Salmonella
enterica serovar Typhimurium.

Balbontín R(1), Rowley G, Pucciarelli MG, López-Garrido J, Wormstone Y, Lucchini 
S, García-Del Portillo F, Hinton JC, Casadesús J.

Author information: 
(1)Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 
Seville, Spain.

Transcriptomic analyses during growth in Luria-Bertani medium were performed in
strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic
derivatives lacking Dam methylase. More genes were repressed than were activated 
by Dam methylation (139 versus 37). Key genes that were differentially regulated 
by Dam methylation were verified independently. The largest classes of
Dam-repressed genes included genes belonging to the SOS regulon, as previously
described in Escherichia coli, and genes of the SOS-inducible Salmonella
prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were 
also identified. Invasion genes in pathogenicity island SPI-1 were activated by
Dam methylation, while the fimbrial operon std was repressed by Dam methylation. 
Certain flagellar genes were repressed by Dam methylation, and Dam(-) mutants of 
S. enterica showed reduced motility. Altered expression patterns in the absence
of Dam methylation were also found for the chemotaxis genes cheR (repressed by
Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB
(activated by Dam). The requirement for DNA adenine methylation in the regulation
of specific virulence genes suggests that certain defects of Salmonella Dam(-)
mutants in the mouse model may be caused by altered patterns of gene expression.

DOI: 10.1128/JB.00847-06 
PMCID: PMC1698197
PMID: 16997949  [Indexed for MEDLINE]


944. J Clin Microbiol. 2006 Dec;44(12):4376-83. Epub 2006 Oct 4.

Development of a serotype-specific DNA microarray for identification of some
Shigella and pathogenic Escherichia coli strains.

Li Y(1), Liu D, Cao B, Han W, Liu Y, Liu F, Guo X, Bastin DA, Feng L, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, 23
Hong Da Street, TEDA, Tianjin 300457, China.

Shigella and pathogenic Escherichia coli are major causes of human infectious
diseases and are responsible for millions of cases of diarrhea worldwide every
year. A convenient and rapid method to identify highly pathogenic serotypes of
Shigella and E. coli is needed for large-scale epidemiologic study, timely
clinical diagnosis, and reliable quarantine of the pathogens. In this study, a
DNA microarray targeting O-serotype-specific genes was developed to detect 15
serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri
type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types
4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was
tested against 186 representative strains of all Shigella and E. coli O
serotypes, 38 clinical isolates, and 9 strains of other bacterial species that
are commonly present in stool samples and was shown to be specific and
reproducible. The detection sensitivity was 50 ng genomic DNA or 10(4) CFU per ml
in mock stool specimens. This is the first report of a microarray for serotyping 
Shigella and pathogenic E. coli. The method has a number of advantages over
traditional bacterial culture and antiserum agglutination methods and is
promising for applications in basic microbiological research, clinical diagnosis,
food safety, and epidemiological surveillance.

DOI: 10.1128/JCM.01389-06 
PMCID: PMC1698391
PMID: 17021058  [Indexed for MEDLINE]


945. BMC Vet Res. 2006 Nov 29;2:34.

Characterization of ovine hepatic gene expression profiles in response to
Escherichia coli lipopolysaccharide using a bovine cDNA microarray.

Cao H(1), Kabaroff LC, You Q, Rodriguez A, Boermans HJ, Karrow NA.

Author information: 
(1)Department of Animal and Poultry Science, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada. hcao@uoguelph.ca <hcao@uoguelph.ca>

BACKGROUND: During systemic gram-negative bacterial infections,
lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex
induces the production of hepatic acute phase proteins that are involved in the
host response to infection and limit the associated inflammatory process.
Identifying the genes that regulate this hepatic response to LPS in ruminants may
provide insight into the pathogenesis of bacterial diseases and eventually
facilitate breeding of more disease resistant animals. The objective of this
research was to profile the expression of ovine hepatic genes in response to
Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray
and quantitative real-time PCR (qRT-PCR).
RESULTS: Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400
ng/kg) and liver biopsies were collected 4-5 hours post-challenge to assess
hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. 
The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down
regulated, whereas the expression of ACTHR, IFNalphaR, CD1, MCP-1 and GH was
increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7
of these genes confirmed the microarray results and demonstrated that GAPDH is
not a suitable housekeeping gene in LPS challenged sheep.
CONCLUSION: We have identified several potentially important genes by bovine cDNA
microarray and qRT-PCR analyses that are differentially expressed during the
ovine hepatic response to systemic LPS challenge. Their potential role in
regulating the inflammatory response to LPS warrants further investigation.

DOI: 10.1186/1746-6148-2-34 
PMCID: PMC1684251
PMID: 17134499  [Indexed for MEDLINE]


946. J Biol Chem. 2006 Nov 3;281(44):33115-26. Epub 2006 Sep 5.

Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification
pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive
NarQ-NarP.

Overton TW(1), Whitehead R, Li Y, Snyder LA, Saunders NJ, Smith H, Cole JA.

Author information: 
(1)School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15
2TT, UK.

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide
catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P(aniA) is
activated by FNR (regulator of fumarate and nitrate reduction), the two-component
regulatory system NarQ-NarP, and induced by nitrite; P(norB) is induced by NO
independently of FNR by an uncharacterized mechanism. We report the results of
microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation,
which revealed that only five genes with readily identified NarP-binding sites
are differentially expressed in narP(+) and narP strains. These include three
genes implicated in the truncated gonococcal denitrification pathway: aniA, norB,
and narQ. We also report that (i) nitrite induces aniA transcription in a narP
mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of 
a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; 
(iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive
nitrogen species response protein) were expressed constitutively in the absence
of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in
N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR
responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E.
coli and gonococcal NarQ are ligand-insensitive and constitutively active: a
"locked-on" phenotype. We conclude that genes involved in the truncated
denitrification pathway of N. gonorrhoeae are key components of the small NarQP
regulon, that NarP indirectly regulates P(norB) by stimulating NO production by
AniA, and that NsrR plays a critical role in enabling gonococci to evade NO
generated as a host defense mechanism.

DOI: 10.1074/jbc.M607056200 
PMID: 16954205  [Indexed for MEDLINE]


947. Antimicrob Agents Chemother. 2006 Nov;50(11):3562-7. Epub 2006 Aug 28.

Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi.

Gomez MJ(1), Neyfakh AA.

Author information: 
(1)Center for Pharmaceutical Biotechnology (M/C 870), University of Illinois at
Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA. jgomez6@uic.edu

Bacterial genes defining intrinsic resistance to antibiotics encode proteins that
can be targeted by antibiotic potentiators. To find such genes, a transposon
insertion library of Acinetobacter baylyi was screened with subinhibitory
concentrations of various antibiotics to find supersusceptible mutants. A DNA
microarray printer was used to replica plate 10,000 individual library clones to 
select mutants unable to grow at 1/10 the MICs of 12 different antibiotics.
Transposon insertions in 11 genes were found to cause an eightfold or higher
hypersusceptibility to at least one antibiotic. Most of the mutants identified
exhibited hypersusceptibility to beta-lactam antibiotics. These included mutants 
with disruptions of genes encoding proteins involved in efflux (acrB and oprM) as
well as genes pertaining to peptidoglycan synthesis and modification (ampD, mpl, 
and pbpG). However, disruptions of genes encoding proteins with seemingly
unrelated functions (gph, argH, hisF, and ACIAD0795) can also render cells
hypersusceptible to beta-lactam antibiotics. A knockout of gshA, involved in
glutathione biosynthesis, enhanced the susceptibility to metronidazole, while a
knockout of recD, involved in recombination and repair, made the bacteria
hypersusceptible to ciprofloxacin. Disruption of acrB in Escherichia coli
rendered the cells hypersusceptible to several antibiotics. However, knockout
mutants of other homologous genes in E. coli showed no significant changes in
antibiotic MICs, indicating that the intrinsic resistance genes are species
specific.

DOI: 10.1128/AAC.00579-06 
PMCID: PMC1635227
PMID: 16940057  [Indexed for MEDLINE]


948. Insect Biochem Mol Biol. 2006 Nov;36(11):857-68. Epub 2006 Sep 3.

A microarray approach identifies ANT, OS-D and takeout-like genes as
differentially regulated in alate and apterous morphs of the green peach aphid
Myzus persicae (Sulzer).

Ghanim M(1), Dombrovsky A, Raccah B, Sherman A.

Author information: 
(1)Department of Entomology, The Volcani Center, P.O. Box 6 Bet Dagan 50250,
Israel. ghanim@agri.gov.il

Aphids exhibit a complex life cycle in which their phenotypes vary in response to
environmental changes. Wing polyphenism is one of the phenotypes that have not
been adequately studied. We developed a cDNA microarray from the green peach
aphid Myzus persicae (Sulzer), and compared mRNA samples derived from alate and
apterous adults. The microarray experiment resulted in 108 significantly changing
clones that represented 31 unique ESTs. Among the highest and significantly
expressed clones in alate adults, seven clones showed portions of a new adenine
nucleotide translocase (ANT) gene from M. persicae and four clones contained
sequences of the OS-D gene. Another clone showed significant homology with the
takeout (TO) and TO-like proteins described from other insects. A full-length
cDNA clone of M. persicae ANT (Mp ANT) was isolated and characterized. An reverse
transcription-polymerase chain reaction (RT-PCR) analysis showed significant
differences in Mp ANT, OS-D and Mp takeout-like genes (Mp TOL) expression in
alate compared to apterous adults. A polyclonal antibody against the beef heart
mitochondrial ANT reacted with a recombinant Mp ANT expressed in Escherichia
coli, as it did with aphid extracts, showing higher amounts of the expected 33kDa
band in alate adults. Spatial expression analysis showed higher Mp ANT expression
in the thorax compared to head, abdomen, leg and antennae. OS-D and Mp TOL showed
ubiquitous expression across the body, with OS-D being significantly higher in
legs and antennae while Mp TOL being significantly higher in the head and
abdomen. The possible causes for higher expression of ANT, OS-D and TOL in alates
and the physiological effects on the aphid flight and dispersal are discussed.

DOI: 10.1016/j.ibmb.2006.08.007 
PMID: 17046599  [Indexed for MEDLINE]


949. J Bacteriol. 2006 Nov;188(22):7713-21. Epub 2006 Sep 8.

Design of a seven-genome Escherichia coli microarray for comparative genomic
profiling.

Willenbrock H(1), Petersen A, Sekse C, Kiil K, Wasteson Y, Ussery DW.

Author information: 
(1)Center for Biological Sequence Analysis, Technical University of Denmark,
Building 301, DK-2800 Kgs. Lyngby, Denmark.

We describe the design and evaluate the use of a high-density oligonucleotide
microarray covering seven sequenced Escherichia coli genomes in addition to
several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and
virulence genes. Its utility is demonstrated for comparative genomic profiling of
two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Deltastx(2)::cat) as well 
as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using
fluorescently labeled genomic DNA to query the microarrays and subsequently
analyze common virulence genes and phage elements and perform whole-genome
comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed
that its phi3538 (Deltastx(2)::cat) phage element originated from the E. coli
3538 (Deltastx(2)::cat) strain, with which it shares a substantial proportion of 
phage elements. Moreover, a number of genes involved in DNA transfer and
recombination was identified in both new strains, providing a likely explanation 
for their capability to transfer phi3538 (Deltastx(2)::cat) between them.
Analyses of control samples demonstrated that results using our custom-designed
microarray were representative of the true biology, e.g., by confirming the
presence of all known chromosomal phage elements as well as 98.8 and 97.7% of
queried chromosomal genes for the two control strains. Finally, we demonstrate
that use of spatial information, in terms of the physical chromosomal locations
of probes, improves the analysis.

DOI: 10.1128/JB.01043-06 
PMCID: PMC1636325
PMID: 16963574  [Indexed for MEDLINE]


950. J Bacteriol. 2006 Nov;188(21):7364-77. Epub 2006 Aug 25.

Selective promoter recognition by chlamydial sigma28 holoenzyme.

Shen L(1), Feng X, Yuan Y, Luo X, Hatch TP, Hughes KT, Liu JS, Zhang YX.

Author information: 
(1)Department of Medicine, Boston Medical Center, Boston University School of
Medicine, MA 02118, USA. lshen@bu.edu

The sigma transcription factor confers the promoter recognition specificity of
RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma 
factors, sigma(66), sigma(54), and sigma(28). We developed two methods to
facilitate the characterization of promoter sequences recognized by C.
trachomatis sigma(28) (sigma(28)(Ct)). One involved the arabinose-induced
expression of plasmid-encoded sigma(28)(Ct) in a strain of Escherichia coli
defective in the sigma(28) structural gene, fliA. The second was an analysis of
transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP
core and recombinant sigma(28)(Ct). These approaches were used to investigate the
interactions of sigma(28)(Ct) with the sigma(28)(Ct)-dependent hctB promoter and 
selected E. coli sigma(28) (sigma(28)(Ec))-dependent promoters, in parallel,
compared with the promoter recognition properties of sigma(28)(EC). Our results
indicate that RNAP containing sigma(28)(Ct) has at least three characteristics:
(i) it is capable of recognizing some but not all sigma(28)(EC)-dependent
promoters; (ii) it can distinguish different promoter structures, preferentially 
activating promoters with upstream AT-rich sequences; and (iii) it possesses a
greater flexibility than sigma(28)(EC) in recognizing variants with different
spacing lengths separating the -35 and -10 elements of the core promoter.

DOI: 10.1128/JB.01014-06 
PMCID: PMC1636291
PMID: 16936033  [Indexed for MEDLINE]


951. J Clin Microbiol. 2006 Nov;44(11):4125-35. Epub 2006 Aug 30.

Comparative genomic analysis of Campylobacter jejuni strains reveals diversity
due to genomic elements similar to those present in C. jejuni strain RM1221.

Parker CT(1), Quiñones B, Miller WG, Horn ST, Mandrell RE.

Author information: 
(1)U.S. Department of Agriculture, Agricultural Research Service, Western
Regional Research Center, Produce Safety and Microbiology Unit, 800 Buchanan
Street, Albany, CA 94710, USA. parker@pw.usda.gov

Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221
identified four large genomic elements, Campylobacter jejuni-integrated elements 
(CJIEs), that were absent from C. jejuni strain NCTC 11168. To further
investigate the genomic diversity of Campylobacter, we conducted a comparative
genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli
strains isolated from various geographical locations and clinical and veterinary 
sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains
examined were positive for at least one RM1221-like genomic element and 27% were 
positive for two or more of these CJIEs. Furthermore, many C. coli strains were
positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for 
the presence or absence of several genes that comprise the various CJIEs, we
developed a multistrain C. jejuni DNA microarray that contained most of the
putative coding sequences for strains NCTC 11168 and RM1221. A comparative
genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed 
the presence of genomic elements similar to those in strain RM1221.
Interestingly, the DNA microarray analysis demonstrated that these genomic
elements in the other C. jejuni strains often exhibited modular patterns with
some regions of the CJIEs present and other regions either absent or highly
divergent compared to strain RM1221. Our CGH method also identified 18 other
intraspecies hypervariable regions, such as the capsule and lipooligosaccharide
biosynthesis regions. Thus, the inclusion of genes from these integrated genomic 
elements and the genes from the other intraspecies hypervariable regions
contributes to a better assessment of the diversity in C. jejuni and may increase
the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally,
we also showed that in CJIE1, a Campylobacter Mu-like phage, is located
differentially in other strains of C. jejuni, suggesting that it may integrate
essentially randomly.

DOI: 10.1128/JCM.01231-06 
PMCID: PMC1698300
PMID: 16943349  [Indexed for MEDLINE]


952. Mol Microbiol. 2006 Nov;62(4):1014-34. Epub 2006 Sep 29.

Cyclic-di-GMP-mediated signalling within the sigma network of Escherichia coli.

Weber H(1), Pesavento C, Possling A, Tischendorf G, Hengge R.

Author information: 
(1)Institut für Biologie, Mikrobiologie, Freie Universität Berlin, 14195 Berlin, 
Germany.

Bis-(3'-5')-cyclic-di-guanosine monophosphate (c-di-GMP) is a bacterial
signalling molecule produced by diguanylate cyclases (DGC, carrying GGDEF
domains) and degraded by specific phosphodiesterases (PDE, carrying EAL domains).
Neither its full physiological impact nor its effector mechanisms are currently
understood. Also, the existence of multiple GGDEF/EAL genes in the genomes of
most species raises questions about output specificity and robustness of c-di-GMP
signalling. Using microarray and gene fusion analyses, we demonstrate that at
least five of the 29 GGDEF/EAL genes in Escherichia coli are not only stationary 
phase-induced under the control of the general stress response master regulator
sigma(S) (RpoS), but also exhibit differential control by additional
environmental and temporal signals. Two of the corresponding proteins, YdaM
(GGDEF only) and YciR (GGDEF + EAL), which in vitro show DGC and PDE activity,
respectively, play an antagonistic role in the expression of the
biofilm-associated curli fimbriae. This control occurs at the level of
transcription of the curli and cellulose regulator CsgD. Moreover, we show that
H-NS positively affects curli expression by inversely controlling the expression 
of ydaM and yciR. Furthermore, we demonstrate a temporally fine-tuned GGDEF
cascade in which YdaM controls the expression of another GGDEF protein, YaiC. By 
genome-wide microarray analysis, evidence is provided that YdaM and YciR strongly
and nearly exclusively control CsgD-regulated genes. We conclude that specific
GGDEF/EAL proteins have very distinct expression patterns, and when present in
physiological amounts, can act in a highly precise, non-global and perhaps
microcompartmented manner on a few or even a single specific target(s).

DOI: 10.1111/j.1365-2958.2006.05440.x 
PMID: 17010156  [Indexed for MEDLINE]


953. Plant Mol Biol. 2006 Nov;62(4-5):715-33. Epub 2006 Sep 26.

Functional analysis of members of the isoflavone and isoflavanone
O-methyltransferase enzyme families from the model legume Medicago truncatula.

Deavours BE(1), Liu CJ, Naoumkina MA, Tang Y, Farag MA, Sumner LW, Noel JP, Dixon
RA.

Author information: 
(1)Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble
Parkway, Ardmore, OK 73401, USA.

Previous studies have identified two distinct O-methyltransferases (OMTs)
implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating 
the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the
B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs 
from the model legume Medicago truncatula cluster as separate branches of the
type I plant small molecule OMT family. To better understand the possible
functions of these related OMTs in secondary metabolism in M. truncatula, seven
of the OMTs were expressed in E. coli, purified, and their in vitro substrate
preferences determined. Many of the enzymes display promiscuous activities, and
some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the
isoflavonoid nucleus. Protein structure homology modeling was used to help
rationalize these catalytic activities. Transcripts encoding the different OMT
genes exhibited differential tissue-specific and infection- or elicitor-induced
expression, but not always in parallel with changes in expression of confirmed
genes of the isoflavonoid pathway. The results are discussed in relation to the
potential in vivo functions of these OMTs based on our current understanding of
the phytochemistry of M. truncatula, and the difficulties associated with gene
annotation in plant secondary metabolism.

DOI: 10.1007/s11103-006-9050-x 
PMCID: PMC2862459
PMID: 17001495  [Indexed for MEDLINE]


954. J Biol Chem. 2006 Oct 13;281(41):31119-30. Epub 2006 Aug 15.

Contribution of interferon-beta to the murine macrophage response to the
toll-like receptor 4 agonist, lipopolysaccharide.

Thomas KE(1), Galligan CL, Newman RD, Fish EN, Vogel SN.

Author information: 
(1)Department of Microbiology and Immunology, University of Maryland, Baltimore, 
Maryland 21201, USA.

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced 
via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in
macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In
this study, we analyzed the responses of macrophages derived from wild-type
(IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-))
to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and
LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and
Affymetrix microarray analyses) resulted in the identification of four distinct
patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset
of each group of differentially regulated genes by computer-assisted promoter
analysis revealed putative IFN-responsive elements in all genes examined.
LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1
Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly
diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of
IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly
increased levels of LPS-induced gene expression for induction of monocyte
chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40
mRNA, whereas no increase or relatively small increases were observed for
IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally,
IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when 
compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta
contributes to LPS-induced lethality; however, not to the extent that one
observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or
TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory
roles for IFN-beta in response to LPS in vitro and in vivo.

DOI: 10.1074/jbc.M604958200 
PMID: 16912041  [Indexed for MEDLINE]


955. Infect Immun. 2006 Oct;74(10):5609-16.

Effects of ompA deletion on expression of type 1 fimbriae in Escherichia coli K1 
strain RS218 and on the association of E. coli with human brain microvascular
endothelial cells.

Teng CH(1), Xie Y, Shin S, Di Cello F, Paul-Satyaseela M, Cai M, Kim KS.

Author information: 
(1)Division of Clinical Research, National Health Research Institutes, Tainan,
Taiwan.

We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae
are the bacterial determinants involved in Escherichia coli K1 binding to human
brain microvascular endothelial cells (HBMEC), which constitute the blood-brain
barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we
showed for the first time that ompA deletion decreased the expression of type 1
fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA
deletion mutant was largely the result of driving the fim promoter toward the
type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to
be decreased with the OmpA mutant compared to the parent strain. Of interest, the
ompA deletion further decreased the abilities of E. coli K1 to bind to and invade
HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or
phase-OFF status. These findings suggest that the decreased ability of the OmpA
mutant to interact with HBMEC is not entirely due to its decreased type 1
fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction 
of E. coli K1 with HBMEC at least in an additive manner.

DOI: 10.1128/IAI.00321-06 
PMCID: PMC1594875
PMID: 16988236  [Indexed for MEDLINE]


956. J Bacteriol. 2006 Oct;188(20):7230-41.

The Rgg regulator of Streptococcus pyogenes influences utilization of nonglucose 
carbohydrates, prophage induction, and expression of the NAD-glycohydrolase
virulence operon.

Dmitriev AV(1), McDowell EJ, Kappeler KV, Chaussee MA, Rieck LD, Chaussee MS.

Author information: 
(1)Division of Basic Biomedical Sciences, Sanford School of Medicine of the
University of South Dakota, Lee Medical Building, 414 East Clark Street,
Vermillion, SD 57069-2390, USA.

The expression of many virulence-associated genes in Streptococcus pyogenes is
controlled in a growth phase-dependent manner. Unlike the model organisms
Escherichia coli and Bacillus subtilis, such regulation is apparently not
dependent upon alternative sigma factors but appears to rely on complex
interactions among several transcriptional regulators, including Rgg. The purpose
of this study was to identify changes in gene expression associated with
inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this 
end, the transcriptomes of wild-type and rgg mutant strains were analyzed during 
both the exponential and postexponential phases of growth using Affymetrix
NimbleExpress gene chips. Genomewide differences in transcript levels were
identified in both phases of growth. Inactivation of rgg disrupted coordinate
expression of genes associated with the metabolism of nonglucose carbon sources, 
such as fructose, mannose, and sucrose. The changes were associated with an
inability of the mutant strain to grow using these compounds as the primary
carbon source. Bacteriophage transcript levels were also altered in the mutant
strain and were associated with decreased induction of at least one prophage.
Finally, transcripts encoding virulence factors involved in cytolysin-mediated
translocation of NAD-glycohydrolase, including the immunity factor IFS and the
cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which 
correlated with the amount of NADase and SLO activities in culture supernatant
fluids. The results provide further evidence that Rgg contributes to growth
phase-dependent gene regulation in strain NZ131.

DOI: 10.1128/JB.00877-06 
PMCID: PMC1636216
PMID: 17015662  [Indexed for MEDLINE]


957. J Clin Microbiol. 2006 Oct;44(10):3703-11.

Identification of virulence genes linked with diarrhea due to atypical
enteropathogenic Escherichia coli by DNA microarray analysis and PCR.

Afset JE(1), Bruant G, Brousseau R, Harel J, Anderssen E, Bevanger L, Bergh K.

Author information: 
(1)Department of Medical Microbiology, St. Olavs University Hospital, N-7006
Trondheim, Norway. jan.afset@ntnu.no.

The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood
diarrhea is controversial. The aim of the present study was to search for genes
linked with diarrhea in atypical EPEC strains from a case-control study among
Norwegian children. Using DNA microarray analysis, genomic DNAs from strains
isolated from children with (n = 37) and without (n = 20) diarrhea were
hybridized against 242 different oligonucleotide probes specific for 182
virulence genes or markers from all known E. coli pathotypes. PCR was performed
to test the strains for seven putative virulence genes not included in the
microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest
statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent, 
nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were
also associated with diarrheal disease. The phylogenetic marker gene yjaA was
negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be 
classified in two main virulence groups based on their content of OI-122, lpfA,
and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to
virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common,
while the majority of isolates from healthy children were classified as virulence
group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In
conclusion, using DNA microarray analysis to determine the virulence gene profile
of atypical EPEC isolates, several genes were found to be significantly
associated with diarrhea. Based on their composition of virulence genes, the
majority of strains could be classified in two virulence groups, of which one was
seen mainly in children with diarrhea.

DOI: 10.1128/JCM.00429-06 
PMCID: PMC1594803
PMID: 17021100  [Indexed for MEDLINE]


958. J Ind Microbiol Biotechnol. 2006 Oct;33(10):801-14. Epub 2006 May 6.

Global transcriptome response of recombinant Escherichia coli to heat-shock and
dual heat-shock recombinant protein induction.

Harcum SW(1), Haddadin FT.

Author information: 
(1)Department of Bioengineering, Clemson University, 401 Rhodes Engineering
Research Center, Clemson, SC 29634-0905, USA. harcum@clemson.edu

Recombinant Escherichia coli cultures are used to manufacture numerous
therapeutic proteins and industrial enzymes, where many of these processes use
elevated temperatures to induce recombinant protein production. The heat-shock
response in wild-type E. coli has been well studied. In this study, the
transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a 
dual heat-shock recombinant protein induction were examined. Most classical
heat-shock protein genes were identified as regulated in both conditions. The
major transcriptome differences between the recombinant and reported wild-type
cultures were heavily populated by hypothetical and putative genes, which
indicates recombinant cultures utilize many unique genes to respond to a
heat-shock. Comparison of the dual stressed culture data with literature
recombinant protein induced culture data revealed numerous differences. The dual 
stressed response encompassed three major response patterns: induced-like,
in-between, and greater than either individual stress response. Also, there were 
no genes that only responded to the dual stress. The most interesting difference 
between the dual stressed and induced cultures was the amino acid-tRNA gene
levels. The amino acid-tRNA genes were elevated for the dual cultures compared to
the induced cultures. Since, tRNAs facilitate protein synthesis via translation, 
this observed increase in amino acid-tRNA transcriptome levels, in concert with
elevated heat-shock chaperones, might account for improved productivities often
observed for thermo-inducible systems. Most importantly, the response of the
recombinant cultures to a heat-shock was more profound than wild-type cultures,
and further, the response to recombinant protein induction was not a simple
additive response of the individual stresses.

DOI: 10.1007/s10295-006-0122-3 
PMID: 16680459  [Indexed for MEDLINE]


959. Mol Microbiol. 2006 Oct;62(2):469-79.

Induction of expression of hfq by DksA is essential for Shigella flexneri
virulence.

Sharma AK(1), Payne SM.

Author information: 
(1)Section of Molecular Genetics and Microbiology, The University of Texas at
Austin, Austin, TX 78712, USA.

Erratum in
    Mol Microbiol. 2006 Dec;62(5):1498.

Hfq plays an important role in cellular physiology by regulating the expression
of several genes. Hfq synthesis in Escherichia coli is subject to auto-repression
at translational level. Studies with Shigella flexneri show that hfq
transcription is regulated by a pleiotropic regulator, DksA. Comparison of gene
expression profiles of wild type and dksA mutant S. flexneri determined that hfq 
expression was reduced in the dksA mutant. As DksA is required for stress
resistance and plaque formation in cultured cell monolayers, a measure of
virulence, we assessed the role of Hfq in the dksA virulence phenotype.
Expression of hfq in the dksA mutant restored plaque formation, and an hfq mutant
failed to form plaques. Thus, DksA plays a role in regulating hfq gene expression
and this regulation is important for S. flexneri virulence. In an in vitro
transcription assay, addition of DksA increased transcription of hfq and this
effect was greatest with one of the known hfq promoters. Addition of ppGpp, a
stringent response molecule, along with DksA in the in vitro transcription assay 
resulted in a further increase in transcription of hfq, indicating that DksA is
required for maximal transcription of hfq during both exponential and stringent
response growth conditions.

DOI: 10.1111/j.1365-2958.2006.05376.x 
PMID: 17020583  [Indexed for MEDLINE]


960. BMC Genomics. 2006 Sep 8;7:230.

Global gene expression during stringent response in Corynebacterium glutamicum in
presence and absence of the rel gene encoding (p)ppGpp synthase.

Brockmann-Gretza O(1), Kalinowski J.

Author information: 
(1)Institut für Genomforschung, Centrum für Biotechnologie, Universität
Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany.
ogretza@genetik.uni-bielefeld.de

BACKGROUND: The stringent response is the initial reaction of microorganisms to
nutritional stress. During stringent response the small nucleotides (p)ppGpp act 
as global regulators and reprogram bacterial transcription. In this work, the
genetic network controlled by the stringent response was characterized in the
amino acid-producing Corynebacterium glutamicum.
RESULTS: The transcriptome of a C. glutamicum rel gene deletion mutant, unable to
synthesize (p)ppGpp and to induce the stringent response, was compared with that 
of its rel-proficient parent strain by microarray analysis. A total of 357 genes 
were found to be transcribed differentially in the rel-deficient mutant strain.
In a second experiment, the stringent response was induced by addition of
DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of 
the maximal effect on transcription was determined by real-time RT-PCR using the 
histidine and serine biosynthetic genes. Transcription of all of these genes
reached a maximum at 10 minutes after SHX addition. Microarray experiments were
performed comparing the transcriptomes of SHX-induced cultures of the
rel-proficient strain and the rel mutant. The differentially expressed genes were
grouped into three classes. Class A comprises genes which are differentially
regulated only in the presence of an intact rel gene. This class includes the
non-essential sigma factor gene sigB which was upregulated and a large number of 
genes involved in nitrogen metabolism which were downregulated. Class B comprises
genes which were differentially regulated in response to SHX in both strains,
independent of the rel gene. A large number of genes encoding ribosomal proteins 
fall into this class, all being downregulated. Class C comprises genes which were
differentially regulated in response to SHX only in the rel mutant. This class
includes genes encoding putative stress proteins and global transcriptional
regulators that might be responsible for the complex transcriptional patterns
detected in the rel mutant when compared directly with its rel-proficient parent 
strain.
CONCLUSION: In C. glutamicum the stringent response enfolds a fast answer to an
induced amino acid starvation on the transcriptome level. It also showed some
significant differences to the transcriptional reactions occurring in Escherichia
coli and Bacillus subtilis. Notable are the rel-dependent regulation of the
nitrogen metabolism genes and the rel-independent regulation of the genes
encoding ribosomal proteins.

DOI: 10.1186/1471-2164-7-230 
PMCID: PMC1578569
PMID: 16961923  [Indexed for MEDLINE]


961. Am J Obstet Gynecol. 2006 Sep;195(3):822-8.

Uterine transcriptomes of bacteria-induced and ovariectomy-induced preterm labor 
in mice are characterized by differential expression of arachidonate metabolism
genes.

Haddad R(1), Gould BR, Romero R, Tromp G, Farookhi R, Edwin SS, Kim MR, Zingg HH.

Author information: 
(1)Perinatology Research Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Department of Health and Human
Services, Bethesda, MD, USA. haddadr@karmanos.org

OBJECTIVE: The purpose of this study was to identify changes in gene expression
that are associated with preterm labor induced by either bacteria or ovariectomy.
STUDY DESIGN: Pregnant mice (14.5 days of gestation) were allocated to: (1)
intrauterine injection of heat-inactivated Escherichia coli; (2) media alone; (3)
ovariectomy; or (4) sham operation. The uterine transcriptome was studied with
photolithographic, very short oligonucleotide-based microarrays, and arachidonate
metabolism genes were assayed with quantitative reverse transcriptase-polymerase 
chain reaction. Significance was determined by analysis of variance.
RESULTS: Microarray-based gene expression changes in the arachidonate metabolism 
pathway are associated globally with bacteria-induced preterm labor (P < or =
.0031) and ovariectomy-induced preterm labor (P < or = .00036). Quantitative
real-time reverse transcriptase-polymerase chain reaction measurements
demonstrated that bacteria-induced preterm labor substantially increased the
expression of genes involved in prostaglandin synthesis. In contrast,
ovariectomy-induced preterm labor increased the expression of genes involved in
lipoxin, leukotriene, and hydroxyeicosatetraenoic acid synthesis.
CONCLUSION: Bacteria-induced and ovariectomy-induced preterm labor each express a
different balance of genes that are required for the synthesis of prostaglandins,
lipoxins, leukotrienes, and hydroxyeicosatetraenoic acids.

DOI: 10.1016/j.ajog.2006.06.053 
PMID: 16949419  [Indexed for MEDLINE]


962. Bioinformatics. 2006 Sep 1;22(17):2175-7. Epub 2006 Jun 9.

NetAlign: a web-based tool for comparison of protein interaction networks.

Liang Z(1), Xu M, Teng M, Niu L.

Author information: 
(1)Hefei National Laboratory for Physical Sciences at Microscale and School of
Life Sciences, University of Science & Technology of China, 96 Jinzhai Road,
Hefei, Anhui 230027, China.

NetAlign is a web-based tool designed to enable comparative analysis of protein
interaction networks (PINs). NetAlign compares a query PIN with a target PIN by
combining interaction topology and sequence similarity to identify conserved
network substructures (CoNSs), which may derive from a common ancestor and
disclose conserved topological organization of interactions in evolution. To
exemplify the application of NetAlign, we perform two genome-scale comparisons
with (1) the Escherichia coli PIN against the Helicobacter pylori PIN and (2) the
Saccharomyces cerevisiae PIN against the Caenorrhabditis elegans PIN. Many of the
identified CoNSs correspond to known complexes; therefore, cross-species PIN
comparison provides a way for discovery of conserved modules. In addition, based 
on the species-to-species differences in CoNSs, we reformulate the problems of
protein-protein interaction (PPI) prediction and species divergence from a
network perspective.AVAILABILITY: http://www1.ustc.edu.cn/lab/pcrystal/NetAlign.

DOI: 10.1093/bioinformatics/btl287 
PMID: 16766562  [Indexed for MEDLINE]


963. Environ Microbiol. 2006 Sep;8(9):1635-47.

Gene expression of Escherichia coli in continuous culture during adaptation to
artificial sunlight.

Berney M(1), Weilenmann HU, Egli T.

Author information: 
(1)Swiss Federal Institute for Aquatic Science and Technology, Eawag, PO Box 611,
CH-8600 Dübendorf, Switzerland.

Escherichia coli growing in continuous culture under continuous UVA irradiation
exhibits growth inhibition with a subsequent adaptation to the stress.
Transcriptome analysis was performed during transient growth inhibition and in
the UVA light-adapted growth state. The results indicate that UVA light induces
stringent response and an additional response that includes the upregulation of
the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and
glutamate. The induction of several SOS response-genes strongly points to DNA
damage as a result of UVA exposure. The involvement of oxidative stress was
observed with the induction of ahpCF. Taken together it supports the hypothesis
of the production of reactive oxygen species by UVA light. In the UVA-adapted
cell population strong repression of the acid tolerance response was found. We
identified the enzyme chorismate mutase as a possible chromophore for UVA
light-inactivation and found strong repression of the pyrBI operon and the gene
mgtA encoding for an ATP-dependent Mg2+ transporter. Furthermore, our results
indicate that the role of RpoS may not be as important in the adaptation of E.
coli to UVA light as it was implicated by previous results with starved cells,
but that RpoS might be of crucial importance for the resistance under transient
light exposure.

DOI: 10.1111/j.1462-2920.2006.01057.x 
PMID: 16913923  [Indexed for MEDLINE]


964. J Bacteriol. 2006 Sep;188(18):6709-13.

Transcriptional response of Escherichia coli to TPEN.

Sigdel TK(1), Easton JA, Crowder MW.

Author information: 
(1)Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University,
Oxford, OH 45056, USA.

DNA microarrays were used to probe the transcriptional response of Escherichia
coli to N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Fifty-five
transcripts were significantly up-regulated, including all of the genes that are 
regulated by Zur and many that are regulated by Fur. In the same TPEN-treated
cells, 46 transcripts were significantly down-regulated.

DOI: 10.1128/JB.00680-06 
PMCID: PMC1595494
PMID: 16952965  [Indexed for MEDLINE]


965. Mol Microbiol. 2006 Sep;61(5):1335-51.

Construction and analysis of chromosomal Clostridium difficile mutants.

O'Connor JR(1), Lyras D, Farrow KA, Adams V, Powell DR, Hinds J, Cheung JK, Rood 
JI.

Author information: 
(1)Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash 
University, Vic. 3800, Australia.

Clostridium difficile is an emerging nosocomial pathogen of increasing importance
and virulence but our ability to study the molecular mechanisms underlying the
pathogenesis of C. difficile-associated disease has been limited because of a
lack of tools for its genetic manipulation. We have now developed a reproducible 
method for the targeted insertional inactivation of chromosomal C. difficile
genes. The approach relies on the observation that an Escherichia
coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can
be used as a form of conditional lethal vector to deliver gene constructs to the 
chromosome. We have used this methodology to insertionally inactivate two
putative response regulator genes, rgaR and rgbR, which encode proteins with
similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic
analysis demonstrated that the C. difficile RgaR protein positively regulated
four genes, including a putative agrBD operon. The RgaR protein was also purified
and shown to bind specifically to sites that contained two consensus VirR boxes
located just upstream of the putative promoters of these genes. The development
of this methodology will significantly enhance our ability to use molecular
approaches to develop a greater understanding of the ability of C. difficile to
cause disease.

DOI: 10.1111/j.1365-2958.2006.05315.x 
PMID: 16925561  [Indexed for MEDLINE]


966. Mol Microbiol. 2006 Sep;61(5):1267-82.

Proteomic and microarray characterization of the AggR regulon identifies a pheU
pathogenicity island in enteroaggregative Escherichia coli.

Dudley EG(1), Thomson NR, Parkhill J, Morin NP, Nataro JP.

Author information: 
(1)Center for Vaccine Development, University of Maryland School of Medicine, 685
W. Baltimore St., Baltimore, MD 21201, USA.

Enteroaggregative Escherichia coli (EAEC) is defined by aggregative adherence
(AA) to HEp-2 cells, where bacteria display adherence to cell surfaces and also
to the intervening substratum in a stacked-brick configuration. We previously
showed that an AraC homologue designated AggR is required for the expression of
plasmid-encoded genes that mediate AA of EAEC strain 042. In this study, we
hypothesized that AggR also controls the expression of other virulence
determinants in EAEC 042. Using proteomic and microarray analysis, we identified 
for the first time that AggR activates the expression of chromosomal genes,
including 25 contiguous genes (aaiA-Y), which are localized to a 117 kb
pathogenicity island (PAI) inserted at pheU. Many of these genes have homologues 
in other Gram-negative bacteria and were recently proposed to constitute a type
VI secretion system (T6SS). AaiC was identified as a secreted protein that has no
apparent homologues within GenBank. EAEC strains carrying in-frame deletions of
aaiB, aaiG, aaiO or aaiP still synthesized AaiC; however, AaiC secretion was
abolished. Cloning of aai genes into E. coli HB101 suggested that aaiA-P are
sufficient for AaiC secretion. A second T6SS was identified within the pheU PAI
that secretes a protein unrelated by sequence identity to AaiC. Distribution
studies indicated that aaiA and aaiC are commonly found in EAEC isolates
worldwide, particularly in strains defined as typical EAEC. These data support
the hypothesis that AggR is a global regulator of EAEC virulence determinants,
and builds on the hypothesis that T6SS is an importance mediator of pathogenesis.

DOI: 10.1111/j.1365-2958.2006.05281.x 
PMID: 16925558  [Indexed for MEDLINE]


967. DNA Res. 2006 Aug 31;13(4):141-53. Epub 2006 Oct 17.

Escherichia coli histone-like protein H-NS preferentially binds to horizontally
acquired DNA in association with RNA polymerase.

Oshima T(1), Ishikawa S, Kurokawa K, Aiba H, Ogasawara N.

Author information: 
(1)Graduate School of Information Science, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara, Japan.

Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid
proteins expressed in exponentially growing Escherichia coli cells. In addition
to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of 
gene expression. The genome-wide distribution of H-NS, compared with the
distribution of RNA polymerase and transcriptionally active genes, was
investigated using a high-density oligonucleotide chip. The new approach utilized
in this study revealed that H-NS binds specifically to approximately 250 loci,
covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase
was detected in >65% of H-NS binding sites with low or no transcriptional
activity, indicating that the association of RNA polymerase to promoter regions
is a general mode of transcription repression by H-NS. This study also revealed
that most H-NS bound DNA have been horizontally acquired, which indicates that
repression of inappropriate gene expression by H-NS plays an important role in
the diversification of the E. coli genome. This study presents a comprehensive
assessment of the distribution of H-NS within the E. coli genome, sheds light on 
the mechanism underlying the transcriptional regulation by H-NS, and provides new
insight into bacterial genome evolution.

DOI: 10.1093/dnares/dsl009 
PMID: 17046956  [Indexed for MEDLINE]


968. Chin Med J (Engl). 2006 Aug 20;119(16):1353-8.

A novel full-length gene of human ribosomal protein L14.22 related to human
glioma.

Qi ZY(1), Hui GZ, Li Y, Zhou ZX, Gu SH, Xie Y.

Author information: 
(1)Department of Neurosurgery, First Affiliated Hospital of Soochow University,
Suzhou 215006, China. qizhenyu@yahoo.com

BACKGROUND: This study was undertaken to obtain differentially expressed genes
related to human glioma by cDNA microarray and the characterization of a novel
full-length gene.
METHODS: Total RNA was extracted form human glioma and normal brain tissue, and
mRNA was used as a probe. The results of hybridization procedure were scanned
with the computer system. The gene named 507E08 cone was subsequently analyzed by
northern blot, bioinformatic approach, and protein expression.
RESULTS: Fifteen differentially expressed genes were obtained from human glioma
by hybridization and scanning for four times. Northern blot analysis confirmed
that the 507E08 clone was low expressed in human brain tissue and over expressed 
in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08
clone was a novel full-length gene, which codes 203 amino acid of protein and is 
called human ribosomal protein 14.22 gene. The nucleotide sequence had been
submitted to the GenBank with the accession number of AF329277. After expression 
in E. coli., protein yielded a major band of apparent molecular mass 22 kDa on an
SDS-PAGE gel.
CONCLUSIONS: cDNA microarray technology can be successfully used to identify
differentially expressed genes. The novel full-length gene of human ribosomal
protein 13.22 may be correlated with the development of human glioma.


PMID: 16934181  [Indexed for MEDLINE]


969. BMC Bioinformatics. 2006 Aug 14;7:377.

A novel data mining method to identify assay-specific signatures in functional
genomic studies.

Rollins DK(1), Zhai D, Joe AL, Guidarelli JW, Murarka A, Gonzalez R.

Author information: 
(1)Department of Chemical and Biological Engineering, Iowa State University,
Ames, Iowa 50011, USA. drollins@iastate.edu

BACKGROUND: The highly dimensional data produced by functional genomic (FG)
studies makes it difficult to visualize relationships between gene products and
experimental conditions (i.e., assays). Although dimensionality reduction methods
such as principal component analysis (PCA) have been very useful, their
application to identify assay-specific signatures has been limited by the lack of
appropriate methodologies. This article proposes a new and powerful PCA-based
method for the identification of assay-specific gene signatures in FG studies.
RESULTS: The proposed method (PM) is unique for several reasons. First, it is the
only one, to our knowledge, that uses gene contribution, a product of the loading
and expression level, to obtain assay signatures. The PM develops and exploits
two types of assay-specific contribution plots, which are new to the application 
of PCA in the FG area. The first type plots the assay-specific gene contribution 
against the given order of the genes and reveals variations in distribution
between assay-specific gene signatures as well as outliers within assay groups
indicating the degree of importance of the most dominant genes. The second type
plots the contribution of each gene in ascending or descending order against a
constantly increasing index. This type of plots reveals assay-specific gene
signatures defined by the inflection points in the curve. In addition, sharp
regions within the signature define the genes that contribute the most to the
signature. We proposed and used the curvature as an appropriate metric to
characterize these sharp regions, thus identifying the subset of genes
contributing the most to the signature. Finally, the PM uses the full dataset to 
determine the final gene signature, thus eliminating the chance of gene exclusion
by poor screening in earlier steps. The strengths of the PM are demonstrated
using a simulation study, and two studies of real DNA microarray data--a study of
classification of human tissue samples and a study of E. coli cultures with
different medium formulations.
CONCLUSION: We have developed a PCA-based method that effectively identifies
assay-specific signatures in ranked groups of genes from the full data set in a
more efficient and simplistic procedure than current approaches. Although this
work demonstrates the ability of the PM to identify assay-specific signatures in 
DNA microarray experiments, this approach could be useful in areas such as
proteomics and metabolomics.

DOI: 10.1186/1471-2105-7-377 
PMCID: PMC1599756
PMID: 16907975  [Indexed for MEDLINE]


970. Analyst. 2006 Aug;131(8):907-15. Epub 2006 Jun 5.

An electronic DNA microarray technique for detection and differentiation of
viable Campylobacter species.

Zhang H(1), Gong Z, Pui O, Liu Y, Li XF.

Author information: 
(1)Environmental Health Sciences, Department of Public Health Sciences,
University of Alberta, Edmonton, Alberta, CanadaT6G 2G3.

An electronic oligonucleotide microarray technique was developed for detection
and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and 
C. lari. This development consisted of four major components: identification of
single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers, 
design of fluorescently labelled SNP-based reporters, development of an
electronic microarray detection, and application of the integrated technique to
analysis of Campylobacter species in food samples. A unique capability of this
technique is the specific detection of viable cells and not dead ones. This is
achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker. 
The identification of two unique SNPs closely located at positions 291 and 294 of
the hsp60 gene enabled the differentiation of the three Campylobacter species.
This technique was able to detect as few as two viable Campylobacter cells. The
analysis of 19 blind Campylobacter samples showed 100% agreement with their
identities obtained using pulsed-field gel electrophoresis. The analysis of six
chicken samples revealed the presence of C. coli in one of the samples.

DOI: 10.1039/b603315f 
PMID: 17028724  [Indexed for MEDLINE]


971. Appl Environ Microbiol. 2006 Aug;72(8):5469-77.

Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in
the basidiomycetous fungus Ustilago maydis.

Hewald S(1), Linne U, Scherer M, Marahiel MA, Kämper J, Bölker M.

Author information: 
(1)Fachbereich Biologie, Philipps-Universität Marburg, 35032 Marburg, Germany.

Many microorganisms produce surface-active substances that enhance the
availability of water-insoluble substrates. Although many of these biosurfactants
have interesting potential applications, very little is known about their
biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts
of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We
recently described a putative glycosyltransferase, Emt1, which is essential for
MEL biosynthesis and whose expression is strongly induced by nitrogen limitation.
We used DNA microarray analysis to identify additional genes involved in MEL
biosynthesis. Here we show that emt1 is part of a gene cluster which comprises
five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and
Mat1, contain short sequence motifs characteristic for acyl- and
acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential
for MEL production, which suggests that they are involved in the acylation of
mannosylerythritol. Deletion of mat1 resulted in the secretion of completely
deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in
Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme
A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity
and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl
groups. Based on these results, we propose a biosynthesis pathway for the
generation of mannosylerythritol lipids in U. maydis.

DOI: 10.1128/AEM.00506-06 
PMCID: PMC1538720
PMID: 16885300  [Indexed for MEDLINE]


972. Appl Microbiol Biotechnol. 2006 Aug;71(6):963-9. Epub 2006 Mar 15.

A novel system for large-scale gene expression analysis: bacterial colonies
array.

Barsalobres-Cavallari C(1), De Rosa Júnior V, Nogueira F, Ferro J, Di Mauro S,
Menossi M, Ulian E, Silva-Filho M.

Author information: 
(1)Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Av. 
Pádua Dias 11, C.P. 83, 13400-970, Piracicaba, SP, Brazil. cfbarsal@ibb.unesp.br

In the present work, we report the use of bacterial colonies to optimize
macroarray technique. The devised system is significantly cheaper than other
methods available to detect large-scale differential gene expression. Recombinant
Escherichia coli clones containing plasmid-encoded copies of 4,608 individual
expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that 
were incubated for 6 and 12 h to allow the bacteria to grow and, consequently,
amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase
gene specific probe from the recombinant plasmid and, subsequently,
phosphorimaged to quantify the microbial cells. Variance analysis demonstrated
that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%)
after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for
12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to
noise ratio was fivefold higher. Taken together, the results of this study
indicate that it is possible to investigate large-scale gene expression using
macroarrays based on bacterial colonies grown for 6 h onto membranes.

DOI: 10.1007/s00253-006-0348-z 
PMID: 16538485  [Indexed for MEDLINE]


973. Biotechnol Lett. 2006 Aug;28(16):1241-6. Epub 2006 Jul 1.

Optimal production of poly-gamma-glutamic acid by metabolically engineered
Escherichia coli.

Jiang H(1), Shang L, Yoon SH, Lee SY, Yu Z.

Author information: 
(1)State Key Laboratory of Agricultural Microbiology, National Engineering
Research Centre of Microbial Pesticides, Huazhong Agricultural University, Wuhan 
430070, P.R. China. yz41@public.wh.hb.cn

Metabolically-engineered Escherichia coli strains were developed by cloning
poly-gamma-glutamic acid (gamma-PGA) biosynthesis genes, consisting of pgsB, pgsC
and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of
gamma-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible 
trc promoter and a constitutive promoter (P(HCE)) derived from the D-amino acid
aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The
constitutive HCE promoter was more efficient than inducible trc promoter for the 
expression of gamma-PGA biosynthesis genes. DNA microarray analysis showed that
the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY,
yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were
down-regulated. When (NH(4))(2)SO(4 )was added at 40 g/l into the feeding
solution, the final gamma-PGA concentration reached 3.7 g/l in the fed-batch
culture of recombinant E. coli/pCOpgs.

DOI: 10.1007/s10529-006-9080-0 
PMID: 16816893  [Indexed for MEDLINE]


974. Infect Immun. 2006 Aug;74(8):4778-92.

Adherent and invasive Escherichia coli is associated with granulomatous colitis
in boxer dogs.

Simpson KW(1), Dogan B, Rishniw M, Goldstein RE, Klaessig S, McDonough PL, German
AJ, Yates RM, Russell DG, Johnson SE, Berg DE, Harel J, Bruant G, McDonough SP,
Schukken YH.

Author information: 
(1)Department of Clinical Sciences, College of Veterinary Medicine, VMC2001,
Cornell University, Ithaca, New York 14853, USA. kws5@cornell.edu

The mucosa-associated microflora is increasingly considered to play a pivotal
role in the pathogenesis of inflammatory bowel disease. This study explored the
possibility that an abnormal mucosal flora is involved in the etiopathogenesis of
granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected
dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ
hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal
DNA sequencing, and histochemistry were used to guide subsequent FISH.
GCB-associated Escherichia coli isolates were evaluated for their ability to
invade and persist in cultured epithelial cells and macrophages as well as for
serotype, phylogenetic group, genome size, overall genotype, and presence of
virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of 
GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E.
coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and
replicated within cultured epithelial cells. Invasion triggered a "splash"-type
response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin,
and paralleled the behavior of the Crohn's disease-associated strain E. coli LF
82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but
similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2,
chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. 
coli, and in the presence of novel multilocus sequence types. We conclude that
GCB is associated with selective intramucosal colonization by E. coli. E. coli
strains associated with GCB and Crohn's disease have an adherent and invasive
phenotype and novel multilocus sequence types and resemble E. coli associated
with extraintestinal disease in phylogeny and virulence gene profile.

DOI: 10.1128/IAI.00067-06 
PMCID: PMC1539603
PMID: 16861666  [Indexed for MEDLINE]


975. Microbiology. 2006 Aug;152(Pt 8):2233-45.

Global analysis of candidate genes important for fitness in a competitive biofilm
using DNA-array-based transposon mapping.

Junker LM(1), Peters JE, Hay AG.

Author information: 
(1)Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.

Escherichia coli strain PHL628 was subjected to saturating Tn5 transposon
mutagenesis and then grown under competitive planktonic or biofilm conditions.
The locations of transposon insertions from the remaining cells were then mapped 
on a gene array. The results from the array mapping indicated that 4.5 % of the
E. coli genome was important under these conditions. Specifically, 114 genes were
identified as important for the biofilm lifestyle, whereas 80 genes were
important for the planktonic lifestyle. Four broad functional categories were
identified as biofilm-important. These included genes encoding cell structures,
small-molecule transport, energy metabolism and regulatory functions. For one of 
these genes, arcA, an insertion mutant was generated and its biofilm-related
phenotype was examined. Results from both the transposon array and insertion
mutagenesis indicated that arcA, which is known to be a negative response
regulator of genes in aerobic pathways, was important for competitiveness in E.
coli PHL628 biofilms. This work also demonstrated that ligation-mediated PCR,
coupled with array-based transposon mapping, was an effective tool for
identifying a large variety of candidate genes that are important for biofilm
fitness.

DOI: 10.1099/mic.0.28767-0 
PMID: 16849790  [Indexed for MEDLINE]


976. Mol Cell Proteomics. 2006 Aug;5(8):1471-83. Epub 2006 May 29.

Proteomics-based validation of genomic data: applications in colorectal cancer
diagnosis.

Madoz-Gúrpide J(1), López-Serra P, Martínez-Torrecuadrada JL, Sánchez L,
Lombardía L, Casal JI.

Author information: 
(1)Protein Technology, Biotechnology Programme, Centro Nacional de
Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain.

Multiple factors are involved in the translation of functional genomic results
into proteins for proteome research and target validation on tumoral tissues. In 
this report, genes were selected by using DNA microarrays on a panel of
colorectal cancer (CRC) paired samples. A large number of up-regulated genes in
colorectal cancer patients were investigated for cellular location, and those
corresponding to membrane or extracellular proteins were used for a non-biased
expression in Escherichia coli. We investigated different sources of cDNA clones 
for protein expression as well as the influence of the protein size and the
different tags with respect to protein expression levels and solubility in E.
coli. From 29 selected genes, 21 distinct proteins were finally expressed as
soluble proteins with, at least, one different fusion protein. In addition, seven
of these potential markers (ANXA3, BMP4, LCN2, SPARC, SPP1, MMP7, and MMP11) were
tested for antibody production and/or validation. Six of the seven proteins (all 
except SPP1) were confirmed to be overexpressed in colorectal tumoral tissues by 
using immunoblotting and tissue microarray analysis. Although none of them could 
be associated to early stages of the tumor, two of them (LCN2 and MMP11) were
clearly overexpressed in late Dukes' stages (B and C). This proteomic study
reveals novel clues for the assembly of a robust and highly efficient high
throughput system for the validation of genomic data. Moreover it illustrates the
different difficulties and bottlenecks encountered for performing a quick
conversion of genomic results into clinically useful proteins.

DOI: 10.1074/mcp.M600048-MCP200 
PMID: 16733264  [Indexed for MEDLINE]


977. Anal Chem. 2006 Jul 15;78(14):4794-802.

Automated detection and quantitation of bacterial RNA by using electrical
microarrays.

Elsholz B(1), Wörl R, Blohm L, Albers J, Feucht H, Grunwald T, Jürgen B, Schweder
T, Hintsche R.

Author information: 
(1)Fraunhofer Institute for Silicon Technology (ISiT), Fraunhoferstrasse 1,
D-25524 Itzehoe, Germany. bruno.elsholz@isit.fraunhofer.de

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an
automated analysis system have been developed for the identification and
quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas
aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus
epidermidis, which are typically involved in urinary tract infections.
Interdigitated gold array electrodes (IDA-electrodes), which have structures in
the nanometer range, have been used for very sensitive analysis. Thiol-modified
oligonucleotides are immobilized on the gold IDA as capture probes. They mediate 
the specific recognition of the target 16S rRNA by hybridization. Additionally
three unlabeled oligonucleotides are hybridized in close proximity to the
capturing site. They are supporting molecules, because they improve the RNA
hybridization at the capturing site. A biotin labeled detector oligonucleotide is
also allowed to hybridize to the captured RNA sequence. The biotin labels enable 
the binding of avidin alkaline phophatase conjugates. The phosphatase liberates
the electrochemical mediator p-aminophenol from its electrically inactive
phosphate derivative. The electrical signals were generated by amperometric redox
cycling and detected by a unique multipotentiostat. The read out signals of the
microarray are position specific current and change over time in proportion to
the analyte concentration. If two additional biotins are introduced into the
affinity binding complex via the supporting oligonucleotides, the sensitivity of 
the assays increase more than 60%. The limit of detection of Escherichia coli
total RNA has been determined to be 0.5 ng/microL. The control of fluidics for
variable assay formats as well as the multichannel electrical read out and data
handling have all been fully automated. The fast and easy procedure does not
require any amplification of the targeted nucleic acids by PCR.

DOI: 10.1021/ac0600914 
PMID: 16841897  [Indexed for MEDLINE]


978. Appl Microbiol Biotechnol. 2006 Jul;71(3):323-8. Epub 2006 Apr 28.

Growth of Escherichia coli MG1655 on LB medium: determining metabolic strategy
with transcriptional microarrays.

Baev MV(1), Baev D, Radek AJ, Campbell JW.

Author information: 
(1)Integrated Genomics Incorporated, Chicago, IL 60612, USA.
mark@integratedgenomics.com

Expression profiles of genes related to stress responses, substrate assimilation,
acetate metabolism, and biosynthesis were obtained by monitoring growth of
Escherichia coli MG1655 in Luria-Bertani (LB) medium with transcriptional
microarrays. Superimposing gene expression profiles on a plot of specific growth 
rate demonstrates that the cells pass through four distinct physiological states 
during fermentation before entering stationary phase. Each of these states can be
characterized by specific patterns of substrate utilization and cellular
biosynthesis corresponding to the nutrient status of the medium. These data allow
the growth phases of the classical microbial growth curve to be redefined in
terms of the physiological states and environmental changes commonly occurring
during bacterial growth in batch culture on LB medium.

DOI: 10.1007/s00253-006-0392-8 
PMID: 16645822  [Indexed for MEDLINE]


979. Appl Microbiol Biotechnol. 2006 Jul;71(3):310-6. Epub 2006 Apr 21.

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars,
alcohols, and organic acids with transcriptional microarrays.

Baev MV(1), Baev D, Radek AJ, Campbell JW.

Author information: 
(1)Integrated Genomics, Inc., Chicago, IL 60612, USA. mark@integratedgenomics.com

Microorganisms respond to environmental changes by reprogramming their metabolism
primarily through altered patterns of gene expression. DNA microarrays provide a 
tool for exploiting microorganisms as living sensors of their environment. The
potential of DNA microarrays to reflect availability of nutrient components
during fermentations on complex media was examined by monitoring global gene
expression throughout batch cultivation of Escherichia coli MG1655 on
Luria-Bertani (LB) medium. Gene expression profiles group into pathways that
clearly demonstrate the metabolic changes occurring in the course of
fermentation. Functional analysis of the gene expression related to metabolism of
sugars, alcohols, and organic acids revealed that E. coli growing on LB medium
switches from a sequential mode of substrate utilization to the simultaneous one 
in the course of the growth. Maltose and maltodextrins are the first of these
substrates to support growth. Utilization of these nutrients associated with the 
highest growth rate of the culture was followed by simultaneous induction of
enzymes involved in assimilation of a large group of other carbon sources
including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol,
amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of
these nutrients to the cells was monitored by induction of corresponding
transport and/or catabolic systems specific for each of the compounds.

DOI: 10.1007/s00253-006-0317-6 
PMID: 16628448  [Indexed for MEDLINE]


980. Appl Microbiol Biotechnol. 2006 Jul;71(3):317-22. Epub 2006 Mar 31.

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino
acids, peptides, and nucleotides with transcriptional microarrays.

Baev MV(1), Baev D, Radek AJ, Campbell JW.

Author information: 
(1)Integrated Genomics Inc, Chicago, IL 60612, USA. mark@integratedgenomics.com

Analysis of gene expression data related to assimilation and biosynthesis of
nitrogen-containing compounds amino acids, peptides, and nucleotides was used to 
monitor availability of these nutrients to Escherichia coli MG1655 growing on
Luria-Bertani medium. The data indicate that free amino acids and nucleotides
only transiently support the nitrogen requirement for growth and are no longer
available by 3.5 h of fermentation. The resulting shortage of available nitrogen 
sources induces the Ntr response, which involves induction of the glnALG,
glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer
membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The
increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF,
ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth.

DOI: 10.1007/s00253-005-0310-5 
PMID: 16575570  [Indexed for MEDLINE]


981. EMBO Rep. 2006 Jul;7(7):710-5. Epub 2006 Jun 16.

Homeostatic regulation of supercoiling sensitivity coordinates transcription of
the bacterial genome.

Blot N(1), Mavathur R, Geertz M, Travers A, Muskhelishvili G.

Author information: 
(1)International University Bremen, Campus Ring 1, 28759 Bremen, Germany.

Regulation of cellular growth implies spatiotemporally coordinated programmes of 
gene transcription. A central question, therefore, is how global transcription is
coordinated in the genome. The growth of the unicellular organism Escherichia
coli is associated with changes in both the global superhelicity modulated by
cellular topoisomerase activity and the relative proportions of the abundant
DNA-architectural chromatin proteins. Using a DNA-microarray-based approach that 
combines mutations in the genes of two important chromatin proteins with induced 
changes of DNA superhelicity, we demonstrate that genomic transcription is
tightly associated with the spatial distribution of supercoiling sensitivity,
which in turn depends on chromatin proteins. We further demonstrate that
essential metabolic pathways involved in the maintenance of growth respond
distinctly to changes of superhelicity. We infer that a homeostatic mechanism
organizing the supercoiling sensitivity is coordinating the
growth-phase-dependent transcription of the genome.

DOI: 10.1038/sj.embor.7400729 
PMCID: PMC1500834
PMID: 16799466  [Indexed for MEDLINE]


982. Int J Food Microbiol. 2006 Jul 1;110(1):62-8. Epub 2006 May 19.

Interaction of bifidobacteria with Caco-2 cells-adhesion and impact on expression
profiles.

Riedel CU(1), Foata F, Goldstein DR, Blum S, Eikmanns BJ.

Author information: 
(1)Department of Microbiology and Biotechnology, University of Ulm, 89068 Ulm,
Germany. c.riedel@ucc.ie

The aim of the present study was to study different strains of bifidobacteria for
adhesion to Caco-2 intestinal epithelial cells (IECs) and to test for the mRNA
response of these cells following interaction with bifidobacteria. Adhesion was
tested at different pH conditions using model epithelia consisting of transwell
cultures of fully differentiated Caco-2 cells. Microarrays were used to
characterize changes in global expression profiles of Caco-2 cells co-cultured
with peripheral blood mononuclear cells (PBMCs) and challenged with
non-pathogenic Escherichia coli D2241 or four different strains of
bifidobacteria. Furthermore, cytokine mRNA of IECs in responses to challenge with
Bifidobacterium bifidum S17 or E. coli D2241 was tested in PBMC-sensitised Caco-2
cells using RT-PCR. Bifidobacteria showed strain-specific adhesion to Caco-2.
Shift of apical pH from 7 to 4.5 resulted in strain-specific changes of adhesion.
Global expression profiles of PBMC-sensitised Caco-2 cells revealed differential 
expression of a significant number of genes only after challenge with E. coli
D2241 while cells were essentially unresponsive to challenge with four strains of
bifidobacteria showing different adhesion properties. Using a RT-PCR approach, in
the same system a similar differential expression after challenge with E. coli
D2241 or B. bifidum S17 was observed for various immune markers. The presented
results suggest that Caco-2 cells might be specifically unresponsive to challenge
with bifidobacteria irrespective of the level of adhesion.

DOI: 10.1016/j.ijfoodmicro.2006.01.040 
PMID: 16713644  [Indexed for MEDLINE]


983. J Clin Microbiol. 2006 Jul;44(7):2389-97.

Identification and characterization of bacterial pathogens causing bloodstream
infections by DNA microarray.

Cleven BE(1), Palka-Santini M, Gielen J, Meembor S, Krönke M, Krut O.

Author information: 
(1)Institute for Medical Microbiology, Immunology and Hygiene, Medical Center,
University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany.

Bloodstream infections are potentially life-threatening and require rapid
identification and antibiotic susceptibility testing of the causative pathogen in
order to facilitate specific antimicrobial therapy. We developed a prototype DNA 
microarray for the identification and characterization of three important
bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes
200 to 800 bp in length that were amplified from recombinant plasmids. These
probes represented genes encoding housekeeping proteins, virulence factors, and
antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3
reference strains, and 13 positive blood cultures revealed that the DNA
microarray was highly specific in identifying S. aureus, E. coli, and P.
aeruginosa strains and in discriminating them from closely related gram-positive 
and gram-negative bacterial strains also known to be etiological agents of
bacteremia. We found a nearly perfect correlation between phenotypic antibiotic
resistance determined by conventional susceptibility testing and genotypic
antibiotic resistance by hybridization to the S. aureus resistance gene probes
mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA 
(erythromycin resistance), and blaZ (penicillin resistance) and the E. coli
resistance gene probes blaTEM-106 (penicillin resistance) and aacC2
(aminoglycoside resistance). Furthermore, antibiotic resistance and virulence
gene probes permitted genotypic discrimination within a species. This novel DNA
microarray demonstrates the feasibility of simultaneously identifying and
characterizing bacteria in blood cultures without prior amplification of target
DNA or preidentification of the pathogen.

DOI: 10.1128/JCM.02291-05 
PMCID: PMC1489523
PMID: 16825354  [Indexed for MEDLINE]


984. Lab Chip. 2006 Jul;6(7):886-95. Epub 2006 May 2.

Microchip-based one step DNA extraction and real-time PCR in one chamber for
rapid pathogen identification.

Lee JG(1), Cheong KH, Huh N, Kim S, Choi JW, Ko C.

Author information: 
(1)Bio Lab, Samsung Advanced Institute of Technology, P.O. Box 111, Suwon
440-600, Korea. biogun.lee@samsung.com

Optimal detection of a pathogen present in biological samples depends on the
ability to extract DNA molecules rapidly and efficiently. In this paper, we
report a novel method for efficient DNA extraction and subsequent real-time
detection in a single microchip by combining laser irradiation and magnetic
beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we
demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli
and Gram-positive bacterial cells as well as the hepatitis B virus mixed with
human serum. We further demonstrate that the real-time pathogen detection was
performed with pre-mixed PCR reagents in a real-time PCR machine using the same
microchip, after laser irradiation in a hand-held device equipped with a small
laser diode. These results suggest that the new sample preparation method is well
suited to be integrated into lab-on-a-chip application of the pathogen detection 
system.

DOI: 10.1039/b515876a 
PMID: 16804593  [Indexed for MEDLINE]


985. Biotechnol Bioeng. 2006 Jun 20;94(3):585-95.

Effect of rpoS gene knockout on the metabolism of Escherichia coli during
exponential growth phase and early stationary phase based on gene expressions,
enzyme activities and intracellular metabolite concentrations.

Rahman M(1), Hasan MR, Oba T, Shimizu K.

Author information: 
(1)Department of Bioscience and Bioinformatics, Kyushu Institute of Technology,
Iizuka, Fukuoka 820-8502, Japan.

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of
a large number of genes in Escherichia coli under stress conditions. The present 
study investigated the growth characteristics and metabolic pathways of rpoS gene
knockout mutant of E. coli growing in LB media under aerobic condition. The
analyses were made based on gene expressions obtained by DNA microarray and
RT-PCR, enzyme activities and intracellular metabolite concentrations at the
exponential and early stationary phases of growth. Although the glucose
utilization pattern of the mutant was similar to the parent strain, the mutant
failed to utilize acetate throughout the cultivation period. Microarray data
indicated that the expression levels of several important genes of acetate
metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in
the absence of rpoS gene. Interestingly, there was an increased activity of TCA
cycle during the exponential growth phase, which was gradually diminished at the 
onset of stationary phase. Moreover, rpoS mutation had profound effect on the
expression of several other genes of E. coli metabolic pathways that were not
described earlier. The changes in the gene expressions, enzyme activities and
intracellular metabolite concentrations of the rpoS mutant are discussed in
details with reference to the major metabolic pathways of E. coli.

2006 Wiley Periodicals, Inc.

DOI: 10.1002/bit.20858 
PMID: 16511888  [Indexed for MEDLINE]


986. BMC Microbiol. 2006 Jun 12;6:53.

Persisters: a distinct physiological state of E. coli.

Shah D(1), Zhang Z, Khodursky A, Kaldalu N, Kurg K, Lewis K.

Author information: 
(1)Department of Biology, Northeastern University, 134 Mugar Hall, 360 Huntington
Ave, Boston, MA 02115, USA. devang@rcn.com

BACKGROUND: Bacterial populations contain persisters, phenotypic variants that
constitute approximately 1% of cells in stationary phase and biofilm cultures.
Multidrug tolerance of persisters is largely responsible for the inability of
antibiotics to completely eradicate infections. Recent progress in understanding 
persisters is encouraging, but the main obstacle in understanding their nature
was our inability to isolate these elusive cells from a wild-type population
since their discovery in 1944.
RESULTS: We hypothesized that persisters are dormant cells with a low level of
translation, and used this to physically sort dim E. coli cells which do not
contain sufficient amounts of unstable GFP expressed from a promoter whose
activity depends on the growth rate. The dim cells were tolerant to antibiotics
and exhibited a gene expression profile distinctly different from those observed 
for cells in exponential or stationary phases. Genes coding for toxin-antitoxin
module proteins were expressed in persisters and are likely contributors to this 
condition.
CONCLUSION: We report a method for persister isolation and conclude that these
cells represent a distinct state of bacterial physiology.

DOI: 10.1186/1471-2180-6-53 
PMCID: PMC1557402
PMID: 16768798  [Indexed for MEDLINE]


987. Br J Cancer. 2006 Jun 5;94(11):1672-7.

Securin (hPTTG1) expression is regulated by beta-catenin/TCF in human colorectal 
carcinoma.

Hlubek F(1), Pfeiffer S, Budczies J, Spaderna S, Jung A, Kirchner T, Brabletz T.

Author information: 
(1)Department of Pathology, Ludwig-Maximilians University of Munich, Thalkirchner
Str. 36, 80337 Munich, Germany. Falk.Hlubek@med.uni-muenchen.de

Overexpression of the transcriptional activator beta-catenin, mostly owing to
loss-of-function mutations of the adenomatous polyposis coli (APC) tumour
suppressor gene, is crucial for the initiation and progression of human
colorectal carcinogenesis. Securin is a regulator of chromosome separation and
its overexpression has been shown to be involved in different tumour-promoting
processes, like transformation, hyperproliferation and angiogenesis, and
correlates with tumour cell invasion. However, the molecular mechanism leading to
securin overexpression in human colorectal cancer is unknown. Here we show a
correlated high expression of beta-catenin and securin (hPTTG1) in colorectal
adenomas and carcinomas and further demonstrate that securin is a target of
beta-catenin transcriptional activation. This implies that deregulation of the
beta-catenin/T-cell factor-signalling pathway leads to overexpression of securin 
in human colorectal cancer, which subsequently may contribute to tumour
progression.

DOI: 10.1038/sj.bjc.6603155 
PMCID: PMC2361298
PMID: 16705313  [Indexed for MEDLINE]


988. BMC Bioinformatics. 2006 Jun 2;7:280.

Integrated biclustering of heterogeneous genome-wide datasets for the inference
of global regulatory networks.

Reiss DJ(1), Baliga NS, Bonneau R.

Author information: 
(1)Institute for Systems Biology, 1441 N, 34th St, Seattle, WA 98103-8904, USA.
dreiss@systemsbiology.org

BACKGROUND: The learning of global genetic regulatory networks from expression
data is a severely under-constrained problem that is aided by reducing the
dimensionality of the search space by means of clustering genes into putatively
co-regulated groups, as opposed to those that are simply co-expressed. Be cause
genes may be co-regulated only across a subset of all observed experimental
conditions, biclustering (clustering of genes and conditions) is more appropriate
than standard clustering. Co-regulated genes are also often functionally
(physically, spatially, genetically, and/or evolutionarily) associated, and such 
a priori known or pre-computed associations can provide support for appropriately
grouping genes. One important association is the presence of one or more common
cis-regulatory motifs. In organisms where these motifs are not known, their de
novo detection, integrated into the clustering algorithm, can help to guide the
process towards more biologically parsimonious solutions.
RESULTS: We have developed an algorithm, cMonkey, that detects putative
co-regulated gene groupings by integrating the biclustering of gene expression
data and various functional associations with the de novo detection of sequence
motifs.
CONCLUSION: We have applied this procedure to the archaeon Halobacterium NRC-1,
as part of our efforts to decipher its regulatory network. In addition, we used
cMonkey on public data for three organisms in the other two domains of life:
Helicobacter pylori, Saccharomyces cerevisiae, and Escherichia coli. The
biclusters detected by cMonkey both recapitulated known biology and enabled novel
predictions (some for Halobacterium were subsequently confirmed in the
laboratory). For example, it identified the bacteriorhodopsin regulon, assigned
additional genes to this regulon with apparently unrelated function, and detected
its known promoter motif. We have performed a thorough comparison of cMonkey
results against other clustering methods, and find that cMonkey biclusters are
more parsimonious with all available evidence for co-regulation.

DOI: 10.1186/1471-2105-7-280 
PMCID: PMC1502140
PMID: 16749936  [Indexed for MEDLINE]


989. Antimicrob Agents Chemother. 2006 Jun;50(6):2038-41.

DNA microarray for detection of macrolide resistance genes.

Cassone M(1), D'Andrea MM, Iannelli F, Oggioni MR, Rossolini GM, Pozzi G.

Author information: 
(1)LAMMB, Università di Siena, Policlinico Le Scotte/V Lotto, Italy.

A DNA microarray was developed to detect bacterial genes conferring resistance to
macrolides and related antibiotics. A database containing 65 nonredundant genes
selected from publicly available DNA sequences was constructed and used to design
100 oligonucleotide probes that could specifically detect and discriminate all 65
genes. Probes were spotted on a glass slide, and the array was reacted with DNA
templates extracted from 20 reference strains of eight different bacterial
species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis,
Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus,
Escherichia coli, and Bacteroides fragilis) known to harbor 29 different
macrolide resistance genes. Hybridization results showed that probes reacted
with, and only with, the expected DNA templates and allowed discovery of three
unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not
yet been described for gram-negative bacteria.

DOI: 10.1128/AAC.01574-05 
PMCID: PMC1479117
PMID: 16723563  [Indexed for MEDLINE]


990. Appl Environ Microbiol. 2006 Jun;72(6):4200-6.

A virulence and antimicrobial resistance DNA microarray detects a high frequency 
of virulence genes in Escherichia coli isolates from Great Lakes recreational
waters.

Hamelin K(1), Bruant G, El-Shaarawi A, Hill S, Edge TA, Bekal S, Fairbrother JM, 
Harel J, Maynard C, Masson L, Brousseau R.

Author information: 
(1)Biotechnology Research Institute, National Research Council of Canada, 6100
Royalmount Ave., Montréal, Québec, Canada H4P 2R2.

Escherichia coli is generally described as a commensal species with occasional
pathogenic strains. Due to technological limitations, there is currently little
information concerning the prevalence of pathogenic E. coli strains in the
environment. For the first time, using a DNA microarray capable of detecting all 
currently described virulence genes and commonly found antimicrobial resistance
genes, a survey of environmental E. coli isolates from recreational waters was
carried out. A high proportion (29%) of 308 isolates from a beach site in the
Great Lakes carried a pathotype set of virulence-related genes, and 14% carried
antimicrobial resistance genes, findings consistent with a potential risk for
public health. The results also showed that another 8% of the isolates had
unusual virulence gene combinations that would be missed by conventional
screening. This new application of a DNA microarray to environmental waters will 
likely have an important impact on public health, epidemiology, and microbial
ecology in the future.

DOI: 10.1128/AEM.00137-06 
PMCID: PMC1489589
PMID: 16751532  [Indexed for MEDLINE]


991. Arch Microbiol. 2006 Jun;185(5):373-82. Epub 2006 Mar 23.

Indole-3-acetic acid improves Escherichia coli's defences to stress.

Bianco C(1), Imperlini E, Calogero R, Senatore B, Amoresano A, Carpentieri A,
Pucci P, Defez R.

Author information: 
(1)Institute of Genetics and Biophysics, Adriano Buzzati Traverso, via P.
Castellino 111, 80131 Naples, Italy.

Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in
many living organisms. To elucidate the physiological changes induced by IAA
treatment, we used Escherichia coli K-12 as a model system. By microarray
analysis we found that 16 genes showed an altered expression level in IAA-treated
cells. One-third of these genes encode cell envelope components, or proteins
involved in bacterial adaptation to unfavourable environmental conditions. We
thus investigated the effect of IAA treatment on some of the structural
components of the envelope that may be involved in cellular response to stresses.
This showed that IAA-treated cells had increased the production of trehalose,
lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated
further that IAA triggers an increased tolerance to several stress conditions
(heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative
stress) and different toxic compounds (antibiotics, detergents and dyes) and this
correlates with higher levels of the heat shock protein DnaK. We suggest that IAA
triggers an increased level of alert and protection against external adverse
conditions by coordinately enhancing different cellular defence systems.

DOI: 10.1007/s00203-006-0103-y 
PMID: 16555073  [Indexed for MEDLINE]


992. Biochem J. 2006 Jun 1;396(2):227-34.

Transcript analysis reveals an extended regulon and the importance of
protein-protein co-operativity for the Escherichia coli methionine repressor.

Marincs F(1), Manfield IW, Stead JA, McDowall KJ, Stockley PG.

Author information: 
(1)Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds
LS2 9JT, UK.

We have used DNA arrays to investigate the effects of knocking out the methionine
repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the
effects in the knockout strain of supplying wild-type or mutant MetJ repressors
from an expression plasmid, thus establishing a rapid assay for in vivo effects
of mutations characterized previously in vitro. Repression is largely restricted 
to known genes involved in the biosynthesis and uptake of methionine. However, we
identified a number of additional genes that are significantly up-regulated in
the absence of repressor. Sequence analysis of the 5' promoter regions of these
genes identified plausible matches to met-box sequences for three of these, and
subsequent electrophoretic mobility-shift assay analysis showed that for two such
loci their repressor affinity is higher than or comparable with the known metB
operator, suggesting that they are directly regulated. This can be rationalized
for one of the loci, folE, by the metabolic role of its encoded enzyme; however, 
the links to the other regulated loci are unclear, suggesting both an extension
to the known met regulon and additional complexity to the role of the repressor. 
The plasmid gene replacement system has been used to examine the importance of
protein-protein co-operativity in operator saturation using the structurally
characterized mutant repressor, Q44K. In vivo, there are detectable reductions in
the levels of regulation observed, demonstrating the importance of balancing
protein-protein and protein-DNA affinity.

DOI: 10.1042/BJ20060021 
PMCID: PMC1462706
PMID: 16515535  [Indexed for MEDLINE]


993. Biomol Eng. 2006 Jun;23(2-3):59-76. Epub 2006 Mar 9.

The affinity concept in bioseparation: evolving paradigms and expanding range of 
applications.

Mondal K(1), Gupta MN.

Author information: 
(1)Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New 
Delhi 110016, India.

The meaning of the word affinity in the context of protein separation has
undergone evolutionary changes over the years. The exploitation of molecular
recognition phenomenon is no longer limited to affinity chromatography modes.
Affinity based separations today include precipitation, membrane based
purification and two-phase/three-phase extractions. Apart from the affinity
ligands, which have biological relationship (in vivo) with the target protein, a 
variety of other ligands are now used in the affinity based separations. These
include dyes, chelated metal ions, peptides obtained by phage display technology,
combinatorial synthesis, ribosome display methods and by systematic evolution of 
ligands by exponential enrichment (SELEX). Molecular modeling techniques have
also facilitated the designing of biomimetic ligands. Fusion proteins obtained by
recombinatorial methods have emerged as a powerful approach in bioseparation.
Overexpression in E. coli often result in inactive and insoluble inclusion
bodies. A number of interesting approaches are used for simultaneous refolding
and purification in such cases. Proteomics also needs affinity chromatography to 
reduce the complexity of the system before analysis by electrophoresis and mass
spectrometry are made. At industrial level, validation, biosafety and process
hygiene are also important aspects. This overview looks at these evolving
paradigms and various strategies which utilize affinity phenomenon for protein
separations.

DOI: 10.1016/j.bioeng.2006.01.004 
PMID: 16527537  [Indexed for MEDLINE]


994. Infect Immun. 2006 Jun;74(6):3565-75.

Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli
strain 83972 in the human urinary tract.

Roos V(1), Klemm P.

Author information: 
(1)Microbial Adhesion Group, Center for Biomedical Microbiology, BioCentrum-DTU, 
Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.

Urinary tract infections (UTIs) are an important health problem worldwide, with
many million cases each year. Escherichia coli is the most common organism
causing UTIs in humans. The asymptomatic bacteriuria E. coli strain 83972 is an
excellent colonizer of the human urinary tract, where it causes long-term bladder
colonization. The strain has been used for prophylactic purposes in patients
prone to more severe and recurrent UTIs. For this study, we used DNA microarrays 
to monitor the expression profile of strain 83972 in the human urinary tract.
Significant differences in expression levels were seen between the in vivo
expression profiles of strain 83972 in three patients and the corresponding in
vitro expression profiles in lab medium and human urine. The data revealed an in 
vivo lifestyle of microaerobic growth with respiration of nitrate coupled to
degradation of sugar acids and amino acids, with no signs of attachment to host
tissues. Interestingly, genes involved in NO protection and metabolism showed
significant up-regulation in the patients. This is one of the first studies to
address bacterial whole-genome expression in humans and the first study to
investigate global gene expression of an E. coli strain in the human urinary
tract.

DOI: 10.1128/IAI.01959-05 
PMCID: PMC1479258
PMID: 16714589  [Indexed for MEDLINE]


995. J Microbiol Methods. 2006 Jun;65(3):453-67. Epub 2005 Oct 18.

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide
microarray and real-time quantitative PCR.

Lee DY(1), Shannon K, Beaudette LA.

Author information: 
(1)Wastewater Technology Centre, Environment Canada, Burlington, Ontario.

As a first step toward building a comprehensive microarray, two low density DNA
microarrays were constructed and evaluated for the accurate detection of
wastewater pathogens. The first one involved the direct hybridization of
wastewater microbial genomic DNA to the functional gene probes while the second
involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray
employed 10 functional genes as detection targets. Sensitivity of the microarray 
was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 
2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the
microarray assay using municipal raw sewage. Sensitivity of the microarray was
enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene
sequences were PCR amplified with a novel universal primer set and allowed
hybridization to 24 species-specific oligonucleotide probes. The minimum
detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 
10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully 
detected multiple bacterial pathogens in wastewater. As a parallel study to
verify efficiency of the DNA microarray, a real-time quantitative PCR assay was
also developed based on the fluorescent TaqMan probes (Applied Biosystems).

DOI: 10.1016/j.mimet.2005.09.008 
PMID: 16239042  [Indexed for MEDLINE]


996. Microbiology. 2006 Jun;152(Pt 6):1799-806.

Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the
foc locus and is unable to express F1C fimbriae.

Roos V(1), Schembri MA, Ulett GC, Klemm P.

Author information: 
(1)Microbial Adhesion Group, Centre for Biomedical Microbiology, BioCentrum-DTU, 
Technical University of Denmark, DK-2800 Lyngby, Denmark.

Escherichia coli is the most common organism associated with asymptomatic
bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes
symptomatic urinary tract infection (UTI), very little is known about the
mechanisms by which these strains colonize the urinary tract. Bacterial adhesion 
conferred by specific surface-associated adhesins is normally considered as a
prerequisite for colonization of the urinary tract. The prototype ABU E. coli
strain 83972 was originally isolated from a girl who had carried it
asymptomatically for 3 years. This study characterized the molecular status of
one of the primary adhesion factors known to be associated with UTI, namely F1C
fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors
present in the human kidney and bladder. Expression of the foc genes was found to
be up-regulated in human urine. It was also shown that although strain 83972
contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed.
Sequencing and genetic complementation revealed that the focD gene, encoding a
component of the F1C transport and assembly system, was non-functional,
explaining the inability of strain 83972 to express this adhesin. The data imply 
that E. coli 83972 has lost its ability to express this important colonization
factor as a result of host-driven evolution. The ancestor of the strain seems to 
have been a pyelonephritis strain of phylogenetic group B2. Strain 83972
therefore represents an example of bacterial adaptation from pathogenicity to
commensalism through virulence factor loss.

DOI: 10.1099/mic.0.28711-0 
PMID: 16735742  [Indexed for MEDLINE]


997. Mol Cell Probes. 2006 Jun-Aug;20(3-4):239-44. Epub 2006 Mar 14.

Escherichia coli O antigen typing using DNA microarrays.

Liu Y(1), Fratamico P.

Author information: 
(1)Microbial Biophysics and Residue Chemistry and Core Technologies, US
Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA.

DNA microarrays were developed for rapid identification of different serogroups
of Escherichia coli in a single platform. Oligonucleotides, as well as PCR
products from genes in the O antigen gene clusters of E. coli serogroups O7,
O104, O111, and O157 were spotted onto glass slides. This was followed by
hybridization with labeled long PCR products of the entire O antigen gene
clusters of these serogroups. Results demonstrated that microarrays consisting of
either oligonucleotides or PCR products generated specific signals for each
serogroup. This is the first report describing the development of model DNA
microarrays for determining the serogroup of E. coli strains.

DOI: 10.1016/j.mcp.2006.01.001 
PMID: 16537102  [Indexed for MEDLINE]


998. PLoS Genet. 2006 Jun;2(6):e96. Epub 2006 Jun 23.

The life-cycle of operons.

Price MN(1), Arkin AP, Alm EJ.

Author information: 
(1)Lawrence Berkeley National Laboratory, Berkeley, California, USA.

Erratum in
    PLoS Genet. 2006 Jul;2(7):e126.

Operons are a major feature of all prokaryotic genomes, but how and why operon
structures vary is not well understood. To elucidate the life-cycle of operons,
we compared gene order between Escherichia coli K12 and its relatives and
identified the recently formed and destroyed operons in E. coli. This allowed us 
to determine how operons form, how they become closely spaced, and how they die. 
Our findings suggest that operon evolution may be driven by selection on gene
expression patterns. First, both operon creation and operon destruction lead to
large changes in gene expression patterns. For example, the removal of lysA and
ruvA from ancestral operons that contained essential genes allowed their
expression to respond to lysine levels and DNA damage, respectively. Second, some
operons have undergone accelerated evolution, with multiple new genes being added
during a brief period. Third, although genes within operons are usually closely
spaced because of a neutral bias toward deletion and because of selection against
large overlaps, genes in highly expressed operons tend to be widely spaced
because of regulatory fine-tuning by intervening sequences. Although operon
evolution may be adaptive, it need not be optimal: new operons often comprise
functionally unrelated genes that were already in proximity before the operon
formed.

DOI: 10.1371/journal.pgen.0020096 
PMCID: PMC1480536
PMID: 16789824  [Indexed for MEDLINE]

Conflict of interest statement: Competing interests. The authors have declared
that no competing interests exist.


999. Wei Sheng Wu Xue Bao. 2006 Jun;46(3):341-6.

[Sequence of Escherichia coli O11 O-antigen gene cluster and identification of
molecular markers specific to O11].

[Article in Chinese]

Wang W(1), Peng X, Wang Q, Cheng JS, Wang L.

Author information: 
(1)TEDA School of Biological Sciences, Nankai University, Tianjin 300457, China. 
wanglei@nankai.edu.cn

Escherichia coli O11 belongs to Shiga toxin-producing Escherichia coli (STEC),
which can cause food-borne disease, hemorrhagic colitis, and hemolytic-uremic
syndrome (HUS) in humans. Because of its character of specificity, the O-antigen 
gene cluster provides the best material for the selection of molecular markers
which can be used for rapid genotyping of bacterial strain. In this study, the
E.coli O11 O-antigen gene cluster was amplified by Long-range PCR and was
sequenced using Shotgun-sequencing approach. Twelve open reading frames were
assigned functions on the basis of homology in the E. coli O11 O-antigen gene
cluster, including UDP-N-acetyl glucosamine-4-epimerase gene (gne), genes
responsible for the biosynthesis of GDP-L-fucose (gmd, fcl, gmm, manC, manB),
glycosyl transferase genes, O-unit flippase gene (wzx) and O-antigen polymerase
gene (wzy). By polymerase chain reaction against representative stains for all
the 166 E. coli and 43 Shigella O serotypes, two genes and four pairs of primers 
were identified to be specific to E. coli O11. Further PCR was done to detect E. 
coli O11 from the environmental specimens, and the sensitivities for detecting
E.coli O11 from the pork and dejecta specimens were 0.25 cfu/g and 2.5 x 10(3)
cfu/g, respectively. Moreover, eight probes were designed and proved to be unique
to E. coli O11, which provides the basis for a sensitive test of the rapid
detection of E. coli O11 by DNA microarray method.


PMID: 16933598  [Indexed for MEDLINE]


1000. Cancer Res. 2006 May 15;66(10):5047-55.

Antiangiogenic antithrombin induces global changes in the gene expression profile
of endothelial cells.

Zhang W(1), Chuang YJ, Jin T, Swanson R, Xiong Y, Leung L, Olson ST.

Author information: 
(1)Center for Molecular Biology of Oral Diseases and Department of Oral Biology, 
College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612,
USA. zhang98@uic.edu

Antithrombin, a serpin family protease inhibitor crucial to hemostasis, acquires 
antiangiogenic properties on undergoing conformational alterations induced by
limited proteolysis or elevated temperature. To better understand the biochemical
mechanisms underlying antithrombin antiangiogenic activity, we did genome-wide
expression profiling, coupled with quantitative reverse transcription-PCR,
Northern blot, and Western blot analyses, to characterize the gene expression
patterns that are induced by antiangiogenic antithrombin in cultured primary
human umbilical vein endothelial cells. Overall, 35 genes with significantly
increased expression and 93 genes with significantly reduced expression (> or
=2-fold changes) due to antiangiogenic antithrombin treatment were identified.
More than half of the down-regulated genes have well-established proangiogenic
functions in endothelial cells, including cell-surface and matrix proteoglycans
(e.g., perlecan, biglycan, and syndecans 1 and 3) and mitogenesis-related
signaling proteins (e.g., mitogen-activated protein kinase 3, signal transducers 
and activators of transcription 2, 3, and 6, and early growth response factor 1).
In contrast, most up-regulated genes (e.g., caspase-3, p21, tissue inhibitor of
metalloproteinases 1, 2, and 3, and adenomatosis polyposis coli) are known for
their antiangiogenic functions which include the promotion of cell apoptosis and 
cell cycle arrest and the inhibition of tumor growth and metastasis. These
results show that the antiangiogenic activity of antithrombin is mediated at
least in part by a global genetic reprogramming of endothelial cells and strongly
implicate an endothelial cell ligand-receptor signaling mechanism in this
reprogramming.

DOI: 10.1158/0008-5472.CAN-05-4449 
PMID: 16707426  [Indexed for MEDLINE]


1001. Appl Environ Microbiol. 2006 May;72(5):3780-4.

Development and validation of an oligonucleotide microarray for detection of
multiple virulence and antimicrobial resistance genes in Escherichia coli.

Bruant G(1), Maynard C, Bekal S, Gaucher I, Masson L, Brousseau R, Harel J.

Author information: 
(1)Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine
Vétérinaire, Université de Montréal, 3200 rue Sicotte, Saint-Hyacinthe, Québec
J2S 7C6, Canada.

An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or
markers and 30 antimicrobial resistance genes was designed and validated using
DNA from known reference strains. This microarray was confirmed to be a powerful 
diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial
resistance, as well as for environmental, epidemiological, and phylogenetic
studies including the evaluation of genome plasticity.

DOI: 10.1128/AEM.72.5.3780-3784.2006 
PMCID: PMC1472324
PMID: 16672535  [Indexed for MEDLINE]


1002. J Vet Med Sci. 2006 May;68(5):447-52.

A novel gene encoding a thrombin inhibitory protein in a cDNA library from
Haemaphysalis longicornis salivary gland.

Nakajima C(1), Imamura S, Konnai S, Yamada S, Nishikado H, Ohashi K, Onuma M.

Author information: 
(1)Laboratory of Infectious Diseases, Department of Disease Control, Graduate
School of Veterinary Medicine, Hokkaido University, Japan.

A novel thrombin inhibitory protein coding gene was identified from a cDNA
library derived from salivary gland of partially-fed Haemaphysalis longicornis
(hard tick). The gene encoded a 93-amino acid protein, designated chimadanin,
which had a signal peptide sequence and was predicted to be a secretory protein. 
It showed no similarity to any other previously identified proteins or conserved 
domain sequences. The gene was expressed during blood feeding and suggested to be
expressed mainly in the salivary gland. The predicted mature region of chimadanin
was expressed in Escherichia coli and characteristics of the recombinant
chimadanin were determined. The activated partial thromboplastin time and the
prothrombin time in sheep plasma were significantly prolonged by chimadanin in a 
dose dependent manner. Amidolytic activity of thrombin was also inhibited by
chimadanin in a dose dependent manner and it suggested that chimadanin was an
anticoagulant with thrombin inhibitory activity. This newly identified thrombin
inhibitor may play an important role in tick blood feeding.


PMID: 16757887  [Indexed for MEDLINE]


1003. Proteomics. 2006 May;6(9):2650-5.

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with
a single DNA address.

Jongsma MA(1), Litjens RH.

Author information: 
(1)Plant Research International, Wageningen, The Netherlands.
maarten.jongsma@wur.nl

The high-throughput deposition of recombinant proteins on chips, beads or
biosensor devices would be greatly facilitated by the implementation of
self-assembly concepts. DNA-directed immobilization via conjugation of proteins
to an oligonucleotide would be preeminently suited for this purpose. Here, we
present a unique method to attach a single DNA address to proteins in one step
during the purification from the E. coli lysate by fusion to human
O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the
conjugates in converting a DNA chip into a protein chip by self assembly is
demonstrated.

DOI: 10.1002/pmic.200500654 
PMID: 16596705  [Indexed for MEDLINE]


1004. J Biol Chem. 2006 Apr 28;281(17):11496-505. Epub 2006 Feb 20.

In prostate cancer cells the interaction of C/EBPalpha with Ku70, Ku80, and
poly(ADP-ribose) polymerase-1 increases sensitivity to DNA damage.

Yin H(1), Glass J.

Author information: 
(1)Feist-Weiller Cancer Center and Department of Medicine, Health Sciences
Center, Shreveport, Louisiana 71130-3932, USA. hyin@lsuhsc.edu

Prostate cancer cell lines were examined for proteins that partnered with the
transcription factor C/EBPalpha by use of a pull-down assay with S-tagged
C/EBPalpha combined with matrix-assisted laser desorption ionization
time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose)
polymerase-1 (PARP-1) were identified as proteins that associated with
C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins
was further demonstrated by glutathione S-transferase (GST) pull-downs using
purified protein expressed in Escherichia coli. The strongest binding was between
C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate
cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of
C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the
interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the
interaction between C/EBPalpha and the Ku proteins as well as PARP-1 showed that 
cells exhibiting these interactions had increased radiation sensitivity and
decreased ability to repair double strand DNA breaks. Deficient DNA repair was
dependent on the prostate cancer cell line tested, suggesting a complex process. 
We conclude that the association of C/EBPalpha with Ku proteins and PARP-1 raises
the likelihood that C/EBPalpha-expressing prostate cancer cells may be more
sensitive to DNA-damaging agents and may be important in the design of new
prostate cancer therapies.

DOI: 10.1074/jbc.M511138200 
PMID: 16490787  [Indexed for MEDLINE]


1005. Nucleic Acids Res. 2006 Apr 13;34(7):e52.

An antibody-based microarray assay for small RNA detection.

Hu Z(1), Zhang A, Storz G, Gottesman S, Leppla SH.

Author information: 
(1)Bacterial Toxins and Therapeutics Section, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Detection of RNAs on microarrays is rapidly becoming a standard approach for
molecular biologists. However, current methods frequently discriminate against
structured and/or small RNA species. Here we present an approach that bypasses
these problems. Unmodified RNA is hybridized directly to DNA microarrays and
detected with the high-affinity, nucleotide sequence-independent, DNA/RNA
hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a
fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG
together with fluorescently labeled streptavidin produces a signal that can be
measured in a standard microarray scanner. The antibody-based method was able to 
detect low abundance small RNAs of Escherichia coli much more efficiently than
the commonly-used cDNA-based method. A specific small RNA was detected in amounts
of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is
an efficient, robust and inexpensive technique that allows quantitative analysis 
of gene expression and does not discriminate against short or structured RNAs.

DOI: 10.1093/nar/gkl142 
PMCID: PMC1435976
PMID: 16614443  [Indexed for MEDLINE]


1006. Appl Environ Microbiol. 2006 Apr;72(4):2661-71.

Genes of Escherichia coli O157:H7 that are involved in high-pressure resistance.

Malone AS(1), Chung YK, Yousef AE.

Author information: 
(1)Department of Food Science and Technology, The Ohio State University, 2015
Fyffe Road, Columbus, OH 43210, USA.

Seventeen Escherichia coli O157:H7 strains were treated with ultrahigh pressure
at 500 MPa and 23 +/- 2 degrees C for 1 min. This treatment inactivated 0.6 to
3.4 log CFU/ml, depending on the strain. The diversity of these strains was
confirmed by pulsed-field gel electrophoresis (PFGE) analysis, and there was no
apparent association between PFGE banding patterns and pressure resistance. The
pressure-resistant strain E. coli O157:H7 EC-88 (0.6-log decrease) and the
pressure-sensitive strain ATCC 35150 (3.4-log decrease) were treated with a
sublethal pressure (100 MPa for 15 min at 23 +/- 2 degrees C) and subjected to
DNA microarray analysis using an E. coli K-12 antisense gene chip. High pressure 
affected the transcription of many genes involved in a variety of intracellular
mechanisms of EC-88, including the stress response, the thiol-disulfide redox
system, Fe-S cluster assembly, and spontaneous mutation. Twenty-four E. coli
isogenic pairs with mutations in the genes regulated by the pressure treatment
were treated with lethal pressures at 400 MPa and 23 +/- 2 degrees C for 5 min.
The barotolerance of the mutants relative to that of the wild-type strains helped
to explain the results obtained by DNA microarray analysis. This study is the
first report to demonstrate that the expression of Fe-S cluster assembly proteins
and the fumarate nitrate reductase regulator decreases the resistance to
pressure, while sigma factor (RpoE), lipoprotein (NlpI), thioredoxin (TrxA),
thioredoxin reductase (TrxB), a trehalose synthesis protein (OtsA), and a
DNA-binding protein (Dps) promote barotolerance.

DOI: 10.1128/AEM.72.4.2661-2671.2006 
PMCID: PMC1449011
PMID: 16597971  [Indexed for MEDLINE]


1007. Appl Environ Microbiol. 2006 Apr;72(4):2449-59.

YliH (BssR) and YceP (BssS) regulate Escherichia coli K-12 biofilm formation by
influencing cell signaling.

Domka J(1), Lee J, Wood TK.

Author information: 
(1)Artie McFerrin Department of Chemical Engineering, 220 Jack E. Brown Building,
Texas A&M University, College Station, TX 77843-3122, USA.

We previously discovered that yliH and yceP are induced in Escherichia coli
biofilms (D. Ren, L. A. Bedzyk, S. M. Thomas, R. W. Ye, and T. K. Wood, Appl.
Microbiol. Biotechnol. 64:515-524, 2004). Here, it is shown that deletion of yceP
(b1060) and yliH (b0836) increases biofilm formation in continuous-flow chambers 
with minimal glucose medium by increasing biofilm mass (240- to 290-fold),
surface coverage (16- to 31-fold), and mean thickness (2,800-fold). To determine 
the genetic basis of the increase in biofilm formation, we examined the
differential gene expression profile in biofilms for both the mutants relative to
the wild-type strain in rich medium with glucose and found that 372 to 882 genes 
were induced and that 76 to 337 were repressed consistently >2-fold (P < or =
0.05). The increase in biofilm formation was related to differential expression
of genes related to stress response (8 to 64 genes) for both mutants, including
rpoS and sdiA. More importantly, 42 to 130 genes related to autoinducer 2 cell
signaling were also differentially expressed, including gadAB and flgBCEGHIJLMN, 
as well as signaling through indole, since 17 to 26 indole-related genes were
differentially expressed, including phoAER, gltBD, mtr (encodes protein for
indole import), and acrEF (encodes proteins for indole export). Increased biofilm
formation in the yliH and yceP mutants in LB supplemented with 0.2% glucose (LB
glu) occurred through a reduction in extracellular and intracellular indole
concentrations in both mutants (50- to 140-fold), and the addition of indole to
the culture restored the wild-type biofilm phenotype; hence, indole represses
biofilms. Additionally, both mutants regulate biofilms through quorum sensing,
since deletion of either yliH or yceP increased extracellular autoinducer 2
concentrations 50-fold when grown in complex medium (most notably in the
stationary phase). Both proteins are involved in motility regulation, since YliH 
(127 amino acids) and YceP (84 amino acids) repressed motility two to sevenfold
(P < or = 0.05) in LB, and YceP repressed motility sevenfold (P < or = 0.05) in
LB glu. Heightened motility in the yceP mutant occurred, due to increased
transcription of the flagella and motility loci, including fliC, motA, and qseB
(3- to 86-fold). We propose new names for these two loci: bssR for yliH and bssS 
for yceP, based on the phrase "regulator of biofilm through signal secretion."

DOI: 10.1128/AEM.72.4.2449-2459.2006 
PMCID: PMC1448992
PMID: 16597943  [Indexed for MEDLINE]


1008. J Clin Microbiol. 2006 Apr;44(4):1495-501.

Differentiation of Escherichia coli pathotypes by oligonucleotide spotted array.

Palaniappan RU(1), Zhang Y, Chiu D, Torres A, Debroy C, Whittam TS, Chang YF.

Author information: 
(1)Department of Population Medicine and Diagnostic Sciences, College of
Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

To accurately determine the pathotypes of Escherichia coli strains, a
comprehensive assessment of each strain that targets multiple genes is required. 
A new approach to the identification and characterization of E. coli pathotypes
was developed by constructing gene-specific probes (70-mers) for not only the
virulence genes associated with each E. coli pathotype but also the O157-,
CFT073-, and K-12-specific and common genes of each pathotype. Analysis of
oligonucleotide probes with reference and clinical isolates of E. coli pathotypes
indicated that the array could differentiate the pathotypes on the basis of their
virulence and specific gene patterns. Probes targeting common genes of E. coli
were present in all the reference and clinical strains. Salmonella enterica
subsp. enterica-specific genes and Salmonella core genes were used as negative
controls. The entire E. coli pathotype showed reactivity to only 4 of the 81
Salmonella-specific gene probes. Characterization of the genetic and virulence
profiles of a single strain by using probes for virulence factors and specific
and common genes in the spotted array is an ideal diagnostic tool for
determination of E. coli pathotypes and could also have a significant impact on
the epidemiological analysis of E. coli infections.

DOI: 10.1128/JCM.44.4.1495-1501.2006 
PMCID: PMC1448672
PMID: 16597882  [Indexed for MEDLINE]


1009. Microbiology. 2006 Apr;152(Pt 4):989-1000.

The response of Escherichia coli to exposure to the biocide polyhexamethylene
biguanide.

Allen MJ(1), White GF, Morby AP.

Author information: 
(1)School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff 
CF10 3US, UK.

The global response of Escherichia coli to the broad-spectrum biocide
polyhexamethylene biguanide (PHMB) was investigated using transcriptional
profiling. The transcriptional analyses were validated by direct determination of
the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either
insertionally inactivated genes and/or plasmids expressing the cognate open
reading frames from a heterologous promoter in the corresponding chromosomally
inactivated strains. The results showed that a wide range of genes was altered in
transcriptional activity and that all of the corresponding knockout strains
subsequently challenged with biocide were altered in tolerance. Of particular
interest was the induction of the rhs genes and the implication of enzymes
involved in the repair/binding of nucleic acids in the generation of tolerance,
suggesting a novel dimension in the mechanism of action of PHMB based on its
interaction with nucleic acids.

DOI: 10.1099/mic.0.28643-0 
PMID: 16549663  [Indexed for MEDLINE]


1010. Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5114-9. Epub 2006 Mar 15.

A previously undescribed pathway for pyrimidine catabolism.

Loh KD(1), Gyaneshwar P, Markenscoff Papadimitriou E, Fong R, Kim KS, Parales R, 
Zhou Z, Inwood W, Kustu S.

Author information: 
(1)Department of Plant and Microbial Biology, 111 Koshland Hall, University of
California, Berkeley, CA 94720-3102, USA.

Comment in
    Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5637-8.

The b1012 operon of Escherichia coli K-12, which is composed of seven
unidentified ORFs, is one of the most highly expressed operons under control of
nitrogen regulatory protein C. Examination of strains with lesions in this operon
on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated
that they failed to use uridine or uracil as the sole nitrogen source and that
the parental strain could use them at room temperature but not at 37 degrees C. A
strain carrying an ntrB(Con) mutation, which elevates transcription of genes
under nitrogen regulatory protein C control, could also grow on thymidine as the 
sole nitrogen source, whereas strains with lesions in the b1012 operon could not.
Growth-yield experiments indicated that both nitrogens of uridine and thymidine
were available. Studies with [(14)C]uridine indicated that a three-carbon waste
product from the pyrimidine ring was excreted. After trimethylsilylation and gas 
chromatography, the waste product was identified by mass spectrometry as
3-hydroxypropionic acid. In agreement with this finding,
2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of 
available nitrogens and the waste products distinguished the pathway encoded by
the b1012 operon from pyrimidine catabolic pathways described previously. We
propose that the genes of this operon be named rutA-G for pyrimidine utilization.
The product of the divergently transcribed gene, b1013, is a tetracycline
repressor family regulator that controls transcription of the b1012 operon
negatively.

DOI: 10.1073/pnas.0600521103 
PMCID: PMC1458803
PMID: 16540542  [Indexed for MEDLINE]


1011. J Biol Chem. 2006 Mar 24;281(12):8090-9. Epub 2006 Jan 17.

Genome-wide transcriptional profile of Escherichia coli in response to high
levels of the second messenger 3',5'-cyclic diguanylic acid.

Méndez-Ortiz MM(1), Hyodo M, Hayakawa Y, Membrillo-Hernández J.

Author information: 
(1)Laboratorio de Microbiología y Genética Molecular, Departamento de Biología
Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad
Nacional Autónoma de México, Mexico City 04510, Mexico.

Erratum in
    J Biol Chem. 2007 Jul 27;282(30):22248.

Cyclic diguanylic acid (c-di-GMP; cGpGp) is a global second messenger controlling
motility and adhesion in bacterial cells. Intracellular concentrations of
c-di-GMP depend on two opposite activities: diguanylate cyclase, recently
assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase,
associated with proteins harboring the EAL domain. To date, little is known about
the targets of c-di-GMP in the cell or if it affects transcriptional regulation
of certain genes. In order to expand our knowledge of the effect of this molecule
on the bacterial metabolism, here we report on the Escherichia coli
transcriptional profile under high levels of c-di-GMP. We show that an important 
number of genes encoding cell surface and membrane-bound proteins are altered in 
their transcriptional activity. On the other hand, genes encoding several
transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and
others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of 
motility and cell division genes were altered, and consistent with this was the
physiological analysis of cells overexpressing yddV, a diguanylate cyclase; these
cells displayed an abnormal cell division process when high levels of c-di-GMP
were present. We also show evidence that the diguanylate cyclase gene yddV is
co-transcribed with dos, a heme base oxygen sensor with c-di-GMP-specific
phosphodiesterase activity. A delta dos::kan mutation rendered the cells unable
to divide properly, suggesting that dos and yddV may be part of a fine-tuning
mechanism for regulating the intracellular levels of c-di-GMP.

DOI: 10.1074/jbc.M510701200 
PMID: 16418169  [Indexed for MEDLINE]


1012. J Biol Chem. 2006 Mar 24;281(12):8024-33. Epub 2005 Nov 30.

Latent pathway activation and increased pathway capacity enable Escherichia coli 
adaptation to loss of key metabolic enzymes.

Fong SS(1), Nanchen A, Palsson BO, Sauer U.

Author information: 
(1)Department of Chemical and Life Science Engineering, Virginia Commonwealth
University, Richmond 28284-3028, USA. ssfong@vcu.edu

The ability of biological systems to adapt to genetic and environmental
perturbations is a fundamental but poorly understood process at the molecular
level. By quantifying metabolic fluxes and global mRNA abundance, we investigated
the genetic and metabolic mechanisms that underlie adaptive evolution of four
metabolic gene deletion mutants of Escherichia coli (delta pgi, delta ppc, delta 
pta, and delta tpi) in parallel evolution experiments of each mutant. The initial
response to the gene deletions was flux rerouting through local bypass reactions 
or normally latent pathways. The principal effect of evolution was improved
capacity of already active pathways, whereas new flux distributions were not
observed. Combinatorial changes in capacity and pathway activation, however, led 
to different intracellular flux states that enabled evolution in three of the
four parallel cases tested. The molecular bases of the evolved phenotypes were
then elucidated by global mRNA transcript analyses. Activation of latent pathways
and flux changes in the tricarboxylic acid cycle were found to correlate well
with molecular changes at the transcriptional level. Flux alterations in other
central metabolic pathways, in contrast, were apparently not connected to changes
in the transcriptional network. These results give new insight into the dynamics 
of the evolutionary process by demonstrating the flexibility of the metabolic
network of E. coli to compensate for genetic perturbations and the utility of
combining multiple high throughput data sets to differentiate between causal and 
noncausal mechanistic changes.

DOI: 10.1074/jbc.M510016200 
PMID: 16319065  [Indexed for MEDLINE]


1013. BMC Bioinformatics. 2006 Mar 20;7 Suppl 1:S11.

A regression-based K nearest neighbor algorithm for gene function prediction from
heterogeneous data.

Yao Z(1), Ruzzo WL.

Author information: 
(1)Department of Computer Science and Engineering, AC101 Paul G. Allen Center,
University of Washington, Seattle WA 98195, USA. yzizhen@cs.washington.edu

BACKGROUND: As a variety of functional genomic and proteomic techniques become
available, there is an increasing need for functional analysis methodologies that
integrate heterogeneous data sources.
METHODS: In this paper, we address this issue by proposing a general framework
for gene function prediction based on the k-nearest-neighbor (KNN) algorithm. The
choice of KNN is motivated by its simplicity, flexibility to incorporate
different data types and adaptability to irregular feature spaces. A weakness of 
traditional KNN methods, especially when handling heterogeneous data, is that
performance is subject to the often ad hoc choice of similarity metric. To
address this weakness, we apply regression methods to infer a similarity metric
as a weighted combination of a set of base similarity measures, which helps to
locate the neighbors that are most likely to be in the same class as the target
gene. We also suggest a novel voting scheme to generate confidence scores that
estimate the accuracy of predictions. The method gracefully extends to multi-way 
classification problems.
RESULTS: We apply this technique to gene function prediction according to three
well-known Escherichia coli classification schemes suggested by biologists, using
information derived from microarray and genome sequencing data. We demonstrate
that our algorithm dramatically outperforms the naive KNN methods and is
competitive with support vector machine (SVM) algorithms for integrating
heterogenous data. We also show that by combining different data sources,
prediction accuracy can improve significantly
CONCLUSION: Our extension of KNN with automatic feature weighting, multi-class
prediction, and probabilistic inference, enhance prediction accuracy
significantly while remaining efficient, intuitive and flexible. This general
framework can also be applied to similar classification problems involving
heterogeneous datasets.

DOI: 10.1186/1471-2105-7-S1-S11 
PMCID: PMC1810312
PMID: 16723004  [Indexed for MEDLINE]


1014. Bioinformatics. 2006 Mar 15;22(6):747-54. Epub 2006 Jan 10.

Using a state-space model with hidden variables to infer transcription factor
activities.

Li Z(1), Shaw SM, Yedwabnick MJ, Chan C.

Author information: 
(1)Department of Chemical Engineering and Material Science, Michigan State
University East Lansing, 48824, USA.

MOTIVATION: In a gene regulatory network, genes are typically regulated by
transcription factors (TFs). Transcription factor activity (TFA) is more
difficult to measure than gene expression levels are. Other models have extracted
information about TFA from gene expression data, but without explicitly modeling 
feedback from the genes. We present a state-space model (SSM) with hidden
variables. The hidden variables include regulatory motifs in the gene network,
such as feedback loops and auto-regulation, making SSM a useful complement to
existing models.
RESULTS: A gene regulatory network incorporating, for example, feed-forward
loops, auto-regulation and multiple-inputs was constructed with an SSM model.
First, the gene expression data were simulated by SSM and used to infer the TFAs.
The ability of SSM to infer TFAs was evaluated by comparing the profiles of the
inferred and simulated TFAs. Second, SSM was applied to gene expression data
obtained from Escherichia coli K12 undergoing a carbon source transition and from
the Saccharomyces cerevisiae cell cycle. The inferred activity profile for each
TF was validated either by measurement or by activity information from the
literature. The SSM model provides a probabilistic framework to simulate gene
regulatory networks and to infer activity profiles of hidden variables.
AVAILABILITY: Supplementary data and Matlab code will be made available at the
URL below.
SUPPLEMENTARY INFORMATION: http://www.chems.msu.edu/groups/chan/ssm.zip.

DOI: 10.1093/bioinformatics/btk034 
PMID: 16403793  [Indexed for MEDLINE]


1015. Bioinformatics. 2006 Mar 15;22(6):739-46. Epub 2005 Dec 20.

Bayesian sparse hidden components analysis for transcription regulation networks.

Sabatti C(1), James GM.

Author information: 
(1)Department of Human Genetics, UCLA, Los Angeles, CA 90095-7088, USA.
csabatti@mednet.ucla.edu

MOTIVATION: In systems like Escherichia Coli, the abundance of sequence
information, gene expression array studies and small scale experiments allows one
to reconstruct the regulatory network and to quantify the effects of
transcription factors on gene expression. However, this goal can only be achieved
if all information sources are used in concert.
RESULTS: Our method integrates literature information, DNA sequences and
expression arrays. A set of relevant transcription factors is defined on the
basis of literature. Sequence data are used to identify potential target genes
and the results are used to define a prior distribution on the topology of the
regulatory network. A Bayesian hidden component model for the expression array
data allows us to identify which of the potential binding sites are actually used
by the regulatory proteins in the studied cell conditions, the strength of their 
control, and their activation profile in a series of experiments. We apply our
methodology to 35 expression studies in E.Coli with convincing results.
AVAILABILITY: www.genetics.ucla.edu/labs/sabatti/software.html
SUPPLEMENTARY INFORMATION: The supplementary material are available at
Bioinformatics online.

DOI: 10.1093/bioinformatics/btk017 
PMID: 16368767  [Indexed for MEDLINE]


1016. BMC Microbiol. 2006 Mar 15;6:30.

Microarray based comparison of two Escherichia coli O157:H7 lineages.

Dowd SE(1), Ishizaki H.

Author information: 
(1)Livestock Issues Research Unit, USDA-ARS, Lubbock, TX, USA.
sdowd@lbk.ars.usda.gov

BACKGROUND: Previous research has identified the potential for the existence of
two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to
cluster primarily within one of these two lineages. To determine if there are
virulence related genes differentially expressed between the two lineages we
chose to utilize microarray technology to perform an initial screening.
RESULTS: Using a 610 gene microarray, designed against the E. coli O157 EDL 933
transcriptome, targeting primarily virulence systems, we chose 3 representative
Lineage I isolates (LI groups mostly clinical isolates) and 3 representative
Lineage II isolates (LII groups mostly bovine isolates). Using standard dye swap 
experimental designs, statistically different expression (P < 0.05) of 73 genes
between the two lineages was revealed. Result highlights indicate that under in
vitro anaerobic growth conditions, there is up-regulation of stx2b, ureD, curli
(csgAFEG), and stress related genes (hslJ, cspG, ibpB, ibpA) in Lineage I, which 
may contribute to enhanced virulence or transmission potential. Lineage II
exhibits significant up-regulation of type III secretion apparatus, LPS, and
flagella related transcripts.
CONCLUSION: These results give insight into comparative regulation of virulence
genes as well as providing directions for future research. Ultimately, evaluating
the expression of key virulence factors among different E. coli O157 isolates has
inherent value and the interpretation of such expression data will continue to
evolve as our understanding of virulence, pathogenesis and transmission improves.

DOI: 10.1186/1471-2180-6-30 
PMCID: PMC1431545
PMID: 16539702  [Indexed for MEDLINE]


1017. Int J Food Microbiol. 2006 Mar 15;107(2):131-7. Epub 2005 Dec 28.

Identification of Bacillus spp., Escherichia coli, Salmonella spp.,
Staphylococcus spp. and Vibrio spp. with 16S ribosomal DNA-based oligonucleotide 
array hybridization.

Chiang YC(1), Yang CY, Li C, Ho YC, Lin CK, Tsen HY.

Author information: 
(1)Department of Food Science, National Chung-Hsing University, Taichung, Taiwan,
ROC.

Rapid identification of the genus and species of bacteria in foods and clinical
specimens is important. In this report, DNA sequences of bacterial 16S rDNA were 
used to develop the oligonucleotide array for the identification of bacterial
strains of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. 
and Vibrio spp. Most of these bacterial strains may cause food-borne outbreaks or
sporadic cases. A rapid (<4 h) detection method that used universal PCR primers
to amplify the variable regions of bacterial 16S rDNA, followed by reverse
hybridization of the PCR products, which were biotin labeled, to the
oligonucleotides arrayed on the chip was developed. Fifteen oligonucleotide
probes were selected and spotted on the nylon strip to determine the array
hybridization patterns. It was successful in discriminating Bacillus spp., E.
coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with identification,
in general, to the genus level, not species level. As 182 randomly selected
strains were assayed, the detection rate was found higher than 98%. Except for 3 
strains, the remaining 179 strains were correctly identified and no cross
reactions were observed. These 179 strains generated five hybridization patterns.
Adding more oligonucleotide probes to the array may allow the detection of more
bacterial genera and species without significantly increasing the complexity or
cost.

DOI: 10.1016/j.ijfoodmicro.2005.04.028 
PMID: 16386323  [Indexed for MEDLINE]


1018. J Bacteriol. 2006 Mar;188(6):2233-43.

Identification of new flagellar genes of Salmonella enterica serovar Typhimurium.

Frye J(1), Karlinsey JE, Felise HR, Marzolf B, Dowidar N, McClelland M, Hughes
KT.

Author information: 
(1)Sidney Kimmel Cancer Center, San Diego, California 92121, USA.

RNA levels of flagellar genes in eight different genetic backgrounds were
compared to that of the wild type by DNA microarray analysis. Cluster analysis
identified new, potential flagellar genes, three putative methyl-accepting
chemotaxis proteins, STM3138 (McpA), STM3152 (McpB), and STM3216(McpC), and a
CheV homolog, STM2314, in Salmonella, that are not found in Escherichia coli.
Isolation and characterization of Mud-lac insertions in cheV, mcpB, mcpC, and the
previously uncharacterized aer locus of S. enterica serovar Typhimurium revealed 
them to be controlled by sigma28-dependent flagellar class 3 promoters. In
addition, the srfABC operon previously isolated as an SsrB-regulated operon
clustered with the flagellar class 2 operon and was determined to be under FlhDC 
control. The previously unclassified fliB gene, encoding flagellin methylase,
clustered as a class 2 gene, which was verified using reporter fusions, and the
fliB transcriptional start site was identified by primer extension analysis. RNA 
levels of all flagellar genes were elevated in flgM or fliT null strains. RNA
levels of class 3 flagellar genes were elevated in a fliS null strain, while
deletion of the fliY, fliZ, or flk gene did not affect flagellar RNA levels
relative to those of the wild type. The cafA (RNase G) and yhjH genes clustered
with flagellar class 3 transcribed genes. Null alleles in cheV, mcpA, mcpB, mcpC,
and srfB did not affect motility, while deletion of yhjH did result in reduced
motility compared to that of the wild type.

DOI: 10.1128/JB.188.6.2233-2243.2006 
PMCID: PMC1428135
PMID: 16513753  [Indexed for MEDLINE]


1019. J Water Health. 2006 Mar;4(1):67-75.

Detection of Salmonella spp. in water using magnetic capture hybridization
combined with PCR or real-time PCR.

Thompson DE(1), Rajal VB, De Batz S, Wuertz S.

Author information: 
(1)Department of Civil and Environmental Engineering, University of California,
Davis, One Shields Avenue, Davis, CA 95616, USA.

The removal of target DNA by magnetic capture hybridization (MCH) from
constituents inhibitory to amplification by polymerase chain reaction (PCR) was
evaluated using Salmonella as the test pathogen. Hybrids were subjected to both
conventional and quantitative real-time PCR (qPCR). When PCR inhibitors commonly 
found in water were added to the reaction, MCH-PCR increased the detection
sensitivity on the order of 8 to 2,000-fold compared with the system using only
PCR. To determine the selectivity of MCH for target DNA (Salmonella), different
amounts of non-target DNA (Escherichia coli) were added to the qPCR reaction. The
highest non-target DNA concentration interfered with the amplification by qPCR
alone, while MCH-qPCR was unaffected. Average recovery of Salmonella DNA by
MCH-qPCR was 31% using optimized buffers, washing solutions and enzymatic
digestion. A recovery function was proposed in order to calculate the real cell
number based on the measured value. Preliminary testing confirmed the suitability
of this method for analysis of natural waters.


PMID: 16604839  [Indexed for MEDLINE]


1020. Mol Microbiol. 2006 Mar;59(6):1807-17.

A pathway branching in transcription initiation in Escherichia coli.

Susa M(1), Kubori T, Shimamoto N.

Author information: 
(1)Structural Biology Center, National Institute of Genetics, The Graduate
University for Advanced Studies, Mishima, Shizuoka, Japan.

In transcription initiation, all RNA polymerase molecules bound to a promoter
have been conventionally supposed to proceed into elongation of transcript.
However, for Escherichia coli RNA polymerase, evidence has been accumulated for a
view that only its fraction can proceed into elongation and the rest is retained 
at a promoter in non-productive form: a pathway branching in transcription
initiation. Proteins such as GreA and GreB affect these fractions at several
promoters in vitro. To reveal the ubiquitous existence of the branched mechanism 
in E. coli, we searched for candidate genes whose transcription decreased by
disruption of greA and greB using a DNA array. Among the arbitrarily selected 11 
genes from over 100, the atpC, cspA and rpsA passed the test by Northern
blotting. The Gre factors activated transcription initiation from their promoters
in vitro, and the results demonstrated that the branched mechanism is exploited
in vivo regulation. Consistently, decrease in the level of the GreA in an
anaerobic stationary condition accompanied a decrease in the levels of
transcripts of these genes.

DOI: 10.1111/j.1365-2958.2006.05058.x 
PMCID: PMC1413587
PMID: 16553885  [Indexed for MEDLINE]


1021. DNA Res. 2006 Feb 28;13(1):3-14. Epub 2006 Feb 22.

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157
revealed by the combinational use of the O157 Sakai OligoDNA microarray and the
Whole Genome PCR scanning.

Ogura Y(1), Kurokawa K, Ooka T, Tashiro K, Tobe T, Ohnishi M, Nakayama K,
Morimoto T, Terajima J, Watanabe H, Kuhara S, Hayashi T.

Author information: 
(1)Division of Bioenvironmental Science, Frontier Science Research Center,
University of Miyazaki, Miyazaki, Japan.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic 
uremic syndrome, is one of the leading worldwide public health threats. Genome
sequencing of two O157 strains have revealed that the chromosome is comprised of 
a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences.
Most of the large O157-specific sequences are prophages and prophage-like
elements, which have carried many virulence genes into the O157 genome. This
suggests that bacteriophages have played the key roles in the emergence of O157. 
The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the
other hand, revealed a high level of genomic diversity in O157. Variation of
prophages has also been suggested as a major factor generating such diversity. In
this study, we analyzed the gene content of O157 strains, by an oligoDNA
microarray, using the same set of strains as examined by the WGPScanning method. 
Although most of the strains were typical O157 : H7, they differed remarkably in 
gene composition, particularly in those on prophages, and we identified more than
400 'variably absent or present' genes which included virulence-related genes.
This confirms the role of prophages in generating the genomic diversity, and
raises a possibility that some level of variation in potential virulence is
present among O157 strains. Fine comparison of the two datasets obtained by
microarray and WGPScanning provided much further details on the O157 genome
diversity than illustrated by each method alone, indicating the usefulness of
this combinational approach in the genomic comparison of closely related strains.

DOI: 10.1093/dnares/dsi026 
PMID: 16766508  [Indexed for MEDLINE]


1022. J Biol Chem. 2006 Feb 24;281(8):4802-15. Epub 2005 Dec 22.

A reassessment of the FNR regulon and transcriptomic analysis of the effects of
nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to
anaerobic growth.

Constantinidou C(1), Hobman JL, Griffiths L, Patel MD, Penn CW, Cole JA, Overton 
TW.

Author information: 
(1)School of Biosciences, University of Birmingham, Birmingham B15 2TT, United
Kingdom.

The transcription factor FNR, the regulator of fumarate and nitrate reduction,
regulates major changes as Escherichia coli adapts from aerobic to anaerobic
growth. In an anaerobic glycerol/trimethylamine N-oxide/fumarate medium, the fnr 
mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to
reveal the response to oxygen, nitrate, and nitrite in the absence of glucose
repression or artifacts because of variations in growth rate. Hence, many of the 
discrepancies between previous microarray studies of the E. coli FNR regulon were
resolved. The current microarray data confirmed 31 of the previously
characterized FNR-regulated operons. Forty four operons not previously known to
be included in the FNR regulon were activated by FNR, and a further 28 operons
appeared to be repressed. For each of these operons, a match to the consensus
FNR-binding site sequence was identified. The FNR regulon therefore minimally
includes at least 103, and possibly as many as 115, operons. Comparison of
transcripts in the parental strain and a narXL deletion mutant revealed that
transcription of 51 operons is activated, directly or indirectly, by NarL, and a 
further 41 operons are repressed. The narP gene was also deleted from the narXL
mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen
promoters were more active in the narP+ strain than in the mutant, and a further 
37 were strongly repressed. This is the first report that NarP might function as 
a global repressor as well as a transcription activator. The data also revealed
possible new defense mechanisms against reactive nitrogen species.

DOI: 10.1074/jbc.M512312200 
PMID: 16377617  [Indexed for MEDLINE]


1023. Biochem Soc Trans. 2006 Feb;34(Pt 1):200-2.

Nitric oxide-sensing mechanisms in Escherichia coli.

Spiro S(1).

Author information: 
(1)School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, 
GA 30332-0230, USA. stephen.spiro@biology.gatech.edu

Exposure of Escherichia coli to nitric oxide (NO) or nitrosating agents causes
significant changes in patterns of gene expression. Three recent studies have
used microarrays to analyse the response of the E. coli transcriptome to NO and
nitrosative stress. Drawing on the array data, I review our current understanding
of the E. coli regulatory systems that are involved.

DOI: 10.1042/BST0340200 
PMID: 16417522  [Indexed for MEDLINE]


1024. Biochem Soc Trans. 2006 Feb;34(Pt 1):104-7.

Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and
NarP during anaerobic growth of Escherichia coli: new insights into microbial
physiology.

Overton TW(1), Griffiths L, Patel MD, Hobman JL, Penn CW, Cole JA, Constantinidou
C.

Author information: 
(1)School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives 
defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic 
growth in the presence or absence of nitrate or nitrite, in non-repressing media 
in which both strain MG1655 and an fnr deletion mutant grew at similar rates.
Glycerol was used as the non-repressing carbon source and both
trimethylamine-N-oxide and fumarate were added as terminal electron acceptors.
Microarray data supplemented with bioinformatic data revealed that the FNR
(fumarate and nitrate reductase regulator) regulon includes at least 104, and
possibly as many as 115, operons, 68 of which are activated and 36 are repressed 
during anaerobic growth. A total of 51 operons were directly or indirectly
activated by NarL in response to nitrate; a further 41 operons were repressed.
Four subgroups of genes implicated in management of reactive nitrogen compounds, 
NO and products of NO metabolism, were identified; they included proteins of
previously unknown function. Global repression by the nitrate- and
nitrite-responsive two-component system, NarQ-NarP, was shown for the first time.
In contrast with the frdABCD, aspA and ansB operons that are repressed only by
NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

DOI: 10.1042/BST0340104 
PMID: 16417494  [Indexed for MEDLINE]


1025. Cancer Res. 2006 Feb 1;66(3):1354-62.

Fibroblast growth factor 9 has oncogenic activity and is a downstream target of
Wnt signaling in ovarian endometrioid adenocarcinomas.

Hendrix ND(1), Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR.

Author information: 
(1)Department of Pathology, University of Michigan Medical School, 210 Washtenaw 
Avenue, Ann Arbor, MI 48109, USA.

Wnt signaling plays a key role in development and adult tissues via effects on
cell proliferation, motility, and differentiation. The cellular response to Wnt
ligands largely depends on their ability to stabilize beta-catenin and the
ability of beta-catenin to bind and activate T-cell factor (TCF) transcription
factors. Roughly 40% of ovarian endometrioid adenocarcinomas (OEA) have
constitutive activation of Wnt signaling as a result of oncogenic mutations in
the beta-catenin protein or inactivating mutations in key negative regulators of 
beta-catenin, such as the adenomatous polyposis coli and Axin tumor suppressor
proteins. We used oligonucleotide microarrays to identify genes of which
expression was activated in OEAs with beta-catenin dysregulation compared with
OEAs lacking Wnt/beta-catenin pathway defects. Using microarray and quantitative 
PCR-based approaches, we found that fibroblast growth factor (FGF9) expression
was increased >6-fold in primary OEAs with Wnt/beta-catenin pathway defects
compared with OEAs lacking such defects. Evidence that beta-catenin and TCFs
regulate FGF9 expression in several epithelial cell lines was obtained. We found 
FGF9 was mitogenic for epithelial cells and fibroblasts and FGF9 could stimulate 
invasion of epithelial and endothelial cells through Matrigel in transwell
assays. Furthermore, FGF9 could promote neoplastic transformation of the
E1A-immortalized RK3E epithelial cell line, and short hairpin RNA-mediated
inhibition of endogenous FGF9 expression in the OEA cell line TOV112D, which
carries a beta-catenin mutation, inhibited neoplastic growth properties of the
cells. Our findings support the notion that FGF9 is a key factor contributing to 
the cancer phenotype of OEAs carrying Wnt/beta-catenin pathway defects.

DOI: 10.1158/0008-5472.CAN-05-3694 
PMID: 16452189  [Indexed for MEDLINE]


1026. Clin Chem. 2006 Feb;52(2):192-201. Epub 2005 Dec 29.

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of
the LEE pathogenicity island of human and animal Shiga toxin-producing
Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains.

Garrido P(1), Blanco M, Moreno-Paz M, Briones C, Dahbi G, Blanco J, Blanco J,
Parro V.

Author information: 
(1)Laboratorio de Ecología Molecular, Centro de Astrobiología (INTA-CSIC),
Madrid, Spain.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E.
coli (EPEC) are important emerging pathogens that can cause a severe and
sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene
variants of the locus of enterocyte effacement (LEE) pathogenicity island
represents an important tool for typing in routine diagnostics as well as in
pathogenesis, epidemiologic, clonal, and immunologic studies.
METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed
and constructed for the detection and typing of genetic variants of the LEE
genes. Oligonucleotide probes were tested for their specificity against the
corresponding type strain by microarray hybridization using fluorescent DNA,
either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified
chromosomal DNA.
RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to
distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta,
delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae
gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4
variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1,
beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the
espD gene. We found a total of 12 different combinations of tir, espA, espB, and 
espD genes among the 25 typed strains.
CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are
effective tools to rapidly screen multiple virulence genes and their variants in 
E. coli strains isolated from human and animal infections. The results
demonstrate the great genetic diversity among LEE genes of human and animal STEC 
and EPEC strains.

DOI: 10.1373/clinchem.2005.059766 
PMID: 16384888  [Indexed for MEDLINE]


1027. J Bacteriol. 2006 Feb;188(3):1199-204.

Transcriptome analysis applied to survival of Shewanella oneidensis MR-1 exposed 
to ionizing radiation.

Qiu X(1), Daly MJ, Vasilenko A, Omelchenko MV, Gaidamakova EK, Wu L, Zhou J,
Sundin GW, Tiedje JM.

Author information: 
(1)Center for Microbial Ecology, PSSB 540, Michigan State University, East
Lansing, MI 48824, USA.

The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella
oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli
and Deinococcus radiodurans, respectively. Transcriptome analysis was used to
identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction
of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period
after irradiation. The genomic response of MR-1 to IR is very similar to its
response to UV radiation (254 nm), which included induction of systems involved
in DNA repair and prophage synthesis and the absence of differential regulation
of tricarboxylic acid cycle activity, which occurs in IR-irradiated D.
radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes 
in MR-1 was observed. DNA damage may not be the principal cause of high
sensitivity to IR, considering that MR-1 carries genes encoding a complex set of 
DNA repair systems and 40 Gy IR induces less than one double-strand break in its 
genome. Instead, a combination of oxidative stress, protein damage, and
prophage-mediated cell lysis during irradiation and recovery might underlie this 
organism's great sensitivity to IR.

DOI: 10.1128/JB.188.3.1199-1204.2006 
PMCID: PMC1347324
PMID: 16428429  [Indexed for MEDLINE]


1028. J Leukoc Biol. 2006 Feb;79(2):339-50. Epub 2005 Nov 10.

Mast cells, which interact with Escherichia coli, up-regulate genes associated
with innate immunity and become less responsive to Fc(epsilon)RI-mediated
activation.

Kulka M(1), Fukuishi N, Rottem M, Mekori YA, Metcalfe DD.

Author information: 
(1)Allergy-Immunology Division, Northwestern University Feinberg School of
Medicine, Chicago, IL, USA.

Mast cells, which are associated with T helper cell type 2-dependent
inflammation, have now been implicated in the innate immune response. To further 
characterize how mast cells are programmed to respond to infectious organisms, we
used expression profiling using DNA microarray analysis of gene expression by
human mast cells (huMC) during ingestion of Escherichia coli and examined
immunoglobulin E (IgE)-mediated degranulation. Analysis of data revealed that
specific groups of genes were modulated, including genes encoding transcription
factors, cell signaling molecules, cell cycle regulators, enzymes, cytokines,
novel chemokines of the CC family, adhesion molecules, and costimulatory
molecules. Enzyme-linked immunosorbent assay analysis confirmed the production of
tumor necrosis factor and the chemokines CC chemokine ligand (CCL)-1/I-309,
CCL-19/macrophage-inflammatory protein-3beta (MIP-3beta), and CCL-18/MIP-4; flow 
cytometry confirmed the up-regulation of carcinoembryonic antigen-related cell
adhesion molecule 1, the integrin CD49d, and CD80. Coincubation with E. coli
down-regulated Fc receptor for IgE I (FcepsilonRI) expression and
FcepsilonRI-mediated huMC degranulation. These data are consistent with the
concept that bacterial exposure directs mast cell responses toward innate
immunity and away from IgE-mediated effects.

DOI: 10.1189/jlb.1004600 
PMID: 16282532  [Indexed for MEDLINE]


1029. Mol Biochem Parasitol. 2006 Feb;145(2):147-57. Epub 2005 Oct 12.

Cloning and characterization of angiotensin converting enzyme related
dipeptidylcarboxypeptidase from Leishmania donovani.

Goyal N(1), Duncan R, Selvapandiyan A, Debrabant A, Baig MS, Nakhasi HL.

Author information: 
(1)Division of Biochemistry, Central Drug Research Institute, Lucknow 226001,
Uttar Pradesh, India. neenacdri@yahoo.com

We report the first identification, gene cloning, recombinant expression and
biochemical characterization of an angiotensin converting enzyme (ACE) related
dipeptidylcarboxypeptidase (DCP) in a protozoan parasite. The mammalian
counterpart of this enzyme, peptidyl dipeptidase A (a carboxyl dipeptidase) also 
known as ACE leads to the cleavage of angiotensin I to produce a potent
vasopressor. The catalytic enzyme activity of its Escherichia coli DCP counter
part can be inhibited by the antihypertensive drug captopril, suggesting that
this class of enzymes constitutes a novel target for drugs and vaccines. By
utilizing a DNA microarray expression profiling approach, we identified a gene
encoding a DCP enzyme for the kinetoplast protozoan Leishmania donovani (LdDCP)
that was differentially expressed in promastigote and amastigote stages of the
parasite life cycle. Both RNA and protein levels of LdDCP are higher in axenic
amastigotes compared to promastigotes. Immuno-fluorescence analysis revealed the 
cytosolic expression of the protein. Primary structure analysis of LdDCP revealed
the presence of an active Zn binding site. When expressed in E. coli, the
recombinant enzyme showed carboxy-dipeptidase activity with synthetic substrates.
Replacement of two histidine and one glutamic acid at positions 466, 470 and 467,
respectively, with alanine residues in its active site resulted in loss of enzyme
activity. Captopril, an ACE specific inhibitor was able both to reduce
significantly LdDCP enzyme activity and to inhibit promastigote growth. Both its 
cytosolic location and close homology to DCPs from bacterial species suggests a
role in parasite nutrition. Further, identification of LdDCP now provides an
opportunity to investigate Leishmania peptidases for their potential as drug and 
vaccine targets.

DOI: 10.1016/j.molbiopara.2005.09.014 
PMID: 16257064  [Indexed for MEDLINE]


1030. Sci China C Life Sci. 2006 Feb;49(1):46-52.

Genomic compositions and phylogenetic analysis of Shigella boydii subgroup.

Wang J(1), Zhang X, Peng J, Yang E, Bin W, Yang J, Dong J, Sun L, Xu X, Jin Q.

Author information: 
(1)State Key Laboratory for Molecular Virology and Genetic Engineering, Beijing
100176, China.

Comparative Genomic Hybridization (CGH) microarray analysis was used to compare
the genomic compositions of all eighteen Shigella boydii serotype representative 
strains. The results indicated the genomic "backbone" of this subgroup contained 
2552 ORFs homologous to nonpathogenic E. coli K12. Compared with the genome of
K12199 ORFs were found to be absent in all S. boydii serotype representatives,
including mainly outer membrane protein genes and O-antigen biosynthesis genes.
Yet the specific ORFs of S. boydii subgroup contained basically bacteriophage
genes and the function unknown (FUN) genes. Some iron metabolism, transport and
type II secretion system related genes were found in most representative strains.
According to the CGH phylogenetic analysis, the eighteen S. boydii serotype
representatives were divided into four groups, in which serotype C13 strain was
remarkably distinguished from the other serotype strains. This grouping result
corresponded to the distribution of some metabolism related genes. Furthermore,
the analysis of genome backbone genes, specific genes, and the phylogenetic trees
allowed us to discover the evolution laws of S. boydii and to find out important 
clues to pathogenesis research, vaccination and the therapeutic medicine
development.


PMID: 16544575  [Indexed for MEDLINE]


1031. Biochemistry. 2006 Jan 17;45(2):581-93.

Facilitating RNA structure prediction with microarrays.

Kierzek E(1), Kierzek R, Turner DH, Catrina IE.

Author information: 
(1)Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego
12/14, 61-704 Poznan, Poland.

Determining RNA secondary structure is important for understanding
structure-function relationships and identifying potential drug targets. This
paper reports the use of microarrays with heptamer 2'-O-methyl
oligoribonucleotides to probe the secondary structure of an RNA and thereby
improve the prediction of that secondary structure. When experimental constraints
from hybridization results are added to a free-energy minimization algorithm, the
prediction of the secondary structure of Escherichia coli 5S rRNA improves from
27 to 92% of the known canonical base pairs. Optimization of buffer conditions
for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding
and improve discrimination between AU and GU pairs are also described. The
results suggest that probing RNA with oligonucleotide microarrays can facilitate 
determination of secondary structure.

DOI: 10.1021/bi051409+ 
PMCID: PMC4070881
PMID: 16401087  [Indexed for MEDLINE]


1032. Biosens Bioelectron. 2006 Jan 15;21(7):989-97.

Manipulation and extraction of genomic DNA from cell lysate by functionalized
magnetic particles for lab on a chip applications.

Yeung SW(1), Hsing IM.

Author information: 
(1)Department of Chemical Engineering, Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon, Hong Kong.

A novel approach for extracting living cells' genomic DNA materials utilizing
functionalized magnetic particles (MPs) is reported in this investigation. This
strategy is amenable to handle bio-samples in a miniaturized environment and it
offers a possibility to separate and purify DNA from other cell lysate mixtures
"on-chip", which is known to be a bottle-neck step in an integrated
micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest
is captured by the MPs based on the hybridization interaction between the
biotinylated probes modified MPs and a complementary region of the targeted
genome. The genome DNA anchored on the particles can be separated from the rest
of cellular mixtures by a simple buffer washing upon the exertion of external
magnetic force. Surface modifications of MPs and hybridization conditions
affecting the genome capturing efficiency are investigated. Extraction of genomic
DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned
with a platinum heater and sensors. On-chip extraction and manipulation of
genomic DNAs illustrated in this study is a step forward toward a total
integrated bioanalytical microsystem for crude cells/sample analysis.

DOI: 10.1016/j.bios.2005.03.008 
PMID: 16368479  [Indexed for MEDLINE]


1033. Biosens Bioelectron. 2006 Jan 15;21(7):1178-85. Epub 2005 Jun 13.

A nanoparticle amplification based quartz crystal microbalance DNA sensor for
detection of Escherichia coli O157:H7.

Mao X(1), Yang L, Su XL, Li Y.

Author information: 
(1)Department of Biological and Agricultural Engineering, University of Arkansas,
Fayetteville, AR 72701, USA.

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle
amplification method, was developed for detection of Escherichia coli O157:H7. A 
thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene
was immobilized onto the QCM sensor surface through self-assembly. The
hybridization was induced by exposing the ssDNA probe to the complementary target
DNA, and resulted in the mass change and therefore frequency change of the QCM.
Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were
used as "mass enhancers" to amplify the frequency change. Synthesized
biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151
bases) amplified using asymmetric PCR with biotin labeled primers were tested. As
low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit
(CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation 
between frequency change and logarithmic number of bacterial cell concentration
was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.

DOI: 10.1016/j.bios.2005.04.021 
PMID: 15951163  [Indexed for MEDLINE]


1034. BMC Bioinformatics. 2006 Jan 12;7:17.

An approach for clustering gene expression data with error information.

Tjaden B(1).

Author information: 
(1)Computer Science Department, Wellesley College, Wellesley, MA 02481, USA.
btjaden@wellesley.edu

BACKGROUND: Clustering of gene expression patterns is a well-studied technique
for elucidating trends across large numbers of transcripts and for identifying
likely co-regulated genes. Even the best clustering methods, however, are
unlikely to provide meaningful results if too much of the data is unreliable.
With the maturation of microarray technology, a wealth of research on statistical
analysis of gene expression data has encouraged researchers to consider error and
uncertainty in their microarray experiments, so that experiments are being
performed increasingly with repeat spots per gene per chip and with repeat
experiments. One of the challenges is to incorporate the measurement error
information into downstream analyses of gene expression data, such as traditional
clustering techniques.
RESULTS: In this study, a clustering approach is presented which incorporates
both gene expression values and error information about the expression
measurements. Using repeat expression measurements, the error of each gene
expression measurement in each experiment condition is estimated, and this
measurement error information is incorporated directly into the clustering
algorithm. The algorithm, CORE (Clustering Of Repeat Expression data), is
presented and its performance is validated using statistical measures. By using
error information about gene expression measurements, the clustering approach is 
less sensitive to noise in the underlying data and it is able to achieve more
accurate clusterings. Results are described for both synthetic expression data as
well as real gene expression data from Escherichia coli and Saccharomyces
cerevisiae.
CONCLUSION: The additional information provided by replicate gene expression
measurements is a valuable asset in effective clustering. Gene expression
profiles with high errors, as determined from repeat measurements, may be
unreliable and may associate with different clusters, whereas gene expression
profiles with low errors can be clustered with higher specificity. Results
indicate that including error information from repeat gene expression
measurements can lead to significant improvements in clustering accuracy.

DOI: 10.1186/1471-2105-7-17 
PMCID: PMC1360687
PMID: 16409635  [Indexed for MEDLINE]


1035. Appl Environ Microbiol. 2006 Jan;72(1):946-9.

Butyrate specifically down-regulates salmonella pathogenicity island 1 gene
expression.

Gantois I(1), Ducatelle R, Pasmans F, Haesebrouck F, Hautefort I, Thompson A,
Hinton JC, Van Immerseel F.

Author information: 
(1)Department of Pathology, Bacteriology and Avian Diseases, Research Group
Veterinary Public Health and Zoonoses, Faculty of Veterinary Medicine, Ghent
University, Salisburylaan 133, B-9820 Merelbeke, Belgium. inne.gantois@UGent.be

Invasion of intestinal epithelial cells by Salmonella enterica is decreased after
exposure to butyric acid. To understand the molecular mechanisms of this
phenomenon, a comparative transcriptomic analysis of Salmonella enterica serovar 
Enteritidis and Salmonella enterica serovar Typhimurium grown in medium
supplemented with butyrate was performed. We found that butyrate down-regulated
the expression of 19 genes common to both serovars by a factor of twofold or
more, and 17 of these genes localized to the Salmonella pathogenicity island 1
(SPI1). These included the SPI1 regulatory genes hilD and invF. Of the remaining 
two genes, ampH has 91% homology to an Escherichia coli penicillin-binding
protein and sopE2 encodes a type III-secreted effector protein associated with
invasion but located at a separate site on the chromosome from SPI1.

DOI: 10.1128/AEM.72.1.946-949.2006 
PMCID: PMC1352287
PMID: 16391141  [Indexed for MEDLINE]


1036. Curr Microbiol. 2006 Jan;52(1):50-4. Epub 2006 Jan 2.

Gene expression profiling of intrinsic thermotolerance in Escherichia coli.

Friedman SM(1), Hossain M, Hasson TH, Kawamura A.

Author information: 
(1)Department of Biological Sciences, Hunter College of The City University of
New York, 695 Park Avenue, New York, NY 10021, USA.
friedman@genectr.hunter.cuny.edu

DNA microarrays were employed to compare gene expression in a thermotolerant,
nalidixic acid-resistant mutant of Escherichia coli with that of the parental
strain. When grown at 37 degrees C, up-regulated genes in the mutant included
those coding for multiple antibiotic resistance proteins and enzymes for the
degradation of small molecules, whereas among the down-regulated genes were those
coding for fimbrial, flagellar, and outer membrane proteins as well as sigma 38. 
When the mutant grown at 42 degrees C was compared to the mutant grown at 37
degrees C, enhanced expression of several genes coding for flagellar proteins was
detected. Reverse transcriptase-polymerase chain reaction analysis of selected
genes confirmed results obtained with microarrays.

DOI: 10.1007/s00284-005-4578-6 
PMID: 16392005  [Indexed for MEDLINE]


1037. Dev Biol (Basel). 2006;126:213-8; discussion 326-7.

Development of a new integrated diagnostic test for identification and
characterization of pathogens.

Martinez G(1), Bruant G, Brousseau R, Masson L, Harel J.

Author information: 
(1)Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine
vétérinaire, Université de Montreal, Saint-Hyacinthe, Qc, Canada.

Animal diseases directly cause multi-million dollar losses world-wide. Therefore 
a rapid, highly specific, cost-effective diagnostic test for detecting a large
set of bacterial virulence and antimicrobial resistance genes simultaneously is
necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group,
proposes developing a powerful molecular tool (DNA microarray) to detect a broad 
range of infectious agents, their endogenous main virulence factors and
antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide
microarray capable of detecting the presence or absence of 169 Escherichia coli
virulence genes or virulence marker genes as well as their variants, in addition 
to 30 principal antimicrobial resistance genes previously characterized in E.
coli strains was developed by our group. This microarray was validated with a
large collection of well characterized pathogenic and reference E. coli strains. 
Moreover, we are developing a new powerful clinical diagnostic microarray tool,
to identify pathogenic bacteria of veterinary interest. The commercialization of 
this assay would allow same day diagnosis of infectious agents and their
antibiotic resistance resulting in early treatment. In addition, this technology 
is also applicable to microbial quality control of food and water.


PMID: 17058497  [Indexed for MEDLINE]


1038. Genome Biol. 2006;7(6):R46.

Analysis of gene expression in operons of Streptomyces coelicolor.

Laing E(1), Mersinias V, Smith CP, Hubbard SJ.

Author information: 
(1)Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT,
UK.

BACKGROUND: Recent studies have shown that microarray-derived gene-expression
data are useful for operon prediction. However, it is apparent that genes within 
an operon do not conform to the simple notion that they have equal levels of
expression.
RESULTS: To investigate the relative transcript levels of intra-operonic genes,
we have used a Z-score approach to normalize the expression levels of all genes
within an operon to expression of the first gene of that operon. Here we
demonstrate that there is a general downward trend in expression from the first
to the last gene in Streptomyces coelicolor operons, in contrast to what we
observe in Escherichia coli. Combining transcription-factor binding-site
prediction with the identification of operonic genes that exhibited higher
transcript levels than the first gene of the same operon enabled the discovery of
putative internal promoters. The presence of transcription terminators and
abundance of putative transcriptional control sequences in S. coelicolor operons 
are also described.
CONCLUSION: Here we have demonstrated a polarity of expression in operons of S.
coelicolor not seen in E. coli, bringing caution to those that apply operon
prediction strategies based on E. coli 'equal-expression' to divergent species.
We speculate that this general difference in transcription behavior could reflect
the contrasting lifestyles of the two organisms and, in the case of Streptomyces,
might also be influenced by its high G+C content genome. Identification of
putative internal promoters, previously thought to cause problems in operon
prediction strategies, has also been enabled.

DOI: 10.1186/gb-2006-7-6-r46 
PMCID: PMC1779546
PMID: 16749941  [Indexed for MEDLINE]


1039. Genome Biol. 2006;7(4):R32. Epub 2006 Apr 20.

A classification based framework for quantitative description of large-scale
microarray data.

Sangurdekar DP(1), Srienc F, Khodursky AB.

Author information: 
(1)Department of Chemical Engineering and Materials Science, University of
Minnesota, Saint Paul, MN 55108, USA.

Genome-wide surveys of transcription depend on gene classifications for the
purpose of data interpretation. We propose a new information-theoretical-based
method to: assess significance of co-expression within any gene group;
quantitatively describe condition-specific gene-class activity; and
systematically evaluate conditions in terms of gene-class activity. We applied
this technique to describe microarray data tracking Escherichia coli
transcriptional responses to more than 30 chemical and physiological
perturbations. We correlated the nature and breadth of the responses with the
nature of perturbation, identified gene group proxies for the perturbation
classes and quantitatively compared closely related physiological conditions.

DOI: 10.1186/gb-2006-7-4-r32 
PMCID: PMC1557986
PMID: 16626502  [Indexed for MEDLINE]


1040. Genome Res. 2006 Jan;16(1):132-9. Epub 2005 Dec 12.

Development of a microscopic platform for real-time monitoring of biomolecular
interactions.

Sasuga Y(1), Tani T, Hayashi M, Yamakawa H, Ohara O, Harada Y.

Author information: 
(1)The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo
113-8613, Japan.

We developed a new microscopic platform for the real-time analysis of molecular
interactions by combining microbead-tagging techniques with total internal
reflection fluorescent microscopy (TIRFM). The optical manipulation of probe
microbeads, followed by photo immobilization on a solid surface, enabled us to
generate arrays with extremely high density (>100 microbeads in a 25 microm x 25 
microm area), and TIRFM made it possible to monitor the binding reactions of
fluorescently labeled targets onto probe microbeads without removal of free
targets. We demonstrated the high performance of this platform through analyses
of interactions between antigen and antibody and between small compounds and
proteins. Then, recombinant protein levels in total cellular lysates of
Escherichia coli were quantified from the association kinetics using
antibody-immobilized microbead arrays, which served as a model for a
protein-profiling array. Furthermore, in combination with in vitro
synthesis-coupled protein labeling, we could kinematically analyze the
interaction of nuclear factor kappaB (p50) with DNA. These results demonstrated
that this platform enabled us to: (1) monitor binding processes of fluorescently 
labeled targets to multiple probes in real-time without removal of free targets, 
(2) determine concentrations of free targets only from the association kinetics
at an early phase, and (3) greatly reduce the required volume of the target
solution, in principle to subnanoliter, for molecular interaction analysis. The
unique features of this microbead-based microarray system open the way to explore
molecular interactions with a wide range of affinities in extremely small volumes
of target solutions, such as extracts from single cells.

DOI: 10.1101/gr.4235806 
PMCID: PMC1356137
PMID: 16344567  [Indexed for MEDLINE]


1041. Infect Immun. 2006 Jan;74(1):615-24.

The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes
uropathogenic E. coli strains in human urine.

Roos V(1), Ulett GC, Schembri MA, Klemm P.

Author information: 
(1)Microbial Adhesion Group, Center for Biomedical Microbiology, BioCentrum-DTU, 
Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.

Escherichia coli is the most common organism associated with asymptomatic
bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes
symptomatic urinary tract infections (UTI), very little is known about the
mechanisms by which these strains colonize the human urinary tract. The prototype
ABU E. coli strain 83972 was originally isolated from a girl who had carried it
asymptomatically for 3 years. Deliberate colonization of UTI-susceptible
individuals with E. coli 83972 has been used successfully as an alternative
approach for the treatment of patients who are refractory to conventional
therapy. Colonization with strain 83972 appears to prevent infection with UPEC
strains in such patients despite the fact that this strain is unable to express
the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we
investigated the growth characteristics of E. coli 83972 in human urine and show 
that it can outcompete a representative spectrum of UPEC strains for growth in
urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was
also demonstrated in a murine model of human UTI, confirming the selective
advantage over UPEC in vivo. Comparison of global gene expression profiles of E. 
coli 83972 grown in lab medium and human urine revealed significant differences
in expression levels in the two media; significant down-regulation of genes
encoding virulence factors such as hemolysin, lipid A, and capsular
polysaccharides was observed in cells grown in urine. Clearly, divergent
abilities of ABU E. coli and UPEC to exploit human urine as a niche for
persistence and survival suggest that these key differences may be exploited for 
preventative and/or therapeutic approaches.

DOI: 10.1128/IAI.74.1.615-624.2006 
PMCID: PMC1346649
PMID: 16369018  [Indexed for MEDLINE]


1042. J Appl Microbiol. 2006;100(1):15-28.

Assessing genetic diversity in plasmids from Escherichia coli and Salmonella
enterica using a mixed-plasmid microarray.

Call DR(1), Kang MS, Daniels J, Besser TE.

Author information: 
(1)Department of Veterinary Microbiology and Pathology, Washington State
University, WA 99164-7040, USA. drcall@wsu.edu

AIMS: To compare genetic composition of plasmids using microarrays composed of
randomly selected fragments of plasmid DNA.
METHODS AND RESULTS: Separate shotgun libraries were constructed from plasmid DNA
pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used 
as probes for microarrays. Plasmid targets were labelled, hybridized overnight,
and bound targets were imaged after enzymatic signal amplification. Control
hybridizations demonstrated significantly higher signal when probes and targets
shared >95% sequence identity. Diagnostic sensitivity and specificity of the
assay was 95 and 99%, respectively. Cluster analysis showed close matches for
replicate experiments with a high correlation between replicates (r = 0.91)
compared with the correlation for nonreplicates (r = 0.09). Analysis of
hybridization data from 43 plasmids generated five distinct clusters, two for
known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and 
two for plasmids harboring a recently disseminated antibiotic resistance gene
(bla(CMY-2)).
CONCLUSION: Mixed-plasmid microarrays are suitable for comparing genetic content 
of wild-type plasmids and hybridization results from this study suggest several
novel hypotheses about plasmid gene exchange between E. coli and S. enterica.
SIGNIFICANCE AND IMPACT OF STUDY: Mixed-plasmid microarrays permit rapid, low
cost analysis and comparison of many plasmids. This ability is critical to
understanding the source, fate, and transport of plasmids amongst commensal and
pathogenic bacteria.

DOI: 10.1111/j.1365-2672.2005.02775.x 
PMID: 16405681  [Indexed for MEDLINE]


1043. Methods Enzymol. 2006;410:342-59.

DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine 
methyltransferase.

Greil F(1), Moorman C, van Steensel B.

Author information: 
(1)Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The
Netherlands.

A large variety of proteins bind to specific parts of the genome to regulate gene
expression, DNA replication, and chromatin structure. DamID is a powerful method 
used to map the genomic interaction sites of these proteins in vivo. It is based 
on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase
(dam). Expression of this fusion protein in vivo leads to preferential
methylation of adenines in DNA surrounding the native binding sites of the dam
fusion partner. Because adenine methylation does not occur endogenously in most
eukaryotes, it provides a unique tag to mark protein interaction sites. The
adenine-methylated DNA fragments are isolated by selective polymerase chain
reaction amplification and can be identified by microarray hybridization. We and 
others have successfully applied DamID to the genome-wide identification of
interaction sites of several transcription factors and other chromatin-associated
proteins. This chapter discusses DamID technology in detail, and a step-by-step
experimental protocol is provided for use in Drosophila cell lines.

DOI: 10.1016/S0076-6879(06)10016-6 
PMID: 16938559  [Indexed for MEDLINE]


1044. Mol Microbiol. 2006 Jan;59(2):551-66.

Transcriptional regulation of fatty acid biosynthesis in Streptococcus
pneumoniae.

Lu YJ(1), Rock CO.

Author information: 
(1)Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, TN
38105-2794, USA.

The transcriptional regulation of membrane fatty acid composition in the human
pathogen Streptococcus pneumoniae is distinct from the systems utilized in the
model organisms Escherichia coli and Bacillus subtilis. The genes encoding the
components of type II fatty acid biosynthesis cluster at a single location within
the S. pneumoniae genome, and the second gene in this cluster (SPR0376) encodes a
transcription factor (FabT) that belongs to the MarR superfamily. Derivatives of 
S. pneumoniae strain D39 were constructed that lacked functional FabT. This
strain had significantly elevated levels of saturated fatty acids and longer
chain lengths than the control strain, was unable to grow at pH 5.5 and had
increased sensitivity to detergent. Eliminating FabT function increased the
expression levels of all of fab genes with the notable exception of fabM. FabT
was purified and bound to the DNA palindrome located within the promoter regions 
of the fabT and fabK genes within the cluster. The analysis of cells with
increased expression of individual genes leads to a model where the physical
properties of the S. pneumoniae membrane is controlled primarily by the activity 
of FabK, the enoyl reductase, which diverts intermediates to saturated fatty acid
formation, in contrast to E. coli where FabB, an elongation condensing enzyme,
pulls the pathway in the direction of unsaturated acid synthesis.

DOI: 10.1111/j.1365-2958.2005.04951.x 
PMID: 16390449  [Indexed for MEDLINE]


1045. Mol Microbiol. 2006 Jan;59(2):528-40.

The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in
Bacillus subtilis.

Uicker WC(1), Schaefer L, Britton RA.

Author information: 
(1)Department of Microbiology and Molecular Genetics, Michigan State University, 
East Lansing, 48824, USA.

In this paper the essential GTPase YlqF is shown to participate in the biogenesis
of the 50S ribosomal subunit in Bacillus subtilis. Cells depleted of YlqF
displayed gene expression profiles and nucleoid morphologies that were consistent
with a function for YlqF in translation. In addition, YlqF is evolutionarily
linked to two eukaryotic GTPases, Nog2p and Nug1p, that are involved in the
biogenesis and the nuclear export of the 60S ribosomal subunit. Analysis of
ribosomes from cells depleted of YlqF demonstrated that the formation of 70S
ribosomes was greatly reduced and the large subunit sedimented at 45S. Cells
grown with varying depleted levels of YlqF, yielding doubling times ranging from 
38 min to 150 min, all displayed the 45S intermediate. Purified YlqF-His(6)
protein associates with the 45S intermediate, but not the mature 50S subunit in
vitro. Analysis of proteins from the 45S intermediate indicated that ribosomal
protein L16, which is added late during in vitro Escherichia coli 50S ribosome
biogenesis, was missing from the 45S intermediate. These results support a model 
in which YlqF participates in the formation of active 70S ribosomes in the cell
by functioning in a late step of 50S subunit biogenesis. Based on these results
we propose to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).

DOI: 10.1111/j.1365-2958.2005.04948.x 
PMID: 16390447  [Indexed for MEDLINE]


1046. Nucleic Acids Res. 2006;34(19):5695-704. Epub 2006 Oct 12.

The majority of Escherichia coli mRNAs undergo post-transcriptional modification 
in exponentially growing cells.

Mohanty BK(1), Kushner SR.

Author information: 
(1)Department of Genetics, University of Georgia, Athens, GA 30602, USA.

Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays
a significant role in mRNA decay and general RNA quality control. However, many
important features of this system, including the prevalence of polyadenylated
mRNAs in the bacterium, are still poorly understood. By comparing the
transcriptomes of wild-type and pcnB deletion strains using macroarray analysis, 
we demonstrate that >90% of E.coli open reading frames (ORFs) transcribed during 
exponential growth undergo some degree of polyadenylation by PAP I, either as
full-length transcripts or decay intermediates. Detailed analysis of over 240
transcripts suggests that Rho-independent transcription terminators serve as
polyadenylation signals. Conversely, mRNAs terminated in a Rho-dependent fashion 
are probably not substrates for PAP I, but can be modified by the addition of
long polynucleotide tails through the biosynthetic activity of polynucleotide
phosphorylase (PNPase). Furthermore, real-time PCR analysis indicates that the
extent of polyadenylation of individual full-length transcripts such as lpp and
ompA varies significantly in wild-type cells. The data presented here
demonstrates that polyadenylation in E.coli occurs much more frequently than
previously envisioned.

DOI: 10.1093/nar/gkl684 
PMCID: PMC1636475
PMID: 17040898  [Indexed for MEDLINE]


1047. Nucleic Acids Res. 2006;34(18):e118. Epub 2006 Sep 25.

A DNA biochip for on-the-spot multiplexed pathogen identification.

Yeung SW(1), Lee TM, Cai H, Hsing IM.

Author information: 
(1)Department of Chemical Engineering, The Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon, Hong Kong.

Miniaturized integrated DNA analysis systems have largely been based on a
multi-chamber design with microfluidic control to process the sample sequentially
from one module to another. This microchip design in connection with optics
involved hinders the deployment of this technology for point-of-care
applications. In this work, we demonstrate the implementation of sample
preparation, DNA amplification, and electrochemical detection in a single silicon
and glass-based microchamber and its application for the multiplexed detection of
Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film
heater and temperature sensor patterned on the silicon substrate. An array of
indium tin oxide (ITO) electrodes was constructed within the microchamber as the 
transduction element. Oligonucleotide probes specific to the target amplicons are
individually positioned at each ITO surface by electrochemical copolymerization
of pyrrole and pyrrole-probe conjugate. These immobilized probes were stable to
the thermal cycling process and were highly selective. The DNA-based
identification of the two model pathogens involved a number of steps including a 
thermal lysis step, magnetic particle-based isolation of the target genomes,
asymmetric PCR, and electrochemical sequence-specific detection using
silver-enhanced gold nanoparticles. The microchamber platform described here
offers a cost-effective and sample-to-answer technology for on-site monitoring of
multiple pathogens.

DOI: 10.1093/nar/gkl702 
PMCID: PMC1636451
PMID: 17000638  [Indexed for MEDLINE]


1048. Nucleic Acids Res. 2006;34(16):4642-52. Epub 2006 Sep 8.

Association of nucleoid proteins with coding and non-coding segments of the
Escherichia coli genome.

Grainger DC(1), Hurd D, Goldberg MD, Busby SJ.

Author information: 
(1)School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15
2TT, UK.

The Escherichia coli chromosome is condensed into an ill-defined structure known 
as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in
maintaining this structure and in mediating chromosome compaction. We have
exploited chromatin immunoprecipitation and high-density microarrays to study the
binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in
vivo. Our results show that the distribution of these proteins is biased to
intergenic parts of the genome, and that the binding profiles overlap. Hence some
targets are associated with combinations of bound FIS, H-NS and IHF. In addition,
many regions associated with FIS and H-NS are also associated with RNA
polymerase.

DOI: 10.1093/nar/gkl542 
PMCID: PMC1636352
PMID: 16963779  [Indexed for MEDLINE]


1049. Water Sci Technol. 2006;54(1):247-55.

Effect of phosphorus limitation on microbial floc structure and gene expression
in activated sludge.

Liu JR(1), Liu CT, Edwards EA, Liss SN.

Author information: 
(1)Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, 
Toronto, Ontario, Canada M5B 2K3.

The effect of limiting phosphorus (P) in activated sludge was investigated in
laboratory-scale sequencing batch reactors (SBRs). Correlative microscopy
revealed that P-limitation (COD:N:P = 100:5:0.05) leads to morphological changes 
in floc structure and the composition of extracellular polymeric substances
(EPS). This was found to be accompanied by expression of quorum-sensing in an
acyl homoserine lactone bioassay. Differential gene expression in relation to
P-limitation was examined in a global profile using the Affymetrix Escherichia
coli antisense genomic microarray. Three separate experiments were conducted
where the impact of P-limitation was examined under batch conditions and in SBRs 
at stable operating conditions and within 3-7 days following a down-shift in P.
Significant changes in open reading frames (ORF) and intergenic regions based on 
the E. coli microarray were observed. Several genes associated with cell
structure, including slt, wbbH, fimH, amB, rfaJ and slp were found to be
expressed. Quorum regulated genes were also found to be expressed including psiF 
which is known to be induced by P-starvation (92% confidence level; 1.45 log
ratio).


PMID: 16898158  [Indexed for MEDLINE]


1050. World J Gastroenterol. 2005 Dec 28;11(48):7615-9.

Detection and identification of intestinal pathogenic bacteria by hybridization
to oligonucleotide microarrays.

Jin LQ(1), Li JW, Wang SQ, Chao FH, Wang XW, Yuan ZQ.

Author information: 
(1)Tianjin Institute of Hygiene and Environmental Medicine 1, Da Li Road, Tianjin
300050, China. jinlianqun@sina.com

AIM: To detect the common intestinal pathogenic bacteria quickly and accurately.
METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene
chip technology. Target genes were amplified and hybridized by oligonucleotide
microarrays.
RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to
11 genera were successfully discriminated under comparatively same conditions,
and a series of specific hybridization maps corresponding to each kind of
bacteria were obtained. When this method was applied to 26 divided cultures, 25
(96.2%) were identified.
CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria
monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp.,
Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica,
and Campylobacter jejuni can be detected and identified by our microarrays. The
accuracy, range, and discrimination power of this assay can be continually
improved by adding further oligonucleotides to the arrays without any significant
increase of complexity or cost.


PMCID: PMC4727218
PMID: 16437687  [Indexed for MEDLINE]


1051. Proc Natl Acad Sci U S A. 2005 Dec 27;102(52):19103-8. Epub 2005 Dec 15.

The global transcriptional regulatory network for metabolism in Escherichia coli 
exhibits few dominant functional states.

Barrett CL(1), Herring CD, Reed JL, Palsson BO.

Author information: 
(1)Bioengineering Department, University of California at San Diego, 9500 Gilman 
Drive, La Jolla, CA 92093-0412, USA.

A principal aim of systems biology is to develop in silico models of whole cells 
or cellular processes that explain and predict observable cellular phenotypes.
Here, we use a model of a genome-scale reconstruction of the integrated metabolic
and transcriptional regulatory networks for Escherichia coli, composed of 1,010
gene products, to assess the properties of all functional states computed in
15,580 different growth environments. The set of all functional states of the
integrated network exhibits a discernable structure that can be visualized in
3-dimensional space, showing that the transcriptional regulatory network
governing metabolism in E. coli responds primarily to the available electron
acceptor and the presence of glucose as the carbon source. This result is
consistent with recently published experimental data. The observation that a
complex network composed of 1,010 genes is organized to achieve few dominant
modes demonstrates the utility of the systems approach for consolidating large
amounts of genome-scale molecular information about a genome and its regulation
to elucidate an organism's preferred environments and functional capabilities.

DOI: 10.1073/pnas.0505231102 
PMCID: PMC1323155
PMID: 16357206  [Indexed for MEDLINE]


1052. Anal Chem. 2005 Dec 15;77(24):8020-6.

Screening of target-specific stress-responsive genes for the development of
cell-based biosensors using a DNA microarray.

Kim BC(1), Youn CH, Ahn JM, Gu MB.

Author information: 
(1)Advanced Environmental Monitoring Research Center (ADEMRC), Gwangju Institute 
of Science and Technology (GIST), 1, Oryoung-dong, Puk-gu, Gwangju 500-712,
Republic of Korea.

In this study, we describe a straightforward strategy to develop whole cell-based
biosensors using fusions of the bacterial bioluminescence genes and the promoters
from chemically responsive genes within Escherichia coli, in which chemical
target-responsive genes were screened by using the information of gene expression
data obtained from DNA microarray analysis. Paraquat was used as a model chemical
to trigger gene expression changes of E. coli and to show the DNA
microarray-assisted development of whole cell-based biosensors. Gene expression
data from the DNA microarray were obtained by time course analysis (10, 30, and
60 min) after exposure to paraquat. After clustering gene expression data
obtained by time course analysis, a group of highly expressed genes over the all 
time courses could be classified. Within this group, three genes expressed highly
for overall time points were selected and promoters of these genes were used as
fusion partners with reporter genes, lux CDABE, to construct whole cell-based
biosensors. The constructed biosensors recognized the presence of model inducer, 
paraquat, and structural analogue chemicals of paraquat with a high specificity, 
and the results were reconfirmed by using DNA microarray experiments for those
structural analogues. This strategy to develop whole cell-based biosensors
assisted by DNA microarray information should be useful in general for
constructing chemical-specific or stress-specific biosensors with a
high-throughput manner.

DOI: 10.1021/ac0514218 
PMID: 16351151  [Indexed for MEDLINE]


1053. Appl Environ Microbiol. 2005 Dec;71(12):8548-57.

Waterborne pathogen detection by use of oligonucleotide-based microarrays.

Maynard C(1), Berthiaume F, Lemarchand K, Harel J, Payment P, Bayardelle P,
Masson L, Brousseau R.

Author information: 
(1)National Research Council of Canada, Biotechnology Research Institute, 6100
Ave. Royalmount, Montreal, Quebec, Canada H4P 2R2.

A small-oligonucleotide microarray prototype was designed with probes specific
for the universal 16S rRNA and cpn60 genes of several pathogens that are usually 
encountered in wastewaters. In addition to these two targets, wecE-specific
oligonucleotide probes were included in the microarray to enhance its
discriminating power within the Enterobacteriaceae family. Universal PCR primers 
were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly
in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA
mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia
enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified
products were fluorescently labeled and hybridized on the prototype chip. The
detection sensitivity for S. enterica serovar Typhimurium was estimated to be on 
the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the
combination of PCR followed by microarray hybridization. The sensitivity of the
prototype could be increased by hybridizing amplicons generated by PCR targeting 
genes specific for a bacterial subgroup, such as wecE genes, instead of universal
taxonomic amplicons. However, there was evidence of PCR bias affecting the
detection limits of a given pathogen as increasing amounts of a different
pathogen were spiked into the test samples. These results demonstrate the
feasibility of using DNA microarrays in the detection of waterborne pathogens
within mixed populations but also raise the problem of PCR bias in such
experiments.

DOI: 10.1128/AEM.71.12.8548-8557.2005 
PMCID: PMC1317427
PMID: 16332846  [Indexed for MEDLINE]


1054. Biol Chem. 2005 Dec;386(12):1219-38.

How to find small non-coding RNAs in bacteria.

Vogel J(1), Sharma CM.

Author information: 
(1)Max Planck Institute for Infection Biology, RNA Biology, Schumannstr. 21/22,
D-10117 Berlin, Germany. vogel@mpiib-berlin.mpg.de

Small non-coding RNAs (sRNAs) have attracted considerable attention as an
emerging class of gene expression regulators. In bacteria, a few regulatory RNA
molecules have long been known, but the extent of their role in the cell was not 
fully appreciated until the recent discovery of hundreds of potential sRNA genes 
in the bacterium Escherichia coli. Orthologs of these E. coli sRNA genes, as well
as unrelated sRNAs, were also found in other bacteria. Here we review the
disparate experimental approaches used over the years to identify sRNA molecules 
and their genes in prokaryotes. These include genome-wide searches based on the
biocomputational prediction of non-coding RNA genes, global detection of
non-coding transcripts using microarrays, and shotgun cloning of small RNAs
(RNomics). Other sRNAs were found by either co-purification with RNA-binding
proteins, such as Hfq or CsrA/RsmA, or classical cloning of abundant small RNAs
after size fractionation in polyacrylamide gels. In addition, bacterial genetics 
offers powerful tools that aid in the search for sRNAs that may play a critical
role in the regulatory circuit of interest, for example, the response to stress
or the adaptation to a change in nutrient availability. Many of the techniques
discussed here have also been successfully applied to the discovery of eukaryotic
and archaeal sRNAs.

DOI: 10.1515/BC.2005.140 
PMID: 16336117  [Indexed for MEDLINE]


1055. FEMS Microbiol Lett. 2005 Dec 1;253(1):119-24. Epub 2005 Oct 10.

Use of a microarray to assess the distribution of plasmid and chromosomal
virulence genes in strains of enteroaggregative Escherichia coli.

Jenkins C(1), van Ijperen C, Dudley EG, Chart H, Willshaw GA, Cheasty T, Smith
HR, Nataro JP.

Author information: 
(1)Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London
NW9 5HT, UK. Claire.Jenkins@royalfree.nhs.uk

A DNA microarray was used to analyze the distribution of plasmid and chromosomal 
genes among strains of enteroaggregative Escherichia coli (EAEC) isolated from a 
prospective diarrhoea surveillance study in the United Kingdom. Target genes were
extracted from existing databases and from the genome sequence of prototype EAEC 
strain 042. We found that strains exhibiting the aggregative adherence (AA)
phenotype could be broadly divided into two groups depending upon whether they
harboured genes from the EAEC virulence plasmid (pAA) and a set of chromosomal
genes found in EAEC strain 042. Several chromosomal loci were inherited en bloc, 
and were more common in strains which we designated Group 1; genes at the pheU
locus were particularly conserved. Genes encoded on the pAA plasmid and those
under control of the master regulator AggR were also concentrated in the Group 1 
EAEC. A gene encoding a type 1 pilin allele was detected more frequently in Group
2 EAEC. Our data suggest that strains previously designated as typical EAEC
harbour a large number of conserved plasmid and chromosomal loci, further
illuminating a package of virulence genes common to the most important EAEC.

DOI: 10.1016/j.femsle.2005.09.040 
PMID: 16243450  [Indexed for MEDLINE]


1056. Infect Immun. 2005 Dec;73(12):7894-905.

Identification of core and variable components of the Salmonella enterica
subspecies I genome by microarray.

Anjum MF(1), Marooney C, Fookes M, Baker S, Dougan G, Ivens A, Woodward MJ.

Author information: 
(1)Department of Food and Environmental Safety, Veterinary Laboratories
Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
m.anjum@vla.defra.gsi.gov.uk

We have performed microarray hybridization studies on 40 clinical isolates from
12 common serovars within Salmonella enterica subspecies I to identify the
conserved chromosomal gene pool. We were able to separate the core invariant
portion of the genome by a novel mathematical approach using a decision tree
based on genes ranked by increasing variance. All genes within the core component
were confirmed using available sequence and microarray information for S.
enterica subspecies I strains. The majority of genes within the core component
had conserved homologues in Escherichia coli K-12 strain MG1655. However, many
genes present in the conserved set which were absent or highly divergent in K-12 
had close homologues in pathogenic bacteria such as Shigella flexneri and
Pseudomonas aeruginosa. Genes within previously established virulence
determinants such as SPI1 to SPI5 were conserved. In addition several genes
within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were
conserved within all S. enterica strains included in this study. Although many
phage and insertion sequence elements were missing from the core component,
approximately half the pseudogenes present in S. enterica serovar Typhi were
conserved. Furthermore, approximately half the genes conserved in the core set
encoded hypothetical proteins. Separation of the core and variant gene sets
within S.enterica subspecies I has offered fundamental biological insight into
the genetic basis of phenotypic similarity and diversity across S. enterica
subspecies I and shown how the core genome of these pathogens differs from the
closely related E. coli K-12 laboratory strain.

DOI: 10.1128/IAI.73.12.7894-7905.2005 
PMCID: PMC1307019
PMID: 16299280  [Indexed for MEDLINE]


1057. J Bacteriol. 2005 Dec;187(24):8350-60.

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic
expression profiling.

Wang L(1), Li J, March JC, Valdes JJ, Bentley WE.

Author information: 
(1)Center for Biosystems Research, University of Maryland Biotechnology
Institute, College Park, MD 20742, USA.

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest
because the gene for its synthase, luxS, is common among a large number of
bacterial species. We have identified luxS-controlled genes in Escherichia coli
under two different growth conditions using DNA microarrays. Twenty-three genes
were affected by luxS deletion in the presence of glucose, and 63 genes were
influenced by luxS deletion in the absence of glucose. Minimal overlap among
these gene sets suggests the role of luxS is condition dependent. Under the
latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of 
the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly
induced genes by luxS. The E. coli lsr operon includes an additional gene, tam,
encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and
lsrK belong to the same operon, lsrRK, which is positively regulated by the
cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is
additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS
was also shown to affect genes involved in methionine biosynthesis, methyl
transfer reactions, iron uptake, and utilization of carbon. It was surprising,
however, that so few genes were affected by luxS deletion in this E. coli K-12
strain under these conditions. Most of the highly induced genes are related to
AI-2 production and transport. These data are consistent with the function of
LuxS as an important metabolic enzyme but appear not to support the role of AI-2 
as a true signal molecule for E. coli W3110 under the investigated conditions.

DOI: 10.1128/JB.187.24.8350-8360.2005 
PMCID: PMC1316998
PMID: 16321939  [Indexed for MEDLINE]


1058. Plant Physiol. 2005 Dec;139(4):1597-611.

Genome analysis and functional characterization of the E2 and RING-type E3 ligase
ubiquitination enzymes of Arabidopsis.

Kraft E(1), Stone SL, Ma L, Su N, Gao Y, Lau OS, Deng XW, Callis J.

Author information: 
(1)Section of Molecular and Cellular Biology, Division of Biological Sciences ,
University of California, Davis, California 95616, USA.

Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes
E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one
functional proteins with a UBC domain and active-site cysteine are predicted in
the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are
predicted or shown to function with ubiquitin-like proteins. Only nine were
previously characterized biochemically as ubiquitin E2s. We obtained soluble
protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia
coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three
previously characterized ubiquitin E2s, were also tested for the ability to
catalyze ubiquitination in vitro in the presence of one of 65 really interesting 
new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of
E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely
related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and 
with all RING types. Expression analysis was performed to determine whether E2s
or E3s were expressed in specific organs or under specific environmental
conditions. Closely related E2s show unique patterns of expression and most
express ubiquitously. Some RING E3s are also ubiquitously expressed; however,
others show organ-specific expression. Of all the organs tested, RING mRNAs are
most abundant in floral organs. This study demonstrates that E2 diversity
includes examples with broad and narrow specificity toward RINGs, and that most
ubiquitin E2s are broadly expressed with each having a unique spatial and
developmental pattern of expression.

DOI: 10.1104/pp.105.067983 
PMCID: PMC1310545
PMID: 16339806  [Indexed for MEDLINE]


1059. Cell Microbiol. 2005 Nov;7(11):1565-78.

Aggregative adherence fimbriae contribute to the inflammatory response of
epithelial cells infected with enteroaggregative Escherichia coli.

Harrington SM(1), Strauman MC, Abe CM, Nataro JP.

Author information: 
(1)Department of Microbiology, The University of Maryland, Baltimore, MD 21201,
USA.

Enteroaggregative Escherichia coli (EAEC) causes watery diarrhoea that is often
mildly inflammatory. Previous studies have reported that the flagellin of EAEC
induces IL-8 from intestinal epithelial cells (IECs) in culture. To characterize 
more fully the inflammatory response to EAEC, we infected IECs with EAEC
prototype strain 042 and assessed cellular responses by macroarray and reverse
transcriptase polymerase chain reaction (RT-PCR). Genes upregulated in
042-infected non-polarized T84 cells included IL-8, IL-6, TNF-alpha, GRO-alpha,
GRO-gamma, ICAM-1, GM-CSF and IL-1alpha. RT-PCR analyses performed with cDNA from
T84 and HT-29 cells infected with an aflagellar mutant (042fliC) suggested that
these responses were primarily mediated by flagellin. To better reproduce the
conditions of the infection for this non-invasive pathogen, we assessed the
responses of polarized IECs to strain 042 infection. As expected, 042 induced
IL-8 production from both polarized T84 and HT-29 cells. However, significant
IL-8 secretion was induced in polarized T84 cells infected with 042fliC,
suggesting that a factor other than flagellin contributes to inflammation in this
model. This non-flagellar IL-8 response required expression of the aggregative
adherence fimbria (AAF) adhesin, and was related to the presence of the minor
fimbria-associated protein AafB. Our data suggest that multiple factors
contribute to EAEC-induced inflammation, and further characterization of the
nature of EAEC proinflammatory factors will greatly advance our understanding of 
this emerging pathogen.

DOI: 10.1111/j.1462-5822.2005.00588.x 
PMID: 16207244  [Indexed for MEDLINE]


1060. Infect Immun. 2005 Nov;73(11):7588-96.

Coordinate expression of fimbriae in uropathogenic Escherichia coli.

Snyder JA(1), Haugen BJ, Lockatell CV, Maroncle N, Hagan EC, Johnson DE, Welch
RA, Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, 5641 Medical Science Building II, 1150 West Medical Center Drive, Ann
Arbor, MI 48109, USA.

Uropathogenic Escherichia coli is the most common etiological agent of urinary
tract infections. Bacteria can often express multiple adhesins during infection
in order to favor attachment to specific niches within the urinary tract. We have
recently demonstrated that type 1 fimbria, a phase-variable virulence factor
involved in adherence, was the most highly expressed adhesin during urinary tract
infection. Here, we examine whether the expression of type 1 fimbriae can affect 
the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli
strain CFT073, which harbors genes for numerous adhesins, were employed in this
study. CFT073-specific DNA microarray analysis of these strains demonstrates that
the expression of type 1 fimbriae coordinately affects the expression of P
fimbriae in an inverse manner. This represents evidence for direct communication 
between genes relating to pathogenesis, perhaps to aid the sequential occupation 
of different urinary tract tissues. While the role of type 1 fimbriae during
infection has been clear, the role of P fimbriae must be further defined to
assert the relevance of coordinated regulation in vivo. Therefore, we examined
the ability of P fimbrial isogenic mutants, constructed in a type 1
fimbrial-negative background, to compete in the murine urinary tract over a
period of 168 h. No differences in the colonization of these mutants were
observed. However, comparison of these results with previous studies suggests
that inversely coordinated expression of adhesin gene clusters does occur in
vivo. Interestingly, the mutant that was incapable of expressing either type 1 or
P fimbriae compensated by synthesizing F1C fimbriae.

DOI: 10.1128/IAI.73.11.7588-7596.2005 
PMCID: PMC1273908
PMID: 16239562  [Indexed for MEDLINE]


1061. J Bacteriol. 2005 Nov;187(22):7655-66.

Genetic composition of the Bacillus subtilis SOS system.

Au N(1), Kuester-Schoeck E, Mandava V, Bothwell LE, Canny SP, Chachu K, Colavito 
SA, Fuller SN, Groban ES, Hensley LA, O'Brien TC, Shah A, Tierney JT, Tomm LL,
O'Gara TM, Goranov AI, Grossman AD, Lovett CM.

Author information: 
(1)Department of Chemistry, Williams College, Williamstown, MA 01267, USA.

The SOS response in bacteria includes a global transcriptional response to DNA
damage. DNA damage is sensed by the highly conserved recombination protein RecA, 
which facilitates inactivation of the transcriptional repressor LexA.
Inactivation of LexA causes induction (derepression) of genes of the LexA
regulon, many of which are involved in DNA repair and survival after DNA damage. 
To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched
the genome for putative LexA binding sites within 300 bp upstream of the start
codons of all annotated open reading frames. We found 62 genes that could be
regulated by putative LexA binding sites. Using mobility shift assays, we found
that LexA binds specifically to DNA in the regulatory regions of 54 of these
genes, which are organized in 34 putative operons. Using DNA microarray analyses,
we found that 33 of the genes with LexA binding sites exhibit RecA-dependent
induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there
are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of
the distinct LexA binding sites reveals an expanded consensus sequence for the B.
subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled 
by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only
eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E.
coli.

DOI: 10.1128/JB.187.22.7655-7666.2005 
PMCID: PMC1280312
PMID: 16267290  [Indexed for MEDLINE]


1062. J Food Prot. 2005 Nov;68(11):2278-86.

Use of oligonucleotide array for identification of six foodborne pathogens and
Pseudomonas aeruginosa grown on selective media.

Lin MC(1), Huang AH, Tsen HY, Wong HC, Chang TC.

Author information: 
(1)Department of Medical Laboratory Science and Biotechnology, School of
Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.

Identification of presumptive foodborne pathogens grown on selective media may
take one to several days and requires a different battery of biochemical tests
for each microorganism. A molecular identification method was developed in which 
universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of
target microorganisms, and PCR products were hybridized to a panel of
species-specific oligonucleotides that were immobilized on a nylon membrane. The 
seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria
monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and
Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 
to 51 strains) and nontarget bacteria (> 500 strains), the performances
(sensitivity and specificity) of the oligonucleotide array were as follows: B.
cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. 
aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%),
and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group
cross-hybridized to the probes used for identification of B. cereus, and positive
results should be confirmed by additional morphological observation of colonies. 
Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but
a simple hemolysis test was used to differentiate the two species. Some strains
of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for
identification of V. parahaemolyticus and caused false-positive reactions. The
advantage of the array is that a common protocol was used to identify the seven
target microorganisms and multiple different microorganisms could be
simultaneously identified on a single array.


PMID: 16300063  [Indexed for MEDLINE]


1063. J Biol Chem. 2005 Oct 28;280(43):36079-87. Epub 2005 Aug 31.

A global regulatory role of gluconeogenic genes in Escherichia coli revealed by
transcriptome network analysis.

Kao KC(1), Tran LM, Liao JC.

Author information: 
(1)Department of Chemical and Biomolecular Engineering, University of California,
Los Angeles, California 90095, USA.

In bacterial adaptation to the dynamic environment, metabolic genes are typically
thought to be the executors, whereas global transcription regulators are regarded
as the decision makers. Although the feedback from metabolic consequence is
believed to be important, much less is understood. This work demonstrates that
the gluconeogenic genes in Escherichia coli, ppsA, sfcA, and maeB, provide a
feedback loop to the global regulator, cAMP receptor protein (CRP), in carbon
source transition. Disruption of one of the gluconeogenic pathways has no
phenotype in balanced growth, but causes a significant delay in the diauxic
transition from glucose to acetate. To investigate the underlying mechanism, we
measured the transcriptome profiles during the transition using DNA microarray,
and network component analysis was employed to obtain the transcription factor
activities. Results showed that one of the global regulators, CRP, was
insufficiently activated during the transition in the ppsA deletion mutant.
Indeed, addition of cAMP partially rescued the delay in transition. These results
suggest that the gluconeogenic flux to phosphoenolpyruvate is important for full 
activation of adenylate cyclase through the phosphorylated enzyme IIA(glu) of the
phosphotransferase system. Reduction of this flux causes insufficient activation 
of CRP and a global metabolic deficiency, which exemplifies a significant
feedback interaction from metabolism to the a global regulatory system.

DOI: 10.1074/jbc.M508202200 
PMID: 16141204  [Indexed for MEDLINE]


1064. World J Gastroenterol. 2005 Oct 7;11(37):5811-5.

Microarray analysis of Escherichia coli O157:H7.

Jin HY(1), Tao KH, Li YX, Li FQ, Li SQ.

Author information: 
(1)Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002,
Jiangsu Province, China. laoyingj@hotmail.com

AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7
with DNA microchips.
METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and
virulent genes of E. coli O157:H7 and other related pathogen genes were
pre-synthesized and immobilized on a solid support to make microchips. The four
genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and
toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific
primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR
with Cy3-labeled single prime. Hybridization was performed for 60 min at 45
degrees. Microchip images were taken using a confocal fluorescent scanner.
RESULTS: Twelve different bacterial strains were detected with various
combinations of four virulent genes. All the O157:H7 strains yielded positive
results by multiplex PCR. The size of the PCR products generated with these
primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes
specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or 
strains containing O157 and H7 genes. No cross hybridization of O157:H7
fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to
yield any signal under comparable conditions. If the Cy3-labeled fluorescent
product of O157 single PCR was diluted 50-fold, no signal was found in agarose
gel electrophoresis, but a positive signal was found in microarray hybridization.
CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient
method for identification and detection of bacterial pathogens.


PMCID: PMC4479681
PMID: 16270390  [Indexed for MEDLINE]


1065. EMBO J. 2005 Oct 5;24(19):3423-34. Epub 2005 Sep 15.

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the
Drosophila Imd pathway.

Kleino A(1), Valanne S, Ulvila J, Kallio J, Myllymäki H, Enwald H, Stöven S,
Poidevin M, Ueda R, Hultmark D, Lemaitre B, Rämet M.

Author information: 
(1)Institute of Medical Technology, University of Tampere, Tampere, Finland.

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway,
controls antimicrobial peptide expression in Drosophila. We performed a
large-scale RNAi screen to identify novel components of the Imd pathway in
Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library
were analyzed for their effect on Attacin promoter activity in response to
Escherichia coli. We identified seven gene products required for the Attacin
response in vitro, including two novel Imd pathway components: inhibitor of
apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1
(TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response
also by the fat body in vivo. Both these factors function downstream of Imd.
Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in
Relish nuclear localization in vitro, suggesting a novel mode of regulation of
the Imd pathway. Our results show that an RNAi-based approach is suitable to
identify genes in conserved signaling cascades.

DOI: 10.1038/sj.emboj.7600807 
PMCID: PMC1276168
PMID: 16163390  [Indexed for MEDLINE]


1066. IEEE/ACM Trans Comput Biol Bioinform. 2005 Oct-Dec;2(4):289-301.

A generalized framework for network component analysis.

Boscolo R(1), Sabatti C, Liao JC, Roychowdhury VP.

Author information: 
(1)Electrical Engineering Department, University of California, Los Angeles, 420 
Westwood Plaza, Los Angeles, CA 90095, USA. riccardo@ee.ucla.edu

The authors recently introduced a framework, named Network Component Analysis
(NCA), for the reconstruction of the dynamics of transcriptional regulators'
activities from gene expression assays. The original formulation had certain
shortcomings that limited NCA's application to a wide class of network dynamics
reconstruction problems, either because of limitations in the sample size or
because of the stringent requirements imposed by the set of identifiability
conditions. In addition, the performance characteristics of the method for
various levels of data noise or in the presence of model inaccuracies were never 
investigated. In this article, the following aspects of NCA have been addressed, 
resulting in a set of extensions to the original framework: 1) The sufficient
conditions on the a priori connectivity information (required for successful
reconstructions via NCA) are made less stringent, allowing easier verification of
whether a network topology is identifiable, as well as extending the class of
identifiable systems. Such a result is accomplished by introducing a set of
identifiability requirements that can be directly tested on the regulatory
architecture, rather than on specific instances of the system matrix. 2) The
two-stage least square iterative procedure used in NCA is proven to identify
stationary points of the likelihood function, under Gaussian noise assumption,
thus reinforcing the statistical foundations of the method. 3) A framework for
the simultaneous reconstruction of multiple regulatory subnetworks is introduced,
thus overcoming one of the critical limitations of the original formulation of
the decomposition, for example, occurring for poorly sampled data (typical of
microarray experiments). A set of monte carlo simulations we conducted with
synthetic data suggests that the approach is indeed capable of accurately
reconstructing regulatory signals when these are the input of large-scale
networks that satisfy the suggested identifiability criteria, even under fairly
noisy conditions. The sensitivity of the reconstructed signals to inaccuracies in
the hypothesized network topology is also investigated. We demonstrate the
feasibility of our approach for the simultaneous reconstruction of multiple
regulatory subnetworks from the same data set with a successful application of
the technique to gene expression measurements of the bacterium Escherichia coli.

DOI: 10.1109/TCBB.2005.47 
PMID: 17044167  [Indexed for MEDLINE]


1067. J Bioinform Comput Biol. 2005 Oct;3(5):1191-205.

Mining time-dependent gene features.

Capobianco E(1).

Author information: 
(1)Department of Biomedical Engineering, Boston University, 44 Cummington Street,
Boston MA 02215, USA. ecapob@bu.edu

This paper presents an application of the Independent Component Analysis (ICA)
method to genomic data. In particular, experimentally produced perturbation
effects over the E.coli bacterium are monitored through the changes of gene
expression values observed at regular times, and until steady state has been
reached. The aim is to control the response of the SOS system to DNA damage. We
might assume that only part of the genetic regulatory network is affected
directly by the perturbation conditions, as indirect cascade effects might also
be present, and some genes may change just because of randomness. ICA decomposes 
the gene matrix and identifies groups of genes belonging to a certain estimated
component by virtue of co-expression; it is of course of interest to establish
co-regulation dynamics, which might underlie the captured correlation. Stronger
forms of dependence, like Mutual Information, are thus computed and compared with
linear correlation in order to validate the results and establish the role of the
identified components in determining the network dynamics.


PMID: 16278954  [Indexed for MEDLINE]


1068. Lab Chip. 2005 Oct;5(10):1053-8. Epub 2005 Aug 31.

Integrated microsystem for dielectrophoretic cell concentration and genetic
detection.

Lagally ET(1), Lee SH, Soh HT.

Author information: 
(1)California Nanosystems Institute and Department of Mechanical and
Environmental Engineering, University of California-Santa Barbara, Santa Barbara,
CA 93106, USA.

We have directly integrated a continuous-flow, electrokinetic method of bacterial
cell concentration with room temperature, sequence-specific genetic detection.
The system we have developed traps cells from a continuous-flow sample stream via
dielectrophoresis, providing a 160-fold increase in cell concentration. PDMS
microvalves then define a 100 nL volume around the trapped cells to which cell
lysis buffer and genetic detection components are introduced. Direct, optical
detection of Escherichia coli MC1061 cells is then accomplished via the
sequence-specific hybridization of an rRNA-directed optical molecular beacon.
This integrated microsystem is capable of sequence-specific genetic detection of 
25 cells within 30 min.

DOI: 10.1039/b505915a 
PMID: 16175260  [Indexed for MEDLINE]


1069. Microbiology. 2005 Oct;151(Pt 10):3287-98.

Regulation of type 1 fimbriae synthesis and biofilm formation by the
transcriptional regulator LrhA of Escherichia coli.

Blumer C(1), Kleefeld A, Lehnen D, Heintz M, Dobrindt U, Nagy G, Michaelis K,
Emödy L, Polen T, Rachel R, Wendisch VF, Unden G.

Author information: 
(1)Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg Universität
Mainz, Becherweg 15, 55099 Mainz, Germany.

Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and 
promote biofilm formation on abiotic surfaces. The transcriptional regulator
LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, 
regulates biofilm formation and expression of type 1 fimbriae. Whole-genome
expression profiling revealed that inactivation of lrhA results in an increased
expression of structural components of type 1 fimbriae. In vitro, LrhA bound to
the promoter regions of the two fim recombinases (FimB and FimE) that catalyse
the inversion of the fimA promoter, and to the invertible element itself.
Translational lacZ fusions with these genes and quantification of fimE transcript
levels by real-time PCR showed that LrhA influences type 1 fimbrial phase
variation, primarily via activation of FimE, which is required for the ON-to-OFF 
transition of the fim switch. Enhanced type 1 fimbrial expression as a result of 
lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. 
Biofilm formation was stimulated by lrhA inactivation and completely suppressed
upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted
via the changed levels of surface molecules, most probably both flagella and type
1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1
fimbrial expression, and thus as a regulator of the initial stages of biofilm
development and, presumably, bacterial adherence to epithelial host cells also.

DOI: 10.1099/mic.0.28098-0 
PMID: 16207912  [Indexed for MEDLINE]


1070. Mol Microbiol. 2005 Oct;58(1):334-44.

Requirement of the dephospho-form of enzyme IIANtr for derepression of
Escherichia coli K-12 ilvBN expression.

Lee CR(1), Koo BM, Cho SH, Kim YJ, Yoon MJ, Peterkofsky A, Seok YJ.

Author information: 
(1)Laboratory of Macromolecular Interactions, Department of Biological Sciences
and Institute of Microbiology, Seoul National University, Seoul 151-742, Korea.

While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase
system (carbohydrate PTS) have been shown to regulate numerous targets, little
such information is available for the nitrogen-metabolic phosphotransferase
system (nitrogen-metabolic PTS). To elucidate the physiological role of the
nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with
Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first 
enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies
revealed that a ptsN (encoding enzyme IIA(Ntr)) mutant became extremely sensitive
to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I(Ntr))
and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity 
of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA(Ntr) 
was found to be necessary to neutralize leucine toxicity. Further studies showed 
that the dephospho-form of enzyme IIA(Ntr) is required for derepression of the
ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step
common to the biosynthesis of the branched-chain amino acids.

DOI: 10.1111/j.1365-2958.2005.04834.x 
PMID: 16164569  [Indexed for MEDLINE]


1071. RNA. 2005 Oct;11(10):1579-87. Epub 2005 Aug 30.

RNA sequence and secondary structure participate in high-affinity CsrA-RNA
interaction.

Dubey AK(1), Baker CS, Romeo T, Babitzke P.

Author information: 
(1)Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, PA 16802, USA.

The global Csr regulatory system controls bacterial gene expression
post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that
plays a central role in repressing several stationary phase processes and
activating certain exponential phase functions. CsrA regulates translation
initiation of several genes by binding to the mRNA leaders and blocking ribosome 
binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of
sequestering CsrA and antagonizing its activity. Each of the known target
transcripts contains multiple CsrA binding sites, although considerable sequence 
variation exists among these RNA targets, with GGA being the most highly
conserved element. High-affinity RNA ligands containing single CsrA binding sites
were identified from a combinatorial library using systematic evolution of
ligands by exponential enrichment (SELEX). The SELEX-derived consensus was
determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and 
the GU sequence being present in all but one ligand. The majority (51/55) of the 
RNAs contained GGA in the loop of a hairpin within the most stable predicted
structure, an arrangement similar to several natural CsrA binding sites.
Strikingly, the identity of several nucleotides that were predicted to form base 
pairs in each stem were 100% conserved, suggesting that primary sequence
information was embedded within the base-paired region. The affinity of CsrA for 
several selected ligands was measured using quantitative gel mobility shift
assays. A mutational analysis of one selected ligand confirmed that the conserved
ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary
structure participates in CsrA-RNA recognition.

DOI: 10.1261/rna.2990205 
PMCID: PMC1370842
PMID: 16131593  [Indexed for MEDLINE]


1072. Langmuir. 2005 Sep 27;21(20):9103-9.

Nanografting de novo proteins onto gold surfaces.

Hu Y(1), Das A, Hecht MH, Scoles G.

Author information: 
(1)Chemistry Department, Princeton Institute for the Science and Technology of
Materials (PRISM), Princeton University, Princeton, NJ 08544, USA.

The immobilization of novel proteins onto addressable locations on a flat surface
has potential applications in a range of biotechnologies. Here we describe the
nanopatterning of a de novo protein onto a gold surface. Patterning was achieved 
using a technique called nanografting, in which the tip of an atomic force
microscope is used to disrupt a preexisting monolayer of alkanethiol molecules on
a gold surface, thereby facilitating exchange with alternative thiol-linked
molecules from the surrounding solution. The protein used for these studies was
chosen from a designed combinatorial library of de novo sequences expressed in E.
coli and was engineered to have a glycine-glycine-cysteine tag at its C-terminus,
thereby enabling attachment to the gold surface through a single cysteine thiol. 
The average height of the grafted protein patterns was found to be somewhat
higher than expected from the known NMR structure of the protein. Compression of 
the nanografted patches by an external force (below 10 nN) was reversible but
showed some hysteresis. Interestingly, both the energy required to deform the
immobilized protein patterns and the energy defined by the hysteresis loop were
found to be of the same order as the energy required to unfold the monomeric
protein in solution. These studies demonstrate the possibility of preparing
nanometer scale protein arrays, lowering significantly the volume requirements of
the protein samples necessary to fabricate protein-based biosensor arrays and
thereby providing a base for increasing their sensitivity.

DOI: 10.1021/la046857h 
PMID: 16171339  [Indexed for MEDLINE]


1073. Biochem Biophys Res Commun. 2005 Sep 16;335(1):71-5.

Analysis of 5' UTR composition and gene expression: canonical versus
non-canonical start codons.

Fuglsang A(1).

Author information: 
(1)Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100
Copenhagen Ø, Denmark. anfu@dfuni.dk

The overall composition upstream of start codons in Escherichia coli was
evaluated and viewed in connection with global transcriptome data. Genes starting
with AUG as initiation codon tended to be expressed at higher levels than the
non-AUG genes, and the upstream region of the non-AUG genes showed negligible
signs of Shine-Dalgarno sequences. The latter is in sharp contrast to the AUG
genes. Viewing these findings in connection with the current literature, it is
proposed that a distinct mechanism for initiation of translation might exist for 
non-AUG genes that are not preceded by a Shine-Dalgarno sequence. A survey
covering a range of other eubacteria (Firmicutes, Proteoacteria, and
Actinobacteria) reveals that it is mainly among the Proteobacteria that non-AUG
genes do not display clear signs of Shine-Dalgarno regions.

DOI: 10.1016/j.bbrc.2005.07.047 
PMID: 16051189  [Indexed for MEDLINE]


1074. Genes Cells. 2005 Sep;10(9):907-18.

Systematic search for the Cra-binding promoters using genomic SELEX system.

Shimada T(1), Fujita N, Maeda M, Ishihama A.

Author information: 
(1)Nippon Institute for Biological Science, Division of Molecular Biology, Ome,
Tokyo 198-0024, Japan.

Cra (or FruR), a global transcription factor with both repression and activation 
activities, controls a large number of the genes for glycolysis and
gluconeogenesis. To get insights into the entire network of transcription
regulation of the E. coli genome by Cra, we isolated a set of Cra-binding
sequences using an improved method of genomic SELEX. From the DNA sequences of 97
independently isolated DNA fragments by SELEX, the Cra-binding sequences were
identified in a total of ten regions on the E. coli genome, including promoters
of six known genes and four hitherto-unidentified genes. All six known promoters 
are repressed by Cra, but none of the activation-type promoters were cloned after
two cyles of SELEX, because the Cra-binding affinity to the repression-type
promoters is higher than the activation-type promoters, as determined by the
quantitative gel shift assay. Of a total of four newly identified Cra-binding
sequences, two are associated with promoter regions of the gapA (glyceraldehyde
3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar
metabolism. The regulation of newly identified genes by Cra was confirmed by the 
in vivo promoter strength assay using a newly developed TFP (two-fluorescent
protein) vector for promoter assay or by in vitro transcription assay in the
presence of Cra protein.

DOI: 10.1111/j.1365-2443.2005.00888.x 
PMID: 16115199  [Indexed for MEDLINE]


1075. Infect Immun. 2005 Sep;73(9):5706-19.

Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in
biofilm formation.

Mey AR(1), Craig SA, Payne SM.

Author information: 
(1)Institute for Cellular and Molecular Biology, The University of Texas, Austin,
TX 78712-1095, USA.

Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like 
E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is
required for repression of genes encoding the superoxide dismutase SodB and
multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is
considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and
it regulates the expression of a variety of genes that are not known to be
regulated by RyhB in E. coli, including genes involved in motility, chemotaxis,
and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic
motility in low-iron medium and was unable to form wild-type biofilms. The defect
in biofilm formation was suppressed by growing the mutant in the presence of
excess iron or succinate. The wild-type strain showed reduced biofilm formation
in iron-deficient medium, further supporting a role for iron in normal biofilm
formation. The ryhB mutant was not defective for colonization in a mouse model
and appeared to be at a slight advantage when competing with the wild-type
parental strain. Other genes whose expression was influenced by RyhB included
those encoding the outer membrane porins OmpT and OmpU, several iron transport
systems, and proteins containing heme or iron-sulfur clusters. These data
indicate that V. cholerae RyhB has diverse functions, ranging from iron
homeostasis to the regulation of biofilm formation.

DOI: 10.1128/IAI.73.9.5706-5719.2005 
PMCID: PMC1231101
PMID: 16113288  [Indexed for MEDLINE]


1076. J Bacteriol. 2005 Sep;187(17):6046-57.

Heat shock response of Archaeoglobus fulgidus.

Rohlin L(1), Trent JD, Salmon K, Kim U, Gunsalus RP, Liao JC.

Author information: 
(1)Department of Chemical Engineering, 5531 Boelter Hall, University of
California, Los Angeles, Los Angeles, CA 90095, USA.

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus 
strain VC-16 was studied using whole-genome microarrays. On the basis of the
resulting expression profiles, approximately 350 of the 2,410 open reading frames
(ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These
span a range of cell functions, including energy production, amino acid
metabolism, and signal transduction, where the majority are uncharacterized. One 
ORF called AF1298 was identified that contains a putative helix-turn-helix DNA
binding motif. The gene product, HSR1, was expressed and purified from
Escherichia coli and was used to characterize specific DNA recognition regions
upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that
AF1298 is autoregulated and is part of an operon with two downstream genes that
encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I 
footprints using HSR1 suggest the presence of a cis-binding motif upstream of
AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in
response to heat shock and encodes a protein only distantly related to the
N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results
suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family 
involved in heat shock regulation in hyperthermophilic and mesophilic Archaea
organisms.

DOI: 10.1128/JB.187.17.6046-6057.2005 
PMCID: PMC1196131
PMID: 16109946  [Indexed for MEDLINE]


1077. J Microbiol Methods. 2005 Sep;62(3):303-16.

Automated methods for multiplexed pathogen detection.

Straub TM(1), Dockendorff BP, Quiñonez-Díaz MD, Valdez CO, Shutthanandan JI,
Tarasevich BJ, Grate JW, Bruckner-Lea CJ.

Author information: 
(1)Interfacial Chemistry and Engineering Group, Pacific Northwest National
Laboratory, 902 Battelle Blvd., P.O. Box 999, Mail stop K4-12 Richland, WA 99352,
USA. timothy.straub@pnl.gov

Detection of pathogenic microorganisms in environmental samples is a difficult
process. Concentration of the organisms of interest also co-concentrates
inhibitors of many end-point detection methods, notably, nucleic acid methods. In
addition, sensitive, highly multiplexed pathogen detection continues to be
problematic. The primary function of the BEADS (Biodetection Enabling Analyte
Delivery System) platform is the automated concentration and purification of
target analytes from interfering substances, often present in these samples, via 
a renewable surface column. In one version of BEADS, automated immunomagnetic
separation (IMS) is used to separate cells from their samples. Captured cells are
transferred to a flow-through thermal cycler where PCR, using labeled primers, is
performed. PCR products are then detected by hybridization to a DNA suspension
array. In another version of BEADS, cell lysis is performed, and community RNA is
purified and directly labeled. Multiplexed detection is accomplished by direct
hybridization of the RNA to a planar microarray. The integrated IMS/PCR version
of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river 
water samples. Multiplexed PCR assays for the simultaneous detection of E. coli
O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for 
the detection of as few as 100 cells for each organism. Results for the RNA
version of BEADS are also showing promising results. Automation yields highly
purified RNA, suitable for multiplexed detection on microarrays, with microarray 
detection specificity equivalent to PCR. Both versions of the BEADS platform show
great promise for automated pathogen detection from environmental samples. Highly
multiplexed pathogen detection using PCR continues to be problematic, but may be 
required for trace detection in large volume samples. The RNA approach solves the
issues of highly multiplexed PCR and provides "live vs. dead" capabilities.
However, sensitivity of the method will need to be improved for RNA analysis to
replace PCR.

DOI: 10.1016/j.mimet.2005.04.012 
PMID: 15979746  [Indexed for MEDLINE]


1078. Mol Microbiol. 2005 Sep;57(6):1636-52.

A genetic selection for supercoiling mutants of Escherichia coli reveals proteins
implicated in chromosome structure.

Hardy CD(1), Cozzarelli NR.

Author information: 
(1)Department of Molecular and Cell Biology, 16 Barker Hall, University of
California, Berkeley, CA 94720-3204, USA.

Chromosomes are divided into topologically independent regions, called domains,
by the action of uncharacterized barriers. With the goal of identifying domain
barrier components, we designed a genetic selection for mutants with reduced
negative supercoiling of the Escherichia coli chromosome. We employed a strain
that contained two chromosomally located reporter genes under the control of a
supercoiling-sensitive promoter and used transposon mutagenesis to generate a
wide range of mutants. We subjected the selected mutants to a series of secondary
screens and identified five proteins as modulators of chromosomal supercoiling in
vivo. Three of these proteins: H-NS, Fis and DksA, have clear ties to chromosome 
biology. The other two proteins, phosphoglucomutase (Pgm) and transketolase
(TktA), are enzymes involved in carbohydrate metabolism and have not previously
been shown to affect DNA. Deletion of any of the identified genes specifically
affected chromosome topology, without affecting plasmid supercoiling. We suggest 
that at least H-NS, Fis and perhaps TktA assist directly in the supercoiling of
domains by forming topological barriers on the E. coli chromosome.

DOI: 10.1111/j.1365-2958.2005.04799.x 
PMID: 16135230  [Indexed for MEDLINE]


1079. Mol Microbiol. 2005 Sep;57(5):1252-64.

A master regulator sigmaB governs osmotic and oxidative response as well as
differentiation via a network of sigma factors in Streptomyces coelicolor.

Lee EJ(1), Karoonuthaisiri N, Kim HS, Park JH, Cha CJ, Kao CM, Roe JH.

Author information: 
(1)School of Biological Sciences and Institute of Microbiology, Seoul National
University, Seoul 151-742, Korea.

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma
factors, which support its complex life cycle. sigma(B), a functional homologue
of sigma(S) from Escherichia coli, controls both osmoprotection and
differentiation in S. coelicolor A3(2). Microarray analysis revealed
sigma(B)-dependent induction of more than 280 genes by 0.2 M KCl. These genes
encode several sigma factors, oxidative defence proteins, chaperones, systems to 
provide osmolytes, cysteine, mycothiol, and gas vesicle. sigma(B) controlled
induction of itself and its two paralogues (sigma(L) and sigma(M)) in a
hierarchical order of sigma(B)-->sigma(L)-->sigma(M), as revealed by S1 mapping
and Western blot analyses. The phenotype of each sigma mutant suggested a
sequential action in morphological differentiation; sigma(B) in forming aerial
mycelium, sigma(L) in forming spores and sigma(M) for efficient sporulation.
sigma(B) was also responsible for the increase in cysteine and mycothiol, the
major thiol buffer in actinomycetes, upon osmotic shock, revealing an overlap
between protections against osmotic and oxidative stresses. Proteins in sigB
mutant were more oxidized (carbonylated) than the wild type. These results
support a hypothesis that sigma(B) serves as a master regulator that triggers
other related sigma factors in a cascade, and thus regulates differentiation and 
osmotic and oxidative response in S. coelicolor.

DOI: 10.1111/j.1365-2958.2005.04761.x 
PMID: 16101999  [Indexed for MEDLINE]


1080. Anal Chem. 2005 Aug 1;77(15):5041-7.

Detection of Salmonella spp. using microsphere-based, fiber-optic DNA
microarrays.

Ahn S(1), Walt DR.

Author information: 
(1)Department of Chemistry, Tufts University, Medford, Massachusetts 02155, USA.

Salmonella spp. are one of the most problematic food pathogens in public health, 
as they are responsible for food poisoning associated with contamination of meat,
poultry, and eggs. Thus, rapid and sensitive detection of Salmonella spp. is
required to ensure food safety. In this study, a fiber-optic DNA microarray using
microsphere-immobilized oligonucleotide probes specific for the Salmonella invA
and spvB genes was developed for detection of Salmonella spp. Microarrays were
prepared by randomly distributing DNA probe-functionalized microspheres
(3.1-microm diameter) into microwells created by etching optical fiber bundles.
Hybridization of the probe-functionalized microspheres to target DNA from
Salmonella was performed and visualized using Cy3-labeled secondary probes in a
sandwich-type assay format. In this study, 10(3)-10(4) cfu/mL of the target
organism could be detected after 1-h hybridization without any additional
amplification. The DNA microarray showed no cross-reactivity with other common
food pathogens, including E. coli and Y. enterocolitica, and could even detect
Salmonella spp. from cocktails of bacterial strains with only moderate loss of
sensitivity due to nonspecific binding. This work suggests that fiber-optic DNA
microarrays can be used for rapid and sensitive detection of Salmonella spp.
Since fiber-optic microarrays can be prepared with different probes, this
approach could also enable the simultaneous detection of multiple food pathogens.

DOI: 10.1021/ac0505270 
PMID: 16053320  [Indexed for MEDLINE]


1081. EMBO Rep. 2005 Aug;6(8):736-41.

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation 
of Escherichia coli DNA replication.

Camara JE(1), Breier AM, Brendler T, Austin S, Cozzarelli NR, Crooke E.

Author information: 
(1)Department of Biochemistry and Molecular Biology, Georgetown University
Medical Center, Washington, District of Columbia 20007, USA.

Initiation of DNA replication from the Escherichia coli chromosomal origin is
highly regulated, assuring that replication occurs precisely once per cell cycle.
Three mechanisms for regulation of replication initiation have been proposed:
titration of free DnaA initiator protein by the datA locus, sequestration of
newly replicated origins by SeqA protein and regulatory inactivation of DnaA
(RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA
microarray analyses showed that the level of initiation in rapidly growing cells 
that lack datA was indistinguishable from that in wild-type cells, and that the
absence of SeqA protein caused only a modest increase in initiation, in agreement
with flow-cytometry data. In contrast, cells lacking Hda overinitiated
replication twofold, implicating RIDA as the predominant mechanism preventing
extra initiation events in a cell cycle.

DOI: 10.1038/sj.embor.7400467 
PMCID: PMC1369143
PMID: 16041320  [Indexed for MEDLINE]


1082. J Clin Microbiol. 2005 Aug;43(8):4197-9.

Approaches to bacterial RNA isolation and purification for microarray analysis of
Escherichia coli K1 interaction with human brain microvascular endothelial cells.

Di Cello F(1), Xie Y, Paul-Satyaseela M, Kim KS.

Author information: 
(1)Division of Pediatric Infectious Diseases, Johns Hopkins University School of 
Medicine, 600 North Wolfe Street, Park 256, Baltimore, MD 21287, USA.

We established a protocol for isolation of microarray-grade bacterial RNA from
Escherichia coli K1 interacting with human brain microvascular endothelial cells.
The extracted RNA was free of human RNA contamination. More importantly,
microarray analysis demonstrated that no bias was introduced in the gene
expression pattern during the RNA isolation procedure.

DOI: 10.1128/JCM.43.8.4197-4199.2005 
PMCID: PMC1233938
PMID: 16081976  [Indexed for MEDLINE]


1083. Sci China C Life Sci. 2005 Aug;48(4):406-13.

Comparative genomics and phylogenetic analysis of S. dysenteriae subgroup.

Yang E(1), Bin W, Peng J, Zhang X, Wang J, Yang J, Dong J, Chu Y, Zhang J, Jin Q.

Author information: 
(1)School of Medicine, Xi'an Jiaotong University, China.

Genomic compositions of representatives of thirteen S. dysenteriae serotypes were
investigated by performing comparative genomic hybridization (CGH) with
microarray containing the whole genomic ORFs (open reading frames, ORFs) of E.
coli K12 strain MG1655 and specific ORFs of S. dysenteriae A1 strain Sd51197. The
CGH results indicated the genomes of the serotypes contain 2654 conserved ORFs
originating from E. coli. However, 219 intrinsic genes of E. coli including those
prophage genes, molecular chaperones, synthesis of specific O antigen and so on
were absent. Moreover, some specific genes such as type II secretion system
associated components, iron transport related genes and some others as well were 
acquired through horizontal transfer. According to phylogenic trees based on
genetic composition, it was demonstrated that A1, A2, A8, A10 were distinct from 
the other S. dysenteriae serotypes. Our results in this report may provide new
insights into the physiological process, pathogenicity and evolution of S.
dysenteriae.


PMID: 16248434  [Indexed for MEDLINE]


1084. Biosens Bioelectron. 2005 Jul 15;21(1):135-44.

Construction and characterization of different MutS fusion proteins as
recognition elements of DNA chip for detection of DNA mutations.

Bi LJ(1), Zhou YF, Zhang XE, Deng JY, Wen JK, Zhang ZP.

Author information: 
(1)Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang 
District, Beijing 100101, People's Republic of China.

Three MutS fusion systems were designed as the mutation recognition and signal
elements of DNA chips for detection of DNA mutations. The expression vectors
containing the encoding sequences of three recombinant proteins,
Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep
tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were
constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed 
in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that
each of the expected proteins was approximately 30% of the total bacterial
proteins. The recombinant proteins were purified to the purity over 90% by
immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay
indicated that three fusion proteins retained the mismatch-binding activity and
the functions of other fusion partners. DNA chips arrayed both mismatched and
unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium
tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with
Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation
recognition and labeling elements of DNA chips. The resulting DNA chips were used
to detect the mismatched and unpaired mutations in the synthesized
oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is
resistant to rifamycin.

DOI: 10.1016/j.bios.2004.08.045 
PMID: 15967361  [Indexed for MEDLINE]


1085. Appl Environ Microbiol. 2005 Jul;71(7):4156-9.

Effect of an artificial RNA marker on gene expression in Escherichia coli.

Tucker DL(1), Karouia F, Wang J, Luo Y, Li TB, Willson RC, Fofanov Y, Fox GE.

Author information: 
(1)Dept. Biology and Biochemistry, University of Houston, Houston, TX 77204-5001,
USA.

Transcriptional analysis was used to examine the effect of a genomically encoded 
artificial RNA on Escherichia coli in rich and minimal media. Only the expression
of a single gene, deoC, was unequivocally affected under both conditions. E. coli
marker strains of this type may be useful in monitoring the fate and transport of
bacteria in various applications.

DOI: 10.1128/AEM.71.7.4156-4159.2005 
PMCID: PMC1168998
PMID: 16000839  [Indexed for MEDLINE]


1086. Appl Environ Microbiol. 2005 Jul;71(7):4022-34.

Differential gene expression for investigation of Escherichia coli biofilm
inhibition by plant extract ursolic acid.

Ren D(1), Zuo R, González Barrios AF, Bedzyk LA, Eldridge GR, Pasmore ME, Wood
TK.

Author information: 
(1)Department of Chemical Engineering and Molecular and Cell Biology, University 
of Connecticut, 191 Auditorium Rd., Storrs, CT 06269-3222, USA.

After 13,000 samples of compounds purified from plants were screened, a new
biofilm inhibitor, ursolic acid, has been discovered and identified. Using both
96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10
microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to
20-fold when added upon inoculation and when added to a 24-h biofilm; however,
ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi,
and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm
formation by >87% for P. aeruginosa in both complex and minimal medium and by 57%
for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic
inhibition on a global genetic basis, DNA microarrays were used to study the gene
expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic
acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes,
respectively, and 19 genes were consistently induced. The consistently induced
genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB),
heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and 
yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic
acid/ml, respectively, and 12 genes were consistently repressed that have
functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as
unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms
without interfering with quorum sensing, as shown with the V. harveyi AI-1 and
AI-2 reporter systems. As predicted by the differential gene expression, deleting
motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become
too motile). Based on the differential gene expression, it was also discovered
that sulfur metabolism (through cysB) affects biofilm formation (in the absence
of ursolic acid).

DOI: 10.1128/AEM.71.7.4022-4034.2005 
PMCID: PMC1169008
PMID: 16000817  [Indexed for MEDLINE]


1087. Exp Parasitol. 2005 Jul;110(3):178-83. Epub 2005 Apr 22.

Entamoeba histolytica: construction and applications of subgenomic databases.

Hofer M(1), Duchêne M.

Author information: 
(1)Department of Specific Prophylaxis and Tropical Medicine, Center for
Physiology and Pathophysiology, Medical University of Vienna, Kinderspitalgasse
15, A-1095 Vienna, Austria.

Knowledge about the influence of environmental stress such as the action of
chemotherapeutic agents on gene expression in Entamoeba histolytica is limited.
We plan to use oligonucleotide microarray hybridization to approach these
questions. As the basis for our array, sequence data from the genome project
carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute
were used to annotate parts of the parasite genome. Three subgenomic databases
containing enzymes, cytoskeleton genes, and stress genes were compiled with the
help of the ExPASy proteomics website and the BLAST servers at the two genome
project sites. The known sequences from reference species, mostly human and
Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence
contigs and the homologs were copied into a Microsoft Access database. In a
similar way, two additional databases of cytoskeletal genes and stress genes were
generated. Metabolic pathways could be assembled from our enzyme database, but
sometimes they were incomplete as is the case for the sterol biosynthesis
pathway. The raw databases contained a significant number of duplicate entries
which were merged to obtain curated non-redundant databases. This procedure
revealed that some E. histolytica genes may have several putative functions.
Representative examples such as the case of the delta-aminolevulinate
synthase/serine palmitoyltransferase are discussed.

DOI: 10.1016/j.exppara.2005.03.018 
PMID: 15955309  [Indexed for MEDLINE]


1088. J Bacteriol. 2005 Jul;187(14):4890-9.

Hierarchical control of anaerobic gene expression in Escherichia coli K-12: the
nitrate-responsive NarX-NarL regulatory system represses synthesis of the
fumarate-responsive DcuS-DcuR regulatory system.

Goh EB(1), Bledsoe PJ, Chen LL, Gyaneshwar P, Stewart V, Igo MM.

Author information: 
(1)Section of Microbiology, University of California, Davis, 95616-8665, USA.

Hierarchical control ensures that facultative bacteria preferentially use the
available respiratory electron acceptor with the most positive standard redox
potential. Thus, nitrate is used before other electron acceptors such as fumarate
for anaerobic respiration. Nitrate regulation is mediated by the NarX-NarL
two-component system, which activates the transcription of operons encoding
nitrate respiration enzymes and represses the transcription of operons for other 
anaerobic respiratory enzymes, including enzymes involved in fumarate
respiration. These are fumarate reductase (encoded by the frdABCD operon),
fumarase B, which generates fumarate from malate, and the DcuB permease for
fumarate, malate, and aspartate. The transcription of the corresponding
structural genes is activated by the DcuS-DcuR two-component system in response
to fumarate or its dicarboxylate precursors. We report results from preliminary
transcription microarray experiments that revealed two previously unknown members
of the NarL regulon: the aspA gene encoding aspartate-ammonia lyase, which
generates fumarate; and the dcuSR operon encoding the dicarboxylate-responsive
regulatory system. We measured beta-galactosidase expression from monocopy
aspA-lacZ, frdA-lacZ, and dcuS-lacZ operon fusions in response to added nitrate
and fumarate and with respect to the dcuR and narL genotypes. Nitrate, acting
through the NarX-NarL regulatory system, repressed the transcription of all three
operons. Only frdA-lacZ expression, however, was responsive to added fumarate or 
a dcuR(+) genotype. Phospho-NarL protein protected operator sites in the aspA and
dcuS promoter regions from DNase I cleavage in vitro. The overall results are
consistent with the hypothesis that nitrate represses frdA operon transcription
not only directly, by repressing frdA promoter activity, but also indirectly, by 
repressing dcuS promoter activity.

DOI: 10.1128/JB.187.14.4890-4899.2005 
PMCID: PMC1169511
PMID: 15995204  [Indexed for MEDLINE]


1089. Metab Eng. 2005 Jul;7(4):318-27.

Functional analysis of 1440 Escherichia coli genes using the combination of
knock-out library and phenotype microarrays.

Ito M(1), Baba T, Mori H, Mori H.

Author information: 
(1)Biofrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd., Tokyo 194-8533, Japan.

Escherichia coli is one of the best elucidated organisms. However, about 40% of
E. coli genes have not been assigned to their function yet. We analyzed 1440
single gene knock-out mutants using the GN2-MicroPlate, which permits assay of 95
carbon-source utilizations simultaneously. In the knock-out library there are
1044 of so called y-genes with no apparent function. The raw dataset was analyzed
and genes were interrelated by the clustering method of the GeneSpring software. 
In the resulted dendrogram of genes, a group of genes with known and related
function tended to be assembled into a cluster. Our clustering method would be
useful for functional assignment of so called y-genes with no apparent function, 
since the resulted dendrogram could connect y-genes to phenotype and function of 
well-studied genes.

DOI: 10.1016/j.ymben.2005.06.004 
PMID: 16095938  [Indexed for MEDLINE]


1090. Metab Eng. 2005 Jul;7(4):280-90.

Determination of functional interactions among signalling pathways in Escherichia
coli K-12.

Yang YL(1), Liao JC.

Author information: 
(1)Department of Chemical Engineering, University of California, Los Angeles, CA 
90095, USA.

Interaction among different signalling pathways has been noted repeatedly.
However, no systematic method has been developed to identify and quantify such
interactions. Here we reported that network component analysis (NCA) was able to 
determine interactions among various signalling pathways in Escherichia coli K-12
based on known transcription factor (TF)-promoter connectivity information and
microarray data from genetic knockout strains. The TF activities determined from 
NCA allow the quantitation of functional interactions, barring gross errors in
the connectivity and microarray data. By using a robust statistical test, 37
pairs of functional interactions were identified. Eighteen interaction pairs
confirmed previous implications, while 19 others represent new predictions. These
results demonstrate that the functional interactions among various signalling
pathways may be rather significant. With reasonable TF-promoter connectivity, NCA
coupled with genetic knockouts and microarray experiments provides a systematic
way to elucidate interaction networks. As this approach cannot distinguish
between cross-talks and unidentified direct regulation, the results should
provide incentives for further experimental testing.

DOI: 10.1016/j.ymben.2005.05.002 
PMID: 16002309  [Indexed for MEDLINE]


1091. Mol Psychiatry. 2005 Jul;10(7):669-77.

The tumor suppressor adenomatous polyposis coli gene is associated with
susceptibility to schizophrenia.

Cui DH(1), Jiang KD, Jiang SD, Xu YF, Yao H.

Author information: 
(1)Department of Psychiatry, Fudan University Affiliated Huashan Hospital,
Shanghai, China.

The etiology of schizophrenia is unclear, although family, twin, and linkage
studies implicate genetic factors. Here, we identified adenomatous polyposis coli
(APC), a tumor suppressor gene, as a risk factor for schizophrenia. We compared
leukocytic gene expression patterns of six pairs of patients with schizophrenia
and healthy controls by microarray. APC expression levels were significantly
increased in all patients compared to healthy controls. To confirm the findings
of microarray analysis, we measured expression levels of APC in the leukocytes
from 30 relapse patients taking antipsychotic medication, 29 first-episode
drug-naïve patients, and 30 healthy controls using real-time quantitative reverse
transcription (RT)-polymerase chain reaction (PCR). APC expression levels were
significantly increased in leukocytes of schizophrenics both taking and not
taking antipsychotic medication and hence the increase of APC expression was not 
due to antipsychotic medication. APC is located at 5q21-22, which has been
previously reported to be linked with schizophrenia. Further, we performed the
transmission disequilibrium test (TDT) and TDT based on haplotypes to search for 
the association between schizophrenia and APC by examining 163 parent-offspring
trios of Chinese descent. We analyzed three single-nucleotide polymorphisms
(SNPs) (rs2229992, rs42427, rs465899) at the exon region of APC. TDT showed that 
the three SNPs are significantly associated with schizophrenia (TDT chi(2)=4.23, 
P<0.05; 4.15, P<0.05; 8.49 P<0.01, respectively; HHRRchi(2)=5.54, P<0.05; 4.40,
P<0.05; 9.79, P<0.01, respectively). We found a significant association between
the APC haplotypes from rs2229992-rs42427-rs465899 and schizophrenia (Global
chi(2)=44.376,df=7, P<0.001). The C-A-T haplotype has a frequency of more than
57% and has a strong association with schizophrenia (chi(2)=15.04, P<0.001).
These results indicate that the APC may be a candidate gene conferring
susceptibility to schizophrenia and also may be associated with reduced
vulnerability to cancer in schizophrenia.

DOI: 10.1038/sj.mp.4001653 
PMID: 15768050  [Indexed for MEDLINE]


1092. Pediatr Res. 2005 Jul;58(1):143-8. Epub 2005 Jun 27.

Rapid diagnosis of bacterial sepsis with PCR amplification and microarray
hybridization in 16S rRNA gene.

Shang S(1), Chen G, Wu Y, DU L, Zhao Z.

Author information: 
(1)Department of Neonatology, Affiliated Children's Hospital of Medical College, 
Zhejiang University, Hangzhou 310003, PR China. shangsq@mail.hz.zj.cn

In this study, blood culture and PCR-microarray analysis were used to examine 172
cases of suspected septicemia. Primers and oligonucleotide probes, based on the
sequences of bacterial 16SrRNA gene, were arrayed by imprinting on microarray
slides. Blood specimens collected from 172 cases of suspected septicemia were
cultured and then tested separately by PCR for the bacterial 16S rRNA. Of the 172
clinical cases, 17 cases tested positive by PCR. The number of positives
identified by PCR (9.88%) was significantly higher than the number of positives
identified by the blood culture (4.65%). When blood culture was used as control, 
the sensitivity of PCR was 100%, the specificity was 97.85%, and the index of
accurate diagnosis was 0.979. When the 17 PCR positive specimens were further
analyzed by hybridization against the microarrays, five were found to be probe
positive for E. coli, four were positive for S. epidermidis, four were positive
for CoNS, and two were positive for Bacillus and Propionibacterium, respectively.
In the eight specimens showing positive results by both PCR and blood culture,
the species determined by microarray analysis corresponded with the result
obtained from blood culture. Detection of the bacterial 16SrRNA genes in clinical
specimens by PCR and microarray analysis can be used to accurately diagnose
neonatal sepsis. This method has a higher sensitivity and specificity than blood 
culture and can provide a rapid way for the etiological diagnosis of neonatal
septicemia.

DOI: 10.1203/01.PDR.0000169580.64191.8B 
PMID: 15985688  [Indexed for MEDLINE]


1093. Plant Physiol. 2005 Jul;138(3):1409-21. Epub 2005 Jun 17.

The histidine kinase Hik34 is involved in thermotolerance by regulating the
expression of heat shock genes in synechocystis.

Suzuki I(1), Kanesaki Y, Hayashi H, Hall JJ, Simon WJ, Slabas AR, Murata N.

Author information: 
(1)Division of Cellular Regulation, National Institute for Basic Biology,
Myodaiji, Okazaki 444-8585, Japan. iwanes6803@biol.tsukuba.ac.jp

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the
transduction of signals associated with various kinds of environmental stress. To
examine the potential role in thermotolerance of Hiks, we used genome microarray 
analysis to screen a Hik knockout library for mutations that affected the
expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced 
the levels of transcripts of a number of heat shock genes, including htpG and
groESL1. Overexpression of the hik34 gene repressed the expression of these heat 
shock genes. In addition, the cells with a mutant gene for Hik34 (DeltaHik34
cells) survived incubation at 48 degrees C for 3 h, while wild-type cells and
cells with mutations in other Hiks were killed. However, mutation of the hik34
gene had only an insignificant effect on the global expression of genes upon
incubation of the mutant cells at 44 degrees C for 20 min. Quantitative
two-dimensional gel electrophoresis revealed that levels of GroES and HspA were
elevated in DeltaHik34 cells after incubation of cells at 42 degrees C for 60
min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified 
it. We found that the protein was autophosphorylated in vitro at physiological
temperatures, but not at elevated temperatures, such as 44 degrees C. These
results suggest that Hik34 might negatively regulate the expression of certain
heat shock genes that might be related to thermotolerance in Synechocystis.

DOI: 10.1104/pp.104.059097 
PMCID: PMC1176413
PMID: 15965020  [Indexed for MEDLINE]


1094. Biotechnol Bioeng. 2005 Jun 30;90(7):805-20.

Glucose metabolism at high density growth of E. coli B and E. coli K: differences
in metabolic pathways are responsible for efficient glucose utilization in E.
coli B as determined by microarrays and Northern blot analyses.

Phue JN(1), Noronha SB, Hattacharyya R, Wolfe AJ, Shiloach J.

Author information: 
(1)Biotechnology Unit, NIH NIDDK, Bethesda, Maryland 20892-2715, USA.

Erratum in
    Biotechnol Bioeng. 2005 Sep 5;91(5):649.

In a series of previous reports it was established by implementing metabolic
flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA
cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia
coli BL21 than in Escherichia coli JM109. These differences were accepted as the 
reason for the differences in the glucose metabolism and acetate excretion of
these two strains. Examination of the bacterial metabolism by microarrays and
time course Northern blot showed that in addition to the glyoxylate shunt, the
TCA cycle and the acetate uptake, other metabolic pathways are active differently
in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen
biosynthesis, and fatty acid degradation. It was found that in E. coli JM109,
acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate
rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses 
acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate
synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and
stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is
converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and
aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose
concentration was high, no transcription of these genes was observed and acetate 
concentration increased, but at low glucose concentrations these genes were
expressed and the acetate concentration decreased. It is possible that there is a
major regulatory molecule that controls not only ppsA and aceBAK but also acs.
The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen
accumulation is constitutively active in E. coli BL21 regardless of glucose
feeding strategy.

DOI: 10.1002/bit.20478 
PMID: 15806547  [Indexed for MEDLINE]


1095. BMC Cancer. 2005 Jun 27;5:66.

Differential gene expression profile reveals deregulation of pregnancy specific
beta1 glycoprotein 9 early during colorectal carcinogenesis.

Salahshor S(1), Goncalves J, Chetty R, Gallinger S, Woodgett JR.

Author information: 
(1)Department of Medical Biophysics, Ontario Cancer Institute, 610 University
Ave., Toronto, Ontario, M5G 2M9, Canada. salahsho@uhnres.utoronto.ca

BACKGROUND: APC (Adenomatous polyposis coli) plays an important role in the
pathogenesis of both familial and sporadic colorectal cancer. Patients carrying
germline APC mutations develop multiple colonic adenomas at younger age and
higher frequency than non-carrier cases which indicates that silencing of one APC
allele may be sufficient to initiate the transformation process.
METHODS: To elucidate the biological dysregulation underlying adenoma formation
we examined global gene expression profiles of adenomas and corresponding normal 
mucosa from an FAP patient. Differential expression of the most significant gene 
identified in this study was further validated by mRNA in situ hybridization,
reverse transcriptase PCR and Northern blotting in different sets of adenomas,
tumours and cancer cell lines.
RESULTS: Eighty four genes were differentially expressed between all adenomas and
corresponding normal mucosa, while only seven genes showed differential
expression within the adenomas. The first group included pregnancy specific
beta-1 glycoprotein 9 (PSG9) (p < 0.006). PSG9 is a member of the
carcinoembryonic antigen (CEA)/PSG family and is produced at high levels during
pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and
familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo
by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18) 
of FAP adenomas and 75% (45/60) of sporadic colorectal cancer cases tested.
CONCLUSION: Detection of PSG9 expression in adenomas, and at higher levels in FAP
cases, indicates that germline APC mutations and defects in Wnt signalling
modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult
except in association with cancer, and it appears to be an early molecular event 
associated with colorectal cancer monitoring of its expression may be useful as a
biomarker for the early detection of this disease.

DOI: 10.1186/1471-2407-5-66 
PMCID: PMC1184062
PMID: 15982419  [Indexed for MEDLINE]


1096. Theor Biol Med Model. 2005 Jun 24;2:23.

Predicting transcription factor activities from combined analysis of microarray
and ChIP data: a partial least squares approach.

Boulesteix AL(1), Strimmer K.

Author information: 
(1)Department of Statistics, University of Munich, Ludwigstr. 33, D-80539 Munich,
Germany. anne-laure.boulesteix@stat.uni-muenchen.de

BACKGROUND: The study of the network between transcription factors and their
targets is important for understanding the complex regulatory mechanisms in a
cell. Unfortunately, with standard microarray experiments it is not possible to
measure the transcription factor activities (TFAs) directly, as their own
transcription levels are subject to post-translational modifications.
RESULTS: Here we propose a statistical approach based on partial least squares
(PLS) regression to infer the true TFAs from a combination of mRNA expression and
DNA-protein binding measurements. This method is also statistically sound for
small samples and allows the detection of functional interactions among the
transcription factors via the notion of "meta"-transcription factors. In
addition, it enables false positives to be identified in ChIP data and activation
and suppression activities to be distinguished.
CONCLUSION: The proposed method performs very well both for simulated data and
for real expression and ChIP data from yeast and E. Coli experiments. It
overcomes the limitations of previously used approaches to estimating TFAs. The
estimated profiles may also serve as input for further studies, such as tests of 
periodicity or differential regulation. An R package "plsgenomics" implementing
the proposed methods is available for download from the CRAN archive.

DOI: 10.1186/1742-4682-2-23 
PMCID: PMC1182396
PMID: 15978125  [Indexed for MEDLINE]


1097. BMC Genomics. 2005 Jun 17;6:95.

A generic approach for the design of whole-genome oligoarrays, validated for
genomotyping, deletion mapping and gene expression analysis on Staphylococcus
aureus.

Charbonnier Y(1), Gettler B, François P, Bento M, Renzoni A, Vaudaux P, Schlegel 
W, Schrenzel J.

Author information: 
(1)Genomic Research Laboratory, University Hospitals of Geneva, rue
Micheli-du-Crest 24, CH-1211 Geneva 14, Switzerland. yvan.charbonnier@genomic.ch

BACKGROUND: DNA microarray technology is widely used to determine the expression 
levels of thousands of genes in a single experiment, for a broad range of
organisms. Optimal design of immobilized nucleic acids has a direct impact on the
reliability of microarray results. However, despite small genome size and
complexity, prokaryotic organisms are not frequently studied to validate selected
bioinformatics approaches. Relying on parameters shown to affect the
hybridization of nucleic acids, we designed freely available software and
validated experimentally its performance on the bacterial pathogen Staphylococcus
aureus.
RESULTS: We describe an efficient procedure for selecting 40-60 mer
oligonucleotide probes combining optimal thermodynamic properties with high
target specificity, suitable for genomic studies of microbial species. The
algorithm for filtering probes from extensive oligonucleotides libraries fitting 
standard thermodynamic criteria includes positional information of predicted
target-probe binding regions. This algorithm efficiently selected probes
recognizing homologous gene targets across three different sequenced genomes of
Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes
yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains
N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset
of control Escherichia coli probes was validated for applications in the fields
of comparative genomics and molecular epidemiology, mapping of deletion mutations
and transcription profiling.
CONCLUSION: This generic chip-design process merging sequence information from
several related genomes improves genome coverage even in conserved regions.

DOI: 10.1186/1471-2164-6-95 
PMCID: PMC1183204
PMID: 15963225  [Indexed for MEDLINE]


1098. BMC Genomics. 2005 Jun 6;6:84.

Decoding the nucleoid organisation of Bacillus subtilis and Escherichia coli
through gene expression data.

Carpentier AS(1), Torrésani B, Grossmann A, Hénaut A.

Author information: 
(1)Laboratoire Génome et Informatique, CNRS UMR 8116, Tour Evry2, 523 Place des
Terrasses, 91034 Evry Cedex, France. carpentier@genopole.cnrs.fr

BACKGROUND: Although the organisation of the bacterial chromosome is an area of
active research, little is known yet on that subject. The difficulty lies in the 
fact that the system is dynamic and difficult to observe directly. The advent of 
massive hybridisation techniques opens the way to further studies of the
chromosomal structure because the genes that are co-expressed, as identified by
microarray experiments, probably share some spatial relationship. The use of
several independent sets of gene expression data should make it possible to
obtain an exhaustive view of the genes co-expression and thus a more accurate
image of the structure of the chromosome.
RESULTS: For both Bacillus subtilis and Escherichia coli the co-expression of
genes varies as a function of the distance between the genes along the
chromosome. The long-range correlations are surprising: the changes in the level 
of expression of any gene are correlated (positively or negatively) to the
changes in the expression level of other genes located at well-defined long-range
distances. This property is true for all the genes, regardless of their
localisation on the chromosome. We also found short-range correlations, which
suggest that the location of these co-expressed genes corresponds to DNA turns on
the nucleoid surface (14-16 genes).
CONCLUSION: The long-range correlations do not correspond to the domains so far
identified in the nucleoid. We explain our results by a model of the nucleoid
solenoid structure based on two types of spirals (short and long). The long
spirals are uncoiled expressed DNA while the short ones correspond to coiled
unexpressed DNA.

DOI: 10.1186/1471-2164-6-84 
PMCID: PMC1177944
PMID: 15938745  [Indexed for MEDLINE]


1099. Bioinformatics. 2005 Jun 1;21(11):2730-8. Epub 2005 Mar 29.

Correlation between gene expression profiles and protein-protein interactions
within and across genomes.

Bhardwaj N(1), Lu H.

Author information: 
(1)Bioinformatics Program, Department of Bioengineering, University of Illinois
at Chicago, Chicago, IL 60607, USA.

MOTIVATION: Function annotation of an unclassified protein on the basis of its
interaction partners is well documented in the literature. Reliable predictions
of interactions from other data sources such as gene expression measurements
would provide a useful route to function annotation. We investigate the global
relationship of protein-protein interactions with gene expression. This
relationship is studied in four evolutionarily diverse species, for which
substantial information regarding their interactions and expression is available:
human, mouse, yeast and Escherichia coli.
RESULTS: In E.coli the expression of interacting pairs is highly correlated in
comparison to random pairs, while in the other three species, the correlation of 
expression of interacting pairs is only slightly stronger than that of random
pairs. To strengthen the correlation, we developed a protocol to integrate
ortholog information into the interaction and expression datasets. In all four
genomes, the likelihood of predicting protein interactions from highly correlated
expression data is increased using our protocol. In yeast, for example, the
likelihood of predicting a true interaction, when the correlation is > 0.9,
increases from 1.4 to 9.4. The improvement demonstrates that protein interactions
are reflected in gene expression and the correlation between the two is
strengthened by evolution information. The results establish that co-expression
of interacting protein pairs is more conserved than that of random ones.

DOI: 10.1093/bioinformatics/bti398 
PMID: 15797912  [Indexed for MEDLINE]


1100. BMC Genomics. 2005 Jun 1;6:81.

Genome-wide localization of mobile elements: experimental, statistical and
biological considerations.

Martinez-Vaz BM(1), Xie Y, Pan W, Khodursky AB.

Author information: 
(1)Department of Biochemistry, Molecular Biology and Biophysics and Biotechnology
Institute, University of Minnesota, Saint Paul, MN 55108, USA. marti112@umn.edu

BACKGROUND: The distribution and location of insertion elements in a genome is an
excellent tool to track the evolution of bacterial strains and a useful molecular
marker to distinguish between closely related bacterial isolates. The information
about the genomic locations of IS elements is available in public sequence
databases. However, the locations of mobile elements may vary from strain to
strain and within the population of an individual strain. Tools that allow de
novo localization of IS elements and are independent of existing sequence
information are essential to map insertion elements and advance our knowledge of 
the role that such elements play in gene regulation and genome plasticity in
bacteria.
RESULTS: In this study, we present an efficient and reliable method for linear
mapping of mobile elements using whole-genome DNA microarrays. In addition, we
describe an algorithm for analysis of microarray data that can be applied to find
DNA sequences physically juxtaposed with a target sequence of interest. This
approach was used to map the locations of the IS5 elements in the genome of
Escherichia coli K12. All IS5 elements present in the E. coli genome known from
GenBank sequence data were identified. Furthermore, previously unknown insertion 
sites were predicted with high sensitivity and specificity. Two variants of E.
coli K-12 MG1655 within a population of this strain were predicted by our
analysis. The only significant difference between these two isolates was the
presence of an IS5 element upstream of the main flagella regulator, flhDC.
Additional experiments confirmed this prediction and showed that these isolates
were phenotypically distinct. The effect of IS5 on the transcriptional activity
of motility and chemotaxis genes in the genome of E. coli strain MG1655 was
examined. Comparative analysis of expression profiles revealed that the presence 
of IS5 results in a mild enhancement of transcription of the flagellar genes that
translates into a slight increase in motility.
CONCLUSION: In summary, this work presents a case study of an experimental and
analytical application of DNA microarrays to map insertion elements in bacteria
and gains an insight into biological processes that might otherwise be overlooked
by relying solely on the available genome sequence data.

DOI: 10.1186/1471-2164-6-81 
PMCID: PMC1174868
PMID: 15929794  [Indexed for MEDLINE]


1101. Mol Cell Probes. 2005 Jun;19(3):195-201.

A DNA microarray for identification of virulence and antimicrobial resistance
genes in Salmonella serovars and Escherichia coli.

Chen S(1), Zhao S, McDermott PF, Schroeder CM, White DG, Meng J.

Author information: 
(1)Department of Nutrition and Food Science, University of Maryland, 0112 Skinner
Building, College Park, MD 20742, USA.

Characterization of antimicrobial resistance and virulence gene profiles provides
important information on the potential pathogenicity of bacteria. This
information can be used to facilitate prompt and effective treatment of bacterial
infections. We developed and tested a PCR-based microarray platform for detecting
virulence and antimicrobial resistance genes in Salmonella serovars and
Escherichia coli. Twelve Salmonella and seven E. coli isolates were screened for 
the presence of 25 virulence and 23 antimicrobial resistance genes. All S.
Typhimurium DT104 isolates harbored virulence plasmids. E. coli O157:H7 isolates 
possessed virulence genes typical of enterohemorrhagic E. coli (EHEC), whereas E.
coli O126 isolates contained virulence genes characteristic of enteropathogenic
E. coli (EPEC) and E. coli O111, O78 and O147 isolates had virulence genes
characteristic of enterotoxigenic E. coli (ETEC). Correlation between
antimicrobial resistance phenotype and genotype was observed for each isolate.
The aadA, tetA, and sulI genes were most commonly detected in bacteria resistant 
to streptomycin, tetracycline and sulfonamide, respectively. All isolates
exhibiting resistance to third generation cephalosporins harbored the bla(CMY-2) 
and bla(TEM-1) genes. Microarray analysis is an effective method to rapidly
screen Salmonella and E. coli for multiple antimicrobial resistance and virulence
genes.

DOI: 10.1016/j.mcp.2004.11.008 
PMID: 15797820  [Indexed for MEDLINE]


1102. Mol Microbiol. 2005 Jun;56(5):1274-86.

A member of the cAMP receptor protein family of transcription regulators in
Mycobacterium tuberculosis is required for virulence in mice and controls
transcription of the rpfA gene coding for a resuscitation promoting factor.

Rickman L(1), Scott C, Hunt DM, Hutchinson T, Menéndez MC, Whalan R, Hinds J,
Colston MJ, Green J, Buxton RS.

Author information: 
(1)Division of Mycobacterial Research, National Institute for Medical Research,
Mill Hill, London NW7 1AA, UK.

Erratum in
    Mol Microbiol. 2005 Aug;57(3):869.

Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription 
factor belonging to the cAMP receptor protein (CRP) family caused growth defects 
in laboratory medium, in bone marrow-derived macrophages and in a mouse model of 
tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified
16 genes with significantly altered expression in the mutant compared with the
wild type. Analysis of the DNA sequences upstream of the corresponding open
reading frames revealed that 12 possessed sequences related to a consensus CRP
binding site that could represent the sites of action of Rv3676. These included
rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type,
and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression
in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the
Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the
product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically
with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site.
Although Rv3676 retains four of the six amino acid residues that bind cAMP in
Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA 
promoter in vitro. In summary, it has been shown that Rv3676 is a direct
regulator of rpfA expression, and because rpfA codes for a resuscitation
promoting factor this may implicate Rv3676 in reactivation of dormant M.
tuberculosis infections.

DOI: 10.1111/j.1365-2958.2005.04609.x 
PMCID: PMC2964915
PMID: 15882420  [Indexed for MEDLINE]


1103. Trends Microbiol. 2005 Jun;13(6):246-8.

Blueprinting the regulatory response of Escherichia coli to the urinary tract.

Seed PC(1), Hultgren SJ.

Author information: 
(1)Department of Pediatrics, Washington University School of Medicine, St. Louis,
MO, 63110, USA.

Recent work has shown how comparative genomic and microarray analyses can provide
insights into the transcriptional state of uropathogenic Escherichia coli (UPEC) 
during infection. This study will serve as an important platform from which to
identify virulence determinants and the principle mechanisms of adaptation to the
urinary tract.

DOI: 10.1016/j.tim.2005.03.014 
PMID: 15936653  [Indexed for MEDLINE]


1104. Theor Biol Med Model. 2005 May 31;2:20.

Promoter addresses: revelations from oligonucleotide profiling applied to the
Escherichia coli genome.

Sivaraman K(1), Seshasayee AS, Swaminathan K, Muthukumaran G, Pennathur G.

Author information: 
(1)Centre for Biotechnology, Anna University, Chennai, India. k.sivan@gmail.com

BACKGROUND: Transcription is the first step in cellular information processing.
It is regulated by cis-acting elements such as promoters and operators in the
DNA, and trans-acting elements such as transcription factors and sigma factors.
Identification of cis-acting regulatory elements on a genomic scale requires
computational analysis.
RESULTS: We have used oligonucleotide profiling to predict regulatory regions in 
a bacterial genome. The method has been applied to the Escherichia coli K12
genome and the results analyzed. The information content of the putative
regulatory oligonucleotides so predicted is validated through intra-genomic
analyses, correlations with experimental data and inter-genome comparisons. Based
on the results we have proposed a model for the bacterial promoter. The results
show that the method is capable of identifying, in the E.coli genome, cis-acting 
elements such as TATAAT (sigma70 binding site), CCCTAT (1 base relative of
sigma32 binding site), CTATNN (LexA binding site), AGGA-containing
hexanucleotides (Shine Dalgarno consensus) and CTAG-containing hexanucleotides
(core binding sites for Trp and Met repressors).
CONCLUSION: The method adopted is simple yet effective in predicting upstream
regulatory elements in bacteria. It does not need any prior experimental data
except the sequence itself. This method should be applicable to most known
genomes. Profiling, as applied to the E.coli genome, picks up known cis-acting
and regulatory elements. Based on the profile results, we propose a model for the
bacterial promoter that is extensible even to eukaryotes. The model is that the
core promoter lies within a plateau of bent AT-rich DNA. This bent DNA acts as a 
homing segment for the sigma factor to recognize the promoter. The model thus
suggests an important role for local landscapes in prokaryotic and eukaryotic
gene regulation.

DOI: 10.1186/1742-4682-2-20 
PMCID: PMC1166578
PMID: 15927055  [Indexed for MEDLINE]


1105. Biosens Bioelectron. 2005 May 15;20(11):2310-3.

A novel optical biosensor format for the detection of clinically relevant TP53
mutations.

Wilson PK(1), Jiang T, Minunni ME, Turner AP, Mascini M.

Author information: 
(1)Cranfield University, UK. p.k.wilson.s04@cranfield.ac.uk

The TP53 gene has been the subject of intense research since the realisation that
inactivation of this gene is common to most cancer types. Numerous publications
have linked TP53 mutations in general or at specific locations to patient
prognosis and therapy response. The findings of many studies using general
approaches such as immunohistochemistry or sequencing are contradictory. However,
the detection of specific mutations, especially those occurring in the
structurally important L2 and L3 zinc binding domains, which are the most common 
sites of TP53 mutations, have been linked to patient prognosis and more strongly 
to radiotherapy and chemotherapy resistance in several major cancers. In this
study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments
Spreeta chips were used to develop a DNA biosensor based on thiolated probes
complementary to these domains. The sensors were able to detect these mutations
in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but
the difference in hybridisation signal was small. Preliminary experiments to
enhance the signal using Escherichia coli mismatch repair proteins, MutS and
single strand binding protein were carried out. It was found that MutS was unable
to bind to mismatch oligonucleotides, but single strand binding protein was able 
to bind to single stranded probes, which had not hybridised to the target,
resulting in a three-fold increase in the sensitivity of the biosensor. While
further work needs to be carried out to optimise the system, these preliminary
experiments indicate that the TI-SPR-1 can be used for the detection of
clinically relevant mutations in the TP53 gene and that the sensitivity can be
increased significantly using single strand binding protein. This system has a
number of advantages over current mutation detection technologies, including
lower cost, ease of sensor preparation and measurement procedures, technical
simplicity and increased speed due to the lack of need for gel electrophoresis.

DOI: 10.1016/j.bios.2004.11.020 
PMID: 15797331  [Indexed for MEDLINE]


1106. Vet Immunol Immunopathol. 2005 May 15;105(3-4):317-29.

Development of a porcine small intestinal cDNA micro-array: characterization and 
functional analysis of the response to enterotoxigenic E. coli.

Niewold TA(1), Kerstens HH, van der Meulen J, Smits MA, Hulst MM.

Author information: 
(1)Animal Sciences Group of Wageningen UR, Research Cluster, Animal Physiology
and Health, P.O. Box 65, 8200 AB Lelystad, The Netherlands. theo.niewold@wur.nl

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous
compounds and micro-flora interact, and its condition is influenced by the
complex interaction between these factors and host genetic elements. Furthermore,
interactions of immune cells with the other components of the intestinal mucosa
are essential in the defense against pathogens. The outcomes of these complex
interactions determine resistance to infectious diseases. The development of
genomic tools and techniques allows for analysis of multiple and complex host
responses. We have constructed a porcine small intestinal micro-array, based on
cDNA from jejunal mucosal scrapings. Material from two developmental distinct
stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad
representation of mucosal transcripts. The micro-array consists of 3468 cDNAs
spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs
revealed a differential expression in at least 300 spots. Furthermore, we report 
the early gene expression response of pig small intestine jejunal mucosa to
infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment 
perfusion (SISP) technique. A response pattern was found in which a marker for
innate defense dominated, demonstrating the strength of this applied technology. 
Further analysis of these response patterns will contribute to a better
understanding of enteric health and disease in pigs. The great similarity between
pig and human suggest results from these continuing studies should be applicable 
for both agricultural and human biomedical purposes.

DOI: 10.1016/j.vetimm.2005.02.010 
PMID: 15808309  [Indexed for MEDLINE]


1107. Vet Immunol Immunopathol. 2005 May 15;105(3-4):289-99.

Gene expression profiling of avian macrophage activation.

Bliss TW(1), Dohms JE, Emara MG, Keeler CL Jr.

Author information: 
(1)Department of Animal and Food Sciences, College of Agriculture and Natural
Resources, University of Delaware, Newark, DE 19716-2150, USA.

Through the process of phagocytosis, the macrophage is responsible for the
clearance and destruction of both intracellular and extracellular pathogens. When
stimulated, macrophages undergo a process of activation involving an increase in 
size and motility, enhanced phagocytic, bactericidal, and tumoricidal activity,
and up-regulation of several cell-surface markers. One well characterized method 
of mammalian macrophage activation involves the Toll-like receptor (TLR) pathway.
TLRs are surface molecules that function as direct receptors for microbial
components. Binding of ligand to TLRs results in activation of transcription
factors that regulate genes involved in microbial killing, apoptosis, and antigen
recognition, as well as pro- and anti-inflammatory cytokines and chemokines. We
have constructed a 4906-element (14,718 spot) avian macrophage-specific cDNA
microarray (AMM). The AMM contains 16 of the approximately 44 genes identified
within the mammalian TLR pathway. This array was used to examine the
transcriptional response of avian macrophages to Gram-negative bacteria and their
cell wall components and to evaluate the contribution of the avian TLR pathway to
that response. Of the elements on the AMM, 981 (20%) exhibited significant
(greater than two-fold, p < 0.01) changes in expression during phagocytosis of
Escherichia coli and 243 (5%) exhibited significant expression changes during
exposure to lipopolysaccharide (LPS). A unique set of overlapping elements (154),
were observed to exhibit significant changes in expression for both phagocytosis 
and LPS stimulation, representing a set of core response elements. Of these
elements, 63% were commonly induced, while 32% were commonly repressed. Both LPS 
and bacteria were found to induce NFkappabeta and several end products of the TLR
pathway.

DOI: 10.1016/j.vetimm.2005.02.013 
PMID: 15808307  [Indexed for MEDLINE]


1108. Appl Environ Microbiol. 2005 May;71(5):2762-5.

The gene yggE functions in restoring physiological defects of Escherichia coli
cultivated under oxidative stress conditions.

Kim SY(1), Nishioka M, Hayashi S, Honda H, Kobayashi T, Taya M.

Author information: 
(1)Division of Chemical Engineering, Department of Materials Engineering Science,
Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka
560-8531, Japan.

DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated 
when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was
cultivated under the oxidative stress generated by photoexcited TiO(2), and pYFD,
pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19
vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying
pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the
culture of wild-type strain, E. coli MM294, in the medium with paraquat (10
micromol/l), maximum specific growth rate of the cells with pYGE was about five
times higher than that of the control cells, with a decreased ROS content in the 
former cells. The introduction of pYGE also suppressed the occurrence of the
cells with altered amino acid requirement in the culture of MM294 cells with
paraquat.

DOI: 10.1128/AEM.71.5.2762-2765.2005 
PMCID: PMC1087592
PMID: 15870370  [Indexed for MEDLINE]


1109. Biotechnol Prog. 2005 May-Jun;21(3):689-99.

Transcriptional response of Escherichia coli to temperature shift.

Gadgil M(1), Kapur V, Hu WS.

Author information: 
(1)Department of Chemical Engineering and Materials Science, Biomedical Genomics 
Center, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota
55455-0132, USA.

Temperature shift is often practiced in the cultivation of Escherichia coli to
reduce undesired metabolite formation and to maximize synthesis of correctly
folded heterologous protein. As the culture temperature is decreased below the
optimal 37 degrees C, growth rate decreases and many physiological changes occur.
In this study, we investigated the gene expression dynamics of E. coli on
switching its cultivation temperature from 37 to 33 and 28 degrees C using whole 
genome DNA microarrays. Approximately 9% of the genome altered expression level
on temperature shift. Overall, the alteration of transcription upon the downshift
of temperature is rapid and globally distributed over a wide range of gene
classes. The general trends of transcriptional changes at 28 and 33 degrees C
were similar. The largest functional class among the differentially expressed
genes was energy metabolism. About 12% of genes in energy metabolism show a
decrease in their level of expression, and approximately 6% show an increase.
Consistent with the decrease in the glucose uptake rate, many genes involved in
glycolysis and the PTS sugar transport systems show decreased expression. Genes
encoding enzymes related to amino acid biosynthesis and transport also have
reduced expression levels. Such decrease in expression probably reflects the
reduced growth rate and the accompanying reduction in energy and amino acid
demand at lower temperatures. However, nearly all genes encoding enzymes in the
TCA cycle have increased expression levels, which may well be compensating the
reduction of the activity of TCA cycle enzymes at lower temperatures. Temperature
shift also results in shift of the cytochromes from the high affinity cytochrome 
o system to the low affinity cytochrome d system. There is no evidence that
protein processing genes are selectively altered to create favorable conditions
for heterologous protein synthesis. Our results indicate that the beneficial
effect of temperature shift in many biotechnological processes is likely to be
attributed to the general effect of reduced growth and metabolism.

DOI: 10.1021/bp049630l 
PMID: 15932244  [Indexed for MEDLINE]


1110. Gut. 2005 May;54(5):623-9.

Activation of RegIIIbeta/gamma and interferon gamma expression in the intestinal 
tract of SCID mice: an innate response to bacterial colonisation of the gut.

Keilbaugh SA(1), Shin ME, Banchereau RF, McVay LD, Boyko N, Artis D, Cebra JJ, Wu
GD.

Author information: 
(1)Division of Gastroenterology, University of Pennsylvania School of Medicine,
Philadelphia, USA.

BACKGROUND AND AIMS: The mechanisms by which commensal bacteria provoke
intestinal inflammation in animal models of inflammatory bowel disease (IBD)
remain incompletely defined, leading to increasing interest in the innate immune 
response of the colonic mucosa to bacterial colonisation.
METHODS: Using gene expression profiling of colonic RNA from C.B17.SCID germ free
mice and those colonised with altered Schaedler's flora, we investigated the
innate immune response to bacterial colonisation in vivo. The two most
consistently induced gene groups were RegIIIbeta and gamma as well as interferon 
gamma (IFN-gamma) response genes.
RESULTS: Using quantitative reverse transcription-polymerase chain reaction, we
showed that RegIIIbeta, RegIIIgamma, and IFN-gamma were constitutively expressed 
in the colon of conventionally housed SCID mice compared with either germ free
SCID or conventionally housed BALB/c mice. Induction of these genes was
reproduced by chronic monoassociation of germ free SCID mice with either of two
separate gut commensal bacterial species-segmented filamentous bacteria and
Schaedler's Escherichia coli. The cellular source for IFN-gamma on
monoassociation of SCID mice with Schaedler's E coli was localised to a subset of
intraepithelial natural killer (IENK) cells that express asialo-GM1. In vivo
IFN-gamma immunoneutralisation studies failed to demonstrate any alteration in
RegIIIbeta or gamma expression.
CONCLUSIONS: Thus bacterial colonisation of the colon independently activates two
distinct innate immune cell types at the mucosal interface with the colonic
lumen, intestinal epithelial cells, and IENK cells, a response that may be
regulated by the adaptive immune system. These innate immune responses may play a
role in the pathogenesis of colitis in SCID adoptive transfer models in mice and 
possibly in patients with IBD.

DOI: 10.1136/gut.2004.056028 
PMCID: PMC1774500
PMID: 15831905  [Indexed for MEDLINE]


1111. Infect Immun. 2005 May;73(5):2958-66.

Pseudomonas aeruginosa SoxR does not conform to the archetypal paradigm for
SoxR-dependent regulation of the bacterial oxidative stress adaptive response.

Palma M(1), Zurita J, Ferreras JA, Worgall S, Larone DH, Shi L, Campagne F,
Quadri LE.

Author information: 
(1)Microbiology and Immunology, Medical College of Cornell University, 1300 York 
Avenue, Box 62, W-706, New York, NY 10021, USA.

SoxR is a transcriptional regulator that controls an oxidative stress response in
Escherichia coli. The regulator is primarily activated by superoxide
anion-dependent oxidation. Activated SoxR turns on transcription of a single
gene, soxS, which encodes a transcriptional regulator that activates a regulon
that includes dozens of oxidative stress response genes. SoxR homologues have
been identified in many bacterial species, including the opportunistic pathogen
Pseudomonas aeruginosa. However, the expected SoxR partner, SoxS, has not been
found in P. aeruginosa. Thus, the primary gene target(s) of P. aeruginosa SoxR is
unknown and the involvement of this regulator in the oxidative stress response of
the bacterium remains unclear. We utilized transcriptome profiling to identify
the P. aeruginosa SoxR regulon and constructed and characterized an unmarked P.
aeruginosa DeltasoxR mutant. We provide evidence indicating that P. aeruginosa
SoxR activates a six-gene regulon in response to O(2)(.-)-induced stress. The
regulon includes three transcriptional units: (i) the recently identified
mexGHI-ompD four-gene operon, which encodes a multidrug efflux pump system
involved in quorum-sensing signal homeostasis; (ii) gene PA3718, encoding a
probable efflux pump; and (iii) gene PA2274, encoding a probable monooxygenase.
We also demonstrate that P. aeruginosa SoxR is not a key regulatory player in the
oxidative stress response. Finally, we show that P. aeruginosa SoxR is required
for virulence in a mouse model of intrapulmonary infection. These results
demonstrate that the E. coli-based SoxRS paradigm does not hold in P. aeruginosa 
and foster new hypotheses for the possible physiological role of P. aeruginosa
SoxR.

DOI: 10.1128/IAI.73.5.2958-2966.2005 
PMCID: PMC1087365
PMID: 15845502  [Indexed for MEDLINE]


1112. Infect Immun. 2005 May;73(5):2923-31.

Escherichia coli K1 RS218 interacts with human brain microvascular endothelial
cells via type 1 fimbria bacteria in the fimbriated state.

Teng CH(1), Cai M, Shin S, Xie Y, Kim KJ, Khan NA, Di Cello F, Kim KS.

Author information: 
(1)Division of Pediatric Infectious Diseases, School of Medicine, Johns Hopkins
University, Baltimore, MD 21287, USA.

Escherichia coli K1 is a major gram-negative organism causing neonatal
meningitis. E. coli K1 binding to and invasion of human brain microvascular
endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the
central nervous system in vivo. In the present study, we showed using DNA
microarray analysis that E. coli K1 associated with HBMEC expressed significantly
higher levels of the fim genes compared to nonassociated bacteria. We also showed
that E. coli K1 binding to and invasion of HBMEC were significantly decreased
with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they
were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1
strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e.,
fimbriated) bacteria. Taken together, we showed for the first time that type 1
fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC
and that type 1 fimbria phase-on E. coli is the major population interacting with
HBMEC.

DOI: 10.1128/IAI.73.5.2923-2931.2005 
PMCID: PMC1087349
PMID: 15845498  [Indexed for MEDLINE]


1113. J Bacteriol. 2005 May;187(9):3259-66.

Global gene expression responses to cadmium toxicity in Escherichia coli.

Wang A(1), Crowley DE.

Author information: 
(1)Department of Environmental Sciences, University of Caliifornia, Riverside, CA
92521, USA.

Genome-wide analysis of temporal gene expression profiles in Escherichia coli
following exposure to cadmium revealed a shift to anaerobic metabolism and
induction of several stress response systems. Disruption in the transcription of 
genes encoding ribosomal proteins and zinc-binding proteins may partially explain
the molecular mechanisms of cadmium toxicity.

DOI: 10.1128/JB.187.9.3259-3266.2005 
PMCID: PMC1082819
PMID: 15838054  [Indexed for MEDLINE]


1114. Physiol Biochem Zool. 2005 May-Jun;78(3):299-315. Epub 2005 May 3.

Changes in gene expression following high-temperature adaptation in
experimentally evolved populations of E. coli.

Riehle MM(1), Bennett AF, Long AD.

Author information: 
(1)Department of Ecology and Evolutionary Biology, University of California,
Irvine, CA 92697-2525, USA. mriehle@umn.edu

Transcription profiling (quantitative analysis of RNA abundance) can provide a
genome-wide picture of gene expression changes that accompany organismal
adaptation to a new environment. Here, we used DNA microarrays to characterize
genome-wide changes in transcript abundance in three replicate lines of the
bacterium E. coli grown for 2,000 generations at a stressful high temperature
(41.5 degrees C). Across these lines, 12% of genes significantly changed
expression during high-temperature adaptation; the majority of these changes
(55%) were less than twofold increments or decrements. Thirty-nine genes, four
times the number expected by chance alone, exhibited moderately or highly
replicated expression changes across lines. Expression changes within a priori
defined functional categories showed an even greater level of replication than
did individual genes. Expression changes in the phenotypically defined stress
genes and adaptation functional categories were important in evolutionary
adaptation to high temperature.

DOI: 10.1086/430035 
PMID: 15887077  [Indexed for MEDLINE]


1115. Biotechnol Bioeng. 2005 Apr 20;90(2):127-53.

Transcriptome profiles for high-cell-density recombinant and wild-type
Escherichia coli.

Haddadin FT(1), Harcum SW.

Author information: 
(1)Department of Chemical Engineering, 112 Biosystem Research Complex, Clemson
University, Clemson, South Carolina 29634, USA.

The transcriptome profiles for wild-type (plasmid-free) and recombinant
(plasmid-bearing) Escherichia coli during well-controlled synchronized
high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was
observed that the growth phase significantly affected the transcriptome profiles,
and the transcriptome profiles were significantly different for the recombinant
and wild-type cultures. The response of the wild-type and recombinant cultures to
an isopropyl-1-thio-beta-D-galactopyranoside- (IPTG-) addition was examined,
where IPTG induced recombinant protein production in the plasmid-bearing
cultures. The IPTG-addition significantly altered the transcriptome response of
the wild-type cultures entering the stationary phase. The IPTG-induced
recombinant protein production resulted in a significant down-regulation of many 
energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and 
translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage
(psp, hfl) and transposon-related genes (tra, ins) were significantly regulated
in the recombinant cultures due to the IPTG-induction. These results indicate
that the signaling mechanism, associated with the recombinant protein production,
may induce a metabolic burden in the form of a phage defense mechanism. Taken
together, these results indicated that recombinant protein production initiated a
cascade of transcriptome responses that down-regulated the very genes needed to
sustain productivity.

Copyright 2005 Wiley Periodicals, Inc.

DOI: 10.1002/bit.20340 
PMID: 15742388  [Indexed for MEDLINE]


1116. Exp Cell Res. 2005 Apr 15;305(1):145-55.

Microarray analysis of immune challenged Drosophila hemocytes.

Johansson KC(1), Metzendorf C, Söderhäll K.

Author information: 
(1)Department of Comparative Physiology, Evolutionary Biology Center, Uppsala
University, Norbyvägen 18A, SE-752 36 Uppsala, Sweden.

Insect hemocytes play multiple roles in immunity and carry out cellular responses
like phagocytosis, encapsulation and melanization as well as producing humoral
effector proteins in the first line of defense after injury and invasion of
microorganisms. In this work, we used the Drosophila melanogaster hemocyte-like
cell line mbn-2 and Affymetrix Drosophila GeneChips to investigate the
transcriptome of a single type of immune competent tissue exposed to
Gram-negative cell wall components (crude LPS) or high dose infection with live
Escherichia coli. We found that gene expression profiles of both treatments
overlap but show important differences in expression levels of several genes
involved in immunity. In addition, cell morphology during infection was monitored
and revealed distinct alterations in cell shape and adhesion. Presence of large
numbers of bacteria also increased the number of cells taking on crystal cell
fate. Taken together, our results indicate that hemocytes sense and respond
differently to purified bacterial surface molecules and infection with live and
actively growing bacteria both at the level of gene expression and in cell
behavior.

DOI: 10.1016/j.yexcr.2004.12.018 
PMID: 15777795  [Indexed for MEDLINE]


1117. J Biol Chem. 2005 Apr 15;280(15):15084-96. Epub 2005 Feb 7.

Global gene expression profiling in Escherichia coli K12: effects of oxygen
availability and ArcA.

Salmon KA(1), Hung SP, Steffen NR, Krupp R, Baldi P, Hatfield GW, Gunsalus RP.

Author information: 
(1)Department of Microbiology, Immunology, and Molecular Genetics, University of 
California, Los Angeles, California 90095-1489, USA.

The ArcAB two-component system of Escherichia coli regulates the
aerobic/anaerobic expression of genes that encode respiratory proteins whose
synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of
E. coli was undertaken to identify other potential targets of oxygen and ArcA
regulation. A group of 175 genes generated from this study and our previous study
on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, 
G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our
gold standard gene set, have p values <0.00013 and a posterior probability of
differential expression value of 0.99. These 175 genes clustered into eight
expression patterns and represent genes involved in a large number of cell
processes, including small molecule biosynthesis, macromolecular synthesis, and
aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175
genes were also identified in our previous study of the fnr allele. A MEME/weight
matrix method was used to identify a new putative ArcA-binding site for all genes
of the E. coli genome. 16 new sites were identified upstream of genes in our gold
standard set. The strict statistical analyses that we have performed on our data 
allow us to predict that 1139 genes in the E. coli genome are regulated either
directly or indirectly by the ArcA protein with a 99% confidence level.

DOI: 10.1074/jbc.M414030200 
PMID: 15699038  [Indexed for MEDLINE]


1118. Biotechnol Bioeng. 2005 Apr 5;90(1):64-76.

Physiological response of central metabolism in Escherichia coli to deletion of
pyruvate oxidase and introduction of heterologous pyruvate carboxylase.

Vemuri GN(1), Minning TA, Altman E, Eiteman MA.

Author information: 
(1)Center for Molecular BioEngineering, Driftmier Engineering, University of
Georgia, Athens, Georgia 30602, USA.

We studied the physiological response of Escherichia coli central metabolism to
the expression of heterologous pyruvate carboxylase (PYC) in the presence and
absence of pyruvate oxidase (POX). These studies were complemented with
expression analysis of central and intermediary metabolic genes and conventional 
in vitro enzyme assays to evaluate glucose metabolism at steady-state growth
conditions (chemostats). The absence of POX activity reduced nongrowth-related
energy metabolism (maintenance coefficient) and increased the maximum specific
rate of oxygen consumption. The presence of PYC activity (i.e., with POX
activity) increased the biomass yield coefficient and reduced the maximum
specific oxygen consumption rate compared to the wildtype. The presence of PYC in
a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater
biomass yield compared to the wildtype. Providing E. coli with PYC or removing
POX increased the threshold specific growth rate at which acetate accumulation
began, with an 80% reduction in acetate accumulation observed at a specific
growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests
utilization of energetically less favorable glucose metabolism via glucokinase
and the Entner-Doudoroff pathway in the absence of functional POX, while the
upregulation of the phosphotransferase glucose uptake system and several amino
acid biosynthetic pathways occurs in the presence of PYC. The physiological and
expression changes resulting from these genetic perturbations demonstrate the
importance of the pyruvate node in respiration and its impact on acetate overflow
during aerobic growth.

Copyright (c) 2005 Wiley Periodicals, Inc.

DOI: 10.1002/bit.20418 
PMID: 15736164  [Indexed for MEDLINE]


1119. Bioinformatics. 2005 Apr 1;21(7):1062-8. Epub 2004 Oct 28.

Extracting relations between promoter sequences and their strengths from
microarray data.

Kiryu H(1), Oshima T, Asai K.

Author information: 
(1)Graduate School of Information Sciences, Nara Institute of Science and
Technology 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan.
hisano-k@is.aist-nara.ac.jp

MOTIVATION: The relations between the promoter sequences and their strengths were
extensively studied in the 1980s. Although these studies uncovered strong
sequence-strength correlations, the cost of their elaborate experimental methods 
have been too high to be applied to a large number of promoters. On the contrary,
a recent increase in the microarray data allows us to compare thousands of gene
expressions with their DNA sequences.
RESULTS: We studied the relations between the promoter sequences and their
strengths using the Escherichia coli microarray data. We modeled those relations 
using a simple weight matrix, which was optimized with a novel support vector
regression method. It was observed that several non-consensus bases in the '-35' 
and '-10' regions of promoter sequences act positively on the promoter strength
and that certain consensus bases have a minor effect on the strength. We analyzed
outliers for which the observed gene expressions deviate from the promoter
strength predictions, and identified several genes with enhanced expressions due 
to multiple promoters and genes under strong regulation by transcription factors.
Our method is applicable to other procaryotes for which both the promoter
sequences and the microarray data are available.

DOI: 10.1093/bioinformatics/bti094 
PMID: 15513998  [Indexed for MEDLINE]


1120. FEBS J. 2005 Apr;272(7):1600-15.

Novel target genes of the Wnt pathway and statistical insights into Wnt target
promoter regulation.

Ziegler S(1), Röhrs S, Tickenbrock L, Möröy T, Klein-Hitpass L, Vetter IR, Müller
O.

Author information: 
(1)Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.

The Wnt pathway controls biological processes via the regulation of target gene
expression. The expression of direct Wnt target genes, e.g. cyclin D1 and MYC, is
activated by the transcription factor TCF, which binds to specific sequence
motifs in the promoter. Indirect target genes are regulated via transcription
regulators, which are targets of the Wnt pathway. As an example, MYC regulates
the MYC interacting zinc finger protein-1 (MIZ-1), which is able to inhibit the
expression of the indirect target p21WAF1. We intended to identify new Wnt target
genes and to get a deeper insight into the regulatory mechanisms of Wnt target
gene expression. For this we analyzed the differential expression pattern of
Wnt-1 activated cells by microarray analysis. We identified 43 sequences
including eight expressed sequence tags (ESTs), which showed increased transcript
levels, and 104 sequences including 19 ESTs with decreased RNA levels. Northern
blot and real-time quantitative PCR analysis of the differential expression
levels of 15 genes confirmed the differential expression trends of eight
candidate genes. When the Wnt pathway was regulated at the lower level of
glycogen synthase kinase-3 beta (GSK-3 beta) or adenomatous polyposis coli (APC),
we detected discrepant expression trends. We compared the number of binding sites
of transcription factors in the genomic regions of all candidate target genes
with the number of sites in control genes. We found that the genomic regions of
the down-regulated genes include an increased number of putative MIZ-1 binding
sites. Our study introduces several new Wnt target genes and provides indications
that the specific gene expression pattern depends on the type of the activation
trigger or the level of interference with the Wnt pathway. Furthermore, our data 
indicate that a high proportion of Wnt target genes are regulated by indirect
mechanisms.

DOI: 10.1111/j.1742-4658.2005.04581.x 
PMID: 15794748  [Indexed for MEDLINE]


1121. J Mol Evol. 2005 Apr;60(4):462-74.

Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium.

Monsieurs P(1), De Keersmaecker S, Navarre WW, Bader MW, De Smet F, McClelland M,
Fang FC, De Moor B, Vanderleyden J, Marchal K.

Author information: 
(1)ESAT-SCD, K.U. Leuven, Kasteelpark Arenberg 10, 3001, Leuven-Heverlee,
Belgium.

The PhoPQ two-component system acts as a transcriptional regulator that responds 
to Mg(2+) starvation both in Escherichia coli and Salmonella typhimurium (Garcia 
et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular
Mg(2+), this two-component system allows S. typhimurium to sense the transition
from an extracellular environment to a subcellular location. Concomitantly with
this transition, a set of virulence factors essential for survival in the
intracellular environment is activated by the PhoPQ system (Groisman et al. 1989;
Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the 
PhoPQ regulon in pathogens must contain target genes specifically contributing to
the virulence phenotype. To verify this hypothesis, we compared the composition
of the PhoPQ regulon between E. coli and S. typhimurium using a combination of
expression experiments and motif data. PhoPQ-dependent genes in both organisms
were identified from PhoPQ-related microarray experiments. To distinguish between
direct and indirect targets, we searched for the presence of the regulatory motif
in the promoter region of the identified PhoPQ-dependent genes. This allowed us
to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S.
typhimurium LT2. Comparison of both regulons revealed a very limited overlap of
PhoPQ-dependent genes between both organisms. These results suggest that the
PhoPQ system has acquired a specialized function during evolution in each of
these closely related species that allows adaptation to the specificities of
their lifestyles (e.g., pathogenesis in S. typhimurium).

DOI: 10.1007/s00239-004-0212-7 
PMID: 15883881  [Indexed for MEDLINE]


1122. Microbiology. 2005 Apr;151(Pt 4):1187-98.

The expression profile of Escherichia coli K-12 in response to minimal, optimal
and excess copper concentrations.

Kershaw CJ(1), Brown NL, Constantinidou C, Patel MD, Hobman JL.

Author information: 
(1)School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15
2TT, UK.

The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal
medium supplemented with elevated copper concentrations (as copper-glycine) has
been analysed using whole-genome oligonucleotide microarrays. At 750 muM
copper-glycine, the expression of both the cue and cus copper-export systems is
evident. At near-lethal copper concentrations (2 mM copper-glycine), the
expression of these two regulons increases significantly. Other regulons with
increased transcription in response to elevated concentrations of copper-glycine 
include those for the superoxide stress response, iron homeostasis, and envelope 
stress. Furthermore, a variety of ORFs with decreased expression in response to
increased copper-glycine has been identified, including the zinc ABC transporter 
and genes involved in the chemotactic response.

DOI: 10.1099/mic.0.27650-0 
PMID: 15817786  [Indexed for MEDLINE]


1123. Mol Microbiol. 2005 Apr;56(1):215-27.

Transcriptional response of Escherichia coli to external copper.

Yamamoto K(1), Ishihama A.

Author information: 
(1)Department of Agricultural Chemistry, Kinki University, Nakamachi 3327-204,
Nara 631-8505, Japan.

Transcriptional response of Escherichia coli upon exposure to external copper was
studied using DNA microarray and in vivo and in vitro transcription assays.
Transcription of three hitherto-identified copper-responsive genes, copA (copper 
efflux transporter), cueO (multicopper oxidase) and cusC (tripartite copper pump 
component) became maximum at 5 min after addition of copper sulphate, and
thereafter decreased to the preshift levels within 30 min. Microarray analysis at
5 min after addition of copper indicated that a total of at least 29 genes
including these three known genes were markedly and specifically affected (28
upregulated and one downregulated). Transcription of the divergent operons,
cusCFB and cusRS, was found to be activated by CusR, which bound to a CusR box
between the cusC and cusR promoters. Except for this site, the CusR box was not
identified in the entire E. coli genome. On the other hand, transcription of copA
and cueO was found to be activated by another copper-responsive factor CueR,
which bound to a conserved inverted repeat sequence, CueR box. A total of 197
CueR boxes were identified on the E. coli genome, including the CueR box
associated with the moa operon for molybdenum cofactor synthesis. At least 10
copper-induced genes were found to be under the control of CpxAR two-component
system, indicating that copper is one of the signals for activation of the CpxAR 
system. In addition, transcription of yedWV, a putative two-component system, was
activated by copper in CusR-dependent manner. Taken together we conclude that the
copper-responsive genes are organized into a hierarchy of the regulation network,
forming at least four regulons, i.e. CueR, CusR, CpxR and YedW regulons. These
copper-responsive regulons appear to sense and respond to different
concentrations of external copper.

DOI: 10.1111/j.1365-2958.2005.04532.x 
PMID: 15773991  [Indexed for MEDLINE]


1124. OMICS. 2005 Spring;9(1):13-29.

Identification of putative sulfurtransferase genes in the extremophilic
Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional
characterization of the proteins.

Acosta M(1), Beard S, Ponce J, Vera M, Mobarec JC, Jerez CA.

Author information: 
(1)Laboratory of Molecular Microbiology and Biotechnology, and Millennium
Institute for Advanced Studies in Cell Biology and Biotechnology (CBB),
Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile.

Eight nucleotide sequences containing a single rhodanese domain were found in the
Acidithiobacillus ferrooxidans ATCC 23270 genome: p11, p14, p14.3, p15, p16,
p16.2, p21, and p28. Amino acids sequence comparisons allowed us to identify the 
potentially catalytic Cys residues and other highly conserved rhodanese family
features in all eight proteins. The genomic contexts of some of the
rhodanese-like genes and the determination of their expression at the mRNA level 
by using macroarrays suggested their implication in sulfur oxidation and
metabolism, formation of Fe-S clusters or detoxification mechanisms. Several of
the putative rhodanese genes were successfully isolated, cloned and overexpressed
in E. coli and their thiosulfate:cyanide sulfurtransferase (TST) and
3-mercaptopyruvate/cyanide sulfurtransferase (MST) activities were determined.
Based on their sulfurtransferase activities and on structural comparisons of
catalytic sites and electrostatic potentials between homology- modeled A.
ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST)
and SseA (MST) proteins, two of the rhodanese-like proteins (P15 and P16.2) could
clearly be defined as TSTs, and P14 and P16 could possibly correspond to MSTs.
Nevertheless, several of the eight A. ferrooxidans rhodanese-like proteins may
have some different functional activities yet to be discovered.

DOI: 10.1089/omi.2005.9.13 
PMID: 15805776  [Indexed for MEDLINE]


1125. Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):3942-7. Epub 2005 Feb 28.

Independence of replisomes in Escherichia coli chromosomal replication.

Breier AM(1), Weier HU, Cozzarelli NR.

Author information: 
(1)Graduate Group in Biophysics and Department of Molecular and Cell Biology,
University of California, Berkeley, CA 94720, USA.

In Escherichia coli DNA replication is carried out by the coordinated action of
the proteins within a replisome. After replication initiation, the two
bidirectionally oriented replisomes from a single origin are colocalized into
higher-order structures termed replication factories. The factory model
postulated that the two replisomes are also functionally coupled. We tested this 
hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA
surrounding oriC in single, combed chromosomes showed instead that one replisome,
usually the leftward one, was significantly ahead of the other 70% of the time.
We next used microarrays to follow replication throughout the genome by measuring
DNA copy number. We found in multiple E. coli strains that the replisomes are
independent, with the leftward replisome ahead of the rightward one. The size of 
the bias was strain-specific, varying from 50 to 130 kb in the array results.
When we artificially blocked one replisome, the other continued unabated, again
demonstrating independence. We suggest an improved version of the factory model
that retains the advantages of threading DNA through colocalized replisomes at
about equal rates, but allows the cell flexibility to overcome obstacles
encountered during elongation.

DOI: 10.1073/pnas.0500812102 
PMCID: PMC552787
PMID: 15738384  [Indexed for MEDLINE]


1126. Antimicrob Agents Chemother. 2005 Mar;49(3):931-44.

Global expression of prophage genes in Escherichia coli O157:H7 strain EDL933 in 
response to norfloxacin.

Herold S(1), Siebert J, Huber A, Schmidt H.

Author information: 
(1)Institut für Medizinishe Mikrobioloie und Hygiene, Technische Universität
Dresden, Dresden, Germany.

We investigated the influence of a low concentration of the gyrase inhibitor
norfloxacin on the transcriptome of enterohemorrhagic Escherichia coli O157:H7
strain EDL933. For this purpose, we used a commercial DNA microarray containing
oligonucleotides specific for E. coli O157:H7 strains EDL933 and RIMD0509952 and 
E. coli K-12 strain MG1655. Under the conditions applied, 5,963 spots (94% of all
spots) could be analyzed. Among these, 118 spots (P < 0.05) indicated
transcriptional upregulation and 122 spots (P < 0.05) indicated transcriptional
downregulation of the E. coli genes present on the array. Eighty-five upregulated
EDL933 genes were phage borne. Fifty-two of them could be ascribed to the Shiga
toxin-encoding phages (Stx phages) BP-933W and CP-933V; the other 33 genes
belonged to non-Stx prophage elements in the EDL933 genome. Genes present in the 
BP-933W prophage genome were induced most strongly up to 158-fold in the case of 
stxA(2) upon induction with norfloxacin. Twenty-two additional upregulated genes 
appeared to be E. coli O157:H7 strain RIMD0509952-specific phage elements, and
the remaining 11 genes were related mainly to recombination and stress functions.
Downregulation was indicated predominantly for genes responsible for bacterial
primary metabolism, such as energy production, cell division, and amino acid
biosynthesis. Interestingly, some genes present in the locus of enterocyte
effacement appeared to be downregulated. The results of the study have shown that
a low concentration of norfloxacin has profound effects on the transcriptome of
E. coli O157:H7.

DOI: 10.1128/AAC.49.3.931-944.2005 
PMCID: PMC549229
PMID: 15728886  [Indexed for MEDLINE]


1127. Bioinformatics. 2005 Mar;21(6):765-73. Epub 2004 Oct 14.

Reconstructing biological networks using conditional correlation analysis.

Rice JJ(1), Tu Y, Stolovitzky G.

Author information: 
(1)Computational Biology Center, IBM T.J. Watson Research Center, PO Box 218,
Yorktown Heights, NY 10598, USA.

MOTIVATION: One of the present challenges in biological research is the
organization of the data originating from high-throughput technologies. One way
in which this information can be organized is in the form of networks of
influences, physical or statistical, between cellular components. We propose an
experimental method for probing biological networks, analyzing the resulting data
and reconstructing the network architecture.
METHODS: We use networks of known topology consisting of nodes (genes), directed 
edges (gene-gene interactions) and a dynamics for the genes' mRNA concentrations 
in terms of the gene-gene interactions. We proposed a network reconstruction
algorithm based on the conditional correlation of the mRNA equilibrium
concentration between two genes given that one of them was knocked down. Using
simulated gene expression data on networks of known connectivity, we investigated
how the reconstruction error is affected by noise, network topology, size,
sparseness and dynamic parameters.
RESULTS: Errors arise from correlation between nodes connected through
intermediate nodes (false positives) and when the correlation between two
directly connected nodes is obscured by noise, non-linearity or multiple inputs
to the target node (false negatives). Two critical components of the method are
as follows: (1) the choice of an optimal correlation threshold for predicting
connections and (2) the reduction of errors arising from indirect connections
(for which a novel algorithm is proposed). With these improvements, we can
reconstruct networks with the topology of the transcriptional regulatory network 
in Escherichia coli with a reasonably low error rate.

DOI: 10.1093/bioinformatics/bti064 
PMID: 15486043  [Indexed for MEDLINE]


1128. J Bacteriol. 2005 Mar;187(5):1783-91.

Evolution of genomic content in the stepwise emergence of Escherichia coli
O157:H7.

Wick LM(1), Qi W, Lacher DW, Whittam TS.

Author information: 
(1)Microbial Evolution Laboratory, 165 Food Safety & Toxicology Building,
Michigan State University, East Lansing, MI 48824, USA.

Genome comparisons have demonstrated that dramatic genetic change often underlies
the emergence of new bacterial pathogens. Evolutionary analysis of Escherichia
coli O157:H7, a pathogen that has emerged as a worldwide public health threat in 
the past two decades, has posited that this toxin-producing pathogen evolved in a
series of steps from O55:H7, a recent ancestor of a nontoxigenic pathogenic clone
associated with infantile diarrhea. We used comparative genomic hybridization
with 50-mer oligonucleotide microarrays containing probes from both pathogenic
and nonpathogenic genomes to infer when genes were acquired and lost. Many
ancillary virulence genes identified in the O157 genome were already present in
an O55:H7-like progenitor, with 27 of 33 genomic islands of >5 kb and specific
for O157:H7 (O islands) that were acquired intact before the split from this
immediate ancestor. Most (85%) of variably absent or present genes are part of
prophages or phage-like elements. Divergence in gene content among these closely 
related strains was approximately 140 times greater than divergence at the
nucleotide sequence level. A >100-kb region around the O-antigen gene cluster
contained highly divergent sequences and also appears to be duplicated in its
entirety in one lineage, suggesting that the whole region was cotransferred in
the antigenic shift from O55 to O157. The beta-glucuronidase-positive O157
variants, although phylogenetically closest to the Sakai strain, were divergent
for multiple adherence factors. These observations suggest that, in addition to
gains and losses of phage elements, O157:H7 genomes are rapidly diverging and
radiating into new niches as the pathogen disseminates.

DOI: 10.1128/JB.187.5.1783-1791.2005 
PMCID: PMC1064018
PMID: 15716450  [Indexed for MEDLINE]


1129. J Bacteriol. 2005 Mar;187(5):1763-72.

Genome-wide analyses of Escherichia coli gene expression responsive to the BaeSR 
two-component regulatory system.

Nishino K(1), Honda T, Yamaguchi A.

Author information: 
(1)Department of Cell Membrane Biology, Institute of Scientific and Industrial
Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan.

The BaeSR two-component regulatory system controls expression of exporter genes
conferring drug resistance in Escherichia coli (S. Nagakubo, K. Nishino, T.
Hirata, and A. Yamaguchi, J. Bacteriol. 184:4161-4167, 2002; N. Baranova and H.
Nikaido, J. Bacteriol. 184:4168-4176, 2002). To understand the whole picture of
BaeSR regulation, a DNA microarray analysis of the effect of BaeR overproduction 
was performed. BaeR overproduction activated 59 genes related to two-component
signal transduction, chemotactic responses, flagellar biosynthesis, maltose
transport, and multidrug transport, and BaeR overproduction also repressed the
expression of the ibpA and ibpB genes. All of the changes in the expression
levels were also observed by quantitative real-time reverse transcription-PCR
analysis. The expression levels of 15 of the 59 BaeR-activated genes were
decreased by deletion of baeSR. Of 11 genes induced by indole (a putative inducer
of the BaeSR system), 10 required the BaeSR system for induction. Combination of 
the expression data sets revealed a BaeR-binding site sequence motif,
5'-TTTTTCTCCATDATTGGC-3' (where D is G, A, or T). Several genes up-regulated by
BaeR overproduction, including genes for maltose transport, chemotactic
responses, and flagellar biosynthesis, required an intact PhoBR or CreBC
two-component regulatory system for up-regulation. These data indicate that there
is cross-regulation among the BaeSR, PhoBR, and CreBC two-component regulatory
systems. Such a global analysis should reveal the regulatory network of the BaeSR
system.

DOI: 10.1128/JB.187.5.1763-1772.2005 
PMCID: PMC1063996
PMID: 15716448  [Indexed for MEDLINE]


1130. J Clin Microbiol. 2005 Mar;43(3):1024-31.

Use of diagnostic microarrays for determination of virulence gene patterns of
Escherichia coli K1, a major cause of neonatal meningitis.

Korczak B(1), Frey J, Schrenzel J, Pluschke G, Pfister R, Ehricht R, Kuhnert P.

Author information: 
(1)Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.

Forty Escherichia coli strains isolated primarily from neonatal meningitis,
urinary tract infections and feces were screened for the presence of virulence
genes with a newly developed microarray on the array tube format. A total of 32
gene probes specific for extraintestinal as well as intestinal E. coli pathotypes
were included. Eighty-eight percent of the analyzed strains were positive for the
K1-specific probe on the microarray and could be confirmed with a specific
antiserum against the K1 capsular polysaccharide. The gene for the hemin receptor
ChuA was predominantly found in 95% of strains. Other virulence genes associated 
with K1 and related strains were P, S, and F1C fimbriae specific for
extraintestinal E. coli, the genes for aerobactin, the alpha-hemolysin and the
cytotoxic necrotizing factor. In two strains, the O157-specific catalase gene and
the gene for the low-molecular-weight heat-stable toxin AstA were detected,
respectively. A total of 19 different virulence gene patterns were observed. No
correlation was observed between specific virulence gene patterns and a clinical 
outcome. The data indicate that virulence genes typical of extraintestinal E.
coli are predominantly present in K1 strains. Nevertheless, some of them can
carry virulence genes known to be characteristic of intestinal E. coli. The
distribution and combination of virulence genes show that K1 isolates constitute 
a heterogeneous group of E. coli.

DOI: 10.1128/JCM.43.3.1024-1031.2005 
PMCID: PMC1081230
PMID: 15750055  [Indexed for MEDLINE]


1131. Biosens Bioelectron. 2005 Feb 15;20(8):1482-90.

Microchamber array based DNA quantification and specific sequence detection from 
a single copy via PCR in nanoliter volumes.

Matsubara Y(1), Kerman K, Kobayashi M, Yamamura S, Morita Y, Tamiya E.

Author information: 
(1)School of Materials Science, Japan Advanced Institute of Science and
Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292, Japan.

A novel method for DNA quantification and specific sequence detection in a highly
integrated silicon microchamber array is described. Polymerase chain reaction
(PCR) mixture of only 40 nL volume could be introduced precisely into each
chamber of the mineral oil layer coated microarray by using a nanoliter
dispensing system. The elimination of carry-over and cross-contamination between 
microchambers, and multiple DNA amplification and detection by TaqMan chemistry
were demonstrated, for the first time, by using our system. Five different gene
targets, related to Escherichia coli were amplified and detected simultaneously
on the same chip by using DNA from three different serotypes as the templates.
The conventional method of DNA quantification, which depends on the real-time
monitoring of variations in fluorescence intensity, was not applied to our
system, instead a simple method was established. Counting the number of the
microchambers with a high fluorescence signal as a consequence of TaqMan PCR
provided the precise quantification of trace amounts of DNA. The initial DNA
concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to
12 copies, and quantification was achieved by observing the changes in the
released fluorescence signals of the microchambers on the chip. DNA target could 
be detected as small as 0.4 copies. The amplified DNA was detected with a CCD
camera built-in to a fluorescence microscope, and also evaluated by a DNA
microarray scanner with associated software. This simple method of counting the
high fluorescence signal released in microchambers as a consequence of TaqMan PCR
was further integrated with a portable miniaturized thermal cycler unit. Such a
small device is surely a strong candidate for low-cost DNA amplification, and
detected as little as 0.4 copies of target DNA.

DOI: 10.1016/j.bios.2004.07.002 
PMID: 15626601  [Indexed for MEDLINE]


1132. J Biotechnol. 2005 Feb 9;115(3):221-37. Epub 2004 Nov 19.

The global gene expression response of Escherichia coli to L-phenylalanine.

Polen T(1), Krämer M, Bongaerts J, Wubbolts M, Wendisch VF.

Author information: 
(1)Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich,
Germany. t.polen@fz-juelich.de

We investigated the global gene expression changes of Escherichia coli due to the
presence of different concentrations of phenylalanine or shikimate in the growth 
medium. The response to 0.5 g l(-1) phenylalanine primarily reflected a perturbed
aromatic amino acid metabolism, in particular due to TyrR-mediated regulation.
The addition of 5g l(-1) phenylalanine reduced the growth rate by half and
elicited a great number of likely indirect effects on genes regulated in response
to changed pH, nitrogen or carbon availability. Consistent with the observed gene
expression changes, supplementation with shikimate, tyrosine and tryptophan
relieved growth inhibition by phenylalanine. In contrast to the wild-type, a tyrR
disruption strain showed increased expression of pckA and of tktB in the presence
of phenylalanine, but its growth was not affected by phenylalanine at the
concentrations tested. The absence of growth inhibition by phenylalanine
suggested that at high phenylalanine concentrations TyrR-defective strains might 
perform better in phenylalanine production.

DOI: 10.1016/j.jbiotec.2004.08.017 
PMID: 15639085  [Indexed for MEDLINE]


1133. Nucleic Acids Res. 2005 Feb 8;33(3):880-92. Print 2005.

A novel method for accurate operon predictions in all sequenced prokaryotes.

Price MN(1), Huang KH, Alm EJ, Arkin AP.

Author information: 
(1)Lawrence Berkeley National Lab 1 Cyclotron Road, Mailstop 939R704, Berkeley,
CA 94720, USA.

We combine comparative genomic measures and the distance separating adjacent
genes to predict operons in 124 completely sequenced prokaryotic genomes. Our
method automatically tailors itself to each genome using sequence information
alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and
Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is
similar to the accuracy of methods that use the same features but are trained on 
experimentally characterized transcripts. In Halobacterium NRC-1 and in
Helicobacter pylori, our method correctly infers that genes in operons are
separated by shorter distances than they are in E.coli, and its predictions using
distance alone are more accurate than distance-only predictions trained on a
database of E.coli transcripts. We use microarray data from six phylogenetically 
diverse prokaryotes to show that combining intergenic distance with comparative
genomic measures further improves accuracy and that our method is broadly
effective. Finally, we survey operon structure across 124 genomes, and find
several surprises: H.pylori has many operons, contrary to previous reports;
Bacillus anthracis has an unusual number of pseudogenes within conserved operons;
and Synechocystis PCC 6803 has many operons even though it has unusually wide
spacings between conserved adjacent genes.

DOI: 10.1093/nar/gki232 
PMCID: PMC549399
PMID: 15701760  [Indexed for MEDLINE]


1134. Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):1927-32. Epub 2005 Jan 26.

Identification of the Wnt signaling activator leucine-rich repeat in Flightless
interaction protein 2 by a genome-wide functional analysis.

Liu J(1), Bang AG, Kintner C, Orth AP, Chanda SK, Ding S, Schultz PG.

Author information: 
(1)Department of Chemistry, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA.

The Wnt signaling pathway acts ubiquitously in metazoans to control various
aspects of embryonic development. Wnt ligands bind their receptors Frizzled and
low-density lipoprotein receptor-related protein 5/6 and function through
Disheveled (Dvl), Axin, adenomatous polyposis coli, glycogen synthase kinase
3beta, and casein kinase (CK) 1 to stabilize beta-catenin and induce lymphocyte
enhancer-binding factor (LEF)/T cell factor (TCF)-dependent transcriptional
activities. To identify previously unrecognized Wnt signaling modulators, a
genome-wide functional screen was performed using large-scale arrayed cDNA
collections. From this screen, both known components and previously
uncharacterized regulators of this pathway were identified, including
beta-catenin, Dvl1, Dvl3, Fbxw-1, Cul1, CK1epsilon, CK1delta, and gamma-catenin. 
In particular, a previously unrecognized activator, LRRFIP2 (leucine-rich repeat 
in Flightless interaction protein 2), was found that interacts with Dvl to
increase the cellular levels of beta-catenin and activate
beta-catenin/LEF/TCF-dependent transcriptional activity. The function of LRRFIP2 
is blocked when a dominant negative Dvl (Xdd1) is coexpressed. Expression of
LRRFIP2 in Xenopus embryos induced double axis formation and Wnt target gene
expression; a dominant negative form of LRRFIP2 suppresses ectopic Wnt signaling 
in Xenopus embryos and partially inhibits endogenous dorsal axis formation. These
data suggest that LRRFIP2 plays an important role in transducing Wnt signals.

DOI: 10.1073/pnas.0409472102 
PMCID: PMC548559
PMID: 15677333  [Indexed for MEDLINE]


1135. Appl Environ Microbiol. 2005 Feb;71(2):1093-6.

Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli,
using gene expression profiles from DNA microarrays.

Shimizu K(1), Hayashi S, Kako T, Suzuki M, Tsukagoshi N, Doukyu N, Kobayashi T,
Honda H.

Author information: 
(1)Department of Biotechnology, School of Engineering, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Gene expression profiles were collected from Escherichia coli strains (OST3410,
TK33, and TK31) before and after exposure to organic solvents, and the six genes 
that showed higher gene expression were selected. Among these genes, glpC
encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably
increased the organic solvent tolerance.

DOI: 10.1128/AEM.71.2.1093-1096.2005 
PMCID: PMC546683
PMID: 15691972  [Indexed for MEDLINE]


1136. Extremophiles. 2005 Feb;9(1):65-73. Epub 2004 Aug 31.

Analysis of hydrostatic pressure effects on transcription in Escherichia coli by 
DNA microarray procedure.

Ishii A(1), Oshima T, Sato T, Nakasone K, Mori H, Kato C.

Author information: 
(1)Department of Biological Information, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,
Yokohama, Kanagawa 226-8501, Japan.

Hydrostatic pressure is a well-known physical stimulus, but its effects on cell
physiology have not been clarified. To investigate pressure effects on
Escherichia coli, we carried out DNA microarray analysis of the entire E. coli
genome. The microarray results showed pleiotropic effects on gene expression. In 
particular, heat- and cold-stress responses were induced simultaneously by the
elevated pressure. Upon temperature stress (including both temperature up- and
down-shifts) and other environmental stresses, gene expression adjusts to adapt
to such environmental changes through regulations by several DNA-binding
proteins. An E. coli mutant, which deleted the hns gene encoding one of the
regulator proteins, exhibited great pressure sensitivity. The result suggested
that the H-NS protein was a possible transcriptional regulator for adaptation of 
the high-pressure stress.

DOI: 10.1007/s00792-004-0414-3 
PMID: 15340867  [Indexed for MEDLINE]


1137. Genome Res. 2005 Feb;15(2):260-8.

Selection-driven transcriptome polymorphism in Escherichia coli/Shigella species.

Le Gall T(1), Darlu P, Escobar-Páramo P, Picard B, Denamur E.

Author information: 
(1)Institut National de la Santé et de la Recherche Médicale (INSERM) E0339,
Faculté de Médecine Xavier Bichat, 75018, Paris, France.

To explore the role of transcriptome polymorphism in adaptation of organisms to
their environment, we evaluated this parameter for the Escherichia coli/Shigella 
bacterial species, which is composed of well-characterized phylogenetic groups
that exhibit characteristic life styles ranging from commensalism to
intracellular pathogenicity. Both the genomic content and the transcriptome of 10
strains representative of the major E. coli/Shigella phylogenetic groups were
evaluated using macroarrays displaying the 4290 K12-MG1655 open reading frames
(ORFs). Although Shigella and enteroinvasive E. coli (EIEC) are not monophyletic,
phylogenetic analysis of the binary coded (presence/absence) gene content data
showed that these organisms group together due to similar patterns of
undetectable K12-MG1655 genes. The variation in transcript abundance was then
analyzed using a core genome of 2880 genes present in all strains, after
adjusting RNA hybridization signals for DNA hybridization signals. Nonrandom
changes in gene expression during the evolution of the E. coli/Shigella species
were evidenced. Phylogenetic analysis of transcriptome data again showed that
Shigella and EIEC strains group together in terms of gene expression, and this
convergence involved groups of genes displaying biologically coherent patterns of
functional divergence. Unlike the other E. coli strains evaluated, Shigella and
EIEC are intracellular pathogens, and therefore face similar selective pressures.
Thus, within the E. coli/Shigella species, strains exhibiting a particular life
style have converged toward a specific gene expression pattern in a subset of
genes common to the species, revealing the role of selection in shaping
transcriptome polymorphism.

DOI: 10.1101/gr.2405905 
PMCID: PMC546527
PMID: 15687289  [Indexed for MEDLINE]


1138. J Bacteriol. 2005 Feb;187(3):1074-90.

Sulfur and nitrogen limitation in Escherichia coli K-12: specific homeostatic
responses.

Gyaneshwar P(1), Paliy O, McAuliffe J, Popham DL, Jordan MI, Kustu S.

Author information: 
(1)Department of Plant & Microbial Biology, University of California, Berkeley,
111 Koshland Hall, Berkeley, CA 94720-3102, USA.

We determined global transcriptional responses of Escherichia coli K-12 to sulfur
(S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures
subjected to nutrient shifts. Using two limitations helped to distinguish between
nutrient-specific changes in mRNA levels and common changes related to the growth
rate. Both homeostatic and slow growth responses were amplified upon shifts. This
made detection of these responses more reliable and increased the number of genes
that were differentially expressed. We analyzed microarray data in several ways: 
by determining expression changes after use of a statistical normalization
algorithm, by hierarchical and k-means clustering, and by visual inspection of
aligned genome images. Using these tools, we confirmed known homeostatic
responses to global S limitation, which are controlled by the activators CysB and
Cbl, and found that S limitation propagated into methionine metabolism, synthesis
of FeS clusters, and oxidative stress. In addition, we identified several open
reading frames likely to respond specifically to S availability. As predicted
from the fact that the ddp operon is activated by NtrC, synthesis of cross-links 
between diaminopimelate residues in the murein layer was increased under
N-limiting conditions, as was the proportion of tripeptides. Both of these
effects may allow increased scavenging of N from the dipeptide
D-alanine-D-alanine, the substrate of the Ddp system.

DOI: 10.1128/JB.187.3.1074-1090.2005 
PMCID: PMC545709
PMID: 15659685  [Indexed for MEDLINE]


1139. Microbiology. 2005 Feb;151(Pt 2):385-98.

Characterization of the flexible genome complement of the commensal Escherichia
coli strain A0 34/86 (O83 : K24 : H31).

Hejnova J(1), Dobrindt U, Nemcova R, Rusniok C, Bomba A, Frangeul L, Hacker J,
Glaser P, Sebo P, Buchrieser C.

Author information: 
(1)Unité de Génomique des Microorganismes Pathogènes and CNRS URA 2171, Institut 
Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France.

Colonization by the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31) 
has proved to be safe and efficient in the prophylaxis and treatment of
nosocomial infections and diarrhoea of preterm and newborn infants in Czech
paediatric clinics over the past three decades. In searching for traits
contributing to this beneficial effect related to the gut colonization capacity
of the strain, the authors have analysed its genome by DNA-DNA hybridization to
E. coli K-12 (MG1655) genomic DNA arrays and to 'Pathoarrays', as well as by
multiplex PCR, bacterial artificial chromosome (BAC) library cloning and shotgun 
sequencing. Four hundred and ten E. coli K-12 ORFs were absent from A0 34/86,
while 72 out of 456 genes associated with pathogenicity islands of E. coli and
Shigella were also detected in E. coli A0 34/86. Furthermore, extraintestinal
pathogenic E. coli-related genes involved in iron uptake and adhesion were
detected by multiplex PCR, and genes encoding the HlyA and cytotoxic necrotizing 
factor toxins, together with 21 genes of the uropathogenic E. coli 536
pathogenicity island II, were identified by analysis of 2304 shotgun and 1344 BAC
clone sequences of A0 34/86 DNA. Multiple sequence comparisons identified 31 kb
of DNA specific for E. coli A0 34/86; some of the genes carried by this DNA may
prove to be implicated in the colonization capacity of the strain, enabling it to
outcompete pathogens. Among 100 examined BAC clones roughly covering the A0 34/86
genome, one reproducibly conferred on the laboratory strain DH10B an enhanced
capacity to persist in the intestine of newborn piglets. Sequencing revealed that
this BAC clone carried gene clusters encoding gluconate and mannonate metabolism,
adhesion (fim), invasion (ibe) and restriction/modification functions. Hence, the
genome of this clinically safe and highly efficient colonizer strain appears to
harbour many 'virulence-associated' genes. These results highlight the thin line 
between bacterial 'virulence' and 'fitness' or 'colonization' factors, and
question the definition of enterobacterial virulence factors.

DOI: 10.1099/mic.0.27469-0 
PMID: 15699189  [Indexed for MEDLINE]


1140. Mol Microbiol. 2005 Feb;55(3):724-38.

Environmental and genetic regulation of the phosphorylcholine epitope of
Haemophilus influenzae lipooligosaccharide.

Wong SM(1), Akerley BJ.

Author information: 
(1)Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester, MA 01655, USA.

In response to environmental signals in the host, bacterial pathogens express
factors required during infection and repress those that interfere with specific 
stages of this process. Signalling pathways controlling virulence factors of the 
human respiratory pathogen, Haemophilus influenzae, are predominantly unknown.
The lipooligosaccharide (LOS) outer core represents a prototypical virulence
trait of H. influenzae that enhances virulence but also provides targets for
innate and adaptive immunity. We report regulation of the display of the
virulence-associated phosphorylcholine (PC) epitope on the LOS in response to
environmental conditions. PC display is optimal under microaerobic conditions and
markedly decreased under conditions of high culture aeration. Gene expression
analysis using a DNA microarray was performed to begin to define the metabolic
state of the cell under these conditions and to identify genes potentially
involved in PC epitope modulation. Global gene expression profiling detected
changes in redox responsive genes and in genes of carbohydrate metabolism. The
effects on carbohydrate metabolism led us to examine the role of the putative H. 
influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in
Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae
csrA gene showed increased PC epitope levels under aerobic conditions.
Furthermore, deletion of csrA elevated mRNA expression of galU, an essential
virulence gene that is critical in generating sugar precursors needed for
polysaccharide formation and LOS outer core synthesis. Growth conditions
predicted to alter the redox state of the culture modulated the PC epitope and
galU expression as well. The results are consistent with a multifactorial
mechanism of control of LOS-PC epitope display involving csrA and environmental
signals that coordinately regulate biosynthetic and metabolic genes controlling
the LOS structure.

DOI: 10.1111/j.1365-2958.2004.04439.x 
PMID: 15660999  [Indexed for MEDLINE]


1141. J Biol Chem. 2005 Jan 28;280(4):2636-43. Epub 2004 Nov 16.

New genes implicated in the protection of anaerobically grown Escherichia coli
against nitric oxide.

Justino MC(1), Vicente JB, Teixeira M, Saraiva LM.

Author information: 
(1)Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
Apartado 127, Avenida da República (EAN), 2781-901 Oeiras, Portugal.

Nitric oxide produced by activated macrophages plays a key role as one of the
immune system's weapons against pathogens. Because the lifetime of nitric oxide
is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic
effects of nitric oxide are greatly increased as in the infection/inflammation
processes, it is important to establish which systems are able to detoxify nitric
oxide under anaerobic conditions. In the present work a new set of Escherichia
coli K-12 genes conferring anaerobic resistance to nitric oxide is presented,
namely the gene product of YtfE and a potential transcriptional regulator of the 
helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for
anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide
is shown to cause a significant alteration of the global E. coli gene
transcription profile that includes the increase of the transcript level of genes
encoding for detoxification enzymes, iron-sulfur cluster assembly systems,
DNA-repairing enzymes, and stress response regulators.

DOI: 10.1074/jbc.M411070200 
PMID: 15546870  [Indexed for MEDLINE]


1142. Biotechnol Bioeng. 2005 Jan 20;89(2):195-205.

Engineering HlyA hypersecretion in Escherichia coli based on proteomic and
microarray analyses.

Lee PS(1), Lee KH.

Author information: 
(1)School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin 
Hall, Ithaca, New York 14853-5201, USA.

Escherichia coli is a common host for recombinant protein production for
biotechnology applications. Secretion to the extracellular media has the
potential to reduce protein aggregation and to simplify downstream purification. 
However, the complexity of the mechanisms of protein secretion has confounded
prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was
used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An
activity assay identified a mutant secreting fourfold more active alpha-hemolysin
than the parent strain. The mutant was characterized using both high-density
microarrays for mRNA profiling and a proteomics strategy for protein expression. 
The relative mRNA and protein expression levels of tRNA-synthetases were
decreased in the mutant compared to the parent. A mathematical model of
prokaryotic translation was used to design a variant of the hlyA gene that
encodes the same amino acid sequence but uses rare codons to slow the rate of
translation by altering five bases. Analysis of the parent strain transformed
with a plasmid containing this variant gene resulted in the recovery of, and
further improvement upon, the selected hypersecretion phenotype. These results
present one of the first successful metabolic engineering attempts based on
molecular information provided by mRNA and protein expression profiling
approaches and resulting in a phenotype useful to the biotechnology community.

DOI: 10.1002/bit.20342 
PMID: 15580578  [Indexed for MEDLINE]


1143. Nucleic Acids Res. 2005 Jan 7;33(1):e3.

ArrayOme: a program for estimating the sizes of microarray-visualized bacterial
genomes.

Ou HY(1), Smith R, Lucchini S, Hinton J, Chaudhuri RR, Pallen M, Barer MR,
Rajakumar K.

Author information: 
(1)Department of Infection, Immunity and Inflammation, Leicester Medical School, 
University of Leicester Leicester LE1 9HN, UK.

ArrayOme is a new program that calculates the size of genomes represented by
microarray-based probes and facilitates recognition of key bacterial strains
carrying large numbers of novel genes. Protein-coding sequences (CDS) that are
contiguous on annotated reference templates and classified as 'Present' in the
test strain by hybridization to microarrays are merged into ICs (ICs). These ICs 
are then extended to account for flanking intergenic sequences. Finally, the
lengths of all extended ICs are summated to yield the 'microarray-visualized
genome (MVG)' size. We tested and validated ArrayOme using both experimental and 
in silico-generated genomic hybridization data. MVG sizing of five sequenced
Escherichia coli and Shigella strains resulted in an accuracy of 97-99%, as
compared to true genome sizes, when the comprehensive ShE.coli meta-array gene
sequences (6239 CDS) were used for in silico hybridization analysis. However, the
E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked
probes for many CFT073 CDS. ArrayOme permits rapid recognition of discordances
between PFGE-measured genome and MVG sizes, thereby enabling high-throughput
identification of strains rich in novel genes. Gene discovery studies focused on 
these strains will greatly facilitate characterization of the global gene pool
accessible to individual bacterial species.

DOI: 10.1093/nar/gni005 
PMCID: PMC546176
PMID: 15640440  [Indexed for MEDLINE]


1144. Anal Chem. 2005 Jan 1;77(1):319-26.

Array-based binary analysis for bacterial typing.

Shepard JR(1), Danin-Poleg Y, Kashi Y, Walt DR.

Author information: 
(1)Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA
02155, USA.

An allele-specific oligonucleotide microarray was developed for rapid typing of
pathogens based on analysis of genomic variations. Using a panel of Escherichia
coli strains as a model system, selected loci were sequenced to uncover
differences, such as single- or multiple-nucleotide polymorphisms as well as
insertion/deletions (indels). While typical genomic profiling experiments employ 
specific sequences targeted to genomic DNA unique to a single strain or virulent 
gene, the present array is designed to type bacteria based on a patterned
signature response across multiple loci. In the signature concept, all strains
are interrogated by hybridizing their amplified DNA to an array containing
multiple probe sequences. Allele-specific oligonucleotide probe sequences
targeting each of these variable regions were synthesized and included in a
custom fiber-optic array. For each locus, a set of specific probe sequences is
selected, such that hybridization gives a binary signal/no signal response to
each of the probes. Using this strategy for multiple loci, many pathogens or
microorganisms could be classified using a limited number of probes. Because of
the advantages of the fiber-optic array platform over other array formats,
including sensitivity and speed, the platform described in this paper is capable 
of supporting a high-throughput diagnostic strategy.

DOI: 10.1021/ac0488006 
PMID: 15623311  [Indexed for MEDLINE]


1145. Appl Environ Microbiol. 2005 Jan;71(1):451-9.

Phenotypic screening of Escherichia coli K-12 Tn5 insertion libraries, using
whole-genome oligonucleotide microarrays.

Winterberg KM(1), Luecke J, Bruegl AS, Reznikoff WS.

Author information: 
(1)Department of Biochemistry, University of Wisconsin--Madison, 433 Babcock Dr.,
Madison, WI 53706-1544, USA.

Complete genome sequences in combination with global screening methods allow
parallel analysis of multiple mutant loci to determine the requirement for
specific genes in different environments. In this paper we describe a
high-definition microarray approach for investigating the growth effects of Tn5
insertions in Escherichia coli K-12. Libraries of insertion mutants generated by 
a unique Tn5 mutagenesis system were grown competitively in defined media.
Biotin-labeled runoff RNA transcripts were generated in vitro from transposon
insertions in each population of mutants. These transcripts were then hybridized 
to custom-designed oligonucleotide microarrays to detect the presence of each
mutant in the population. By using this approach, the signal associated with 25
auxotrophic insertions in a 50-mutant pool was not detectable following nine
generations of growth in glucose M9 minimal medium. It was found that individual 
insertion sites could be mapped to within 50 bp of their genomic locations, and
340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions
were detected in 15 genes for which no previous insertions have been reported.
Other applications of this method are discussed.

DOI: 10.1128/AEM.71.1.451-459.2005 
PMCID: PMC544249
PMID: 15640221  [Indexed for MEDLINE]


1146. Can J Microbiol. 2005 Jan;51(1):29-35.

Genome-wide comparison of phage M13-infected vs. uninfected Escherichia coli.

Karlsson F(1), Malmborg-Hager AC, Albrekt AS, Borrebaeck CA.

Author information: 
(1)Department of Immunotechnology, Lund University, Sweden.

To identify Escherichia coli genes potentially regulated by filamentous phage
infection, we used oligonucleotide microarrays. Genome-wide comparison of phage
M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed.
The analysis revealed altered transcription levels of 12 E. coli genes in
response to phage infection, and the observed regulation of phage genes
correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host
genes affected could be grouped into 3 different categories based on cellular
function, suggesting a coordinated response. The significantly upregulated genes 
encode proteins involved in reactions of the energy-generating phosphotransferase
system and transcription processing, which could be related to phage
transcription. No genes belonging to any known E. coli stress response pathways
were scored as upregulated. Furthermore, phage infection led to significant
downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp,
and crl. These downregulated genes are normally part of the host stress response 
mechanisms that protect the bacterium during conditions of acid stress and
stationary phase transition. The phage-infected cells demonstrated impaired
function of the oxidative and the glutamate-dependent acid resistance systems.
Thus, global transcriptional analysis and functional analysis revealed previously
unknown host responses to filamentous phage infection.

DOI: 10.1139/w04-113 
PMID: 15782232  [Indexed for MEDLINE]


1147. Cancer Res. 2005 Jan 1;65(1):166-76.

Transcriptional profiles of intestinal tumors in Apc(Min) mice are unique from
those of embryonic intestine and identify novel gene targets dysregulated in
human colorectal tumors.

Reichling T(1), Goss KH, Carson DJ, Holdcraft RW, Ley-Ebert C, Witte D, Aronow
BJ, Groden J.

Author information: 
(1)Department of Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati College of Medicine, Cincinnati Children's Hospital Medical Center,
Cincinnati, OH 45267, USA.

The adenomatous polyposis coli (APC) tumor suppressor is a major regulator of the
Wnt signaling pathway in normal intestinal epithelium. APC, in conjunction with
AXIN and GSK-3beta, forms a complex necessary for the degradation of
beta-catenin, thereby preventing beta-catenin/T-cell factor interaction and
alteration of growth-controlling genes such as c-MYC and cyclin D1. Inappropriate
activation of the Wnt pathway, via Apc/APC mutation, leads to gastrointestinal
tumor formation in both the mouse and human. In order to discover novel genes
that may contribute to tumor progression in the gastrointestinal tract, we used
cDNA microarrays to identify 114 genes with altered levels of expression in
Apc(Min) mouse adenomas from the duodenum, jejunum, and colon. Changes in the
expression of 24 of these 114 genes were not observed during mouse development at
embryonic day 16.5, postnatal day 1, or postnatal day 14 (relative to normal
adult intestine). These 24 genes are not previously known Wnt targets. Seven
genes were validated by real-time reverse transcription-PCR analysis, whereas
four genes were validated by in situ hybridization to mouse adenomas. Real-time
reverse transcription-PCR analysis of human colorectal cancer cell lines and
adenocarcinomas revealed that altered expression levels were also observed for
six of the genes Igfbp5, Lcn2, Ly6d, N4wbp4 (PMEPA1), S100c, and Sox4.


PMID: 15665292  [Indexed for MEDLINE]


1148. Genome Inform. 2005;16(2):205-14.

Reverse engineering genetic networks using evolutionary computation.

Noman N(1), Iba H.

Author information: 
(1)Department of Frontier Informatics, Graduate School of Frontier Sciences, The 
University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8561, Japan.
noman@iba.k.u-tokyo.ac.jp

This paper proposes an improved evolutionary method for constructing the
underlying network structure and inferring effective kinetic parameters from the 
time series data of gene expression using decoupled S-system formalism. We
employed Trigonometric Differential Evolution (TDE) as the optimization engine of
our algorithm for capturing the dynamics in gene expression data. A more
effective fitness function for attaining the sparse structure, which is the
hallmark of biological networks, has been applied. Experiments on artificial
genetic network show the power of the algorithm in constructing the network
structure and predicting the regulatory parameters. The method is used to
evaluate interactions between genes in the SOS signaling pathway in Escherichia
coli using gene expression data.


PMID: 16901103  [Indexed for MEDLINE]


1149. Infect Immun. 2005 Jan;73(1):88-102.

Transcriptional adaptation of Shigella flexneri during infection of macrophages
and epithelial cells: insights into the strategies of a cytosolic bacterial
pathogen.

Lucchini S(1), Liu H, Jin Q, Hinton JC, Yu J.

Author information: 
(1)The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United
Kingdom.

Shigella flexneri, the etiologic agent of bacillary dysentery, invades epithelial
cells as well as macrophages and dendritic cells and escapes into the cytosol
soon after invasion. Dissection of the global gene expression profile of the
bacterium in its intracellular niche is essential to fully understand the biology
of Shigella infection. We have determined the complete gene expression profiles
for S. flexneri infecting human epithelial HeLa cells and human macrophage-like
U937 cells. Approximately one quarter of the S. flexneri genes showed significant
transcriptional adaptation during infection; 929 and 1,060 genes were up- or
down-regulated within HeLa cells and U937 cells, respectively. The key S.
flexneri virulence genes, ipa-mxi-spa and icsA, were drastically down-regulated
during intracellular growth. This theme seems to be common in bacterial
infection, because the Ipa-Mxi-Spa-like type III secretion systems were also
down-regulated during mammalian cell infection by Salmonella enterica serovar
Typhimurium and Escherichia coli O157. The bacteria experienced restricted levels
of iron, magnesium, and phosphate in both host cell types, as shown by
up-regulation of the sitABCD system, the mgtA gene, and genes of the phoBR
regulon. Interestingly, ydeO and other acid-induced genes were up-regulated only 
in U937 cells and not in HeLa cells, suggesting that the cytosol of U937 cells is
acidic. Comparison with the gene expression of intracellular Salmonella serovar
Typhimurium, which resides within the Salmonella-containing vacuole, indicated
that S. flexneri is exposed to oxidative stress in U937 cells. This work will
facilitate functional studies of hundreds of novel intracellularly regulated
genes that may be important for the survival and growth strategies of Shigella in
the human host.

DOI: 10.1128/IAI.73.1.88-102.2005 
PMCID: PMC538992
PMID: 15618144  [Indexed for MEDLINE]


1150. J Appl Genet. 2005;46(2):171-7.

Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells.

Pareek R(1), Wellnitz O, Van Dorp R, Burton J, Kerr D.

Author information: 
(1)Department of Animal Science, Terrill Hall, University of Vermont, 570 Main
Street, Burlington, VT 05405, USA.

Infection of the bovine mammary gland, in addition to causing animal distress, is
a major economic burden of the dairy industry. Greater understanding of the
initial host response to infection may lead to more accurate selection of
resistant animals or to novel prophylactic or therapeutic intervention
strategies. The epithelial cell plays a role in the host response by alerting the
immune system to the infection and providing a signal as to where the infection
is located. To understand this process better, a cDNA microarray approach was
used to search for potential signals produced by mammary epithelial cells in
response to exposure to Escherichia coli lipopolysaccharide (LPS). Total RNA from
separate cultures of epithelial cells from 4 Holstein cows was harvested 6 h
after LPS challenge or control conditions. For each cow, RNA from control or
LPS-exposed cells was transcribed to cDNA and labeled with Cy3 or Cy5, then
pooled and applied to a bovine total leukocyte (BOTL) microarray slide containing
1278 unique transcripts. Dye reversal was used so that RNA from two of the
control cultures was labeled with Cy3 while RNA from the other two control
cultures was labeled with Cy5. From the resulting microarray data we selected 4
of the 9 genes significantly (P < 0.02) induced (>1.25-fold) in response to LPS
exposure for more detailed analysis. The array signal intensity for 3 of these
genes, RANTES/CCL5, IL-6 and T-PA, was relatively low, but quantitative real-time
RT-PCR (Q-RT-PCR) analysis revealed that they were induced 208-fold, 10-fold and 
3-fold, respectively. The gene that showed the greatest fold induction by
microarray analysis (2.5-fold) was CXCL5. This gene had a relatively strong
signal intensity on the array and was easily detected by northern blot analysis, 
which indicated a 10-fold induction. This cell culture model system provides
evidence for an important role of the mammary epithelial cell in initiating the
innate response to infection.


PMID: 15876684  [Indexed for MEDLINE]


1151. J Biopharm Stat. 2005;15(5):783-97.

Nonparametric methods for microarray data based on exchangeability and borrowed
power.

Lee ML(1), Whitmore GA, Björkbacka H, Freeman MW.

Author information: 
(1)Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts,
USA. meiling@channing.harvard.edu

This article proposes nonparametric inference procedures for analyzing microarray
gene expression data that are reliable, robust, and simple to implement. They are
conceptually transparent and require no special-purpose software. The analysis
begins by normalizing gene expression data in a unique way. The resulting
adjusted observations consist of gene-treatment interaction terms (representing
differential expression) and error terms. The error terms are considered to be
exchangeable, which is the only substantial assumption. Thus, under a family null
hypothesis of no differential expression, the adjusted observations are
exchangeable and all permutations of the observations are equally probable. The
investigator may use the adjusted observations directly in a distribution-free
test method or use their ranks in a rank-based method, where the ranking is taken
over the whole data set. For the latter, the essential steps are as follows: (1) 
Calculate a Wilcoxon rank-sum difference or a corresponding Kruskal-Wallis rank
statistic for each gene. (2) Randomly permute the observations and repeat the
previous step. (3) Independently repeat the random permutation a suitable number 
of times. Under the exchangeability assumption, the permutation statistics are
independent random draws from a null cumulative distribution function (c.d.f)
approximated by the empirical c.d.f Reference to the empirical c.d.f tells if the
test statistic for a gene is outlying and, hence, shows differential expression. 
This feature is judged by using an appropriate rejection region or computing a
p-value for each test statistic, taking into account multiple testing. The
distribution-free analog of the rank-based approach is also available and has
parallel steps which are described in the article. The proposed nonparametric
analysis tends to give good results with no additional refinement, although a few
refinements are presented that may interest some investigators. The
implementation is illustrated with a case application involving differential gene
expression in wild-type and knockout mice of an E. coli lipopolysaccharide (LPS) 
endotoxin treatment, relative to a baseline untreated condition.

DOI: 10.1081/BIP-200067778 
PMID: 16078385  [Indexed for MEDLINE]


1152. J Biosci Bioeng. 2005 Jan;99(1):72-4.

Time-course data analysis of gene expression profiles reveals purR regulon
concerns in organic solvent tolerance in Escherichia coli.

Shimizu K(1), Hayashi S, Doukyu N, Kobayashi T, Honda H.

Author information: 
(1)Department of Biotechnology, School of Engineering, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

A time-course gene-expression profile was generated for Escherichia coli TK31
when it was exposed to an organic solvent mixture, and classified by fuzzy
adaptive resonance theory (Fuzzy ART). It was found that the purR regulon plays
an important role in the organic solvent tolerance (OST) of E. coli.

DOI: 10.1263/jbb.99.072 
PMID: 16233757  [Indexed for MEDLINE]


1153. Neuro Oncol. 2005 Jan;7(1):20-31.

Microarray analysis of pediatric ependymoma identifies a cluster of 112 candidate
genes including four transcripts at 22q12.1-q13.3.

Suarez-Merino B(1), Hubank M, Revesz T, Harkness W, Hayward R, Thompson D,
Darling JL, Thomas DG, Warr TJ.

Author information: 
(1)Department of Molecular Neuroscience, Institute of Neurology, National
Hospital for Neurology and Neurosurgery, University College London, London, UK.

Ependymomas are glial cell-derived tumors characterized by varying degrees of
chromosomal abnormalities and variability in clinical behavior. Cytogenetic
analysis of pediatric ependymoma has failed to identify consistent patterns of
abnormalities, with the exception of monosomy of 22 or structural abnormalities
of 22q. In this study, a total of 19 pediatric ependymoma samples were used in a 
series of expression profiling, quantitative real-time PCR (Q-PCR), and loss of
heterozygosity experiments to identify candidate genes involved in the
development of this type of pediatric malignancy. Of the 12,627 genes analyzed, a
subset of 112 genes emerged as being abnormally expressed when compared to three 
normal brain controls. Genes with increased expression included the oncogene
WNT5A; the p53 homologue p63; and several cell cycle, cell adhesion, and
proliferation genes. Underexpressed genes comprised the NF2 interacting gene
SCHIP-1 and the adenomatous polyposis coli (APC)-associated gene EB1 among
others. We validated the abnormal expression of six of these genes by Q-PCR. The 
subset of differentially expressed genes also included four underexpressed
transcripts mapping to 22q12.313.3. By Q-PCR we show that one of these genes, 7
CBX7(22q13.1), was deleted in 55% of cases. Other genes mapping to cytogenetic
hot spots included two overexpressed and three underexpressed genes mapping to
1q31-41 and 6q21-q24.3, respectively. These genes represent candidate genes
involved in ependymoma tumorigenesis. To the authors' knowledge, this is the
first time microarray analysis and Q-PCR have been linked to identify
heterozygous/homozygous deletions.

DOI: 10.1215/S1152851704000596) 
PMCID: PMC1871622
PMID: 15701279  [Indexed for MEDLINE]


1154. Nucleic Acids Symp Ser (Oxf). 2005;(49):269-70.

In vitro selection of RNA aptamers for the Escherichia coli release factor 1.

Ogawa A(1), Nishi T, Sando S, Aoyama Y.

Author information: 
(1)Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.

We carried out an in vitro selection of RNA aptamers that bind to Escherichia
coli release factor 1 (E. coli RF1). The selected aptamer (class II) showed an
apparent dissociation constant of nM range. The binding of the class II aptamer
with E. coli RF1 is highly specific (orthogonal), allowing selective inhibition
of RF1 activity in the E. coli translation system.

DOI: 10.1093/nass/49.1.269 
PMID: 17150737  [Indexed for MEDLINE]


1155. J Biol Chem. 2004 Dec 31;279(53):55707-14. Epub 2004 Oct 13.

Identification of a new member of the phage shock protein response in Escherichia
coli, the phage shock protein G (PspG).

Lloyd LJ(1), Jones SE, Jovanovic G, Gyaneshwar P, Rolfe MD, Thompson A, Hinton
JC, Buck M.

Author information: 
(1)Department of Biological Sciences, Sir Alexander Fleming Building, Imperial
College London, South Kensington Campus, London SW7 2AZ, United Kingdom.

The phage shock protein operon (pspABCDE) of Escherichia coli is strongly
up-regulated in response to overexpression of the filamentous phage secretin
protein IV (pIV) and by many other stress conditions including defects in protein
export. PspA has an established role in maintenance of the proton-motive force of
the cell under stress conditions. Here we present evidence for a new member of
the phage shock response in E. coli. Using transcriptional profiling, we show
that the synthesis of pIV in E. coli leads to a highly restricted response
limited to the up-regulation of the psp operon genes and yjbO. The psp operon and
yjbO are also up-regulated in response to pIV in Salmonella enterica serovar
Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that
contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a
putative inner membrane protein that is co-controlled with the psp operon genes
and is predicted to be an effector of the psp response in E. coli. We present
evidence that yjbO expression is driven by sigma(54)-RNA polymerase, activated by
PspF and integration host factor, and negatively regulated by PspA. PspF
specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We
found that increased expression of YjbO results in decreased motility of
bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and
is a member of the phage shock protein F regulon, we propose that yjbO be renamed
pspG.

DOI: 10.1074/jbc.M408994200 
PMID: 15485810  [Indexed for MEDLINE]


1156. Nature. 2004 Dec 23;432(7020):1050-4.

Accurate multiplex gene synthesis from programmable DNA microchips.

Tian J(1), Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G.

Author information: 
(1)Harvard Medical School, 77 Ave Louis Pasteur, Boston, Massachusetts 02115,
USA.

Testing the many hypotheses from genomics and systems biology experiments demands
accurate and cost-effective gene and genome synthesis. Here we describe a
microchip-based technology for multiplex gene synthesis. Pools of thousands of
'construction' oligonucleotides and tagged complementary 'selection'
oligonucleotides are synthesized on photo-programmable microfluidic chips,
released, amplified and selected by hybridization to reduce synthesis errors
ninefold. A one-step polymerase assembly multiplexing reaction assembles these
into multiple genes. This technology enabled us to synthesize all 21 genes that
encode the proteins of the Escherichia coli 30S ribosomal subunit, and to
optimize their translation efficiency in vitro through alteration of codon bias. 
This is a significant step towards the synthesis of ribosomes in vitro and should
have utility for synthetic biology in general.

DOI: 10.1038/nature03151 
PMID: 15616567  [Indexed for MEDLINE]


1157. J Bacteriol. 2004 Dec;186(24):8172-80.

Specialized persister cells and the mechanism of multidrug tolerance in
Escherichia coli.

Keren I(1), Shah D, Spoering A, Kaldalu N, Lewis K.

Author information: 
(1)Department of Biology, Northeastern University, 134 Mugar Hall, 360 Huntington
Ave., Boston, MA 02115. k.lewis@neu.edu.

Bacterial populations produce persisters, cells that neither grow nor die in the 
presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The 
mechanisms of MDT and the nature of persisters have remained elusive. Our
previous research has shown that persisters are largely responsible for the
recalcitrance of biofilm infections. A general method for isolating persisters
was developed, based on lysis of regular cells by ampicillin. A gene expression
profile of persisters contained toxin-antitoxin (TA) modules and other genes that
can block important cellular functions such as translation. Bactericidal
antibiotics kill cells by corrupting the target function (for example,
aminoglycosides interrupt translation, producing toxic peptides). We reasoned
that inhibition of translation will lead to a shutdown of cellular functions,
preventing antibiotics from corrupting their targets, giving rise to MDT
persister cells. Overproduction of the RelE toxin, an inhibitor of translation,
caused a sharp increase in persisters. Functional expression of a putative HipA
toxin also increased persisters, while deletion of the hipBA module caused a
sharp decrease in persisters in both stationary and biofilm populations. HipA is 
thus the first validated persister-MDT gene. We suggest that random fluctuation
in the levels of MDT proteins leads to the formation of rare persister cells. The
function of these specialized dormant cells is to ensure the survival of the
population in the presence of lethal factors.

DOI: 10.1128/JB.186.24.8172-8180.2004 
PMCID: PMC532439
PMID: 15576765  [Indexed for MEDLINE]


1158. Mol Genet Genomics. 2004 Dec;272(5):580-91. Epub 2004 Nov 19.

Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12.

Patten CL(1), Kirchhof MG, Schertzberg MR, Morton RA, Schellhorn HE.

Author information: 
(1)Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1,
Canada.

The alternative sigma factor RpoS controls the expression of many
stationary-phase genes in Escherichia coli and other bacteria. Though the RpoS
regulon is a large, conserved system that is critical for adaptation to nutrient 
deprivation and other stresses, it remains incompletely characterized. In this
study, we have used oligonucleotide arrays to delineate the transcriptome that is
controlled by RpoS during entry into stationary phase of cultures growing in rich
medium. The expression of known RpoS-dependent genes was confirmed to be
regulated by RpoS, thus validating the use of microarrays for expression
analysis. The total number of positively regulated stationary-phase genes was
found to be greater than 100. More than 45 new genes were identified as
positively controlled by RpoS. Surprisingly, a similar number of genes were found
to be negatively regulated by RpoS, and these included almost all genes required 
for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a
physically contiguous group of genes located in the Rac prophage region. Negative
regulation by RpoS is thus much more extensive than has previously been
recognized, and is likely to be an important contributing factor to the
competitive growth advantage of rpoS mutants reported in previous studies.

DOI: 10.1007/s00438-004-1089-2 
PMID: 15558318  [Indexed for MEDLINE]


1159. J Biol Chem. 2004 Nov 12;279(46):47543-54. Epub 2004 Aug 30.

Novel phenotypes of Escherichia coli tat mutants revealed by global gene
expression and phenotypic analysis.

Ize B(1), Porcelli I, Lucchini S, Hinton JC, Berks BC, Palmer T.

Author information: 
(1)Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH,
United Kingdom.

The Tat protein export system serves to export folded proteins harboring an
N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this 
study, we have used gene expression profiling of Escherichia coli supported by
phenotypic analysis to investigate how cells respond to a defect in the Tat
pathway. Previous work has demonstrated that strains mutated in genes encoding
essential Tat pathway components are defective in the integrity of their cell
envelope because of the mislocalization of two amidases involved in cell wall
metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol.
Microbiol. 48, 1183-1193). To distinguish between genes that are differentially
expressed specifically because of the cell envelope defect and those that result 
from other effects of the tatC deletion, we also analyzed two different
transposon mutants of the DeltatatC strain that have their outer membrane
integrity restored. Approximately 50% of the genes that were differentially
expressed in the tatC mutant are linked to the envelope defect, with the products
of many of these genes involved in self-defense or protection mechanisms,
including the production of exopolysaccharide. Among the changes that were not
explicitly linked to envelope integrity, we characterized a role for the Tat
system in iron acquisition and copper homeostasis. Finally, we have demonstrated 
that overproduction of the Tat substrate SufI saturates the Tat translocon and
produces effects on global gene expression that are similar to those resulting
from the DeltatatC mutation.

DOI: 10.1074/jbc.M406910200 
PMID: 15347649  [Indexed for MEDLINE]


1160. Appl Environ Microbiol. 2004 Nov;70(11):6435-43.

Survival of Shewanella oneidensis MR-1 after UV radiation exposure.

Qiu X(1), Sundin GW, Chai B, Tiedje JM.

Author information: 
(1)Center for Microbial Ecology, Michigan State University, East Lansing,
Michigan 48824, USA.

We systematically investigated the physiological response as well as DNA damage
repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB,
UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among
Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of
Escherichia coli K-12 values. Stationary cells did not show an increased UVA
resistance compared to exponential-phase cells; instead, they were more sensitive
at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than
Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 
was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of 
UVA m(-2), and 558 J of solar UVB m(-2), respectively. Photoreactivation
conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- 
to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation,
respectively. A significant UV mutability to rifampin resistance was detected in 
both UVC- and UVB-treated samples, with the mutation frequency in the range of
10(-5) to 10(-6). Unlike in E. coli, the expression levels of the nucleotide
excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage
inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA
deficient) with uvrA of MR-1 increased the UVC survival of this strain by more
than 3 orders of magnitude. Loss of damage inducibility of the NER system appears
to contribute to the high sensitivity of this bacterium to UVR as well as to
other DNA-damaging agents.

DOI: 10.1128/AEM.70.11.6435-6443.2004 
PMCID: PMC525172
PMID: 15528503  [Indexed for MEDLINE]


1161. Biosens Bioelectron. 2004 Nov 1;20(4):728-35.

Amplicon secondary structure prevents target hybridization to oligonucleotide
microarrays.

Lane S(1), Evermann J, Loge F, Call DR.

Author information: 
(1)Department of Veterinary Microbiology and Pathology, Washington State
University, 402 Bustad Hall, P.O. Box 647040, Pullman, WA 99164-7040, USA.

DNA microarrays that are used as end-point detectors for PCR assays are typically
composed of short (15-25 mer) oligonucleotide probes bound to glass. When
designing these detectors, we have frequently encountered situations where a
probe would not hybridize to its complementary, terminally labeled PCR amplicon. 
To determine if failures could be explained by general phenomenon such as
secondary structure, we designed a microarray to detect eight regions of the
Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different
lengths using a biotin conjugated, antisense primer. Amplicons were then
hybridized to the microarray and detected using a combination of signal
amplification and fluorescence. In most cases, probe sequences complementary to
the 5' region of the amplified products (sense orientation) did not hybridize to 
their respective amplicon. We tested for positional bias and showed that a biotin
conjugated sense primer mirrored the same probe failures. Nick translated
products, however, hybridized to all probes. Because nick translation generates
many labeled fragments of random length, we concluded that this method disrupted 
secondary structure that otherwise prevented the amplicons from hybridizing to
their respective probes. We also show that nick translation does not compromise
detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp).
Despite the increased cost of the nick translation, we concluded that this
labeling strategy will reduce the time needed to design new assays as well as
avoid possible false negatives during field applications. Alternative labeling
strategies are also discussed.

DOI: 10.1016/j.bios.2004.04.014 
PMID: 15522587  [Indexed for MEDLINE]


1162. Environ Health Perspect. 2004 Nov;112(16):1614-21.

The TAO-Gen algorithm for identifying gene interaction networks with application 
to SOS repair in E. coli.

Yamanaka T(1), Toyoshiba H, Sone H, Parham FM, Portier CJ.

Author information: 
(1)Laboratory of Computational Biology and Risk Analysis, National Institute of
Environmental Health Sciences, National Institutes of Health/DHHS, 111 Alexander 
Drive, Research Triangle Park, NC 27709, USA.

One major unresolved issue in the analysis of gene expression data is the
identification and quantification of gene regulatory networks. Several methods
have been proposed for identifying gene regulatory networks, but these methods
predominantly focus on the use of multiple pairwise comparisons to identify the
network structure. In this article, we describe a method for analyzing gene
expression data to determine a regulatory structure consistent with an observed
set of expression profiles. Unlike other methods this method goes beyond pairwise
evaluations by using likelihood-based statistical methods to obtain the network
that is most consistent with the complete data set. The proposed algorithm
performs accurately for moderate-sized networks with most errors being minor
additions of linkages. However, the analysis also indicates that sample sizes may
need to be increased to uniquely identify even moderate-sized networks. The
method is used to evaluate interactions between genes in the SOS signaling
pathway in Escherichia coli using gene expression data where each gene in the
network is over-expressed using plasmids inserts.


PMCID: PMC1247658
PMID: 15598612  [Indexed for MEDLINE]


1163. Infect Immun. 2004 Nov;72(11):6373-81.

Transcriptome of uropathogenic Escherichia coli during urinary tract infection.

Snyder JA(1), Haugen BJ, Buckles EL, Lockatell CV, Johnson DE, Donnenberg MS,
Welch RA, Mobley HL.

Author information: 
(1)Department of Microbiology and Immunology, University of Maryland School of
Medicine, Baltimore, USA.

A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that
includes each open reading frame was used to analyze the transcriptome of CFT073 
bacteria isolated directly from the urine of infected CBA/J mice. The in vivo
expression profiles were compared to that of E. coli CFT073 grown statically to
exponential phase in rich medium, revealing the strategies this pathogen uses in 
vivo for colonization, growth, and survival in the urinary tract environment. The
most highly expressed genes overall in vivo encoded translational machinery,
indicating that the bacteria were in a rapid growth state despite specific
nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved 
in adherence, was highly upregulated in vivo. Five iron acquisition systems were 
all highly upregulated during urinary tract infection, as were genes responsible 
for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance,
and microcin secretion. Surprisingly, other fimbrial genes, such as pap and
foc/sfa, and genes involved in motility and chemotaxis were downregulated in
vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron
acquisition, capsule, and microcin secretion genes, thus partially mimicking
growth in vivo. On the basis of gene expression levels, the urinary tract appears
to be nitrogen and iron limiting, of high osmolarity, and of moderate
oxygenation. This study represents the first assessment of any E. coli
pathotype's transcriptome in vivo and provides specific insights into the
mechanisms necessary for urinary tract pathogenesis.

DOI: 10.1128/IAI.72.11.6373-6381.2004 
PMCID: PMC523057
PMID: 15501767  [Indexed for MEDLINE]


1164. J Bacteriol. 2004 Nov;186(22):7796-803.

Global transcriptome analysis of the heat shock response of Shewanella
oneidensis.

Gao H(1), Wang Y, Liu X, Yan T, Wu L, Alm E, Arkin A, Thompson DK, Zhou J.

Author information: 
(1)Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 
37831, USA.

Shewanella oneidensis is an important model organism for bioremediation studies
because of its diverse respiratory capabilities. However, the genetic basis and
regulatory mechanisms underlying the ability of S. oneidensis to survive and
adapt to various environmentally relevant stresses is poorly understood. To
define this organism's molecular response to elevated growth temperatures,
temporal gene expression profiles were examined in cells subjected to heat stress
by using whole-genome DNA microarrays for S. oneidensis. Approximately 15% (n =
711) of the total predicted S. oneidensis genes (n = 4,648) represented on the
microarray were significantly up- or downregulated (P < 0.05) over a 25-min
period after shift to the heat shock temperature. As expected, the majority of
the genes that showed homology to known chaperones and heat shock proteins in
other organisms were highly induced. In addition, a number of predicted genes,
including those encoding enzymes in glycolysis and the pentose cycle, serine
proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine 
kinases, and hypothetical proteins were induced. Genes encoding membrane proteins
were differentially expressed, suggesting that cells possibly alter their
membrane composition or structure in response to variations in growth
temperature. A substantial number of the genes encoding ribosomal proteins
displayed downregulated coexpression patterns in response to heat stress, as did 
genes encoding prophage and flagellar proteins. Finally, a putative regulatory
site with high conservation to the Escherichia coli sigma32-binding consensus
sequence was identified upstream of a number of heat-inducible genes.

DOI: 10.1128/JB.186.22.7796-7803.2004 
PMCID: PMC524878
PMID: 15516594  [Indexed for MEDLINE]


1165. J Bacteriol. 2004 Nov;186(21):7196-204.

Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief
valve?

Van Dyk TK(1), Templeton LJ, Cantera KA, Sharpe PL, Sariaslani FS.

Author information: 
(1)DuPont Company CR&D, Rt. 141 and Powdermill Road, P.O. Box 80173, Wilmington, 
DE 19880-0173, USA. Tina.K.Van-Dyk@usa.dupont.com

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in
upregulation of yhcP, encoding a protein of the putative efflux protein family.
Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane
fusion protein family, and yhcR, encoding a small protein without a known or
suggested function. The function of the upstream, divergently transcribed gene
yhcS, encoding a regulatory protein of the LysR family, in regulating expression 
of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic
carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux
function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP
mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression
of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA
hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of
hundreds of diverse compounds tested were defined as substrates of the YhcQP
efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect
their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB,
respectively. The role of pHBA in normal E. coli metabolism and the highly
regulated expression of the AaeAB efflux system suggests that the physiological
role may be as a "metabolic relief valve" to alleviate toxic effects of
imbalanced metabolism.

DOI: 10.1128/JB.186.21.7196-7204.2004 
PMCID: PMC523213
PMID: 15489430  [Indexed for MEDLINE]


1166. J Bacteriol. 2004 Nov;186(21):7186-95.

SigmaS-dependent gene expression at the onset of stationary phase in Escherichia 
coli: function of sigmaS-dependent genes and identification of their promoter
sequences.

Lacour S(1), Landini P.

Author information: 
(1)Swiss Federal Institute of Environmental Technology (EAWAG), Dübendorf,
Switzerland.

The sigma(S) subunit of RNA polymerase, the product of the rpoS gene, controls
the expression of genes responding to starvation and cellular stresses. Using
gene array technology, we investigated rpoS-dependent expression at the onset of 
stationary phase in Escherichia coli grown in rich medium. Forty-one genes were
expressed at significantly lower levels in an rpoS mutant derived from the MG1655
strain; for 10 of these, we also confirmed rpoS and stationary-phase dependence
by reverse transcription-PCR. Only seven genes (dps, osmE, osmY, sodC, rpsV,
wrbA, and yahO) had previously been recognized as rpoS dependent. Several newly
identified rpoS-dependent genes are involved in the uptake and metabolism of
amino acids, sugars, and iron. Indeed, the rpoS mutant strain shows severely
impaired growth on some sugars such as fructose and N-acetylglucosamine. The rpoS
gene controls the production of indole, which acts as a signal molecule in
stationary-phase cells, via regulation of the tnaA-encoded tryptophanase enzyme. 
Genes involved in protein biosynthesis, encoding the ribosome-associated protein 
RpsV (sra) and the initiation factor IF-1 (infA), were also induced in an
rpoS-dependent fashion. Using primer extension, we determined the promoter
sequences of a selection of rpoS-regulated genes representative of different
functional classes. Significant fractions of these promoters carry sequence
features specific for Esigma(S) recognition of the -10 region, such as cytosines 
at positions -13 (70%) and -12 (30%) as well as a TG motif located upstream of
the -10 region (50%), thus supporting the TGN(0-2)C(C/T)ATA(C/A)T consensus
sequence recently proposed for sigma(S).

DOI: 10.1128/JB.186.21.7186-7195.2004 
PMCID: PMC523212
PMID: 15489429  [Indexed for MEDLINE]


1167. J Bacteriol. 2004 Nov;186(21):7112-22.

Classification and strength measurement of stationary-phase promoters by use of a
newly developed promoter cloning vector.

Shimada T(1), Makinoshima H, Ogawa Y, Miki T, Maeda M, Ishihama A.

Author information: 
(1)Nippon Institute for Biological Science, Shinmachi 9-2221, Ome, Tokyo
198-0024, Japan.

When an Escherichia coli culture changes from exponential growth to the
stationary phase, expression of growth-related genes levels off, while a number
of stationary-phase-specific genes are turned on. To gain insight into the growth
phase-dependent global regulation of genome transcription, we analyzed the
strength and specificity of promoters associated with the stationary-phase genes.
For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream
from the translation initiation codon were isolated and inserted into a newly
constructed doubly fluorescent protein (DFP) vector. The activity of test
promoters was determined by measuring the green fluorescent protein (GFP). To
avoid the possible influence of plasmid copy number, the level of transcription
of reference promoter lacUV5 on the same plasmid was determined by measuring the 
red fluorescent protein (RFP). Thus, the activities of test promoters could be
easily and accurately determined by determining the GFP/RFP ratio. Analysis of
the culture time-dependent variation of 100 test promoters indicated that (i) a
major group of the stationary-phase promoters are up-regulated only in the
presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some
promoters takes place even in the absence of RpoS; and (iii) the activity of some
promoters increases in the absence of RpoS. This classification was confirmed by 
testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.

DOI: 10.1128/JB.186.21.7112-7122.2004 
PMCID: PMC523215
PMID: 15489422  [Indexed for MEDLINE]


1168. Nucleic Acids Res. 2004 Nov 1;32(19):5874-93. Print 2004.

Identification of the CRP regulon using in vitro and in vivo transcriptional
profiling.

Zheng D(1), Constantinidou C, Hobman JL, Minchin SD.

Author information: 
(1)School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.

The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that
controls transcription initiation from more than 100 promoters by binding to a
specific DNA sequence within cognate promoters. Many genes in the CRP regulon
have been predicted simply based on the presence of DNA-binding sites within gene
promoters. In this study, we have exploited a newly developed technique, run-off 
transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using
ROMA, we identified 176 operons that were activated by CRP in vitro and 16
operons that were repressed. Using positive control mutants in different regions 
of CRP, we were able to classify the different promoters into class I or class
II/III. A total of 104 operons were predicted to contain Class II CRP-binding
sites. Sequence analysis of the operons that were repressed by CRP revealed
different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional
profiles failed to identify most CRP-dependent regulation because of the
complexity of the regulatory network. Analysis of these operons supports the
hypothesis that CRP is not only a regulator of genes required for catabolism of
sugars other than glucose, but also regulates the expression of a large number of
other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons.

DOI: 10.1093/nar/gkh908 
PMCID: PMC528793
PMID: 15520470  [Indexed for MEDLINE]


1169. J Biotechnol. 2004 Oct 19;114(1-2):107-20.

Gene expression profiling of Escherichia coli expressing double Vitreoscilla
haemoglobin.

Roos V(1), Andersson CI, Bülow L.

Author information: 
(1)Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical
Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.

In a recent investigation, expression of a double Vitreoscilla haemoglobin (two
fused VHb molecules) in Escherichia coli grown in shake flasks resulted in higher
final cell density and considerably higher levels of ribosomes and tRNA. In this 
study, we have investigated the E. coli transcriptome in cells expressing native 
VHb, double VHb and control cells lacking VHb by hybridising mRNA from the
different constructs to high-density oligonucleotide arrays. Within the 95%
confidence interval, 4 and 5% of all detected genes in native VHb cells were up- 
and down-regulated, respectively; in double VHb cells the corresponding numbers
were 6 and 10%, respectively. Dividing the data into different functional groups 
revealed that genes involved in energy metabolism, central intermediary
metabolism and cell processes were the most affected at the mRNA level.
Particularly, the up-regulation of genes involved in translation and
posttranslational modification observed in double VHb cells demonstrates a strong
relationship between the regulation of ribosomal genes and the actual number of
ribosomes.

DOI: 10.1016/j.jbiotec.2004.07.002 
PMID: 15464604  [Indexed for MEDLINE]


1170. Toxicol Appl Pharmacol. 2004 Oct 15;200(2):103-10.

Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) 
phthalate using cDNA microarray and real-time RT-PCR.

Kijima K(1), Toyosawa K, Yasuba M, Matsuoka N, Adachi T, Komiyama M, Mori C.

Author information: 
(1)Safety Research Laboratories, Dainippon Pharmaceutical Co, Ltd, Suita, Osaka
564-0053, Japan.

To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene
expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single
oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h 
after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in
addition to apoptosis-related genes, genes associated with atrophy, APEX
nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2
(TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell
adhesion kinase were differentially expressed. To investigate the relationship
between histopathological alteration and gene expression, we selected genes
associated with apoptosis and analyzed their expression by real-time quantitative
reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP
treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of 
bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be
related to the fact that no morphological apoptotic change was induced after
dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic
activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and
Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, 
these gene regulations might lead the cells into apoptosis in the case of high
exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and
caspase-11/caspase-3 cascade were not increased. These results indicate that the 
cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to
apoptosis with DEHP treatment.

DOI: 10.1016/j.taap.2004.03.015 
PMID: 15476863  [Indexed for MEDLINE]


1171. Appl Environ Microbiol. 2004 Oct;70(10):6098-112.

Transcriptional analysis of biofilm formation processes in the anaerobic,
hyperthermophilic bacterium Thermotoga maritima.

Pysz MA(1), Conners SB, Montero CI, Shockley KR, Johnson MR, Ward DE, Kelly RM.

Author information: 
(1)Department of Chemical Engineering, North Carolina State University, Raleigh, 
NC 27695-7905, USA.

Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was 
found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters 
during chemostat cultivation on maltose-based media at 80 degrees C. A
whole-genome cDNA microarray was used to examine differential expression patterns
between biofilm and planktonic populations. Mixed-model statistical analysis
revealed differential expression (twofold or more) of 114 open reading frames in 
sessile cells (6% of the genome), over a third of which were initially annotated 
as hypothetical proteins in the T. maritima genome. Among the previously
annotated genes in the T. maritima genome, which showed expression changes during
biofilm growth, were several that corresponded to biofilm formation genes
identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli,
and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells
exhibited increased transcription of genes involved in iron and sulfur transport,
as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains
of quinones. These findings were all consistent with the up-regulation of
iron-sulfur cluster assembly and repair functions in biofilm cells. Significant
up-regulation of several beta-specific glycosidases was also noted in biofilm
cells, despite the fact that maltose was the primary carbon source fed to the
chemostat. The reasons for increased beta-glycosidase levels are unclear but are 
likely related to the processing of biofilm-based polysaccharides. In addition to
revealing insights into the phenotype of sessile T. maritima communities, the
methodology developed here can be extended to study other anaerobic biofilm
formation processes as well as to examine aspects of microbial ecology in
hydrothermal environments.

DOI: 10.1128/AEM.70.10.6098-6112.2004 
PMCID: PMC522082
PMID: 15466556  [Indexed for MEDLINE]


1172. Biochem J. 2004 Oct 1;383(Pt 1):149-58.

Sputa nerve growth factor forms a preferable substitute to mouse 7S-beta nerve
growth factor.

Koh DC(1), Armugam A, Jeyaseelan K.

Author information: 
(1)Department of Biochemistry, Faculty of Medicine, National University of
Singapore, 8 Medical Drive, Singapore 117597, Singapore.

The NGF (nerve growth factor) from Naja sputatrix has been purified by gel
filtration followed by reversed-phase HPLC. The protein showed a very high
ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two 
cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF,
sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. 
Sputa NGF has been found to be non-toxic under both in vivo and in vitro
conditions. The induction of neurite outgrowth by this NGF has been found to
involve the high-affinity trkA-p75NTR complex of receptors. The pro-survival
mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB 
gene by a corresponding down-regulation of inhibitory kappaB gene. Real-time PCR 
and protein profiling (by surface-enhanced laser-desorption-ionization
time-of-flight) have confirmed that sputa NGF up-regulates the expression of the 
endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that
sputa NGF is capable of promoting additional beneficial effects such as the
up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type
calcium channel. Hence, sputa NGF forms a new and useful NGF.

DOI: 10.1042/BJ20040569 
PMCID: PMC1134053
PMID: 15225125  [Indexed for MEDLINE]


1173. Infect Immun. 2004 Oct;72(10):6012-22.

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased
expression of BclXL and blockade of mitochondrial cytochrome c release in
macrophages.

Sukumaran SK(1), Selvaraj SK, Prasadarao NV.

Author information: 
(1)Division of Infectious Diseases, The Saban Research Institute, Childrens
Hospital Los Angeles, Los Angeles, California 90027, USA.

Escherichia coli K1 survival in the blood is a critical step for the onset of
meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid
host defense mechanisms by finding a niche to survive and multiply. Our recent
studies have shown that E. coli K1 enters and survives in both monocytes and
macrophages in the newborn rat model of meningitis as well as in macrophage cell 
lines. Here we demonstrate that E. coli K1 not only extends the survival of human
and murine infected macrophage cell lines but also renders them resistant to
apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli
expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are
resistant to DNA fragmentation and phosphatidylserine exposure induced by
staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an
antiapoptotic protein, both at the mRNA level as assessed by gene array analysis 
and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection 
of macrophages induced the release of cytochrome c from mitochondria into the
cytosol and the activation of caspases 3, 6, and 9, events that were
significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+
E. coli-infected cells were resistant to a decrease in the transmembrane
potential of mitochondria induced by staurosporine as measured by the MitoCapture
fluorescence technique. Complementation of OmpA- E. coli with a plasmid
containing the ompA gene restored the ability of OmpA- E. coli to inhibit the
apoptosis of infected macrophages, further demonstrating that E. coli OmpA
expression is critical for inducing macrophage survival and thereby finding a
safe haven for its growth.

DOI: 10.1128/IAI.72.10.6012-6022.2004 
PMCID: PMC517578
PMID: 15385505  [Indexed for MEDLINE]


1174. J Bacteriol. 2004 Oct;186(20):7007-14.

Genome-wide transcriptional analysis of the cold shock response in wild-type and 
cold-sensitive, quadruple-csp-deletion strains of Escherichia coli.

Phadtare S(1), Inouye M.

Author information: 
(1)Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Ln., 
Piscataway, NJ 08854, USA. phadtasa@umdnj.edu.

A DNA microarray-based global transcript profiling of Escherichia coli in
response to cold shock showed that in addition to the known cold shock-inducible 
genes, new genes such as the flagellar operon, those encoding proteins involved
in sugar transport and metabolism, and remarkably, genes encoding certain heat
shock proteins are induced by cold shock. In the light of strong reduction in
metabolic activity of the cell after temperature downshift, the induction of
sugar metabolism machinery is unexpected. The deletion of four csps (cspA, cspB, 
cspG, and cspE) affected cold shock induction of mostly those genes that are
transiently induced in the acclimation phase, emphasizing that CspA homologues
are essential in the acclimation phase. Relevance of these findings with respect 
to the known RNA chaperone function of CspA homologues is discussed.

DOI: 10.1128/JB.186.20.7007-7014.2004 
PMCID: PMC522181
PMID: 15466053  [Indexed for MEDLINE]


1175. J Bacteriol. 2004 Oct;186(20):6938-43.

Genomic studies with Escherichia coli MelR protein: applications of chromatin
immunoprecipitation and microarrays.

Grainger DC(1), Overton TW, Reppas N, Wade JT, Tamai E, Hobman JL, Constantinidou
C, Struhl K, Church G, Busby SJ.

Author information: 
(1)School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15
2TT, United Kingdom. d.grainger@bham.ac.uk.

Escherichia coli MelR protein is a transcription activator that is essential for 
melibiose-dependent expression of the melAB genes. We have used chromatin
immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB 
promoter in vivo. Our results show that MelR is associated with promoter DNA,
both in the absence and presence of the inducer melibiose. In contrast, RNA
polymerase is recruited to the melAB promoter only in the presence of inducer.
The MelR DK261 positive control mutant binds to the melAB promoter but cannot
recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an
Affymetrix GeneChip array, showed that the melAB promoter is the major, if not
the sole, target in E. coli for MelR. This was confirmed by a transcriptomics
experiment to analyze RNA in cells either with or without melR.

DOI: 10.1128/JB.186.20.6938-6943.2004 
PMCID: PMC522211
PMID: 15466047  [Indexed for MEDLINE]


1176. J Bacteriol. 2004 Oct;186(20):6714-20.

Global transcriptional effects of a suppressor tRNA and the inactivation of the
regulator frmR.

Herring CD(1), Blattner FR.

Author information: 
(1)Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall,
Madison, WI 53706, USA.

Expression of an amber suppressor tRNA should result in read-through of the 326
open reading frames (ORFs) that terminate with amber stop codons in the
Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF 
might alter the activities of the protein and have effects on cellular
physiology, while suppression of a pseudogene could lead to a gain of function.
We used oligonucleotide microarrays to determine if any effects were apparent at 
the level of transcription in glucose minimal medium. Surprisingly, only eight
genes had significantly different expression in the presence of the suppressor.
Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon
whose likely function is the degradation of formaldehyde. Expression of wild-type
yaiN was shown to result in repression of the operon, while a
suppression-mimicking allele lacking the amber stop codon and extended 7 amino
acids did not. The operon was shown to be induced by formaldehyde, and the genes 
have been renamed frmR, frmA, and frmB, respectively.

DOI: 10.1128/JB.186.20.6714-6720.2004 
PMCID: PMC522192
PMID: 15466022  [Indexed for MEDLINE]


1177. Mol Microbiol. 2004 Oct;54(2):420-38.

Deletion of the genes encoding the MtrA-MtrB two-component system of
Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics
susceptibility and expression of genes involved in osmoprotection.

Möker N(1), Brocker M, Schaffer S, Krämer R, Morbach S, Bott M.

Author information: 
(1)Institut für Biochemie der Universität zu Köln, D-50674 Köln, Germany.

The MtrAB two-component signal transduction system is highly conserved in
sequence and genomic organization in Mycobacterium and Corynebacterium species,
but its function is completely unknown. Here, the role of MtrAB was studied with 
C. glutamicum as model organism. In contrast to M. tuberculosis, it was possible 
to delete the mtrAB genes in C. glutamicum. The mutant cells showed a radically
different cell morphology and were more sensitive to penicillin, vancomycin and
lysozyme but more resistant to ethambutol. In order to identify the molecular
basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type
were compared with DNA microarrays. Three genes showed a more than threefold
increased RNA level in the mutant, i.e. mepA (NCgl2411) encoding a putative
secreted metalloprotease, ppmA (NCgl2737 ) encoding a putative membrane-bound
protease modulator, and lpqB encoding a putative lipoprotein of unknown function.
Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells
that were hypersensitive to an osmotic downshift, supporting the idea that
peptidoglycan is the target of MepA. The mRNA level of two genes was more than
fivefold decreased in the mutant, i.e. betP and proP which encode transporters
for the uptake of betaine and proline respectively. The microarray results were
confirmed by primer extension and RNA dot blot experiments. In the latter, the
transcript level of genes involved in osmoprotection was tested before and after 
an osmotic upshift. The mRNA level of betP, proP and lcoP was strongly reduced or
undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was
increased. The changes in cell morphology, antibiotics susceptibility and the
mRNA levels of betP, proP, lcoP, mscL and mepA could be reversed by expression of
plasmid-encoded copies of mtrAB in the DeltamtrAB mutant, confirming that these
changes occurred as a consequence of the mtrAB deletion.

DOI: 10.1111/j.1365-2958.2004.04249.x 
PMID: 15469514  [Indexed for MEDLINE]


1178. Biochem Biophys Res Commun. 2004 Sep 10;322(1):347-54.

Genome-scale identification of conditionally essential genes in E. coli by DNA
microarrays.

Tong X(1), Campbell JW, Balázsi G, Kay KA, Wanner BL, Gerdes SY, Oltvai ZN.

Author information: 
(1)Department of Pathology, Northwestern University, Chicago, IL 60611, USA.

Identifying the genes required for the growth or viability of an organism under a
given condition is an important step toward understanding the roles these genes
play in the physiology of the organism. Currently, the combination of global
transposon mutagenesis with PCR-based mapping of transposon insertion sites is
the most common method for determining conditional gene essentiality. In order to
accelerate the detection of essential gene products, here we test the utility and
reliability of a DNA microarray technology-based method for the identification of
conditionally essential genes of the bacterium, Escherichia coli, grown in rich
medium under aerobic or anaerobic growth conditions using two different DNA
microarray platforms. Identification and experimental verification of five
hypothetical E. coli genes essential for anaerobic growth directly demonstrated
the utility of the method. However, the two different DNA microarray platforms
yielded largely non-overlapping results after a two standard deviations cutoff
and were subjected to high false positive background levels. Thus, further
methodological improvements are needed prior to the use of DNA microarrays to
reliably identify conditionally essential genes on genome-scale.

DOI: 10.1016/j.bbrc.2004.07.110 
PMID: 15313213  [Indexed for MEDLINE]


1179. Antimicrob Agents Chemother. 2004 Sep;48(9):3260-7.

Genome-wide transcriptional profiling of the Escherichia coli response to a
proline-rich antimicrobial peptide.

Tomasinsig L(1), Scocchi M, Mettulio R, Zanetti M.

Author information: 
(1)Department of Biomedical Sciences and Technology, University of Udine, P. le
Kolbe 4, I-33100 Udine, Italy.

Most antimicrobial peptides (AMPs) impair the viability of target bacteria by
permeabilizing bacterial membranes. However, the proline-rich AMPs have been
shown to kill susceptible organisms without causing significant membrane
perturbation and may act by inhibiting the activity of bacterial targets. To gain
initial insight into the events that follow interaction of a proline-rich peptide
with bacterial cells, we used DNA macroarray technology to monitor
transcriptional alterations of Escherichia coli in response to challenge with a
subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes
in the expression levels of 70 bacterial genes from various functional categories
were detected. Among these, 26 genes showed decreased expression, while 44 genes,
including genes that are potentially involved in bacterial resistance to
antimicrobials, showed increased expression. The generation of a transcriptional 
response under the experimental conditions used is consistent with the ability of
Bac7(1-35) to interact with bacterial components and affect biological processes 
in this organism.

DOI: 10.1128/AAC.48.9.3260-3267.2004 
PMCID: PMC514742
PMID: 15328082  [Indexed for MEDLINE]


1180. Di Yi Jun Yi Da Xue Xue Bao. 2004 Sep;24(9):1033-6.

[An initial examination of the spermatozoal gene expression profile].

[Article in Chinese]

Mao XM(1), Ma WL, Feng CQ, Zou YG, Zheng WL.

Author information: 
(1)Institute of Molecular Biology, First Military Medical University, Guangzhou
510515, China. mxm@fimmu.com

OBJECTIVE: To collect the normal spermatozoal gene expression sequence tags with 
the restriction display technique for constructing a microarray to understand
spermatozoal gene expression profiles.
METHODS: The total RNA extracted from normal human spermatozoa were reversely
transcribed into cDNAs, which were digested by Sau3AI and linked to universal
adapters (adapter 1) at both ends. According to the sequence of the adapter, a
pair of primers (universal primers 1) was designed, followed by PCR with primers 
1 and the PCR products were transferred into E.coli. The positive clones were
collected after identification to serve as the probes for constructing the gene
expression microarray of spermatozoa by printing those probes on the slides. The 
accomplished microarrays were examined by Cy3-labeled normal spermatozoal
samples.
RESULTS: Altogether 1 859 probes were collected, from which 368 were picked out
randomly for constructing the microarray.
CONCLUSIONS: Human spermatozoa contain a rich repertoire of RNAs, and the probes 
we prepared possess good incredibility and speciality.


PMID: 15447855  [Indexed for MEDLINE]


1181. Infect Immun. 2004 Sep;72(9):5452-9.

Transcriptome of enterohemorrhagic Escherichia coli O157 adhering to eukaryotic
plasma membranes.

Dahan S(1), Knutton S, Shaw RK, Crepin VF, Dougan G, Frankel G.

Author information: 
(1)Centre for Molecular Microbiology and Infection, Department of Biological
Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia
coli O157:H7 gene expression during binding to plasma membranes. Analysis of the 
complete transcriptomes of the bound bacteria revealed increased levels of
stress-associated mRNAs and decreased levels of mRNA encoding proteins involved
in translation and type III secretion.

DOI: 10.1128/IAI.72.9.5452-5459.2004 
PMCID: PMC517483
PMID: 15322044  [Indexed for MEDLINE]


1182. J Bacteriol. 2004 Sep;186(18):6179-85.

The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli.

Lease RA(1), Smith D, McDonough K, Belfort M.

Author information: 
(1)Wadsworth Center, New York State Department of Health, Center for Medical
Sciences, Albany, New York, USA. ral@jhu.edu

DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts 
by RNA-RNA interactions to control translation and turnover of specific mRNAs.
Two targets of DsrA regulation are RpoS, the stationary-phase and stress response
sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global
transcription repressor. Genes regulated globally by RpoS and H-NS include stress
response proteins and virulence factors for pathogenic E. coli. Here, by using
transcription profiling via DNA arrays, we have identified genes induced by DsrA.
Steady-state levels of mRNAs from many genes increased with DsrA overproduction, 
including multiple acid resistance genes of E. coli. Quantitative primer
extension analysis verified the induction of individual acid resistance genes in 
the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic 
E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants.
Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive
dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in
acid resistance. Whether DsrA targets acid resistance genes directly by base
pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in
either event, our results suggest that DsrA RNA may enhance the virulence of
pathogenic E. coli.

DOI: 10.1128/JB.186.18.6179-6185.2004 
PMCID: PMC515158
PMID: 15342588  [Indexed for MEDLINE]


1183. J Bacteriol. 2004 Sep;186(17):5640-8.

Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons:
identification of its binding site and functional analysis of lmrB and yxaGH.

Yoshida K(1), Ohki YH, Murata M, Kinehara M, Matsuoka H, Satomura T, Ohki R,
Kumano M, Yamane K, Fujita Y.

Author information: 
(1)Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima, Japan. 
kenyoshi@kobe-u.ac.jp

The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a
repressor of its own operon, while LmrB acts as a multidrug efflux transporter.
LmrA was produced in Escherichia coli cells and was shown to bind to the lmr
promoter region, in which an LmrA-binding site was identified. Genome-wide
screening involving DNA microarray analysis allowed us to conclude that LmrA also
repressed yxaGH, which was not likely to contribute to the multidrug resistance. 
LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding 
sites were identified. The LmrA regulon was thus determined to comprise lmrAB and
yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence,
TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.

DOI: 10.1128/JB.186.17.5640-5648.2004 
PMCID: PMC516806
PMID: 15317768  [Indexed for MEDLINE]


1184. J Clin Microbiol. 2004 Sep;42(9):4083-91.

Development and validation of a diagnostic DNA microarray to detect
quinolone-resistant Escherichia coli among clinical isolates.

Yu X(1), Susa M, Knabbe C, Schmid RD, Bachmann TT.

Author information: 
(1)Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 
D-70569 Stuttgart, Germany.

The incidence of resistance against fluoroquinolones among pathogenic bacteria
has been increasing in accordance with the worldwide use of this drug.
Escherichia coli is one of the most relevant species for quinolone resistance. In
this study, a diagnostic microarray for single-base-mutation detection was
developed, which can readily identify the most prevalent E. coli genotypes
leading to quinolone resistance. Based on genomic sequence analysis using public 
databases and our own DNA sequencing results, two amino acid positions (83 and
87) on the A subunit of the DNA gyrase, encoded by the gyrA gene, have been
identified as mutation hot spots and were selected for DNA microarray detection. 
Oligonucleotide probes directed against these two positions were designed so that
they could cover the most important resistance-causing and silent mutations. The 
performance of the array was validated with 30 clinical isolates of E. coli from 
four different hospitals in Germany. The microarray results were confirmed by
standard DNA sequencing and were in full agreement with phenotypic antimicrobial 
susceptibility testing.

DOI: 10.1128/JCM.42.9.4083-4091.2004 
PMCID: PMC516282
PMID: 15364994  [Indexed for MEDLINE]


1185. J Clin Microbiol. 2004 Sep;42(9):3985-91.

Use of culture, PCR analysis, and DNA microarrays for detection of Campylobacter 
jejuni and Campylobacter coli from chicken feces.

Keramas G(1), Bang DD, Lund M, Madsen M, Bunkenborg H, Telleman P, Christensen
CB.

Author information: 
(1)MIC-Department of Micro and Nanotechnology, Technical University of Denmark,
DTU-Building 345 east, DK-2800 Kongens Lyngby, Denmark.

A DNA microarray for detection of Campylobacter spp. was recently developed and
applied to detect Campylobacter spp. directly from chicken feces. Sixty-five
pooled chicken cloacal swab samples from 650 individual broiler chickens were
included in the study. The results of Campylobacter sp. detection obtained with
DNA microarrays were compared to those obtained by conventional culture and gel
electrophoresis. By conventional culture, 60% of the samples were positive for
either Campylobacter jejuni or Campylobacter coli. By PCR and capillary
electrophoresis, 95% of the samples were positive for Campylobacter spp., whereas
with DNA microarrays all samples were positive for Campylobacter spp. By
application of DNA microarray analysis, the isolates in 4 samples (6%) could not 
be identified to the species level, whereas by PCR-capillary electrophoresis, the
isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary 
electrophoresis analysis revealed that two (3%) of the samples were positive for 
both C. jejuni and C. coli, while DNA microarray analysis revealed that nine
(14%) of the samples were positive for both species. Of 65 samples, 2 samples
were identified to contain C. coli by conventional culture but were positive for 
C. jejuni by both PCR-capillary electrophoresis and DNA microarray analysis. The 
discrepancy between the methods is discussed.

DOI: 10.1128/JCM.42.9.3985-3991.2004 
PMCID: PMC516363
PMID: 15364980  [Indexed for MEDLINE]


1186. J Microbiol Methods. 2004 Sep;58(3):403-11.

Application of oligonucleotide array technology for the rapid detection of
pathogenic bacteria of foodborne infections.

Hong BX(1), Jiang LF, Hu YS, Fang DY, Guo HY.

Author information: 
(1)Department of Microbiology, School of Medicine, Sun Yatsen University,
Zhongshan Road(II)74, Guangzhou 510089, China.

A rapid and accurate method for detection for common pathogenic bacteria in
foodborne infections was established by using oligonucleotide array technology.
Nylon membrane was used as the array support. A mutation region of the 23S rRNA
gene was selected as the discrimination target from 14 species (genera) of
bacteria causing foodborne infections and two unrelated bacterial species. A pair
of universal primers was designed for PCR amplification of the 23S rRNA gene.
Twenty-one species (genera)-specific oligonucleotide detection probes were
synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification
products of 14 species of pathogenic bacteria were hybridized to the
oligonucleotide array. Hybridization results were analyzed with
digoxigenin-linked enzyme reaction. Results indicated that nine species of
pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella
dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus
cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity
and specificity for the oligonucleotide array. Two other species (Salmonella
enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but 
the reaction did not affect their detection. After redesigning the probes,
positive hybridization results were obtained with Staphylococcus aureus, but not 
with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide
array can also be applied to samples collected in clinical settings of foodborne 
infections. The superiority of oligonucleotide array over other tests lies on its
rapidity, accuracy and efficiency in the diagnosis, treatment and control of
foodborne infections.

DOI: 10.1016/j.mimet.2004.05.005 
PMID: 15279944  [Indexed for MEDLINE]


1187. Zhonghua Er Ke Za Zhi. 2004 Sep;42(9):668-72.

[Rapid diagnosis of common pathogenic bacteria infection in newborn infants by
16SrDNA oligonucleotide array].

[Article in Chinese]

Shen DX(1), Du J, Feng ZC.

Author information: 
(1)Department of Pediatrics, Zhujiang Hospital, The First Military Medical
University, Guangzhou, 510282 China.

OBJECTIVE: The rapid identification of pathogenic bacteria is important for
earlier effective patient management and antimicrobial therapy, especially for
the infant patient, whose immunological system is not fully developed. However
conventional microbiogical techniques of bacterial identification, culture and
isolation of pathogenic bacteria, identification by biochemistry and serological 
assay, are time-consuming and require intensive labor. On the basis of special
gene sequence, PCR provides simple and rapid way to identify bacteria. But it is 
difficult to identify all of bacteria species which are suspicious of pathogenic 
agents. Oligonucleotide arrays provide a powerful tool for parallel detection of 
target genes. The objective of this study was to test a reverse oligonucleotide
assay, which hybridize with the PCR product of 16SrDNA using a pair of universal 
primers, to rapidly identify common infant pathogenic bacteria.
METHODS: By comparison and analysis of the 16SrDNA sequences of common pathogenic
bacteria, a region, which has numerous sequence variations and flanked by highly 
conserved sequences, was found. A pair of universal primers was designed
according to its flanking conservative sequence, and a set of probes specially
targeting to eight species of infant pathogenic bacteria, including
staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Streptococcus faecalis, Hemophilus influenzae, Enterobacter cloacae, Escherichia 
coli, and Acinetobacter baumannii,according to the variable sequences. The probes
were fixed on the nylon membrane with positive electricity, and hybridized them
with the products of PCR using the universal primers.
RESULTS: The universal primers could amplify the target sequence from bacteria
including the eight common infant pathogenic bacteria and Staphylococcus
epidermidis, Enterobacter aerogenes, Streptococcus pneumoniae,beta-hemolytic
streptococcus, Neisseria meningitides, Citrobacter freundii, Bacillus subtilis,
and Salmonella infantis,but could not amplify rotavirus and human DNA as control.
The results showed that the oligonucleotide array could specially hybridize with 
the eight bacteria to be examined and could not hybridize with other bacteria.
The lowest concentration of DNA (product of PCR) for oligonucleotide array was
about 25 ng/ml. The results proved that the probes are highly selective and the
oligonucleotide arrays could parallelly detect the eight common infant pathogenic
bacteria. The results suggested that the oligonucleotide array system was able to
identify the eight common infant pathogenic bacteria from clinical specimens and 
the results were the same as identified by automated bacterial detection machine.
From the further experiments, the oligonucleotide array system could directly
diagnose the common infant pathogenic bacteria from the broths of samples
culture.
CONCLUSIONS: Despite limited number of identifiable bacteria and lack of
information on antibiotic susceptibility of bacteria, the reverse oligonucleotide
assay system, which contains amplification of the segment of 16rDNA from samples 
using the universal primers and parallel detection of PCR products using specific
probes, is an effective method to rapidly identify the eight common infant
pathogenic bacteria.


PMID: 15482667  [Indexed for MEDLINE]


1188. Zhonghua Er Ke Za Zhi. 2004 Sep;42(9):663-7.

[Rapid diagnosis of neonatal sepsis by 16SrRNA genes PCR amplification and
genechip hybridization].

[Article in Chinese]

Tong MQ(1), Shang SQ, Wu YD, Zhao ZY.

Author information: 
(1)Department of Internal Medicine, Children's Hospital Affiliated to Medical
College, Zhejiang University, Hangzhou 310003, China.

OBJECTIVE: To explore a method for rapid diagnosis of sepsis in newborn infants.
METHODS: (1) The primers and oligonucleotide probes were designed and synthesized
based on the sequences of bacterial 16SrRNA gene. The gene chip was prepared
through the probes printed onto special glass slides. The gene chip included 18
special probes: universal probe 1, universal probe 2, Gram positive bacterial
probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2,
Staphylococcus aureus, coagulase negative staphylococcus (CoNS) 1, CoNS 2,
Escherichia coli, Hemophilus influenzae, Listeria monocytogenes, Streptococcus
pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus,
Meningococcus, Corynebacterium, Propionibacterium; (2) Blood specimens from 285
cases of suspected septicemia were cultured and bacterial 16S rRNA gene was
detected separately; DNA isolated from blood specimens and cerebrospinal fluid
was amplified by PCR, and PCR products were hybridized with the probes on the
gene chips. Hybridization results were scanned and read by laser-scanner.
RESULTS: (1) Of the 285 cases, 17 were positive by PCR and the positive rate
(5.96%) was significantly higher than that of blood culture (2.81%) (P < 0.01).
When blood culture was taken as control, the sensitivity of PCR was 100% and
Specificity was 96.75%, the index of accurate diagnosis was 0.968. (2) The 17
specimens which showed positive results by PCR were further hybridized on the
gene chip. All were positive by universal probes. Among all of them, 5 were
positive by E. coli probe; 4 were positive by Staphylococcus epidermidis; two
were positive by Bacillus and Propionibacterium probes, separately; 4 were
positive by CoNS. The 8 specimens which showed positive results by both PCR and
blood culture, the result of gene chip hybridization coincided with the result of
blood culture.
CONCLUSION: Detection of the bacterial 16SrRNA genes in clinical specimens by
gene chip hybridization technology can diagnose neonatal septicemia rapidly. This
method has higher sensitivity and specificity than blood culture or other methods
and can provide a rapid way for the etiological diagnosis of neonatal septicemia.
Therefore the genechip method may be valuable and practical in early diagnosis of
neonatal septicemia.


PMID: 15482666  [Indexed for MEDLINE]


1189. J Biol Chem. 2004 Aug 13;279(33):34397-405. Epub 2004 Jun 9.

The tumor suppressor adenomatous polyposis coli and caudal related homeodomain
protein regulate expression of retinol dehydrogenase L.

Jette C(1), Peterson PW, Sandoval IT, Manos EJ, Hadley E, Ireland CM, Jones DA.

Author information: 
(1)Huntsman Cancer Institute, Department of Oncological Sciences, University of
Utah, 2000 Circle of Hope, Salt Lake City, UT 84112, USA.

Development of normal colon epithelial cells proceeds through a systematic
differentiation of cells that emerge from stem cells within the base of colon
crypts. Genetic mutations in the adenomatous polyposis coli (APC) gene are
thought to cause colon adenoma and carcinoma formation by enhancing colonocyte
proliferation and impairing differentiation. We currently have a limited
understanding of the cellular mechanisms that promote colonocyte differentiation.
Herein, we present evidence supporting a lack of retinoic acid biosynthesis as a 
mechanism contributing to the development of colon adenomas and carcinomas.
Microarray and reverse transcriptase-PCR analyses revealed reduced expression of 
two retinoid biosynthesis genes: retinol dehydrogenase 5 (RDH5) and retinol
dehydrogenase L (RDHL) in colon adenomas and carcinomas as compared with normal
colon. Consistent with the adenoma and carcinomas samples, seven colon carcinoma 
cell lines also lacked expression of RDH5 and RDHL. Assessment of RDH enzymatic
activity within these seven cell lines showed poor conversion of retinol into
retinoic acid when compared with normal cells such as normal human mammary
epithelial cells. Reintroduction of wild type APC into an APC-deficient colon
carcinoma cell line (HT29) resulted in increased expression of RDHL without
affecting RDH5. APC-mediated induction of RDHL was paralleled by increased
production of retinoic acid. Investigations into the mechanism responsible for
APC induction of RDHL indicated that beta-catenin fails to repress RDHL. The
colon-specific transcription factor CDX2, however, activated an RDHL promoter
construct and induced endogenous RDHL. Finally, the induction of RDHL by APC
appears dependent on the presence of CDX2. We propose a novel role for APC and
CDX2 in controlling retinoic acid biosynthesis and in promoting a
retinoid-induced program of colonocyte differentiation.

DOI: 10.1074/jbc.M314021200 
PMID: 15190067  [Indexed for MEDLINE]


1190. Bioinformatics. 2004 Aug 12;20(12):1928-39. Epub 2004 Mar 25.

Hypothesis-driven approach to predict transcriptional units from gene expression 
data.

Steinhauser D(1), Junker BH, Luedemann A, Selbig J, Kopka J.

Author information: 
(1)Max Planck Institute of Molecular Plant Physiology, 14476 Golm, Germany.
Steinhauser@mpimp-golm.mpg.de

MOTIVATION: A major issue in computational biology is the reconstruction of
functional relationships among genes, for example the definition of regulatory or
biochemical pathways. One step towards this aim is the elucidation of
transcriptional units, which are characterized by co-responding changes in mRNA
expression levels. These units of genes will allow the generation of hypotheses
about respective functional interrelationships. Thus, the focus of analysis
currently moves from well-established functional assignment through comparison of
protein and DNA sequences towards analysis of transcriptional co-response. Tools 
that allow deducing common control of gene expression have the potential to
complement and extend routine BLAST comparisons, because gene function may be
inferred from common transcriptional control.
RESULTS: We present a co-clustering strategy of genome sequence information and
gene expression data, which was applied to identify transcriptional units within 
diverse compendia of expression profiles. The phenomenon of prokaryotic operons
was selected as an ideal test case to generate well-founded hypotheses about
transcriptional units. The existence of overlapping and ambiguous operon
definitions allowed the investigation of constitutive and conditional expression 
of transcriptional units in independent gene expression experiments of
Escherichia coli. Our approach allowed identification of operons with high
accuracy. Furthermore, both constitutive mRNA co-response as well as conditional 
differences became apparent. Thus, we were able to generate insight into the
possible biological relevance of gene co-response. We conclude that the suggested
strategy will be amenable in general to the identification of transcriptional
units beyond the chosen example of E.coli operons.
AVAILABILITY: The analyses of E.coli transcript data presented here are available
upon request or at http://csbdb.mpimp-golm.mpg.de/

DOI: 10.1093/bioinformatics/bth182 
PMID: 15044239  [Indexed for MEDLINE]


1191. Genetics. 2004 Aug;167(4):2111-9.

Genome image programs: visualization and interpretation of Escherichia coli
microarray experiments.

Zimmer DP(1), Paliy O, Thomas B, Gyaneshwar P, Kustu S.

Author information: 
(1)Department of Plant and Microbial Biology, University of California, Berkeley,
California 94720, USA.

We have developed programs to facilitate analysis of microarray data in
Escherichia coli. They fall into two categories: manipulation of microarray
images and identification of known biological relationships among lists of genes.
A program in the first category arranges spots from glass-slide DNA microarrays
according to their position in the E. coli genome and displays them compactly in 
genome order. The resulting genome image is presented in a web browser with an
image map that allows the user to identify genes in the reordered image. Another 
program in the first category aligns genome images from two or more experiments. 
These images assist in visualizing regions of the genome with common
transcriptional control. Such regions include multigene operons and clusters of
operons, which are easily identified as strings of adjacent, similarly colored
spots. The images are also useful for assessing the overall quality of
experiments. The second category of programs includes a database and a number of 
tools for displaying biological information about many E. coli genes
simultaneously rather than one gene at a time, which facilitates identifying
relationships among them. These programs have accelerated and enhanced our
interpretation of results from E. coli DNA microarray experiments. Examples are
given.

Copyright 2004 Genetics Society of America

DOI: 10.1534/genetics.104.027532 
PMCID: PMC1471004
PMID: 15342544  [Indexed for MEDLINE]


1192. J Clin Microbiol. 2004 Aug;42(8):3766-74.

Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer
resistance.

Grimm V(1), Ezaki S, Susa M, Knabbe C, Schmid RD, Bachmann TT.

Author information: 
(1)Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 
Stuttgart, Germany.

Erratum in
    J Clin Microbiol. 2004 Oct;42(10):4919.

Standard clinical procedures for pathogen resistance identification are laborious
and usually require 2 days of cultivation before the resistance can be determined
unequivocally. In contrast, clinicians and patients face increasing threats from 
antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels
of resistance. A major class of microbial resistance stems from the occurrence of
beta-lactamases, which, if mutated, can cause the severe extended-spectrum
beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause
resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase
inhibitors. We describe an oligonucleotide microarray for identification of the
single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants 
described to date which are related to the ESBL and/or IRT phenotype. The target 
DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella
pneumoniae cells isolated from clinical samples, was amplified and fluorescently 
labeled by PCR with consensus primers in the presence of cyanine 5-labeled
nucleotides. The total assay, including PCR, hybridization, and image analysis,
could be performed in 3.5 h. The microarray results were validated by standard
clinical procedures. The microarray outperformed the standard procedures in terms
of assay time and the depth of information provided. In conclusion, this array
offers an attractive option for the identification and epidemiologic monitoring
of TEM beta-lactamases in the routine clinical diagnostic laboratory.

DOI: 10.1128/JCM.42.8.3766-3774.2004 
PMCID: PMC497576
PMID: 15297528  [Indexed for MEDLINE]


1193. Mol Cell Proteomics. 2004 Aug;3(8):780-7. Epub 2004 Apr 28.

Only a small subset of the horizontally transferred chromosomal genes in
Escherichia coli are translated into proteins.

Taoka M(1), Yamauchi Y, Shinkawa T, Kaji H, Motohashi W, Nakayama H, Takahashi N,
Isobe T.

Author information: 
(1)Department of Chemistry, Graduate School of Science, Tokyo Metropolitan
University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.

Horizontally transferred genes are believed to play a critical role in the
divergence of bacterial strains from a common ancestor, but whether all of these 
genes express functional proteins in the cell remains unknown. Here, we used an
integrated LC-based protein identification technology to analyze the proteome of 
Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins,
which are equivalent to approximately 35% of the total open reading frames
predicted in the genome. This subset contained proteins with cellular abundance
of several dozens to hundreds of thousands of copies, and included nearly all
types of proteins in terms of chemical characteristics, subcellular distribution,
and function. Interestingly, the subset also contained 138 of 164 gene products
that are currently known to be essential for bacterial viability (84% coverage). 
However, the subset contained only a very small population (10%) of protein
products from genes mapped within K-loops, which are "hot spots" for the
integration of foreign DNAs within the K12 genome. On the other hand, these genes
in K-loops appeared to be transcribed to RNAs almost as efficiently as the native
genes in the bacterial cell as monitored by DNA microarray analysis, raising the 
possibility that most of the recently acquired foreign genes are inadequate for
the translational machinery for the native genes and do not generate functional
proteins within the cell.

DOI: 10.1074/mcp.M400030-MCP200 
PMID: 15115803  [Indexed for MEDLINE]


1194. Trends Biotechnol. 2004 Aug;22(8):381-3.

Extracting novel information from gene expression data.

Li Z(1), Chan C.

Author information: 
(1)Department of Chemical Engineering and Material Science, Michigan State
University, East Lansing, MI 48824, USA.

Data from high throughput technologies, such as DNA microarrays, necessitated the
development of new computational methodologies for analyzing the high dimensional
information contained within the gene expression data. Liao's group suggested the
use of network component analysis to predict transcription factor activities by
integrating gene expression data from Escherichia coli with known connectivity
information between their genes and transcription factors. This introduces an
approach for obtaining novel information from gene expression data.

DOI: 10.1016/j.tibtech.2004.06.007 
PMID: 15283978  [Indexed for MEDLINE]


1195. Biochem J. 2004 Jul 15;381(Pt 2):373-8.

The water- and salt-stress-regulated Asr1 (abscisic acid stress ripening) gene
encodes a zinc-dependent DNA-binding protein.

Kalifa Y(1), Gilad A, Konrad Z, Zaccai M, Scolnik PA, Bar-Zvi D.

Author information: 
(1)Department of Life Sciences and The Doris and Bertie Black Center for
Bioenergetics in Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva
84105, Israel.

Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a 
small plant-specific protein whose cellular mode of action defies deduction based
on its sequence or homology analyses, is one of numerous plant gene products with
unknown biological roles that become over-expressed under water- and salt-stress 
conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are
transiently increased following exposure of plants to poly(ethylene glycol), NaCl
or abscisic acid. Western blot and indirect immunofluorescence analysis with
anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic
and nuclear subcellular compartments; approx. one-third of the total ASR1 protein
could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and 
can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1,
overexpressed in Escherichia coli and purified to homogeneity, possesses
zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX
(systematic evolution of ligands by exponential enrichment) analysis suggest that
ASR1 binds at a preferred DNA sequence.

DOI: 10.1042/BJ20031800 
PMCID: PMC1133842
PMID: 15101820  [Indexed for MEDLINE]


1196. Genes Dev. 2004 Jul 15;18(14):1766-79.

Topological domain structure of the Escherichia coli chromosome.

Postow L(1), Hardy CD, Arsuaga J, Cozzarelli NR.

Author information: 
(1)Department of Molecular and Cell Biology and Department of Mathematics,
University of California at Berkeley, Berkeley, California 94720, USA.

The circular chromosome of Escherichia coli is organized into independently
supercoiled loops, or topological domains. We investigated the organization and
size of these domains in vivo and in vitro. Using the expression of >300
supercoiling-sensitive genes to gauge local chromosomal supercoiling, we
quantitatively measured the spread of relaxation from double-strand breaks
generated in vivo and thereby calculated the distance to the nearest domain
boundary. In a complementary approach, we gently isolated chromosomes and
examined the lengths of individual supercoiled loops by electron microscopy. The 
results from these two very different methods agree remarkably well. By comparing
our results to Monte Carlo simulations of domain organization models, we conclude
that domain barriers are not placed stably at fixed sites on the chromosome but
instead are effectively randomly distributed. We find that domains are much
smaller than previously reported, approximately 10 kb on average. We discuss the 
implications of these findings and present models for how domain barriers may be 
generated and displaced during the cell cycle in a stochastic fashion.

Copyright 2004 Cold Spring Harbor Laboratory Press ISSN

DOI: 10.1101/gad.1207504 
PMCID: PMC478196
PMID: 15256503  [Indexed for MEDLINE]


1197. Bioinformatics. 2004 Jul 10;20(10):1500-5.

Gene expression analysis on biochemical networks using the Potts spin model.

König R(1), Eils R.

Author information: 
(1)Division Intelligent Bioinformatics Systems, German Cancer Research Center
(DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. r.koenig@dkfz.de

MOTIVATION: Microarray technology allows us to profile the expression of a large 
subset or all genes of a cell. Biochemical research over the last three decades
has elucidated an increasingly complete image of the metabolic architecture. For 
less complex organisms, such as Escherichia coli, the biochemical network has
been described in much detail. Here, we investigate the clustering of such
networks by applying gene expression data that define edge lengths in the
network.
RESULTS: The Potts spin model is used as a nearest neighbour based clustering
algorithm to discover fragmentation of the network in mutants or in biological
samples when treated with drugs. As an example, we tested our method with gene
expression data from E.coli treated with tryptophan excess, starvation and
trpyptophan repressor mutants. We observed fragmentation of the tryptophan
biosynthesis pathway, which corresponds well to the commonly known regulatory
response of the cells.

DOI: 10.1093/bioinformatics/bth109 
PMID: 15231542  [Indexed for MEDLINE]


1198. Appl Biochem Biotechnol. 2004 Jul-Sep;118(1-3):215-32.

Genomewide expression analysis in amino acid-producing bacteria using DNA
microarrays.

Polen T(1), Wendisch VF.

Author information: 
(1)Institute of Biotechnology 1, Research Center Jülich, Leo-Brandt-Str, D-52428,
Germany.

DNA microarray technology has become an important research tool for biotechnology
and microbiology. It is now possible to characterize genetic diversity and gene
expression in a genomewide manner. DNA microarrays have been applied extensively 
to study the biology of many bacteria including Escherichia coli, but only
recently have they been developed for the Gram-positive Corynebacterium
glutamicum. Both bacteria are widely used for biotechnological amino acid
production. In this article, in addition to the design and generation of
microarrays as well as their use in hybridization experiments and subsequent data
analysis, we describe recent applications of DNA microarray technology regarding 
amino acid production in C. glutamicum and E. coli. We also discuss the impact of
functional genomics studies on fundamental as well as applied aspects of amino
acid production with C. glutamicum and E. coli.


PMID: 15304751  [Indexed for MEDLINE]


1199. Di Yi Jun Yi Da Xue Xue Bao. 2004 Jul;24(7):738-41.

[Isolation of the target gene from cDNA restriction fragments using 70-mer oligo 
microarray].

[Article in Chinese]

Shi R(1), Ma WL, Liu CH, Song YB, Wu QH, Guo QY, Zheng WL.

Author information: 
(1)Institute of Molecular Biology, First Military Medical University, Guangzhou
510515, China.

OBJECTIVE: To study the method for using a 70-mer oligo microarray as the probe
to isolate target genes from the cDNA restriction fragments.
METHOD: Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock
culture and reversely transcribed into the double-stranded cDNAs, which were
prepared into restriction cDNA fragments using restriction display (RD) method.
The microarray was printed using a single 70-mer specific oligo designed to
according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled 
by PCR method with the Cy5 universal primer before hybridization with the
microarray. The microarray was stripped after washing and scanning, and the strip
solution was collected for another round of PCR amplification using the universal
primer without fluorescence. The PCR product was then cloned into PUC18 T vector 
and transformed into to E.coli JM109 cells for amplification, and the plasmids
were extracted and sequenced for identification.
RESULTS: BLAST results showed that the target gene was cloned successfully.
CONCLUSION: The target gene can be isolated directly using the 70-mer oligo
microarray as the probe from the cDNA fragments prepared by RD method, without
the necessity of building a cDNA library. This method can also be used in further
research to acquire the differentially expressed genes after the oligo microarray
hybridization.


PMID: 15257889  [Indexed for MEDLINE]


1200. Microbiology. 2004 Jul;150(Pt 7):2289-300.

Gene array analysis of Yersinia enterocolitica FlhD and FlhC: regulation of
enzymes affecting synthesis and degradation of carbamoylphosphate.

Kapatral V(1), Campbell JW, Minnich SA, Thomson NR, Matsumura P, Prüss BM.

Author information: 
(1)Integrated Genomics, Inc., 2201 West Campbell Park Dr., Chicago, IL 60612,
USA.

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria.
Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli 
flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC
complex has an effect on metabolism similar to E. coli. To study metabolic gene
regulation, a partial Yersinia enterocolitica 8081c microarray was constructed
and the expression patterns of wild-type cells were compared to an flhDC mutant
strain at 25 and 37 degrees C. The overlap between the E. coli and Y.
enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at
least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate
hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase
(carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a
pathway that is involved in the degradation of L-histidine to L-glutamate and
eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and
carbamoylaspartate. A number of other genes were regulated at a lower rate. In
two additional experiments, the expression of wild-type cells grown at 4 or 25
degrees C was compared to the same strain grown at 37 degrees C. The expression
of the flagella master operon flhD was not affected by temperature, whereas the
flagella-specific sigma factor fliA was highly expressed at 25 degrees C and
reduced at 4 and 37 degrees C. Several other flagella genes, all of which are
under the control of FliA, exhibited a similar temperature profile. These data
are consistent with the hypothesis that temperature regulation of flagella genes 
might be mediated by the flagella-specific sigma factor FliA and not the flagella
master regulator FlhD/FlhC.

DOI: 10.1099/mic.0.26814-0 
PMID: 15256571  [Indexed for MEDLINE]


1201. Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9780-5. Epub 2004 Jun 21.

Microarray analysis of transposition targets in Escherichia coli: the impact of
transcription.

Manna D(1), Breier AM, Higgins NP.

Author information: 
(1)Department of Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, Birmingham, AL 35294, USA.

Transposable elements have influenced the genetic and physical composition of all
modern organisms. Defining how different transposons select target sites is
critical for understanding the biochemical mechanism of this type of
recombination and the impact of mobile genes on chromosome structure and
function. Phage Mu replicates in Gram-negative bacteria using an extremely
efficient transposition reaction. Replicated copies are excised from the
chromosome and packaged into virus particles. Each viral genome plus several
hundred base pairs of host DNA covalently attached to the prophage right end is
packed into a virion. To study Mu transposition preferences, we used DNA
microarray technology to measure the abundance of >4,000 Escherichia coli genes
in purified Mu phage DNA. Insertion hot- and cold-spot genes were found
throughout the genome, reflecting >1,000-fold variation in utilization frequency.
A moderate preference was observed for genes near the origin compared to terminus
of replication. Large biases were found at hot and cold spots, which often
include several consecutive genes. Efficient transcription of genes had a strong 
negative influence on transposition. Our results indicate that local chromosome
structure is more important than DNA sequence in determining Mu target-site
selection.

DOI: 10.1073/pnas.0400745101 
PMCID: PMC470751
PMID: 15210965  [Indexed for MEDLINE]


1202. Anal Chem. 2004 Jun 1;76(11):3162-70.

Integrated portable genetic analysis microsystem for pathogen/infectious disease 
detection.

Lagally ET(1), Scherer JR, Blazej RG, Toriello NM, Diep BA, Ramchandani M,
Sensabaugh GF, Riley LW, Mathies RA.

Author information: 
(1)Chemistry Department, University of California, Berkeley, California 94720,
USA.

An integrated portable genetic analysis microsystem including PCR amplification
and capillary electrophoretic (CE) analysis coupled with a compact instrument for
electrical control and laser-excited fluorescence detection has been developed.
The microdevice contains microfabricated heaters, temperature sensors, and
membrane valves to provide controlled sample positioning and immobilization in
200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal
fluorescence detection optics, electronics for sensing and powering the PCR
reactor, and high-voltage power supplies for conducting CE separations. The
fluorescein-labeled PCR products are amplified and electrophoretically analyzed
in a gel-filled microchannel in <10 min. We demonstrate the utility of this
instrument by performing pathogen detection and genotyping directly from whole
Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay
consists of a triplex PCR amplification targeting genes that encode 16S ribosomal
RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution
demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay
uses a femA marker to identify cells as S. aureus and a mecA marker to probe for 
methicillin resistance. This integrated portable genomic analysis microsystem
demonstrates the feasibility of performing rapid high-quality detection of
pathogens and their antimicrobial drug resistance.

DOI: 10.1021/ac035310p 
PMID: 15167797  [Indexed for MEDLINE]


1203. Extremophiles. 2004 Jun;8(3):209-17. Epub 2004 Feb 27.

Transcriptional analysis of dynamic heat-shock response by the hyperthermophilic 
bacterium Thermotoga maritima.

Pysz MA(1), Ward DE, Shockley KR, Montero CI, Conners SB, Johnson MR, Kelly RM.

Author information: 
(1)Department of Chemical Engineering, North Carolina State University, Raleigh, 
NC 27695-7905, USA.

The thermal stress response of the hyperthermophilic bacterium Thermotoga
maritima was characterized using a 407-open reading frame-targeted cDNA
microarray. Transient gene expression was followed for 90 min, following a shift 
from 80 degrees C to 90 degrees C. While some aspects of mesophilic heat-shock
response were conserved in T. maritima, genome content suggested differentiating 
features that were borne out by transcriptional analysis. Early induction of
predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL
(TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved
CIRCE elements upstream of hrcA and groES. Induction of the T. maritima rpoE/
sigW and rpoD/ sigA homologs suggests a mechanism for global heat-shock response 
in the absence of an identifiable ortholog to a major heat-shock sigma factor. In
contrast to heat-shock response in Escherichia coli, the majority of genes
encoding ATP-dependent proteases were downregulated, including clpP (TM0695),
clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869). Notably, T.
maritima showed indications of a late heat-shock response with the induction of a
marR homolog (TM0816), several other putative transcriptional regulators (TM1023,
TM1069), and two alpha-glucosidases (TM0434 and TM1068). Taken together, the
results reported here indicate that, while T. maritima shares core elements of
the bacterial heat-shock response with mesophiles, the thermal stress regulatory 
strategies of this organism differ significantly. However, it remains to be
elucidated whether these differences are related to thermophilicity or
phylogenetic placement.

Copyright 2004 Springer-Verlag

DOI: 10.1007/s00792-004-0379-2 
PMID: 14991425  [Indexed for MEDLINE]


1204. J Bacteriol. 2004 Jun;186(12):3911-21.

Extensive genomic diversity in pathogenic Escherichia coli and Shigella Strains
revealed by comparative genomic hybridization microarray.

Fukiya S(1), Mizoguchi H, Tobe T, Mori H.

Author information: 
(1)Kyowa Hakko Kogyo, Tokyo Research Laboratories, 3-6-6 Asahimachi, Machidashi, 
Tokyo 194-8533, Japan.

Escherichia coli, including the closely related genus Shigella, is a highly
diverse species in terms of genome structure. Comparative genomic hybridization
(CGH) microarray analysis was used to compare the gene content of E. coli K-12
with the gene contents of pathogenic strains. Missing genes in a pathogen were
detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of
W3110, a commonly used wild-type K-12 strain. For 22 strains subjected to the CGH
microarray analyses 1,424 ORFs were found to be absent in at least one strain.
The common backbone of the E. coli genome was estimated to contain about 2,800
ORFs. The mosaic distribution of absent regions indicated that the genomes of
pathogenic strains were highly diversified because of insertions and deletions.
Prophages, cell envelope genes, transporter genes, and regulator genes in the
K-12 genome often were not present in pathogens. The gene contents of the strains
tested were recognized as a matrix for a neighbor-joining analysis. The
phylogenic tree obtained was consistent with the results of previous studies.
However, unique relationships between enteroinvasive strains and Shigella,
uropathogenic, and some enteropathogenic strains were suggested by the results of
this study. The data demonstrated that the CGH microarray technique is useful not
only for genomic comparisons but also for phylogenic analysis of E. coli at the
strain level.

DOI: 10.1128/JB.186.12.3911-3921.2004 
PMCID: PMC419953
PMID: 15175305  [Indexed for MEDLINE]


1205. Dev Comp Immunol. 2004 May 17;28(6):635-45.

Gene expression profiling of bovine macrophages in response to Escherichia coli
O157:H7 lipopolysaccharide.

Chitko-McKown CG(1), Fox JM, Miller LC, Heaton MP, Bono JL, Keen JE, Grosse WM,
Laegreid WW.

Author information: 
(1)USDA, ARS, Roman L. Hruska US Meat Animal Research Center (MARC), State Spur
18D, P.O. Box 166, Clay Center, Nebraska 68933-0166, USA.

The aim of this study was to identify changes in bovine macrophage gene
expression in response to treatment with Escherichia coli 0157:H7
lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from
control and LPS-treated primary macrophages hybridized to greater than 5644
(79.8%) of the non-control gene targets on a commercially available microarray
containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these
target sequences, 44 were differentially expressed upon exposure to LPS,
including 18 genes not previously reported to exist in cattle. These included a
pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and
inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated
and control macrophages consistently hybridized to targets known to be expressed 
constitutively by macrophages, as expected given the predicted cDNA sequence
homology. That this human system was accurately estimating levels of bovine
transcripts was further verified by real-time quantitative reverse transcriptase 
polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first
report of bovine-human cross-species expression profiling by microarray
hybridization demonstrates the utility of this technique in bovine gene
expression and discovery.

DOI: 10.1016/j.dci.2003.10.002 
PMID: 15177116  [Indexed for MEDLINE]


1206. Am J Respir Crit Care Med. 2004 May 15;169(10):1135-43. Epub 2004 Mar 4.

Differential gene expression in gram-negative and gram-positive sepsis.

Yu SL(1), Chen HW, Yang PC, Peck K, Tsai MH, Chen JJ, Lin FY.

Author information: 
(1)Department of Surgery, National Taiwan University Hospital and National Taiwan
University College of Medicine, Taipei, Taiwan, Republic of China.

Sepsis is the most common cause of death in patients in the intensive care unit. 
Genome-wide gene expression analysis can provide insights into the molecular
alterations of sepsis. Total mRNA was extracted from the livers of 6 uninfected
control mice and 60 septic mice after infusion of either live Escherichia coli or
Staphylococcus aureus. Using a murine complementary DNA microarray system,
changes in gene expression were monitored at six time points (uninfected, 2, 8,
24, 48, and 72 hours). Overall, 4.8% of 6,144 assessed genes were differentially 
regulated with a greater than twofold change across all time points. Most of the 
genes with altered expression were commonly present in gram-negative and
gram-positive sepsis, but the expression levels of 17 genes were different
between both types of sepsis at particular time points after infection. The
microarray results support the hypothesis that both gram-positive and
gram-negative sepsis share a final common pathway involved in the pathogenesis of
sepsis, but certain genes are differentially expressed under distinct regulation.
These results may provide insights into the pathogenesis of sepsis and may also
help identify some altered genes that can serve as new targets for diagnostic
tools and therapeutic strategies.

DOI: 10.1164/rccm.200211-1278OC 
PMID: 15001460  [Indexed for MEDLINE]


1207. Proc Natl Acad Sci U S A. 2004 May 11;101(19):7427-32. Epub 2004 May 3.

Carbon nutrition of Escherichia coli in the mouse intestine.

Chang DE(1), Smalley DJ, Tucker DL, Leatham MP, Norris WE, Stevenson SJ, Anderson
AB, Grissom JE, Laux DC, Cohen PS, Conway T.

Author information: 
(1)Advanced Center for Genome Technology, Department of Botany and Microbiology, 
University of Oklahoma, Norman, OK 73019-0245, USA.

Whole-genome expression profiling revealed Escherichia coli MG1655 genes induced 
by growth on mucus, conditions designed to mimic nutrient availability in the
mammalian intestine. Most were nutritional genes corresponding to catabolic
pathways for nutrients found in mucus. We knocked out several pathways and tested
the relative fitness of the mutants for colonization of the mouse intestine in
competition with their wild-type parent. We found that only mutations in sugar
pathways affected colonization, not phospholipid and amino acid catabolism, not
gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate 
pathway. Gluconate appeared to be a major carbon source used by E. coli MG1655 to
colonize, having an impact on both the initiation and maintenance stages.
N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in
initiation, but not maintenance. Glucuronate, mannose, fucose, and ribose
appeared to be involved in maintenance, but not initiation. The in vitro order of
preference for these seven sugars paralleled the relative impact of the
corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine 
> N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose. The results 
of this systematic analysis of nutrients used by E. coli MG1655 to colonize the
mouse intestine are intriguing in light of the nutrient-niche hypothesis, which
states that the ecological niches within the intestine are defined by nutrient
availability. Because humans are presumably colonized with different commensal
strains, differences in nutrient availability may provide an open niche for
infecting E. coli pathogens in some individuals and a barrier to infection in
others.

DOI: 10.1073/pnas.0307888101 
PMCID: PMC409935
PMID: 15123798  [Indexed for MEDLINE]


1208. Proc Natl Acad Sci U S A. 2004 May 4;101(18):7046-51. Epub 2004 Apr 23.

Three replication origins in Sulfolobus species: synchronous initiation of
chromosome replication and asynchronous termination.

Lundgren M(1), Andersson A, Chen L, Nilsson P, Bernander R.

Author information: 
(1)Department of Molecular Evolution, Evolutionary Biology Center, Uppsala
University, Norbyvägen 18C, SE-752 36 Uppsala, Sweden.

Chromosome replication origins were mapped in vivo in the two hyperthermophilic
archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using
microarray-based marker frequency analysis. Bidirectional replication was found
to be initiated in near synchrony from three separate sites in both organisms.
Two of the three replication origins in each species were located in the vicinity
of a cdc6/orc1 replication initiation gene, whereas no known
replication-associated gene could be identified near the third origin in either
organism. In contrast to initiation, replication termination occurred
asynchronously, such that certain replication forks continued to progress for >40
min after the others had terminated. In each species, all replication forks
advanced at similar DNA polymerization rates; this was found to be an order of
magnitude below that displayed by Escherichia coli and thus closer to eukaryotic 
elongation rates. In S. acidocaldarius, a region containing short regularly
spaced repeats was found to hybridize aberrantly, as compared to the rest of the 
chromosome, raising the possibility of a centromere-like function.

DOI: 10.1073/pnas.0400656101 
PMCID: PMC406463
PMID: 15107501  [Indexed for MEDLINE]


1209. Appl Environ Microbiol. 2004 May;70(5):3047-54.

Nucleic acid amplification strategies for DNA microarray-based pathogen
detection.

Vora GJ(1), Meador CE, Stenger DA, Andreadis JD.

Author information: 
(1)Center for Bio/Molecular Science and Engineering, Naval Research Laboratory,
Washington, DC 20375, USA. gvora@cbmse.nrl.navy.mil

DNA microarray-based screening and diagnostic technologies have long promised
comprehensive testing capabilities. However, the potential of these powerful
tools has been limited by front-end target-specific nucleic acid amplification.
Despite the sensitivity and specificity associated with PCR amplification, the
inherent bias and limited throughput of this approach constrain the principal
benefits of downstream microarray-based applications, especially for pathogen
detection. To begin addressing alternative approaches, we investigated four
front-end amplification strategies: random primed, isothermal Klenow
fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of
each amplification strategy was assessed by hybridizing amplicons to microarrays 
consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic
Escherichia coli O157:H7 and by quantitating their sensitivities for the
detection of O157:H7 in laboratory and environmental samples. Although nearly
identical levels of hybridization specificity were achieved for each method,
multiplex PCR was at least 3 orders of magnitude more sensitive than any
individual random amplification approach. However, the use of Klenow-plus-Klenow 
and phi29 polymerase-plus-Klenow tandem random amplification strategies provided 
better sensitivities than multiplex PCR. In addition, amplification biases among 
the five genetic loci tested were 2- to 20-fold for the random approaches, in
contrast to >4 orders of magnitude for multiplex PCR. The same random
amplification strategies were also able to detect all five diagnostic targets in 
a spiked environmental water sample that contained a 63-fold excess of
contaminating DNA. The results presented here underscore the feasibility of using
random amplification approaches and begin to systematically address the
versatility of these approaches for unbiased pathogen detection from
environmental sources.


PMCID: PMC404398
PMID: 15128566  [Indexed for MEDLINE]


1210. Appl Microbiol Biotechnol. 2004 May;64(4):515-24. Epub 2004 Jan 16.

Gene expression in Escherichia coli biofilms.

Ren D(1), Bedzyk LA, Thomas SM, Ye RW, Wood TK.

Author information: 
(1)Departments of Chemical Engineering and Molecular and Cell Biology, University
of Connecticut, 191 Auditorium Road, Storrs, CT 06269-3222, USA.

DNA microarrays were used to study the gene expression profile of Escherichia
coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 
plates in continuous reactors were used to create the biofilms. For the biofilms 
grown on glass wool, 22 genes were induced significantly (p< or =0.05) compared
to suspension cells, including several genes for the stress response ( hslS,
hslT, hha, and soxS), type I fimbriae ( fimG), metabolism ( metK), and 11 genes
of unknown function ( ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, 
b1601, and b0836). The DNA microarray results were corroborated with RNA dot
blotting. For the biofilm grown on mild steel plates, the DNA microarray data
showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days
in the continuous reactors did not exhibit differential gene expression compared 
to suspension cells although genes were induced at 0.03/h. The present study
suggests that biofilm gene expression is strongly associated with environmental
conditions and that stress genes are involved in E. coli JM109 biofilm formation.

Copyright 2004 Springer-Verlag

DOI: 10.1007/s00253-003-1517-y 
PMID: 14727089  [Indexed for MEDLINE]


1211. J Bacteriol. 2004 May;186(10):3254-8.

Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655.

Brokx SJ(1), Ellison M, Locke T, Bottorff D, Frost L, Weiner JH.

Author information: 
(1)Project CyberCell, Department of Biochemistry, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada.

To gain insight into the cell envelope of Escherichia coli grown under aerobic
and anaerobic conditions, lipoproteins were examined by using functional
genomics. The mRNA expression levels of each of these genes under three growth
conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by
using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR 
(RT-PCR). Many genes showed significant changes in expression level. The RT-PCR
results were in very good agreement with the microarray data. The results of this
study represent the first insights into the possible roles of unknown lipoprotein
genes and broaden our understanding of the composition of the cell envelope under
different environmental conditions. Additionally, these data serve as a test set 
for the refinement of high-throughput bioinformatic and global gene expression
methods.


PMCID: PMC400601
PMID: 15126489  [Indexed for MEDLINE]


1212. J Bacteriol. 2004 May;186(10):3006-14.

Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems
of Escherichia coli.

Eguchi Y(1), Okada T, Minagawa S, Oshima T, Mori H, Yamamoto K, Ishihama A,
Utsumi R.

Author information: 
(1)Department of Bioscience and Biotechnology, Graduate School of Agriculture,
Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.

Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS
two-component system of Escherichia coli resulted in enhanced expression of 13
PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, 
slyB, ybjG, yrbL, and mgrB. This regulatory network between the two systems also 
occurred as a result of overproduction of the EvgA regulator; however, enhanced
transcription of the phoPQ genes did not further activate expression of the
PhoP/PhoQ-regulated genes. These results demonstrated signal transduction from
the EvgA/EvgS system to the PhoP/PhoQ system in E. coli and also identified the
genes that required the two systems for enhanced expression. This is one example 
of the intricate signal transduction networks that are posited to exist in E.
coli.


PMCID: PMC400602
PMID: 15126461  [Indexed for MEDLINE]


1213. Appl Environ Microbiol. 2004 Apr;70(4):2354-66.

Analysis of gene expression in Escherichia coli in response to changes of
growth-limiting nutrient in chemostat cultures.

Hua Q(1), Yang C, Oshima T, Mori H, Shimizu K.

Author information: 
(1)Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.

Studies of steady-state metabolic fluxes in Escherichia coli grown in
nutrient-limited chemostat cultures suggest remarkable flux alterations in
response to changes of growth-limiting nutrient in the medium (Hua et al., J.
Bacteriol. 185:7053-7067, 2003). To elucidate the physiological adaptation of
cells to the nutrient condition through the flux change and understand the
molecular mechanisms underlying the change in the flux, information on gene
expression is of great importance. DNA microarray analysis was performed to
investigate the global transcriptional responses of steady-state cells grown in
chemostat cultures with limited glucose or ammonia while other environmental
conditions and the growth rate were kept constant. In slow-growing cells
(specific growth rate of 0.10 h(-1)), 9.8% of a total of 4,071 genes
investigated, especially those involved in amino acid metabolism, central carbon 
and energy metabolism, transport system and cell envelope, were observed to be
differentially expressed between the two nutrient-limited cultures. One important
characteristic of E. coli grown under nutrient limitation was its capacity to
scavenge carbon or nitrogen from the medium through elevating the expression of
the corresponding transport and assimilation genes. The number of differentially 
expressed genes in faster-growing cells (specific growth rate of 0.55 h(-1)),
however, decreased to below half of that in slow-growing cells, which could be
explained by diverse transcriptional responses to the growth rate under different
nutrient limitations. Independent of the growth rate, 92 genes were identified as
being differentially expressed. Genes tightly related to the culture conditions
were highlighted, some of which may be used to characterize nutrient-limited
growth.


PMCID: PMC383082
PMID: 15066832  [Indexed for MEDLINE]


1214. Lab Chip. 2004 Apr;4(2):152-8. Epub 2004 Jan 16.

Development of a multiplex microarray microsystem.

Keramas G(1), Perozziello G, Geschke O, Christensen CB.

Author information: 
(1)MIC - Department of Micro and Nanotechnology, Technical University of Denmark,
DTU - Building 345 east, DK-2800 Kongens Lyngby, Denmark.

A hybrid multiplex microarray microsystem has been developed that consists of 32 
individually addressable array reaction chambers, supporting the use of
multichannel pipettes for addition of up to 8 samples simultaneously.
Discrimination between Campylobacter jejuni and Campylobacter coli bacteria was
observed in DNA samples containing Campylobacter spp., with the same specificity 
and sensitivity as when compared to a full-size microarray. The spinloaded
multiplex microarray microsystem described provides a novel and convenient test
format for simultaneous low-density microarray analysis and is universally
adaptable to other DNA, protein or small molecule microarray based applications.

DOI: 10.1039/b313472e 
PMID: 15052357  [Indexed for MEDLINE]


1215. J Biol Chem. 2004 Mar 26;279(13):12588-97. Epub 2004 Jan 7.

Probing the ArcA-P modulon of Escherichia coli by whole genome transcriptional
analysis and sequence recognition profiling.

Liu X(1), De Wulf P.

Author information: 
(1)Department of Microbiology and Molecular Genetics, Harvard Medical School,
Boston, MA 02115, USA.

The ArcB/ArcA two-component signal transduction system of Escherichia coli
regulates gene expression in response to the redox conditions of growth. Over the
years, genetic screens have lead to the identification of about 30
ArcA-P-controlled operons that are involved in redox metabolism. However, the
discovery of 3 targets that are not implicated in respiratory metabolism (the tra
operon for plasmid conjugation, psi site for Xer-based recombination, and oriC
site for chromosome replication) suggests that the Arc modulon may comprise
additional operons that are involved in a myriad of functions. To identify these 
operons, we derived the ArcA-P-dependent transcription profile of E. coli using
oligonucleotide-based microarray analysis. The findings indicated that 9% of all 
open reading frames in E. coli are affected either directly or indirectly by
ArcA-P. To identify which operons are under the direct control of ArcA-P, we
developed the ArcA-P recognition weight matrix from footprinting data and used it
to scan the genome, yielding an ArcA-P sequence affinity map. By overlaying both 
methods, we identified 55 new Arc-regulated operons that are implicated in energy
metabolism, transport, survival, catabolism, and transcriptional regulation. The 
data also suggest that the Arc response pathway, which translates into a net
global downscaling of gene expression, overlaps partly with the FNR regulatory
network. A conservative but reasonable assessment is that the Arc pathway
recruits 100-150 operons to mediate a role in cellular adaptation that is more
extensive than hitherto anticipated.

DOI: 10.1074/jbc.M313454200 
PMID: 14711822  [Indexed for MEDLINE]


1216. BMC Microbiol. 2004 Mar 22;4:12.

Library on a slide for bacterial comparative genomics.

Zhang L(1), Srinivasan U, Marrs CF, Ghosh D, Gilsdorf JR, Foxman B.

Author information: 
(1)Department of Epidemiology, University of Michigan School of Public Health,
109 Observatory Street, Ann Arbor, MI48109, USA. lxzhang@umich.edu

BACKGROUND: We describe a novel application of microarray technology for
comparative genomics of bacteria in which libraries of entire genomes rather than
the sequence of a single genome or sets of genes are arrayed on the slide and
then probed for the presence or absence of specific genes and/or gene alleles.
RESULTS: We first adopted a 96-well high throughput working protocol to
efficiently isolate high quality genomic DNA. We then optimized conditions to
print genomic DNA onto a glass slide with high density (up to 15000 spots) and to
sensitively detect gene targets in each genome spot using fluorescently labeled
DNA probe. Finally, we created an E. coli reference collection array and probed
it for the presence or absence of the hemolysin (hly) gene using a dual channel
non-competing hybridization strategy. Results from the array hybridization
matched perfectly with previous tests.
CONCLUSIONS: This new form of microarray technology, Library on a Slide, is an
efficient way for sharing and utilizing large strain collections in comparative
genomic analyses.

DOI: 10.1186/1471-2180-4-12 
PMCID: PMC394321
PMID: 15035675  [Indexed for MEDLINE]


1217. Nucleic Acids Res. 2004 Mar 18;32(5):e51.

Improving the sensitivity and specificity of gene expression analysis in highly
related organisms through the use of electronic masks.

Nagpal S(1), Karaman MW, Timmerman MM, Ho VV, Pike BL, Hacia JG.

Author information: 
(1)The Institute for Genetic Medicine, University of Southern California, 2250
Alcazar Street, IGM 240, Los Angeles, CA 90089, USA.

DNA microarrays are powerful tools for comparing gene expression profiles from
closely related organisms. However, a single microarray design is frequently used
in these studies. Therefore, the levels of certain transcripts can be grossly
underestimated due to sequence differences between the transcripts and the
arrayed DNA probes. Here, we seek to improve the sensitivity and specificity of
oligonucleotide microarray-based gene expression analysis by using genomic
sequence information to predict the hybridization efficiency of orthologous
transcripts to a given microarray. To test our approach, we examine hybridization
patterns from three Escherichia coli strains on E.coli K-12 MG1655 gene
expression microarrays. We create electronic mask files to discard data from
probes predicted to have poor hybridization sensitivity and specificity to cDNA
targets from each strain. We increased the accuracy of gene expression analysis
and identified genes that cannot be accurately interrogated in each strain using 
these microarrays. Overall, these studies provide guidelines for designing
effective electronic masks for gene expression analysis in organisms where
substantial genome sequence information is available.

DOI: 10.1093/nar/gnh048 
PMCID: PMC390348
PMID: 15031318  [Indexed for MEDLINE]


1218. Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2758-63. Epub 2004 Feb 23.

Global analysis of Escherichia coli RNA degradosome function using DNA
microarrays.

Bernstein JA(1), Lin PH, Cohen SN, Lin-Chao S.

Author information: 
(1)Department of Genetics, Stanford University, Stanford, CA 94305, USA.

RNase E, an essential endoribonuclease of Escherichia coli, interacts through its
C-terminal region with multiple other proteins to form a complex termed the RNA
degradosome. To investigate the degradosome's proposed role as an RNA decay
machine, we used DNA microarrays to globally assess alterations in the
steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution
in bacteria carrying mutations in the degradosome constituents RNase E,
polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that
the functions of all four of these proteins are necessary for normal mRNA
turnover. We identified specific transcripts and functionally distinguishable
transcript classes whose half-life and abundance were affected congruently by
multiple degradosome proteins, affected differentially by mutations in
degradosome constituents, or not detectably altered by degradosome mutations. Our
results, which argue that decay of some E. coli mRNAs in vivo depends on the
action of assembled degradosomes, whereas others are acted on by degradosome
proteins functioning independently of the complex, imply the existence of
structural features or biochemical factors that target specific classes of mRNAs 
for decay by degradosomes.

DOI: 10.1073/pnas.0308747101 
PMCID: PMC365694
PMID: 14981237  [Indexed for MEDLINE]


1219. Antimicrob Agents Chemother. 2004 Mar;48(3):890-6.

Killing by ampicillin and ofloxacin induces overlapping changes in Escherichia
coli transcription profile.

Kaldalu N(1), Mei R, Lewis K.

Author information: 
(1)Department of Biology, Northeastern University, Boston, Massachusetts 02115,
USA.

Erratum in
    Antimicrob Agents Chemother. 2004 Aug;48(8):3213.

The basis of bactericidal versus bacteriostatic action of antibiotics and the
mechanism of bacterial cell death are largely unknown. Related to this important 
issue is the essential invulnerability to killing of persisters: cells forming a 
small subpopulation largely responsible for the recalcitrance of biofilms to
chemotherapy. To learn whether death is accompanied by changes in expression of
particular genes, we compared transcription profiles of log-phase Escherichia
coli treated with bactericidal concentrations of two unrelated antibiotics:
ampicillin and ofloxacin. Massive changes in transcription profile were observed 
in response to either agent, and there was a significant overlap in genes whose
transcription was affected. A small group of mostly uncharacterized genes was
induced and a much larger set was transcriptionally repressed by both
antibiotics. Among the repressed genes were those required for flagellar
synthesis, energy metabolism, transport of small molecules, and protein
synthesis.


PMCID: PMC353072
PMID: 14982780  [Indexed for MEDLINE]


1220. Appl Environ Microbiol. 2004 Mar;70(3):1749-57.

Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase
system by DNA microarray profiling.

Hansen EH(1), Schembri MA, Klemm P, Schäfer T, Molin S, Gram L.

Author information: 
(1)Novozymes A/S, DK-2880 Bagsvaerd, Denmark.

A novel antimicrobial enzyme system, the Curvularia haloperoxidase system, was
examined with the aim of elucidating its mechanism of antibacterial action.
Escherichia coli strain MG1655 was stressed with sublethal concentrations of the 
enzyme system, causing a temporary arrest of growth. The expression of genes
altered upon exposure to the Curvularia haloperoxidase system was analyzed by
using DNA microarrays. Only a limited number of genes were involved in the
response to the Curvularia haloperoxidase system. Among the induced genes were
the ibpA and ibpB genes encoding small heat shock proteins, a gene cluster of six
genes (b0301-b0306) of unknown function, and finally, cpxP, a member of the Cpx
pathway. Knockout mutants were constructed with deletions in b0301-b0306, cpxP,
and cpxARP, respectively. Only the mutant lacking cpxARP was significantly more
sensitive to the enzyme system than was the wild type. Our results demonstrate
that DNA microarray technology cannot be used as the only technique to
investigate the mechanisms of action of new antimicrobial compounds. However, by 
combining DNA microarray analysis with the subsequent creation of knockout
mutants, we were able to pinpoint one of the specific responses of E.
coli--namely, the Cpx pathway, which is important for managing the stress
response from the Curvularia haloperoxidase system.


PMCID: PMC368414
PMID: 15006801  [Indexed for MEDLINE]


1221. Clin Cancer Res. 2004 Mar 1;10(5):1698-705.

Epstein-barr virus-positive gastric carcinoma has a distinct protein expression
profile in comparison with epstein-barr virus-negative carcinoma.

Lee HS(1), Chang MS, Yang HK, Lee BL, Kim WH.

Author information: 
(1)Department of Pathology, Seoul National University College of Medicine, Seoul,
Korea.

PURPOSE: EBV has been detected in 2-16% of gastric carcinomas. However, there is 
little information available about the gene expression profile of EBV-positive
gastric carcinomas.
EXPERIMENTAL DESIGN: EBV infection was examined using EBV-encoded small RNAs
(EBERs) in situ hybridization, and 63 (5.6%) of 1127 consecutive gastric
carcinomas were found to be EBV-positive. The expressions of 27 tumor-associated 
proteins were evaluated immunohistochemically in 63 EBV-positive gastric
carcinomas and 287 EBV-negative carcinomas using the tissue array method. In
addition, the genotype of EBV was investigated by PCR amplification of LMP1
(latent membrane protein 1), Epstein-Barr nuclear antigen 2 (EBNA2), and EBNA3B
genes.
RESULTS: EBV-positive gastric carcinomas are characterized by the presence of
lymphoid stroma, proximal location, and predominance in males. In comparison with
EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of
expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the 
expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal
cancer), and some DNA repair proteins (P < 0.05). There was negative association 
between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA,
C-erbB2, and smad7. Using hierarchical cluster analysis, we divided EBV-positive 
gastric carcinomas into two clusters. Those patients with cluster 1 (42 cases)
carcinomas had a better prognosis than those with cluster 2 (12 cases; P =
0.0002) or those with EBV-negative carcinomas (280 cases; P = 0.0251). Fifty-one 
(92.7%) of 55 EBV-positive carcinomas demonstrated the 30-bp deletion in LMP1
gene, and 53 (96.4%) of 55 cases were type 1 for EBNA2 and EBNA3B genes.
CONCLUSION: EBV-positive gastric carcinomas have a distinct protein expression
profile as well as distinct clinicopathological features, as compared with
EBV-negative carcinomas. The subclassification of EBV-positive carcinomas, by
hierarchical cluster analysis, is significantly associated with patient survival.


PMID: 15014022  [Indexed for MEDLINE]


1222. J Bacteriol. 2004 Mar;186(5):1388-97.

Transcriptional organization and regulation of the L-idonic acid pathway (GntII
system) in Escherichia coli.

Bausch C(1), Ramsey M, Conway T.

Author information: 
(1)Department of Botany and Microbiology, University of Oklahoma, Norman,
Oklahoma 73019.

The genetic organization of the idn genes that encode the pathway for L-idonate
catabolism was characterized. The monocistronic idnK gene is transcribed
divergently from the idnDOTR genes, which were shown to form an operon. The
215-bp regulatory region between the idnK and idnD genes contains promoters in
opposite orientation with transcription start sites that mapped to positions -26 
and -29 with respect to the start codons. The regulatory region also contains a
single putative IdnR/GntR binding site centered between the two promoters, a CRP 
binding site upstream of idnD, and an UP element upstream of idnK. The genes of
the L-idonate pathway were shown to be under catabolite repression control.
Analysis of idnD- and idnK-lacZ fusions in a nonpolar idnD mutant that is unable 
to interconvert L-idonate and 5-ketogluconate indicated that either compound
could induce the pathway. The L-idonate pathway was first characterized as a
subsidiary pathway for D-gluconate catabolism (GntII), which is induced by
D-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the 
idnK and idnD operons are induced by D-gluconate in a GntI system mutant,
presumably by endogenous formation of 5-ketogluconate from D-gluconate. Thus, the
regulation of the GntII system is appropriate for this pathway, which is
primarily involved in L-idonate catabolism; the GntII system can be induced by
D-gluconate under conditions that block the GntI system.


PMCID: PMC344402
PMID: 14973046  [Indexed for MEDLINE]


1223. J Biol Chem. 2004 Feb 20;279(8):6840-6. Epub 2003 Dec 1.

Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor 
of transforming growth factor-beta signaling, as a target of the beta-catenin
pathway in colorectal tumor cells.

Sekiya T(1), Adachi S, Kohu K, Yamada T, Higuchi O, Furukawa Y, Nakamura Y,
Nakamura T, Tashiro K, Kuhara S, Ohwada S, Akiyama T.

Author information: 
(1)Laboratory of Molecular and Genetic Information, Institute of Molecular and
Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-0032, Japan.

The Wnt signaling pathway is activated in most human colorectal tumors.
Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC),
as well as activation of beta-catenin, causes the accumulation of beta-catenin,
which in turn associates with the T cell factor/lymphoid enhancer factor
(TCF/LEF) family of transcription factors and activates transcription of their
target genes. Here we show that beta-catenin activates transcription of the BMP
and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of
BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular
carcinomas relative to the corresponding non-cancerous tissues. Expression of
BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant 
of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that
beta-catenin is responsible for the aberrant expression of BAMBI in colorectal
tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor
cells to transforming growth factor-beta signaling. These results suggest that
beta-catenin interferes with transforming growth factor-beta-mediated growth
arrest by inducing the expression of BAMBI, and this may contribute to colorectal
and hepatocellular tumorigenesis.

DOI: 10.1074/jbc.M310876200 
PMID: 14660579  [Indexed for MEDLINE]


1224. J Biol Chem. 2004 Feb 13;279(7):5537-48. Epub 2003 Nov 25.

Phosphotransferase-mediated transport of the osmolyte
2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the
mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as
repressor.

Sampaio MM(1), Chevance F, Dippel R, Eppler T, Schlegel A, Boos W, Lu YJ, Rock
CO.

Author information: 
(1)Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,
2780-156 Oeiras, Portugal.

2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in
hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up
and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake 
is mediated by the P-enolpyruvate-dependent phosphotransferase system with the
MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The
apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65
nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA
phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA
was devoid of MG transport but is complemented by a plasmid harboring mngA.
Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake
and the metabolism of galactarate and glucarate controlled by the CdaR activator.
The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a
protein with alpha-mannosidase activity. farR, the gene upstream of mngA (now
called mngR) had previously been characterized as a fatty acyl-responsive
regulator; however, deletion of mngR resulted in the up-regulation of only two
genes, mngA and mngB. The mngR deletion caused constitutive MG transport that
became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR 
is the regulator (repressor) of the MG transport/metabolism system. Thus, the
mngR mngA mngB gene cluster encodes an MG utilizing system.

DOI: 10.1074/jbc.M310980200 
PMID: 14645248  [Indexed for MEDLINE]


1225. Alcohol. 2004 Feb;32(2):113-27.

Microarray gene analysis of the liver in a rat model of chronic, voluntary
alcohol intake.

Deaciuc IV(1), Peng X, D'Souza NB, Shedlofsky SI, Burikhanov R, Voskresensky IV, 
de Villiers WJ.

Author information: 
(1)College of Medicine, Department of Internal Medicine, 800 Rose Street,
MN649A-0298, University of Kentucky, Lexington, KY 40536, USA.
ion.deaciuc@louisville.edu

The mechanisms underlying alcoholic liver disease are not fully understood. It
has been established that alcohol interferes with transcriptional and
translational regulatory steps of cell function. To understand such an effect,
assessment of alcohol-induced changes in the simultaneous expression of a large
number of genes may prove very useful. The purpose of the current study was to
test a large number of genes ( approximately 8700) for possible changes in
expression induced by alcohol alone or in addition to treatment with
lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver.
Male rats were fed an alcohol-containing liquid diet (Lieber-DeCarli) for 14-15
weeks, injected with Escherichia coli LPS (0.8 mg x kg(-1)), and killed 24 h
later. Blood samples were taken for determination of plasma liver enzyme
activity, and liver samples were obtained for histologic evaluation and total RNA
extraction. Total RNA was analyzed for gene expression (Rat Toxicology U34 Array;
Affymetrix, Santa Clara, CA). Of 8740 genes on the microchip, 2259 were expressed
in the liver. Seven hundred ninety-eight genes underwent significant changes
induced by either alcohol or LPS, but listed in this article are only those that 
significantly increased or decreased expression twofold or more. The genes were
assigned to functional groups and reviewed. Gene changes were discussed from two 
viewpoints: relevance to established hypotheses of alcohol and LPS mechanisms of 
action and revealing of novel mechanisms of alcohol-induced liver injury.
Application of DNA microarray technology to the study of alcohol-induced liver
injury generated novel theoretical and experimental approaches to alcohol-induced
liver injury.

DOI: 10.1016/j.alcohol.2003.12.001 
PMID: 15163562  [Indexed for MEDLINE]


1226. Genome Res. 2004 Feb;14(2):201-8.

A motif co-occurrence approach for genome-wide prediction of
transcription-factor-binding sites in Escherichia coli.

Bulyk ML(1), McGuire AM, Masuda N, Church GM.

Author information: 
(1)Harvard University Graduate Biophysics Program, Harvard Medical School,
Boston, Massachusetts 02115, USA.

Various computational approaches have been developed for predicting
cis-regulatory DNA elements in prokaryotic genomes. We describe a novel method
for predicting transcription-factor-binding sites in Escherichia coli. Our method
takes advantage of the principle that transcription factors frequently coregulate
gene expression, but without requiring prior knowledge of which groups of genes
are coregulated. Using position weight matrices for 49 known transcription
factors, we examined spacings between pairs of matrix hits. These pairs were
assigned probabilities according to the overrepresentation of their separation
distance. The functions of many open reading frames (ORFs) downstream from
predicted binding sites are unknown, and may correspond to novel regulon members.
For five predictions, knockouts with mutated replacements of the predicted
binding sites were created in E. coli MG1655. Quantitative real-time PCR (RT-PCR)
indicates that for each of the knockouts, at least one gene immediately
downstream exhibits a statistically significant change in mRNA expression. This
approach may be useful in analyzing binding sites in a variety of organisms.

DOI: 10.1101/gr.1448004 
PMCID: PMC327095
PMID: 14762058  [Indexed for MEDLINE]


1227. J Bacteriol. 2004 Feb;186(3):880-4.

Inhibiting cell division in Escherichia coli has little if any effect on gene
expression.

Arends SJ(1), Weiss DS.

Author information: 
(1)Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.

DNA microarrays were used to compare gene expression in dividing and nondividing 
(filamentous) cultures of Escherichia coli. Although cells from these cultures
differed profoundly in morphology, their gene expression profiles were nearly
identical. These results extend previous evidence that there is no division
checkpoint in E. coli, and progression through the cell cycle is not regulated by
the transcription of different genes during different parts of the cell cycle.


PMCID: PMC321490
PMID: 14729718  [Indexed for MEDLINE]


1228. Mol Microbiol. 2004 Feb;51(3):659-74.

Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene
expression.

Beloin C(1), Valle J, Latour-Lambert P, Faure P, Kzreminski M, Balestrino D,
Haagensen JA, Molin S, Prensier G, Arbeille B, Ghigo JM.

Author information: 
(1)Groupe de génétique des biofilms-CNRS URA 2172, Institut Pasteur, 25 rue du Dr
Roux, 75724 Paris Cedex 15, France.

The formation of biofilm results in a major lifestyle switch that is thought to
affect the expression of multiple genes and operons. We used DNA arrays to study 
the global effect of biofilm formation on gene expression in mature Escherichia
coli K-12 biofilm. We show that, when biofilm is compared with the exponential
growth phase, 1.9% of the genes showed a consistent up- or downregulation by a
factor greater than two, and that 10% of the E. coli genome is significantly
differentially expressed. The functions of the genes induced in these conditions 
correspond to stress response as well as energy production, envelope biogenesis
and unknown functions. We provide evidence that the expression of stress envelope
response genes, such as the psp operon or elements of the cpx and rpoE pathways, 
is a general feature of E. coli mature biofilms. We also compared biofilm with
the stationary growth phase and showed that the biofilm lifestyle, although
sharing similarities with the stationary growth phase, triggers the expression of
specific sets of genes. Using gene disruption of 54 of the most biofilm-induced
genes followed by a detailed phenotypic study, we validated the biological
relevance of our analysis and showed that 20 of these genes are required for the 
formation of mature biofilm. This group includes 11 genes of previously unknown
function. These results constitute a comprehensive analysis of the global
transcriptional response triggered in mature E. coli biofilms and provide
insights into its physiological signature.


PMID: 14731270  [Indexed for MEDLINE]


1229. Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):745-50. Epub 2004 Jan 12.

Prominent roles of the NorR and Fur regulators in the Escherichia coli
transcriptional response to reactive nitrogen species.

Mukhopadhyay P(1), Zheng M, Bedzyk LA, LaRossa RA, Storz G.

Author information: 
(1)Cell Biology and Metabolism Branch, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

We examined the genomewide transcriptional responses of Escherichia coli treated 
with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium
nitrite (NaNO(2)) during aerobic growth. These assays showed that NorR, a homolog
of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the
global repressor of ferric ion uptake, are major regulators of the response to
reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E. coli 
defenses against superoxide-generating compounds and hydrogen peroxide,
respectively, have minor roles. Moreover, additional regulators of the E. coli
response to reactive nitrogen species remain to be identified because several of 
the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant
strains. We propose that the E. coli transcriptional response to reactive
nitrogen species is a composite response mediated by the modification of multiple
transcription factors containing iron or redox-active cysteines, some
specifically designed to sense NO and its derivatives and others that are
collaterally activated by the reactive nitrogen species.

DOI: 10.1073/pnas.0307741100 
PMCID: PMC321752
PMID: 14718666  [Indexed for MEDLINE]


1230. Anal Chem. 2004 Jan 15;76(2):365-72.

A robust technique for assembly of nucleic acid hybridization chips based on
electrochemically templated chitosan.

Yi H(1), Wu LQ, Ghodssi R, Rubloff GW, Payne GF, Bentley WE.

Author information: 
(1)Center for Biosystems Research, University of Maryland Biotechnology
Institute, 5115 Plant Sciences Building, College Park, Maryland 20742, USA.

A nucleic acid hybridization assay was assembled onto a robust and readily
addressable silicon-based chip using polysaccharide chitosan as a scaffold for
the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique
polymer, ideally suited for this application because its net charge and
solubility are pH dependent. Specifically in this work, gold-patterned electrodes
were created using standard photolithographic techniques, chitosan was
electrodeposited in a spatially resolved manner onto the polarized electrodes,
probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and
DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target 
nucleic acid was quantifiable, reproducible, and robust; the surface was
regenerated and rehybridized up to eight times without loss of signal. Finally,
transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an
indicator of cellular stress, was observed using the hybridization chip sandwich 
assay. Thus, this method enables rapid and facile monitoring of gene expression
in a format that is reusable and requires minimal reagent quantities.

DOI: 10.1021/ac034662t 
PMID: 14719884  [Indexed for MEDLINE]


1231. Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):641-6. Epub 2003 Dec 23.

Transcriptome-based determination of multiple transcription regulator activities 
in Escherichia coli by using network component analysis.

Kao KC(1), Yang YL, Boscolo R, Sabatti C, Roychowdhury V, Liao JC.

Author information: 
(1)Department of Chemical Engineering, University of California, Los Angeles, CA 
90095, USA.

Cells adjust gene expression profiles in response to environmental and
physiological changes through a series of signal transduction pathways. Upon
activation or deactivation, the terminal regulators bind to or dissociate from
DNA, respectively, and modulate transcriptional activities on particular
promoters. Traditionally, individual reporter genes have been used to detect the 
activity of the transcription factors. This approach works well for simple,
non-overlapping transcription pathways. For complex transcriptional networks,
more sophisticated tools are required to deconvolute the contribution of each
regulator. Here, we demonstrate the utility of network component analysis in
determining multiple transcription factor activities based on transcriptome
profiles and available connectivity information regarding network connectivity.
We used Escherichia coli carbon source transition from glucose to acetate as a
model system. Key results from this analysis were either consistent with
physiology or verified by using independent measurements.

DOI: 10.1073/pnas.0305287101 
PMCID: PMC327201
PMID: 14694202  [Indexed for MEDLINE]


1232. Adv Biochem Eng Biotechnol. 2004;89:47-71.

Monitoring of stress responses.

Schweder T(1), Hecker M.

Author information: 
(1)Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität, Jahnstrasse 15,
17487 Greifswald, Germany. Schweder@uni-greifswald.de

New developments in the RNA analysis techniques now enable a comprehensive view
on the bacterial physiology under bioprocess conditions. The DNA-chip technology 
allows a genome wide transcriptional profiling of bacterial cells, whose genome
sequence is available. Although the analyses of microbial bioprocesses have still
been somewhat limited to date, this technique has already been successfully
applied in different laboratories for the investigation of stress responses of
selected industrially relevant bacterial hosts. Transcriptome analyses in
combination with high resolution two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for
the description of general and specific stress and starvation responses of
Escherichia coli and Bacillus subtilis. The consideration of bacterial stress and
starvation responses is of crucial importance for the successful establishment of
an industrial large scale bioprocess. Stress genes can be used as marker genes in
order to monitor the fitness of industrial bacterial hosts during fermentation
processes. This chapter gives an overview of current RNA analysis techniques. The
bacterial stress and starvation responses, which are of potential importance for 
industrial microbial bioprocesses are summarised.


PMID: 15217155  [Indexed for MEDLINE]


1233. Anal Biochem. 2004 Jan 1;324(1):84-91.

Detection of bacteriophage infection and prophage induction in bacterial cultures
by means of electric DNA chips.

Gabig-Ciminska M(1), Los M, Holmgren A, Albers J, Czyz A, Hintsche R, Wegrzyn G, 
Enfors SO.

Author information: 
(1)Department of Biotechnology, Royal Institute of Technology (KTH),
Roslagstullsbacken 21, S-10691 Stockholm, Sweden. magda.gabig@biotech.kth.se

Infections of bacterial cultures by bacteriophages are common and serious
problems in many biotechnological laboratories and factories. A method for
specific, quantitative, and quick detection of phage contamination, based on the 
use of electric DNA chip is described here. Different phages of Escherichia coli 
and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial
culture samples and detected by combination of bead-based sandwich hybridization 
with enzyme-labeled probes and detection of the enzymatic product using silicon
chips. The assay resulted in specific signals from all four tested phages without
significant background. Although high sensitivity was achieved in 4h assay time, 
a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A
multiplex DNA chip technique involving a mixture of probes allows for detection
of various types of phages in one sample. These analyses confirmed the
specificity of the assay.


PMID: 14654049  [Indexed for MEDLINE]


1234. Epidemiol Infect. 2004 Jan;132(1):61-5.

A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli
strains using variable bacterial interspersed mosaic elements.

Horváth R(1), Dendis M, Schlegelová J, Růzicka F, Benedík J.

Author information: 
(1)Genex CZ Ltd, Podstránská 74, 600 00 Brno, Czech Republic.

The original method for molecular typing of E. coli strains was developed using
the polymorphism in chromosomal sequences of bacterial interspersed mosaic
elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The
applicability of the method in the epidemiology of E. coli was tested on a group 
of 524 strains of human and veterinary origin. In the studied group 18 different 
genotypes were detected. Significant differences were found in the frequencies of
the genotypes among various groups of strains, suggesting the method could be a
promising tool in the epidemiology of E. coli.


PMCID: PMC2870079
PMID: 14979591  [Indexed for MEDLINE]


1235. Genome Biol. 2004;5(11):R87. Epub 2004 Nov 1.

Genomic transcriptional response to loss of chromosomal supercoiling in
Escherichia coli.

Peter BJ(1), Arsuaga J, Breier AM, Khodursky AB, Brown PO, Cozzarelli NR.

Author information: 
(1)Department of Molecular and Cell Biology, University of California, Berkeley, 
CA 94720-3204, USA. bpeter@mrc-lmb.cam.ac.uk

BACKGROUND: The chromosome of Escherichia coli is maintained in a negatively
supercoiled state, and supercoiling levels are affected by growth phase and a
variety of environmental stimuli. In turn, supercoiling influences local DNA
structure and can affect gene expression. We used microarrays representing nearly
the entire genome of Escherichia coli MG1655 to examine the dynamics of
chromosome structure.
RESULTS: We measured the transcriptional response to a loss of supercoiling
caused either by genetic impairment of a topoisomerase or addition of specific
topoisomerase inhibitors during log-phase growth and identified genes whose
changes are statistically significant. Transcription of 7% of the genome (306
genes) was rapidly and reproducibly affected by changes in the level of
supercoiling; the expression of 106 genes increased upon chromosome relaxation
and the expression of 200 decreased. These changes are most likely to be direct
effects, as the kinetics of their induction or repression closely follow the
kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by
relaxation have a significantly enriched AT content in both upstream and coding
regions.
CONCLUSIONS: The 306 supercoiling-sensitive genes are functionally diverse and
widely dispersed throughout the chromosome. We propose that supercoiling acts as 
a second messenger that transmits information about the environment to many
regulatory networks in the cell.

DOI: 10.1186/gb-2004-5-11-r87 
PMCID: PMC545778
PMID: 15535863  [Indexed for MEDLINE]


1236. Genome Res. 2004 Jan;14(1):188-96.

A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy
number changes by array comparative genomic hybridization.

Chung YJ(1), Jonkers J, Kitson H, Fiegler H, Humphray S, Scott C, Hunt S, Yu Y,
Nishijima I, Velds A, Holstege H, Carter N, Bradley A.

Author information: 
(1)The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge CB10 1SA, UK.

Microarray-based comparative genomic hybridization (CGH) has become a powerful
method for the genome-wide detection of chromosomal imbalances. Although BAC
microarrays have been used for mouse CGH studies, the resolving power of these
analyses was limited because high-density whole-genome mouse BAC microarrays were
not available. We therefore developed a mouse BAC microarray containing 2803
unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general
amplification of BAC clone DNA prior to spotting, we designed a set of three
novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially
amplify mouse genomic sequences while minimizing unwanted amplification of
contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays
reproducibly identified DNA copy number alterations in cell lines and primary
tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

DOI: 10.1101/gr.1878804 
PMCID: PMC314296
PMID: 14707179  [Indexed for MEDLINE]


1237. J Appl Microbiol. 2004;96(3):419-29.

Strategies for the detection of Escherichia coli O157:H7 in foods.

Deisingh AK(1), Thompson M.

Author information: 
(1)Department of Chemistry, University of Toronto, Toronto, Ontario, Canada.
anildeisingh@aol.com


PMID: 14962121  [Indexed for MEDLINE]


1238. PLoS Biol. 2004 Jan;2(1):E9. Epub 2003 Dec 15.

Similarities and differences in genome-wide expression data of six organisms.

Bergmann S(1), Ihmels J, Barkai N.

Author information: 
(1)Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel.

Comparing genomic properties of different organisms is of fundamental importance 
in the study of biological and evolutionary principles. Although differences
among organisms are often attributed to differential gene expression, genome-wide
comparative analysis thus far has been based primarily on genomic sequence
information. We present a comparative study of large datasets of expression
profiles from six evolutionarily distant organisms: S. cerevisiae, C. elegans, E.
coli, A. thaliana, D. melanogaster, and H. sapiens. We use genomic sequence
information to connect these data and compare global and modular properties of
the transcription programs. Linking genes whose expression profiles are similar, 
we find that for all organisms the connectivity distribution follows a power-law,
highly connected genes tend to be essential and conserved, and the expression
program is highly modular. We reveal the modular structure by decomposing each
set of expression data into coexpressed modules. Functionally related sets of
genes are frequently coexpressed in multiple organisms. Yet their relative
importance to the transcription program and their regulatory relationships vary
among organisms. Our results demonstrate the potential of combining sequence and 
expression data for improving functional gene annotation and expanding our
understanding of how gene expression and diversity evolved.

DOI: 10.1371/journal.pbio.0020009 
PMCID: PMC300882
PMID: 14737187  [Indexed for MEDLINE]

Conflict of interest statement: The authors have declared that no conflicts of
interest exist.


1239. Cell Mol Biol (Noisy-le-grand). 2003 Dec;49(8):1261-7.

The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus
polyphemus.

Cao Z(1), McAdory BS, Wang Y, Peng H, Martinez JL, Townsel JG.

Author information: 
(1)Department of Biological Sciences, Tennessee State University, Nashville, TN
37209-1561, USA.

Semaphorins are a family of soluble and membrane-bound proteins that play a
critical role in axonal guidance and other processes of neuronal development.
Currently, more than twenty semaphorins have been identified, all of which share 
a conserved 500 amino acid domain near the amino terminus. Semaphorins are
divided into eight classes according to species of origin and structural
similarities. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are
present in vertebrates and viruses encode class V semaphorin. Microarray analysis
of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus
polyphemus. Based on these data, we aligned 31 different sequences and designed
degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW). RT-PCR
products were generated using 6 forward primers and 4 reverse primers. The
expected size PCR products (750 bp) was obtained and then ligated with pCR II
TOPO vector and transferred into E. coli Top 10. Five partial semaphorin cDNAs
were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were
partially cloned. Subsequent Northern blot analyses using these Limulus
specific-probes revealed hybridization with total RNAs purified from six
different tissues.


PMID: 14983996  [Indexed for MEDLINE]


1240. Mol Microbiol. 2003 Dec;50(5):1665-82.

The RcsC sensor kinase is required for normal biofilm formation in Escherichia
coli K-12 and controls the expression of a regulon in response to growth on a
solid surface.

Ferrières L(1), Clarke DJ.

Author information: 
(1)Molecular Microbiology Laboratory, Department of Biology and Biochemistry,
University of Bath, Claverton Down, Bath BA2 7AY, UK.

Bacteria are often found associated with surfaces as sessile bacterial
communities called biofilms, and the formation of a biofilm can be split up into 
different stages each requiring the expression of specific genes. The production 
of extracellular polysaccharides (EPS) is important for the maturation of
biofilms and is controlled by the Rcs two-component pathway in Escherichia coli
(and other Gram-negative bacteria). In this study, we show, for the first time,
that the RcsC sensor kinase is required for normal biofilm development in E.
coli. Moreover, using a combination of DNA macroarray technology and
transcriptional fusion analysis, we show that the expression of > 150 genes is
controlled by RcsC in E. coli. In silico analyses of the RcsC regulon predicts
that 50% of the genes encode proteins that are either localized to the envelope
of E. coli or have activities that affect the structure/properties of the
bacterial surface, e.g. the production of colanic acid. Moreover, we also show
that RcsC is activated during growth on a solid surface. Therefore, we suggest
that the RcsC sensor kinase may play an important role in the remodelling of the 
bacterial surface during growth on a solid surface and biofilm formation.


PMID: 14651646  [Indexed for MEDLINE]


1241. Nucleic Acids Res. 2003 Dec 1;31(23):6976-85.

Unexpected correlations between gene expression and codon usage bias from
microarray data for the whole Escherichia coli K-12 genome.

dos Reis M(1), Wernisch L, Savva R.

Author information: 
(1)School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX,
UK.

Escherichia coli has long been regarded as a model organism in the study of codon
usage bias (CUB). However, most studies in this organism regarding this topic
have been computational or, when experimental, restricted to small datasets;
particularly poor attention has been given to genes with low CUB. In this work,
correspondence analysis on codon usage is used to classify E.coli genes into
three groups, and the relationship between them and expression levels from
microarray experiments is studied. These groups are: group 1, highly biased
genes; group 2, moderately biased genes; and group 3, AT-rich genes with low CUB.
It is shown that, surprisingly, there is a negative correlation between codon
bias and expression levels for group 3 genes, i.e. genes with extremely low codon
adaptation index (CAI) values are highly expressed, while group 2 show the lowest
average expression levels and group 1 show the usual expected positive
correlation between CAI and expression. This trend is maintained over all
functional gene groups, seeming to contradict the E.coli-yeast paradigm on CUB.
It is argued that these findings are still compatible with the mutation-selection
balance hypothesis of codon usage and that E.coli genes form a dynamic system
shaped by these factors.


PMCID: PMC290265
PMID: 14627830  [Indexed for MEDLINE]


1242. Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13213-8. Epub 2003 Oct 29.

A specific endoribonuclease, RNase P, affects gene expression of polycistronic
operon mRNAs.

Li Y(1), Altman S.

Author information: 
(1)Department of Molecular, Cellular, and Developmental Biology, Yale University,
New Haven, CT 06520, USA.

The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43
degrees C because of a temperature-sensitive mutation in C5 protein, the protein 
subunit of the enzyme. Microarray analysis reveals the expression of several
noncoding intergenic regions that are increased at 43 degrees C compared with 30 
degrees C. These regions are substrates for RNase P, and they are cleaved less
efficiently than, for example, tRNA precursors. An analysis of the tna, secG,
rbs, and his operons, all of which contain RNase P cleavage sites, indicates that
RNase P affects gene expression for regions downstream of its cleavage sites.

DOI: 10.1073/pnas.2235589100 
PMCID: PMC263755
PMID: 14585931  [Indexed for MEDLINE]


1243. Appl Environ Microbiol. 2003 Nov;69(11):6825-32.

Comparative genomics of insect-symbiotic bacteria: influence of host environment 
on microbial genome composition.

Rio RV(1), Lefevre C, Heddi A, Aksoy S.

Author information: 
(1)Department of Epidemiology and Public Health, Yale University School of
Medicine, New Haven, Connecticut 06510, USA.

Commensal symbionts, thought to be intermediary amid obligate mutualists and
facultative parasites, offer insight into forces driving the evolutionary
transition into mutualism. Using macroarrays developed for a close relative,
Escherichia coli, we utilized a heterologous array hybridization approach to
infer the genomic compositions of a clade of bacteria that have recently
established symbiotic associations: Sodalis glossinidius with the tsetse fly
(Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with
the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within
their hosts currently reflect different forms of symbiotic associations. Their
hosts, members of distant insect taxa, occupy distinct ecological niches and have
evolved to survive on restricted diets of blood for tsetse and cereal for the
rice weevil. Comparison of genome contents between the two microbes indicates
statistically significant differences in the retention of genes involved in
carbon compound catabolism, energy metabolism, fatty acid metabolism, and
transport. The greatest reductions have occurred in carbon catabolism, membrane
proteins, and cell structure-related genes for Sodalis and in genes involved in
cellular processes (i.e., adaptations towards cellular conditions) for SOPE.
Modifications in metabolic pathways, in the form of functional losses
complementing particularities in host physiology and ecology, may have occurred
upon initial entry from a free-living to a symbiotic state. It is possible that
these adaptations, streamlining genomes, act to make a free-living state no
longer feasible for the harnessed microbe.


PMCID: PMC262273
PMID: 14602646  [Indexed for MEDLINE]


1244. Genome Res. 2003 Nov;13(11):2435-43.

Regulatory network of Escherichia coli: consistency between literature knowledge 
and microarray profiles.

Gutiérrez-Ríos RM(1), Rosenblueth DA, Loza JA, Huerta AM, Glasner JD, Blattner
FR, Collado-Vides J.

Author information: 
(1)Program of Computational Genomics, Centro de Investigación sobre Fijación de
Nitrógeno, Univercidad Nacional Autónoma de México (CIFN-UNAM), Morelos 62100,
México.

The transcriptional network of Escherichia coli may well be the most complete
experimentally characterized network of a single cell. A rule-based approach was 
built to assess the degree of consistency between whole-genome microarray
experiments in different experimental conditions and the accumulated knowledge in
the literature compiled in RegulonDB, a data base of transcriptional regulation
and operon organization in E. coli. We observed a high and statistical
significant level of consistency, ranging from 70%-87%. When effector metabolites
of regulatory proteins are not considered in the prediction of the active or
inactive state of the regulators, consistency falls by up to 40%. Similarly,
consistency decreases when rules for multiple regulatory interactions are altered
or when "on" and "off" entries were assigned randomly. We modified the initial
state of regulators and evaluated the propagation of errors in the network that
do not correlate linearly with the connectivity of regulators. We interpret this 
deviation mainly as a result of the existence of redundant regulatory
interactions. Consistency evaluation opens a new space of dialogue between theory
and experiment, as the consequences of different assumptions can be evaluated and
compared.

DOI: 10.1101/gr.1387003 
PMCID: PMC403762
PMID: 14597655  [Indexed for MEDLINE]


1245. J Bacteriol. 2003 Nov;185(21):6392-9.

Genome-scale analysis of the uses of the Escherichia coli genome: model-driven
analysis of heterogeneous data sets.

Allen TE(1), Herrgård MJ, Liu M, Qiu Y, Glasner JD, Blattner FR, Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California-San Diego, La Jolla,
California 92093-0412, USA.

The recent availability of heterogeneous high-throughput data types has increased
the need for scalable in silico methods with which to integrate data related to
the processes of regulation, protein synthesis, and metabolism. A sequence-based 
framework for modeling transcription and translation in prokaryotes has been
established and has been extended to study the expression state of the entire
Escherichia coli genome. The resulting in silico analysis of the expression state
highlighted three facets of gene expression in E. coli: (i) the metabolic
resources required for genome expression and protein synthesis were found to be
relatively invariant under the conditions tested; (ii) effective promoter
strengths were estimated at the genome scale by using global mRNA abundance and
half-life data, revealing genes subject to regulation under the experimental
conditions tested; and (iii) large-scale genome location-dependent expression
patterns with approximately 600-kb periodicity were detected in the E. coli
genome based on the 49 expression data sets analyzed. These results support the
notion that a structured model-driven analysis of expression data yields
additional information that can be subjected to commonly used statistical
analyses. The integration of heterogeneous genome-scale data (i.e., sequence,
expression data, and mRNA half-life data) is readily achieved in the context of
an in silico model.


PMCID: PMC219383
PMID: 14563874  [Indexed for MEDLINE]


1246. J Microbiol Methods. 2003 Nov;55(2):399-409.

Computational methods and evaluation of RNA stabilization reagents for
genome-wide expression studies.

Bhagwat AA(1), Phadke RP, Wheeler D, Kalantre S, Gudipati M, Bhagwat M.

Author information: 
(1)Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville
Agricultural Research Center, Agricultural Research Service, USDA, 10300
Baltimore Ave., Beltsville, MD 20705-2350, USA. bhagwata@ba.ars.usda.gov

Gene expression studies require high quality messenger RNA (mRNA) in addition to 
other factors such as efficient primers and labeling reagents. To prevent RNA
degradation and to improve the quality of gene array expression data, several
commercial reagents have become available. We examined a conventional hot-phenol 
lysis method and RNA stabilization reagents, and generated comparative gene
expression profiles from Escherichia coli cells grown on minimal medium. Our data
indicate that certain RNA stabilization reagents induce stress responses and
proper caution must be exercised during their use. We observed that the
laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high 
quality mRNA and reproducibility was such that reliable gene expression profiles 
were generated. To assist in the analysis of gene expression data, we wrote a
number of macros that use the most recent gene annotation and process data in
accordance with gene function. Scripts were also written to examine the
occurrence of artifacts, based on GC content, length of the individual open
reading frame (ORF), its distribution on plus and minus DNA strands, and the
distance from the replication origin.


PMID: 14529961  [Indexed for MEDLINE]


1247. Mol Cell Biochem. 2003 Nov;253(1-2):167-77.

The bacterial adaptive response gene, barA, encodes a novel conserved histidine
kinase regulatory switch for adaptation and modulation of metabolism in
Escherichia coli.

Sahu SN(1), Acharya S, Tuminaro H, Patel I, Dudley K, LeClerc JE, Cebula TA,
Mukhopadhyay S.

Author information: 
(1)Virginia-Maryland Regional College of Veterinary Medicine, USA.

Histidine kinases are important prokaryotic determinants of cellular adaptation
to environmental conditions, particularly stress. The highly conserved histidine 
kinase, BarA, encoded by the bacterial adaptive response gene, barA, is a member 
of the family of tripartite histidine kinases, and is involved in stress
adaptation. BarA has been implicated to play a role during infection of
epithelial cells. Homologues and orthologues of BarA have been found in
pathogenic yeast, fungi, mould and in plants. The primary aim of this review is
to assimilate evidence present in the current literature linking the role of BarA
in stress response, and to support it with preliminary experimental evidence
indicating that, it is indeed a global response regulator. In particular, the
review focuses on the unusual domain structure of the BarA protein, its role in
oxidative, weak acid, and osmotic stress responses and its role in biofilm
formation. A preliminary genomic approach to identify downstream genes regulated 
by the BarA signaling pathway, using DNA microarray, is reported. The results
demonstrate that BarA plays a global response regulatory role in cell division,
carbon metabolism, iron metabolism and pili formation. The evolutionary
significance of these types of histidine kinase sensors is reviewed in light of
their roles in pathogenesis.


PMID: 14619967  [Indexed for MEDLINE]


1248. Mol Microbiol. 2003 Nov;50(4):1111-24.

Global analysis of small RNA and mRNA targets of Hfq.

Zhang A(1), Wassarman KM, Rosenow C, Tjaden BC, Storz G, Gottesman S.

Author information: 
(1)Cell Biology and Metabolism Branch, National Institute of Child Health and
Development, Bethesda MD 20892, USA.

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for
the action of many small regulatory RNAs that act by basepairing with target
mRNAs. Hfq binds this family of small RNAs efficiently. We have used
co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic
microarrays to identify members of this small RNA family. This approach was
extremely sensitive; even Hfq-binding small RNAs expressed at low levels were
readily detected. At least 15 of 46 known small RNAs in E. coli interact with
Hfq. In addition, high signals in other intergenic regions suggested up to 20
previously unidentified small RNAs bind Hfq; five were confirmed by Northern
analysis. Strong signals within genes and operons also were detected, some of
which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq
appears to compete with RNase E and modulate RNA processing and degradation. Thus
Hfq immunoprecipitation followed by microarray analysis is a highly effective
method for detecting a major class of small RNAs as well as identifying new Hfq
functions.


PMID: 14622403  [Indexed for MEDLINE]


1249. Biosens Bioelectron. 2003 Oct 30;19(1):1-8.

DNA microarray for discrimination between pathogenic 0157:H7 EDL933 and
non-pathogenic Escherichia coli strains.

Wu CF(1), Valdes JJ, Bentley WE, Sekowski JW.

Author information: 
(1)US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD
21010, USA.

The primary technique currently used to detect biological agents is based on
immunoassays. Although sensitive and specific, currently employed immunoassays
generally rely on the detection of a single epitope, and therefore often cannot
discriminate subtle strain-specific differences. Since DNA microarrays can
hybridize hundreds to thousands of genomic targets simultaneously and do not rely
on phenotypic expression of these genetic features for identification purposes,
they have enormous potential to provide inexpensive, flexible and specific
strain-specific detection and identification of pathogens. In this study,
pathogenic Escherichia coli O157:H7-specific genes, non-pathogenic K12-specific
genes, common E. coli genes, and negative control genes were polymerase chain
reaction-amplified and spotted onto the surface of treated glass slides. After
labeled bacterial cDNA samples were hybridized with probes on the microarray,
specific fluorescence patterns were obtained, enabling identification of
pathogenic E. coli O157:H7 and non-pathogenic E. coli K12. To test the utility of
this microarray device to detect genetically engineered bacteria, E. coli BL21 (a
B strain derivative with antibiotic resistance gene, ampR) and E. coli JM107 (a
K12 strain derivative lacking the gene ompT) were also employed. The array
successfully confirmed the strain genotypes and demonstrated that antibiotic
resistance can also be detected. The ability to assess multiple data points makes
this array method more efficient and accurate than a typical immunoassay, which
detects a single protein product.


PMID: 14558993  [Indexed for MEDLINE]


1250. Antimicrob Agents Chemother. 2003 Oct;47(10):3290-5.

Identifying antimicrobial resistance genes with DNA microarrays.

Call DR(1), Bakko MK, Krug MJ, Roberts MC.

Author information: 
(1)Department of Veterinary Microbiology and Pathology, College of Veterinary
Medicine, Washington State University, Pullman, Washington 99164, USA.
drcall@wsu.edu

We developed and tested a glass-based microarray suitable for detecting multiple 
tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the
beta-lactamase bla(TEM-1) gene, and a 16S ribosomal DNA gene (Escherichia coli)
were generated from known controls by PCR. The resulting products (ca. 550 bp)
were applied as spots onto epoxy-silane-derivatized, Teflon-masked slides by
using a robotic spotter. DNA was extracted from test strains, biotinylated,
hybridized overnight to individual microarrays at 65 degrees C, and detected with
Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a
detection threshold of 3x the standard deviation, we correctly identified tet
genes carried by 39 test strains. Nine additional strains were not known to
harbor any genes represented on the microarray, and these strains were negative
for all 17 tet probes as expected. We verified that R741a, which was originally
thought to carry a novel tet gene, tet(I), actually harbored a tet(G) gene.
Microarray technology has the potential for screening a large number of different
antibiotic resistance genes by the relatively low-cost methods outlined in this
paper.


PMCID: PMC201141
PMID: 14506043  [Indexed for MEDLINE]


1251. Bull Environ Contam Toxicol. 2003 Oct;71(4):826-32.

Identification of Escherichia coli O157:H7 with oligonucleotide arrays.

Liu H(1), Wang H, Shi Z, Liu Q, Zhu J, He N, Wang H, Lu Z.

Author information: 
(1)Chien-Shiung Wu Laboratory, Department of Biomedical Engineering, Southeast
University, Nanjing 210096, People's Republic of China.

DOI: 10.1007/s00128-003-0209-8 
PMID: 14672138  [Indexed for MEDLINE]


1252. J Bacteriol. 2003 Oct;185(20):6158-70.

Interfering with different steps of protein synthesis explored by transcriptional
profiling of Escherichia coli K-12.

Sabina J(1), Dover N, Templeton LJ, Smulski DR, Söll D, LaRossa RA.

Author information: 
(1)Central Research and Development, DuPont Company, Wilmington, Delaware
19880-0173, USA

Escherichia coli responses to four inhibitors that interfere with translation
were monitored at the transcriptional level. A DNA microarray method provided a
comprehensive view of changes in mRNA levels after exposure to these agents.
Real-time reverse transcriptase PCRanalysis served to verify observations made
with microarrays, and a chromosomal grpE::lux operon fusion was employed to
specifically monitor the heat shock response. 4-Azaleucine, a competitive
inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock
response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase 
activity, resulted in changes reminiscent of the stringent response. Treatment
with kasugamycin and puromycin (targeting ribosomal subunit association as well
as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal
protein operons. Abundant biosynthetic transcripts were often significantly
diminished after treatment with any of these agents. Exposure of a relA strain to
mupirocin resulted in accumulation of ribosomal protein operon transcripts.
However, the relA strain's response to the other inhibitors was quite similar to 
that of the wild-type strain.


PMCID: PMC225041
PMID: 14526028  [Indexed for MEDLINE]


1253. J Bacteriol. 2003 Oct;185(19):5735-46.

Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB
phosphorelay system in Escherichia coli.

Hagiwara D(1), Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Mizuno T.

Author information: 
(1)Laboratory of Molecular Microbiology, School of Agriculture, Nagoya
University, Chikusa-ku, Nagoya 464-8601, Japan.

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated 
through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked
cps::lacZ reporter gene, we first searched for physiological stimuli that
propagate the Rcs signaling system. The expression of cps::lacZ was activated
when cells were grown at a low temperature (20 degrees C) in the presence of
glucose as a carbon source and in the presence of a relatively high concentration
of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene
product (a putative outer membrane-located lipoprotein) was also an essential
signaling component. Based on the defined signaling pathway and physiological
stimuli for the Rcs signaling system, we conducted genome-wide analyses with
microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two
genes were identified as putative Rcs regulon members; these genes included 15
new genes in addition to 17 of the previously described cps genes. Using a set of
37 two-component system mutants, we performed alternative genome-wide analyses.
The results showed that the propagation of the zinc-responsive Rcs signaling
system was largely dependent on another two-component system, PhoQ/P. Considering
the fact that the PhoQ/P signaling system responds to external magnesium, we
obtained evidence which supports the view that there is a signaling network that 
connects the Rcs system with the PhoQ/P system, which coordinately regulates
extracellular polysaccharide synthesis in response to the external concentrations
of divalent cations.


PMCID: PMC193970
PMID: 13129944  [Indexed for MEDLINE]


1254. J Immunol. 2003 Oct 1;171(7):3668-74.

Flagellin is the major proinflammatory determinant of enteropathogenic
Salmonella.

Zeng H(1), Carlson AQ, Guo Y, Yu Y, Collier-Hyams LS, Madara JL, Gewirtz AT,
Neish AS.

Author information: 
(1)Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine,
Emory University School of Medicine, Atlanta, GA 30322, USA.

The gastroenteritis-causing pathogen Salmonella typhimurium induces profound
transcriptional changes in intestinal epithelia resulting in the recruitment of
neutrophils whose presence is the histopathologic hallmark of salmonellosis. Here
we used cDNA microarray expression profiling to define the molecular determinants
that mediate such changes in model intestinal epithelia. Enteropathogenic
Salmonella induced a classical proinflammatory gene expression program similar to
that activated by the canonical proinflammatory agonist TNF-alpha.
Nonproinflammatory bacteria, both commensals (Escherichia coli) and systemic
pathogens (S. typhi), did not activate this expression profile. While S.
typhimurium strains lacking the SPI-1-encoded type III system were fully
proinflammatory, strains lacking the genes for the flagellar structural component
flagellin were nearly devoid of proinflammatory signaling. Lastly, the epithelial
proinflammatory response could be largely recapitulated by basolateral addition
of purified flagellin. Thus, S. typhimurium flagellin is the major molecular
trigger by which this pathogen activates gut epithelial proinflammatory gene
expression.


PMID: 14500664  [Indexed for MEDLINE]


1255. Nucleic Acids Res. 2003 Oct 1;31(19):5776-88.

Selective inhibitory DNA aptamers of the human RNase H1.

Pileur F(1), Andreola ML, Dausse E, Michel J, Moreau S, Yamada H, Gaidamakov SA, 
Crouch RJ, Toulmé JJ, Cazenave C.

Author information: 
(1)INSERM U386, IFR Pathologies Infectieuses, Université Victor Segalen Bordeaux 
2, 146, rue Léo Saignat, 33076 Bordeaux cedex, France.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA
hybrid via its C-terminal RNase H domain, the latter being closely related to
Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences 
that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human
RNase H1. None of them could fold into a simple perfect double-stranded DNA
hairpin confirming that double-stranded DNA does not constitute a trivial ligand 
for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human
RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite
different in structure with V-2 folding into a large, imperfect but stable
hairpin loop. The VI-2 structure consists of a central region unimolecular
quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3'
tails that form a stem of six base pairs. Base pairing between the 5' and 3'
tails appears crucial for conferring the inhibitory properties to the aptamer.
Finally, the inhibitory aptamers were capable of completely abolishing the action
of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with
human RNase H1, with IC50 ranging from 50 to 100 nM.


PMCID: PMC206449
PMID: 14500841  [Indexed for MEDLINE]


1256. Plant J. 2003 Oct;36(2):189-202.

Roles of OsCKI1, a rice casein kinase I, in root development and plant hormone
sensitivity.

Liu W(1), Xu ZH, Luo D, Xue HW.

Author information: 
(1)National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology
and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, China.

Casein kinases are critical in cell division and differentiation across species. 
A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via
cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length
cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR 
and Northern blot analysis indicated that OsCKI1 was constitutively expressed in 
various rice tissues and upregulated by treatments with BR and abscisic acid
(ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia
coli showed that the protein was capable of phosphorylating casein. The
physiological roles of OsCKI1 were studied through antisense transgenic
approaches, and homozygous transgenic plants showed abnormal root development,
including fewer lateral and adventitious roots, and shortened primary roots as a 
result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a
specific inhibitor of CKI, also confirmed these functions of OsCKI1.
Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA
could restore normal root development, and measurement of free IAA content in
CKI-deficient primary and adventitious roots revealed altered auxin content,
indicating that OsCKI1 is involved in auxin metabolism or that it may affect
auxin levels. Transgenic plants were less sensitive than control plants to ABA or
BR treatment during germination, suggesting that OsCKI1 may be involved in
various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the
localization of OsCKI1 to the nucleus, suggesting a possible involvement in
regulation of gene expression. In OsCKI1-deficient plants, differential gene
expression was investigated using cDNA chip technology, and results indicated
that genes related to signal transduction and hormone metabolism were indeed with
altered expression.


PMID: 14535884  [Indexed for MEDLINE]


1257. Rheumatology (Oxford). 2003 Oct;42(10):1155-63. Epub 2003 May 30.

Protein interaction for an interferon-inducible systemic lupus associated gene,
IFIT1.

Ye S(1), Pang H, Gu YY, Hua J, Chen XG, Bao CD, Wang Y, Zhang W, Qian J, Tsao BP,
Hahn BH, Chen SL, Rao ZH, Shen N.

Author information: 
(1)Shanghai Clinical Centre of Rheumatic Diseases and Institute of Rheumatology, 
Department of Rheumatology, Renji Hospital, Shanghai Second Medical University,
China.

OBJECTIVE: To identify disease-related genes and immune-regulatory pathways in
the pathogenesis of systemic lupus erythematosus (SLE) by using gene expression
profiling and protein-protein interaction analysis.
METHODS: Peripheral white blood cell gene expression profiles of 10 SLE patients 
were determined by oligonucleotide microarray analysis. Clustering of the gene
expression profile was compared with the clinical immune phenotype. SLE-induced
genes that were over- or under-expressed were determined and independently
validated using a real-time polymerase chain reaction (PCR) method. To study
their potential function and the possible pathways involved, a candidate gene was
cloned and a GST (glutathione S-transferase) fusion protein was expressed in
Escherichia coli. The fusion protein was further purified using the glutathione
Sepharose 4B system, and was treated as bait to capture prey from SLE peripheral 
white blood cell lysate. MALDI-TOF (matrix-assisted laser
desorption/ionization-time-of-flight) mass spectrometry was then performed to
determine the prey protein.
RESULTS: Similarity was found between the gene expression profile and the immune 
phenotype clusters of the SLE patients. More than 20 disease-associated genes
were identified, some of which have not been related to SLE previously. Of these 
genes, a cluster of interferon-induced genes were highly correlated. IFIT1
(interferon-induced with tetratricopeptide repeats 1) was one of these genes, and
overexpression of its mRNA was confirmed independently by real-time PCR in a
larger population (40 SLE patients and 29 normal controls). An IFIT1 protein-
protein interaction study showed that IFIT1 may interact with Rho/Rac guanine
nucleotide exchange factor.
CONCLUSION: The gene expression profile seems to be the molecular basis of the
diverse immune phenotype of SLE. On the basis of the SLE-related genes found in
this study, we suggest that the interferon-related immune pathway is important in
the pathogenesis of SLE. IFIT1 is the first gene described as a candidate gene
for SLE, and may function by activating Rho proteins through interaction with
Rho/Rac guanine nucleotide exchange factor. IFIT1 and the interferon-related
pathway may provide potential targets for novel interventions in the treatment of
SLE.

DOI: 10.1093/rheumatology/keg315 
PMID: 12777642  [Indexed for MEDLINE]


1258. Biotechnol Bioeng. 2003 Sep 20;83(6):721-8.

Detection and identification of Escherichia coli, Shigella, and Salmonella by
microarrays using the gyrB gene.

Kakinuma K(1), Fukushima M, Kawaguchi R.

Author information: 
(1)Genomics Research Institute, SRL, Inc., 5-6-50 Shinmachi, Hino-shi, Tokyo
191-0002, Japan.

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for
identifying enteric bacteria. However, it may not always be applicable for
distinguishing closely related bacteria. Therefore, we selected gyrB genes that
encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as
target genes. The molecular evolution rate of gyrB genes is higher than that of
16S rRNA, and gyrB genes are distributed universally among bacterial species.
Microarray technology includes the methods of arraying cDNA or oligonucleotides
on substrates such as glass slides while acquiring a lot of information
simultaneously. Thus, it is possible to identify the enteric bacteria easily
using microarray technology. We devised a simple method of rapidly identifying
bacterial species through the combined use of gyrB genes and microarrays. Closely
related bacteria were not identified at the species level using 16S rRNA sequence
analysis, whereas they were identified at the species level based on the reaction
patterns of oligonucleotides on our microarrays using gyrB genes.

Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 721-728, 2003.

DOI: 10.1002/bit.10709 
PMID: 12889036  [Indexed for MEDLINE]


1259. Cell. 2003 Sep 5;114(5):623-34.

RraA. a protein inhibitor of RNase E activity that globally modulates RNA
abundance in E. coli.

Lee K(1), Zhan X, Gao J, Qiu J, Feng Y, Meganathan R, Cohen SN, Georgiou G.

Author information: 
(1)Department of Genetics, Stanford University School of Medicine, Stanford, CA
94305, USA.

Ribonuclease E (RNase E) has a key role in mRNA degradation and the processing of
catalytic and structural RNAs in E. coli. We report the discovery of an
evolutionarily conserved 17.4 kDa protein, here named RraA (regulator of
ribonuclease activity A) that binds to RNase E and inhibits RNase E
endonucleolytic cleavages without altering cleavage site specificity or
interacting detectably with substrate RNAs. Overexpression of RraA circumvents
the effects of an autoregulatory mechanism that normally maintains the RNase E
cellular level within a narrow range, resulting in the genome-wide accumulation
of RNase E-targeted transcripts. While not required for RraA action, the
C-terminal RNase E region that serves as a scaffold for formation of a
multiprotein degradosome complex modulates the inhibition of RNase E catalytic
activity by RraA. Our results reveal a possible mechanism for the dynamic
regulation of RNA decay and processing by inhibitory RNase binding proteins.


PMID: 13678585  [Indexed for MEDLINE]


1260. Biosci Biotechnol Biochem. 2003 Sep;67(9):2030-3.

Fosmidomycin resistance in adenylate cyclase deficient (cya) mutants of
Escherichia coli.

Sakamoto Y(1), Furukawa S, Ogihara H, Yamasaki M.

Author information: 
(1)Department of Food Science and Technology, College of Bioresouce Sciences,
Nihon University, Fujisawa, Kanagawa, Japan.

Adenylate cyclase deficient (cya) mutants of E. coli K-12 were found to be
resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway,
just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and
also to fosmidomycin. This fact shows that fosmidomycin was transported inside
via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed
that the transcription of glpT and other genes concerning glycerol utilization
were highly dependent on the presence of cAMP.


PMID: 14519998  [Indexed for MEDLINE]


1261. J Bacteriol. 2003 Sep;185(18):5611-26.

Physiological studies of Escherichia coli strain MG1655: growth defects and
apparent cross-regulation of gene expression.

Soupene E(1), van Heeswijk WC, Plumbridge J, Stewart V, Bertenthal D, Lee H,
Prasad G, Paliy O, Charernnoppakul P, Kustu S.

Author information: 
(1)Department of Plant and Microbial Biology, University of California, Berkeley,
California 94720-3102, USA.

Escherichia coli strain MG1655 was chosen for sequencing because the few
mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it
has a number of growth defects. Internal pyrimidine starvation due to polarity of
the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the
isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a
large deletion around the fnr (fumarate-nitrate respiration) regulatory gene.
Although studies on DNA microarrays revealed apparent cross-regulation of gene
expression between galactose and lactose metabolism in the Stock Center isolate
of MG1655, this was due to the occurrence of mutations that increased lacY
expression and suppressed slow growth on galactose. The explanation for apparent 
cross-regulation between galactose and N-acetylglucosamine metabolism was
similar. By contrast, cross-regulation between lactose and maltose metabolism
appeared to be due to generation of internal maltosaccharides in lactose-grown
cells and may be physiologically significant. Lactose is of restricted
distribution: it is normally found together with maltosaccharides, which are
starch degradation products, in the mammalian intestine. Strains designated
MG1655 and obtained from other sources differed from the Stock Center isolate and
each other in several respects. We confirmed that use of other E. coli strains
with MG1655-based DNA microarrays works well, and hence these arrays can be used 
to study any strain of interest. The responses to nitrogen limitation of two
urinary tract isolates and an intestinal commensal strain isolated recently from 
humans were remarkably similar to those of MG1655.


PMCID: PMC193769
PMID: 12949114  [Indexed for MEDLINE]


1262. J Bacteriol. 2003 Sep;185(18):5442-51.

Global analyses of transcriptomes and proteomes of a parent strain and an
L-threonine-overproducing mutant strain.

Lee JH(1), Lee DE, Lee BU, Kim HS.

Author information: 
(1)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, 373-1, Kusung-dong, Yusung-gu, Taejon, 305-701, Korea.

We compared the transcriptome, proteome, and nucleotide sequences between the
parent strain Escherichia coli W3110 and the L-threonine-overproducing mutant E. 
coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the
genes of E. coli, and two-dimensional gel electrophoresis was used to compare
protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited
differential expression profiles. Typically, genes such as aceA, aceB, icdA,
gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the
glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis
(L-glutamine, L-leucine, proline, and L-threonine), were significantly
upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD,
and many ribosomal protein genes were downregulated in TF5015 compared to W3110. 
The differential expression such as upregulation of thr operon and expression of 
yigJ would result in an accumulation of L-threonine in TF5015. Furthermore, two
significant mutations, thrA345 and ilvA97, which are essential for overproduction
of L-threonine, were identified in TF5015 by the sequence analysis. In
particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a
significant incremental effect on L-threonine production. Upregulation of aceBA
and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low
accumulation of acetate in TF5015. Such comprehensive analyses provide
information regarding the regulatory mechanism of L-threonine production and the 
physiological consequences in the mutant stain.


PMCID: PMC193774
PMID: 12949096  [Indexed for MEDLINE]


1263. J Clin Microbiol. 2003 Sep;41(9):4071-80.

Microarray-based identification of thermophilic Campylobacter jejuni, C. coli, C.
lari, and C. upsaliensis.

Volokhov D(1), Chizhikov V, Chumakov K, Rasooly A.

Author information: 
(1)FDA Center for Food Safety and Applied Nutrition, College Park, Maryland
20740, USA.

DNA microarrays are an excellent potential tool for clinical microbiology, since 
this technology allows relatively rapid identification and characterization of
microbial and viral pathogens. In the present study, an oligonucleotide
microarray was developed and used for the analysis of thermophilic Campylobacter 
spp., the primary food-borne pathogen in the United States. We analyzed four
Campylobacter species: Campylobacter jejuni, C. coli, C. lari, and C.
upsaliensis. Our assay relies on the PCR amplification of specific regions in
five target genes (fur, glyA, cdtABC, ceuB-C, and fliY) as a first step, followed
by microarray-based analysis of amplified DNAs. Alleles of two genes, fur and
glyA, which are found in all tested thermophilic Campylobacter spp., were used
for identification and discrimination among four bacterial species, the ceuB-C
gene was used for discrimination between C. jejuni and C. coli, and the fliY and 
cdt genes were used as additional genetic markers specific either for C.
upsaliensis and C. lari or for C. jejuni. The array was developed and validated
by using 51 previously characterized Campylobacter isolates. All isolates were
unambiguously identified on the basis of hybridization patterns with 72
individual species-specific oligoprobes. Microarray identification of C. jejuni
and C. coli was confirmed by PCR amplification of other genes used for
identification (hipO and ask). Our results demonstrate that oligonucleotide
microarrays are suitable for rapid and accurate simultaneous differentiation
among C. jejuni, C. coli, C. lari, and C. upsaliensis.


PMCID: PMC193862
PMID: 12958228  [Indexed for MEDLINE]


1264. J Biol Chem. 2003 Aug 29;278(35):33562-70. Epub 2003 Jun 14.

Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into
double-stranded filaments.

Oliva MA(1), Huecas S, Palacios JM, Martín-Benito J, Valpuesta JM, Andreu JM.

Author information: 
(1)Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones
Cientificas, Madrid, Spain.

We have studied the assembly and GTPase of purified FtsZ from the
hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of
eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His6 (histidine-tagged FtsZ), 
and the new mutants FtsZ-W319Y and FtsZ-W319Y-His6, with light scattering,
nucleotide analyses, electron microscopy, and image processing methods. This has 
revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is
suppressed in Na+-containing buffer, generating stabilized structures that
require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal
FtsZ(wt) assembles into associated and isolated filaments made of two parallel
protofilaments with a 43 A longitudinal spacing between monomers, and this
structure is also observed in bacterial FtsZ from Escherichia coli. The His6
extension facilitates the artificial formation of helical tubes and sheets.
FtsZ-W319Y-His6 is an inactivated GTPase whose assembly remains regulated by GTP 
and Mg2+. It forms two-dimensional crystals made of symmetrical pairs of
tubulin-like protofilaments, which associate in an antiparallel array (similarly 
to the known Ca2+-induced sheets of FtsZ-His6). In contrast to the lateral
interactions of microtubule protofilaments, we propose that the primary assembly 
product of FtsZ is the double-stranded filament, one or several of which might
form the dynamic Z ring during prokaryotic cell division.

DOI: 10.1074/jbc.M303798200 
PMID: 12807911  [Indexed for MEDLINE]


1265. Nucleic Acids Res. 2003 Aug 15;31(16):e95.

Restriction site tagged (RST) microarrays: a novel technique to study the species
composition of complex microbial systems.

Zabarovsky ER(1), Petrenko L, Protopopov A, Vorontsova O, Kutsenko AS, Zhao Y,
Kilosanidze G, Zabarovska V, Rakhmanaliev E, Pettersson B, Kashuba VI, Ljungqvist
O, Norin E, Midtvedt T, Möllby R, Winberg G, Ernberg I.

Author information: 
(1)Microbiology and Tumor Biology Center, Department of Cell and Molecular
Biology, Karolinska Institute, 171 77 Stockholm, Sweden. eugzab@ki.se

We have developed a new type of microarray, restriction site tagged (RST), for
example NotI, microarrays. In this approach only sequences surrounding specific
restriction sites (i.e. NotI linking clones) were used for generating
microarrays. DNA was labeled using a new procedure, NotI representation, where
only sequences surrounding NotI sites were labeled. Due to these modifications,
the sensitivity of RST microarrays increases several hundred-fold compared to
that of ordinary genomic microarrays. In a pilot experiment we have produced NotI
microarrays from Gram-positive and Gram-negative bacteria and have shown that
even closely related Escherichia coli strains can be easily discriminated using
this technique. For example, two E.coli strains, K12 and R2, differ by less than 
0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily 
discriminate between these strains. However, these strains showed distinctly
different hybridization patterns with NotI microarrays. The same technique can be
adapted to other restriction enzymes as well. This type of microarray opens the
possibility not only for studies of the normal flora of the gut but also for any 
problem where quantitative and qualitative analysis of microbial (or large viral)
genomes is needed.


PMCID: PMC169987
PMID: 12907747  [Indexed for MEDLINE]


1266. J Biol Chem. 2003 Aug 8;278(32):29837-55. Epub 2003 May 15.

Global gene expression profiling in Escherichia coli K12. The effects of oxygen
availability and FNR.

Salmon K(1), Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP.

Author information: 
(1)Department of Microbiology, Immunology, and Molecular Genetics and the
Molecular Biology Institute, UCLA, Los Angeles, Cal;ifornia, 90095-1489, USA.

The work presented here is a first step toward a long term goal of systems
biology, the complete elucidation of the gene regulatory networks of a living
organism. To this end, we have employed DNA microarray technology to identify
genes involved in the regulatory networks that facilitate the transition of
Escherichia coli cells from an aerobic to an anaerobic growth state. We also
report the identification of a subset of these genes that are regulated by a
global regulatory protein for anaerobic metabolism, FNR. Analysis of these data
demonstrated that the expression of over one-third of the genes expressed during 
growth under aerobic conditions are altered when E. coli cells transition to an
anaerobic growth state, and that the expression of 712 (49%) of these genes are
either directly or indirectly modulated by FNR. The results presented here also
suggest interactions between the FNR and the leucine-responsive regulatory
protein (Lrp) regulatory networks. Because computational methods to analyze and
interpret high dimensional DNA microarray data are still at an early stage, and
because basic issues of data analysis are still being sorted out, much of the
emphasis of this work is directed toward the development of methods to identify
differentially expressed genes with a high level of confidence. In particular, we
describe an approach for identifying gene expression patterns (clusters) obtained
from multiple perturbation experiments based on a subset of genes that exhibit
high probability for differential expression values.

DOI: 10.1074/jbc.M213060200 
PMID: 12754220  [Indexed for MEDLINE]


1267. J Biol Chem. 2003 Aug 8;278(32):29478-86. Epub 2003 May 13.

Global iron-dependent gene regulation in Escherichia coli. A new mechanism for
iron homeostasis.

McHugh JP(1), Rodríguez-Quinoñes F, Abdul-Tehrani H, Svistunenko DA, Poole RK,
Cooper CE, Andrews SC.

Author information: 
(1)School of Animal & Microbial Sciences, University of Reading, Reading, RG6
6AJ, United Kingdom.

Organisms generally respond to iron deficiency by increasing their capacity to
take up iron and by consuming intracellular iron stores. Escherichia coli, in
which iron metabolism is particularly well understood, contains at least 7
iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent
repression is mediated by a transcriptional repressor, Fur (ferric uptake
regulation), which also controls genes involved in other processes such as iron
storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress
resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene 
expression in E. coli has revealed several novel Fur-repressed genes likely to
specify at least three additional iron-transport pathways. Interestingly, a large
group of energy metabolism genes was found to be iron and Fur induced. Many of
these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent 
regulation appears to represent a novel iron-homeostatic mechanism whereby the
synthesis of many iron-containing proteins is repressed under iron-restricted
conditions. This mechanism thus accounts for the low iron contents of fur mutants
and explains how E. coli can modulate its iron requirements. Analysis of
55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein
composition for the fur mutant, and visible and EPR spectroscopy showed major
reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for 
the chelator-treated wild-type and/or fur mutant, correlating well with the array
and quantitative RT-PCR data. In combination, the results provide compelling
evidence for the regulation of intracellular iron consumption by the Fe2+-Fur
complex.

DOI: 10.1074/jbc.M303381200 
PMID: 12746439  [Indexed for MEDLINE]


1268. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9530-5. Epub 2003 Jul 28.

Clusterin as a biomarker in murine and human intestinal neoplasia.

Chen X(1), Halberg RB, Ehrhardt WM, Torrealba J, Dove WF.

Author information: 
(1)McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI
53706, USA.

Early detection of colorectal cancer is critical for the management of this
disease. Biomarkers for early detection of several cancers have been developed
and applied clinically in recent years. We have sought to discover candidate
biomarkers without the restricted choice of markers placed on microarrays, and
without the biological complications of genetic and environmental heterogeneity. 
We have compared by cDNA subtraction two genetically matched sets of mice, one
developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other
tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then
subjected to a series of validation steps. In situ hybridization and
immunohistochemistry were used to analyze clusterin expression at a cellular
level on a series of murine intestinal and human colonic neoplasms. Elevated
clusterin expression was characterized within certain regions of murine and human
tumors regardless of tumor stage, location, or mode of initiation. The cells
showing high clusterin levels generally lacked differentiation markers and
adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed
low levels of clusterin. Its specific expression patterns and correlation with
cellular events during tumorigenesis make it a useful diagnostic tool in the
mouse and a potential contributor to the set of biomarkers for early detection of
human colon cancer.

DOI: 10.1073/pnas.1233633100 
PMCID: PMC170952
PMID: 12886021  [Indexed for MEDLINE]


1269. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9232-7. Epub 2003 Jul 23.

Toward a protein profile of Escherichia coli: comparison to its transcription
profile.

Corbin RW(1), Paliy O, Yang F, Shabanowitz J, Platt M, Lyons CE Jr, Root K,
McAuliffe J, Jordan MI, Kustu S, Soupene E, Hunt DF.

Author information: 
(1)Department of Chemistry, University of Virginia, Charlottesville, VA 22901,
USA.

High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a
protein profile of Escherichia coli strain MG1655 grown in minimal medium with
glycerol as the carbon source. By using cell lysate from only 3 x 108 cells, at
least four different tryptic peptides were detected for each of 404 proteins in a
short 4-h experiment. At least one peptide with a high reliability score was
detected for 986 proteins. Because membrane proteins were underrepresented, a
second experiment was performed with a preparation enriched in membranes. An
additional 161 proteins were detected, of which from half to two-thirds were
membrane proteins. Overall, 1,147 different E. coli proteins were identified,
almost 4 times as many as had been identified previously by using other tools.
The protein list was compared with the transcription profile obtained on
Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein
products were found was detected at the mRNA level. The arithmetic mean mRNA
signal intensity for these genes was 3-fold higher than that for all 4,300
protein-coding genes of E. coli. Thus, GeneChip data confirmed the high
reliability of the protein list, which contains about one-fourth of the proteins 
of E. coli. Detection of even those membrane proteins and proteins of undefined
function that are encoded by the same operons (transcriptional units) encoding
proteins on the list remained low.

DOI: 10.1073/pnas.1533294100 
PMCID: PMC170901
PMID: 12878731  [Indexed for MEDLINE]


1270. Cancer Res. 2003 Aug 1;63(15):4368-74.

Regulation of caspase expression and apoptosis by adenomatous polyposis coli.

Chen T(1), Yang I, Irby R, Shain KH, Wang HG, Quackenbush J, Coppola D, Cheng JQ,
Yeatman TJ.

Author information: 
(1)Department of Interdisciplinary Oncology and Surgery, University of South
Florida, Tampa, FL 33612, USA.

The adenomatous polyposis coli (APC) gene, a member of the WNT pathway, has been 
shown to assign intestinal epithelial cells to a program of proliferation or
differentiation through regulation of the beta-catenin/TCF-4 complex. Wild-type
APC, in certain cellular contexts, appears to induce differentiation and
apoptosis, although mutant forms of APC, known to produce polyps and ultimately
cancers, may suppress these events. Here, we show that mutant forms of APC can
induce repression of select terminal caspases as a potential means of attenuating
responses to apoptotic stimuli. Using gene expression profiling to interrogate
the intact intestines of Apc(+/min) mice harboring numerous polyps, we identified
a reduction in the mRNA expression of both caspases 3 and 7. We additionally
identified a reduction in protein levels of caspase-3, caspase-7, and caspase-9
in human colon cancer specimens known to harbor APC mutations. A reduction in
caspase protein levels resulted in resistance to apoptotic-inducing agents and
restoration of caspase levels reinstated apoptotic capacities. Consistent with
Wnt pathway involvement, dominant negative TCF/LEF induced caspase protein
expression. These data provide support for the hypothesis that one of the
functions of APC is the regulation of caspase activity and other apoptotic
proteins by controlling their expression levels in the cell.


PMID: 12907606  [Indexed for MEDLINE]


1271. J Bacteriol. 2003 Aug;185(16):4956-72.

Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of 
all two-component systems.

Zhou L(1), Lei XH, Bochner BR, Wanner BL.

Author information: 
(1)Department of Biological Sciences, Purdue University, West Lafayette, Indiana 
47907, USA.

Two-component systems are the most common mechanism of transmembrane signal
transduction in bacteria. A typical system consists of a histidine kinase and a
partner response regulator. The histidine kinase senses an environmental signal, 
which it transmits to its partner response regulator via a series of
autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work 
has been done on particular systems, including several systems with regulatory
roles in cellular physiology, communication, development, and, in the case of
bacterial pathogens, the expression of genes important for virulence. We used two
methods to investigate two-component regulatory systems in Escherichia coli K-12.
First, we systematically constructed mutants with deletions of all two-component 
systems by using a now-standard technique of gene disruption (K. A. Datsenko and 
B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed
these deletion mutants with a new technology called Phenotype MicroArrays, which 
permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we
tested 100 mutants, including mutants with individual deletions of all
two-component systems and several related genes, including creBC-regulated genes 
(cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and
ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000
tests provided a wealth of new information concerning many of these systems. Of
37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes
were expected, and several new phenotypes were also revealed. The results are
discussed in terms of the biological roles and other information concerning these
systems, including DNA microarray data for a large number of the same mutants.
Other mutational effects are also discussed.


PMCID: PMC166450
PMID: 12897016  [Indexed for MEDLINE]


1272. Mol Cell Probes. 2003 Aug;17(4):187-96.

Development of a sensitive DNA microarray suitable for rapid detection of
Campylobacter spp.

Keramas G(1), Bang DD, Lund M, Madsen M, Rasmussen SE, Bunkenborg H, Telleman P, 
Christensen CB.

Author information: 
(1)Mikroelektronik Centret (MIC), Technical University of Denmark, Building 345
East, Kongens 2800, Lyngby, Denmark.

Campylobacter is the most common cause of human acute bacterial gastroenteritis
worldwide, widely distributed and isolated from human clinical samples as well as
from many other different sources. To comply with the demands of consumers for
food safety, there is a need for development of a rapid, sensitive and specific
detection method for Campylobacter. In this study, we present the development of 
a novel sensitive DNA-microarray based detection method, evaluated on
Campylobacter and non-Campylobacter reference strains, to detect Campylobacter
directly from the faecal cloacal swabs. The DNA-microarray method consists of two
steps: first, both universal bacterial sequences and specific Campylobacter
sequences (size range: 149-307 bp) are amplified and fluorescently labeled using 
multiplex-PCR, targeting the 16S rRNA, the 16S-23S rRNA intergenic region and
specific Campylobacter genes. Secondly, the Cy5 labeled PCR-amplicons are
hybridised to immobilised capture probes on the microarray. The method allows
detection of three to thirty genome equivalents (6-60 fg DNA) of Campylobacter
within 3 h, with a hands on time of only 15 min. Using the DNA-microarrays, two
closely related Campylobacter species, Campylobacter jejuni and Campylobacter
coli could be detected and differentiated directly from chicken faeces. The
DNA-microarray method has a high potential for automation and incorporation into 
a dedicated mass screening microsystem.


PMID: 12944122  [Indexed for MEDLINE]


1273. Mol Microbiol. 2003 Aug;49(4):947-63.

Characterization of the roles of NikR, a nickel-responsive pleiotropic
autoregulator of Helicobacter pylori.

Contreras M(1), Thiberge JM, Mandrand-Berthelot MA, Labigne A.

Author information: 
(1)Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 75724-Paris
15, France.

The Helicobacter pylori genome contains a gene (hp1338 or nikR) that encodes a
nickel-dependent regulator that is homologous to the Escherichia coli
nickel-responsive regulator, NikR. The H. pylori nikR product acts as a
pleiotropic metal-dependent regulator. We constructed a non-polar isogenic mutant
deleted for the nikR gene. NikR was essential for the survival of H. pylori in
the presence of high nickel and cobalt ion concentrations in vitro. We screened a
DNA macroarray for genes that were differentially expressed in parental and
nikR-deficient H. pylori strains grown in the presence of excess nickel. We found
that H. pylori NikR mediates the expression of nickel-activated and -repressed
genes. In the presence of excess nickel, NikR activated the transcription of
ureA-ureB (hp72-73), nixA (hp1077 ), copA2 (hp1072), hpn (hp1427 ) and hpn-like
(hp1432) genes and repressed the expression of genes encoding proteins involved
in ferric iron uptake and storage [pfr (hp0653), fur (hp1027 ), frpB4 (hp1512),
exbB/exbD (hp1339-1340), ceuE (hp1561)], motility [cheV (hp616), flaA (hp0601),
flaB (hp0115 )], stress responses [hrcA-grpE-dnaK (hp111-110-109)] and encoding
outer-membrane proteins [omp11(hp0472), omp31 (hp1469), omp32 (hp1501)]. Slot
blot DNA/RNA hybridization experiments using RNA from three independent bacterial
cultures confirmed the transcriptome data for 10 selected genes. The results of
gel shift experiments using purified native NikR, beta-galactosidase assays with 
the region between nikR and the exbB/exbD divergent operon, and the study of exbB
gene expression using a gentamicin/apramycin reporter gene in H. pylori indicated
that NikR is an autorepressor that binds to this intergenic region and also
controls the expression of the exbB/exbD/tonB operon, which provides energy for
ferric iron uptake. Thus, as previously suggested for Fur in H. pylori, NikR
appears to be a global regulator of the metabolism of some divalent cations
within a highly complex regulated network.


PMID: 12890020  [Indexed for MEDLINE]


1274. Appl Environ Microbiol. 2003 Jul;69(7):4144-50.

A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces
ethanol.

Becker J(1), Boles E.

Author information: 
(1)Institut für Mikrobiologie, Heinrich-Heine-Universität, D-40225 Düsseldorf,
Germany.

Metabolic engineering is a powerful method to improve, redirect, or generate new 
metabolic reactions or whole pathways in microorganisms. Here we describe the
engineering of a Saccharomyces cerevisiae strain able to utilize the pentose
sugar L-arabinose for growth and to ferment it to ethanol. Expanding the
substrate fermentation range of S. cerevisiae to include pentoses is important
for the utilization of this yeast in economically feasible biomass-to-ethanol
fermentation processes. After overexpression of a bacterial L-arabinose
utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli
AraB and AraD and simultaneous overexpression of the L-arabinose-transporting
yeast galactose permease, we were able to select an L-arabinose-utilizing yeast
strain by sequential transfer in L-arabinose media. Molecular analysis of this
strain, including DNA microarrays, revealed that the crucial prerequisite for
efficient utilization of L-arabinose is a lowered activity of L-ribulokinase.
Moreover, high L-arabinose uptake rates and enhanced transaldolase activities
favor utilization of L-arabinose. With a doubling time of about 7.9 h in a medium
with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to
0.08 g of ethanol per g (dry weight). h(-1) under oxygen-limiting conditions, and
high ethanol yields, this yeast strain should be useful for efficient
fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.


PMCID: PMC165137
PMID: 12839792  [Indexed for MEDLINE]


1275. J Bacteriol. 2003 Jul;185(13):3696-702.

Identification and molecular characterization of the Mg2+ stimulon of Escherichia
coli.

Minagawa S(1), Ogasawara H, Kato A, Yamamoto K, Eguchi Y, Oshima T, Mori H,
Ishihama A, Utsumi R.

Author information: 
(1)Department of Bioscience and Biotechnology, Graduate School of Agriculture,
Kinki University, 3327-204 Nakamachi, Nara 631-8505Japan.

Transcription profile microarray analysis in Escherichia coli was performed to
identify the member genes of the Mg(2+) stimulon that respond to the availability
of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA
levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and
WQ3007 (phoQ defective) were compared with those of W3110 in the absence of
MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three
strains (the supplemental data are shown at
http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the
PhoP/PhoQ system is involved directly or indirectly in the transcription of these
genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats
of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1
nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon 
genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB,
and mgtA genes reported previously. In gel shift and DNase I footprinting assays,
all of these genes were found to be regulated directly by PhoP. Thus, we
concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL
genes make up the Mg(2+) stimulon in E. coli.


PMCID: PMC161583
PMID: 12813061  [Indexed for MEDLINE]


1276. J Cell Biochem. 2003 Jul 1;89(4):848-61.

Gene expression in human neutrophils during activation and priming by bacterial
lipopolysaccharide.

Tsukahara Y(1), Lian Z, Zhang X, Whitney C, Kluger Y, Tuck D, Yamaga S, Nakayama 
Y, Weissman SM, Newburger PE.

Author information: 
(1)Department of Genetics, Boyer Center for Molecular Medicine, Yale University
School of Medicine, New Haven, CT, USA.

Erratum in
    J Cell Biochem. 2003 Aug 15;89(6):1302.

Circulating neutrophils play a key role both in the systemic inflammatory
response and in complications of bacterial infection such as septic shock and
septic multiple organ dysfunction syndrome. We have analyzed gene expression
patterns in human neutrophils stimulated by E. coli lipopolysaccharide (LPS),
with or without prior exposure to LPS, using differential display and
oligonucleotide chip techniques. We identified 307 genes that were activated or
repressed after treatment with LPS at 10 ng/ml and 385 genes after LPS at 100
ng/ml, compared with untreated neutrophils. The two sets included many
transcription factors, cytokines, chemokines, interleukins, and surface antigens,
as well as members of the toll-like receptor, Rel/NF-kappaB, and immune mediator 
gene families. Time course analysis showed that the early and late neutrophil
responses to LPS share some common mechanisms, but many changes in gene
expression are transient or late to develop. Neutrophils also showed a priming
response to LPS, in which 97 genes significantly changed expression on
re-exposure to lower dose LPS and were analyzed by unsupervised hierarchical
clustering. These findings indicate that the neutrophil is a transcriptionally
active cell responsive to environmental stimuli and capable of a complex series
of both early and late changes in gene expression. Supplementary material for
this article can be found on the Journal of Cellular Biochemistry website
(http://jws-edci.interscience.wiley.com:8998/jpages/0730-2312/suppmat/89/v89.page
.html).

Copyright 2003 Wiley-Liss, Inc.

DOI: 10.1002/jcb.10526 
PMID: 12858349  [Indexed for MEDLINE]


1277. Microbiology. 2003 Jul;149(Pt 7):1859-69.

Production of the signalling molecule, autoinducer-2, by Neisseria meningitidis: 
lack of evidence for a concerted transcriptional response.

Dove JE(1), Yasukawa K, Tinsley CR, Nassif X.

Author information: 
(1)Laboratoire de Microbiologie, INSERM U570, Faculté de Médecine Necker-Enfants 
Malades, 75015 Paris, France.

Neisseria meningitidis is a Gram-negative bacterium which is an important
causative agent of septicaemia and meningitis. LuxS has been shown to be involved
in the biosynthesis of a quorum sensing molecule, autoinducer-2 (AI-2), known to 
play a role in virulence in Escherichia coli, as well as other bacteria. Evidence
that serogroup B of N. meningitidis produces AI-2, along with the observation
that a luxS mutant of this strain had attenuated virulence in an infant rat model
of bacteraemia, led to further investigation of the role of this quorum sensing
molecule in N. meningitidis. In this study, it is demonstrated that AI-2 is not
involved in regulating growth of meningococci, either in culture or in contact
with epithelial cells. Furthermore, transcriptional profiling using DNA
microarrays shows an absence of the concerted regulation seen in other bacteria. 
Taken together, these data suggest that in N. meningitidis, AI-2 may be a
metabolic by-product and not a cell-to-cell signalling molecule.

DOI: 10.1099/mic.0.26185-0 
PMID: 12855737  [Indexed for MEDLINE]


1278. Physiol Genomics. 2003 Jun 24;14(1):47-58.

Evolutionary changes in heat-inducible gene expression in lines of Escherichia
coli adapted to high temperature.

Riehle MM(1), Bennett AF, Lenski RE, Long AD.

Author information: 
(1)Department of Ecology and Evolutionary Biology, University of California at
Irvine, Irvine, California 92697-2525, USA. mriehle@uci.edu

The involvement of heat-inducible genes, including the heat-shock genes, in the
acute response to temperature stress is well established. However, their
importance in genetic adaptation to long-term temperature stress is less clear.
Here we use high-density arrays to examine changes in expression for 35
heat-inducible genes in three independent lines of Escherichia coli that evolved 
at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited
significant changes in heat-inducible gene expression relative to their ancestor,
including parallel changes in fkpA, gapA, and hslT. As a group, the
heat-inducible genes were significantly more likely than noncandidate genes to
have evolved changes in expression. Genes encoding molecular chaperones and
ATP-dependent proteases, key components of the cytoplasmic stress response,
exhibit relatively little expression change; whereas genes with periplasmic
functions exhibit significant expression changes suggesting a key role for the
extracytoplasmic stress response in the adaptation to high temperature. Following
acclimation at 41.5 degrees C, two of the three lines exhibited significantly
improved survival at 50 degrees C, indicating changes in inducible
thermotolerance. Thus evolution at high temperature led to significant changes at
the molecular level in heat-inducible gene expression and at the organismal level
in inducible thermotolerance and fitness.

DOI: 10.1152/physiolgenomics.00034.2002 
PMID: 12672900  [Indexed for MEDLINE]


1279. Mol Microbiol. 2003 Jun;48(6):1491-500.

Consequences of reductive evolution for gene expression in an obligate
endosymbiont.

Wilcox JL(1), Dunbar HE, Wolfinger RD, Moran NA.

Author information: 
(1)Department of Ecology and Evolutionary Biology, University of Arizona,
BioSciences West Rm 310, Tucson 85721, USA. wilcoxjl@email.arizona.edu

The smallest cellular genomes are found in obligate symbiotic and pathogenic
bacteria living within eukaryotic hosts. In comparison with large genomes of
free-living relatives, these reduced genomes are rearranged and have lost most
regulatory elements. To test whether reduced bacterial genomes incur reduced
regulatory capacities, we used full-genome microarrays to evaluate
transcriptional response to environmental stress in Buchnera aphidicola, the
obligate endosymbiont of aphids. The 580 genes of the B. aphidicola genome
represent a subset of the 4500 genes known from the related organism, Escherichia
coli. Although over 20 orthologues of E. coli heat stress (HS) genes are retained
by B. aphidicola, only five were differentially expressed after near-lethal heat 
stress treatments, and only modest shifts were observed. Analyses of upstream
regulatory regions revealed loss or degradation of most HS (sigma32) promoters.
Genomic rearrangements downstream of an intact HS promoter yielded upregulation
of a functionally unrelated and an inactivated gene. Reanalyses of comparable
experimental array data for E. coli and Bacillus subtilis revealed that
genome-wide differential expression was significantly lower in B. aphidicola. Our
demonstration of a diminished stress response validates reports of temperature
sensitivity in B. aphidicola and suggests that this reduced bacterial genome
exhibits transcriptional inflexibility.


PMID: 12791133  [Indexed for MEDLINE]


1280. J Bacteriol. 2003 May;185(10):3190-201.

Genes of the GadX-GadW regulon in Escherichia coli.

Tucker DL(1), Tucker N, Ma Z, Foster JW, Miranda RL, Cohen PS, Conway T.

Author information: 
(1)Advanced Center for Genome Technology, The University of Oklahoma, Norman,
Oklahoma 73019, USA.

Acid in the stomach is thought to be a barrier to bacterial colonization of the
intestine. Escherichia coli, however, has three systems for acid resistance,
which overcome this barrier. The most effective of these systems is dependent on 
transport and decarboxylation of glutamate. GadX regulates two genes that encode 
isoforms of glutamate decarboxylase critical to this system, but additional genes
associated with the glutamate-dependent acid resistance system remained to be
identified. The gadX gene and a second downstream araC-like transcription factor 
gene, gadW, were mutated separately and in combination, and the gene expression
profiles of the mutants were compared to those of the wild-type strain grown in
neutral and acidified media under conditions favoring induction of
glutamate-dependent acid resistance. Cluster and principal-component analyses
identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping
demonstrated that these genes are organized in 10 operons. Analysis of the strain
lacking GadX but possessing GadW confirmed that GadX is a transcriptional
activator under acidic growth conditions. Analysis of the strain lacking GadW but
possessing GadX indicated that GadW exerts negative control over three GadX
target genes. The strain lacking both GadX and GadW was defective in acid
induction of most but not all GadX target genes, consistent with the roles of
GadW as an inhibitor of GadX-dependent activation of some genes and an activator 
of other genes. Resistance to acid was decreased under certain conditions in a
gadX mutant and even more so by combined mutation of gadX and gadW. However,
there was no defect in colonization of the streptomycin-treated mouse model by
the gadX mutant in competition with the wild type, and the gadX gadW mutant was a
better colonizer than the wild type. Thus, E. coli colonization of the mouse does
not appear to require glutamate-dependent acid resistance.


PMCID: PMC154079
PMID: 12730179  [Indexed for MEDLINE]


1281. J Clin Microbiol. 2003 May;41(5):2113-25.

Rapid identification of Escherichia coli pathotypes by virulence gene detection
with DNA microarrays.

Bekal S(1), Brousseau R, Masson L, Prefontaine G, Fairbrother J, Harel J.

Author information: 
(1)Biotechnology Research Institute, National Research Council of Canada,
Montreal, Quebec H4P 2R2.

One approach to the accurate determination of the pathogenic potential
(pathotype) of isolated Escherichia coli strains would be through a complete
assessment of each strain for the presence of all known E. coli virulence
factors. To accomplish this, an E. coli virulence factor DNA microarray composed 
of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays 
corresponding to different E. coli pathotypes was developed. Fluorescently
labeled genomic DNAs from E. coli strains representing known pathotypes were
initially hybridized to the virulence gene microarrays for both chip optimization
and validation. Hybridization pattern analysis with clinical isolates permitted a
rapid assessment of their virulence attributes and determination of the
pathogenic group to which they belonged. Virulence factors belonging to two
different pathotypes were detected in one human E. coli isolate (strain
H87-5406). The microarray was also tested for its ability to distinguish among
phylogenetic groups of genes by using gene probes derived from the
attaching-and-effacing locus (espA, espB, tir). After hybridization with these
probes, we were able to distinguish E. coli strains harboring espA, espB, and tir
sequences closely related to the gene sequences of an enterohemorrhagic strain
(EDL933), a human enteropathogenic strain (E2348/69), or an animal
enteropathogenic strain (RDEC-1). Our results show that the virulence factor
microarray is a powerful tool for diagnosis-based studies and that the concept is
useful for both gene quantitation and subtyping. Additionally, the multitude of
virulence genes present on the microarray should greatly facilitate the detection
of virulence genes acquired by horizontal transfer and the identification of
emerging pathotypes.


PMCID: PMC154688
PMID: 12734257  [Indexed for MEDLINE]


1282. Mol Microbiol. 2003 May;48(3):699-712.

Regulatory network of acid resistance genes in Escherichia coli.

Masuda N(1), Church GM.

Author information: 
(1)Department of Genetics, Warren Alpert Building, Room 513, Harvard Medical
School, 200 Longwood Ave., Boston, MA 02115, USA.

Overexpression of the response regulator EvgA confers an acid-resistant phenotype
to exponentially growing Escherichia coli. This acid resistance is partially
abolished by deletion of ydeP, yhiE or ydeO, genes induced by EvgA
overexpression. Microarray analysis identified two classes of operons (genes).
The first class contains seven operons induced by EvgA overexpression in the
absence of ydeO, an AraC/XylS regulator gene. The second class contains 12
operons induced by YdeO overexpression. Operons in the second class were induced 
by EvgA overexpression only in the presence of ydeO. EvgA is likely to directly
upregulate operons in the first class, and indirectly upregulate operons in the
second class via YdeO. Analysis using the motif-finding program alignace
identified an 18 bp inverted repeat motif in six upstream regions of all seven
operons directly regulated by EvgA. Gel mobility shift assays showed the specific
binding of EvgA to the six sequences. Introduction of mutations into the inverted
repeats upstream of ydeP and b1500-ydeO resulted in reduction in EvgA-induced
ydeP and ydeO expression and acid resistance. These results suggest that EvgA
binds to the inverted repeats and upregulates the downstream genes.
Overexpression of YdeP, YdeO and YhiE conferred acid resistance to exponentially 
growing cells, whereas GadX overexpression did not. Microarray analysis also
identified several GadX-activated genes. Several genes induced by overexpression 
of YdeO and GadX overlapped; however, yhiE was induced only by YdeO. The acid
resistance induced by YdeO overexpression was abolished by deletion of yhiE,
gadC, slp-yhiF, hdeA or hdeD, genes induced by YdeO overexpression, suggesting
that several genes orchestrate YdeO-induced acid resistance. We propose a model
of the regulatory network of the acid resistance genes.


PMID: 12694615  [Indexed for MEDLINE]


1283. RNA. 2003 May;9(5):518-32.

The effect of a single, temperature-sensitive mutation on global gene expression 
in Escherichia coli.

Li Y(1), Cole K, Altman S.

Author information: 
(1)Department of Molecular, Cellular and Developmental Biology, Yale University, 
New Haven, Connecticut 06520, USA.

High-density DNA microarrays have been used to explore the genomic profiling of
gene expression of a defective Escherichia coli strain with a
temperature-sensitive mutation in the protein component of RNase P. A novel gene 
cluster was discovered in which two of the genes are known substrates of RNase P.
The expression pattern of essential genes and gene discovery from intergenic
regions, for which other new transcripts are found, are also discussed.


PMCID: PMC1370418
PMID: 12702811  [Indexed for MEDLINE]


1284. Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4672-7. Epub 2003 Apr 7.

Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray
analysis.

Løbner-Olesen A(1), Marinus MG, Hansen FG.

Author information: 
(1)Department of Life Sciences and Chemistry, Roskilde University, DK-4000
Roskilde, Denmark. lobner@ruc.dk

High-density oligonucleotide arrays were used to monitor global transcription
patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells 
lacking Dam methyltransferase showed a modest increase in transcription of the
genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which
preferentially binds hemimethylated DNA, were found to have a transcriptional
profile almost identical to WT bacteria overexpressing Dam methyltransferase. The
latter two strains differed from WT in two ways. First, the origin proximal genes
were transcribed with increased frequency due to increased gene dosage. Second,
chromosomal domains of high transcriptional activity alternate with regions of
low activity, and our results indicate that the activity in each domain is
modulated in the same way by SeqA deficiency or Dam overproduction. We suggest
that the methylation status of the cell is an important factor in forming and/or 
maintaining chromosome structure.

DOI: 10.1073/pnas.0538053100 
PMCID: PMC153614
PMID: 12682301  [Indexed for MEDLINE]


1285. Biol Bull. 2003 Apr;204(2):196-9.

A(r)Ray of hope in analysis of the function and diversity of microbial
communities.

Polz MF(1), Bertilsson S, Acinas SG, Hunt D.

Author information: 
(1)Department of Civil and Environmental Engineering, Massachusetts Institute of 
Technology, Cambridge, Massachusetts 02139, USA. mpolz@mit.edu

The vast majority of microorganisms in the environment remain uncultured, and
their existence is known only from sequences retrieved by PCR. As a consequence, 
our understanding of the ecological function of dominant microbial populations in
the environment is limited. We will review microbial diversity studies and show
that these may have moved from an extreme underestimation to a potentially severe
overestimation of diversity. The latter results from a simple PCR-generated
artifact: the cloning of heteroduplex molecules followed by Escherichia coli
mismatch repair, which may generate an exponential increase in observed sequence 
diversity. However, simple modifications to current PCR amplification protocols
minimize such artifactual sequences and may bring within our reach estimation of 
bacterial diversity in environmental samples. Such estimates may spur new
culture-independent approaches based on genomic and microarray technology,
allowing correlation of phylogenetic identity with the ecological function of
unculturable organisms. In particular, we are developing a DNA microarray that
enables identification of individual populations active in utilization of
specific organic substrates. The array consists of 16S and 23S rDNA-targeted
oligonucleotides and is hybridized to RNA extracted from samples incubated with
(14)C-labeled organic substrates. Populations that metabolize the substrate can
be identified by the radiolabel incorporated in their rRNA after only one to two 
cell doublings, ensuring realistic preservation of community structure. Thus, the
microarray approach may provide a powerful means to link microbial community
structure with in situ function of individual populations.

DOI: 10.2307/1543558 
PMID: 12700153  [Indexed for MEDLINE]


1286. Brief Funct Genomic Proteomic. 2003 Apr;2(1):47-56.

Beyond expression profiling: next generation uses of high density oligonucleotide
arrays.

Kapranov P(1), Sementchenko VI, Gingeras TR.

Author information: 
(1)Affymetrix, Inc., Santa Clara, CA 95051, USA.

In the past decade, microarray technology has become a major tool for
high-throughput comprehensive analysis of gene expression, genotyping and
resequencing applications. Currently, the most widely employed application of
high-density oligonucleotide arrays (HDOAs) involves monitoring changes in gene
expression. This application has been carried out in a variety of organisms
ranging from Escherichia coli to humans. The recent near completion of the human 
and mouse genome sequences, however, as well as the genomes of other model
experimental species, has allowed for novel applications of HDOAs, such as: the
discovery of novel transcripts, mapping functionally important genomic regions
and identifying functional domains in RNA molecules. Integrating all this
information will provide novel global views of the locations of RNA
transcription, DNA replication and the protein nucleic acid interactions that
regulate these processes.


PMID: 15239943  [Indexed for MEDLINE]


1287. Genes Chromosomes Cancer. 2003 Apr;36(4):361-74.

DNA microarrays for comparative genomic hybridization based on DOP-PCR
amplification of BAC and PAC clones.

Fiegler H(1), Carr P, Douglas EJ, Burford DC, Hunt S, Scott CE, Smith J, Vetrie
D, Gorman P, Tomlinson IP, Carter NP.

Author information: 
(1)Wellcome Trust Sanger Institute/Cancer Research UK Genomic Microarray Group,
Hinxton, Cambridge, CB10 1SA, United Kingdom.

Erratum in
    Genes Chromosomes Cancer. 2003 Jun;37(2):223.

We have designed DOP-PCR primers specifically for the amplification of large
insert clones for use in the construction of DNA microarrays. A bioinformatic
approach was used to construct primers that were efficient in the general
amplification of human DNA but were poor at amplifying E. coli DNA, a common
contaminant of DNA preparations from large insert clones. We chose the three most
selective primers for use in printing DNA microarrays. DNA combined from the
amplification of large insert clones by use of these three primers and spotted
onto glass slides showed more than a sixfold increase in the human to E. coli
hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The
microarrays reproducibly delineated previously characterized gains and deletions 
in a cancer cell line and identified a small gain not detected by use of
conventional CGH. We also describe a method for the bulk testing of the
hybridization characteristics of chromosome-specific clones spotted on
microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we
describe a set of clones selected from the publicly available Golden Path of the 
human genome at 1-Mb intervals and a view in the Ensembl genome browser from
which data required for the use of these clones in array CGH and other
experiments can be downloaded across the Internet.

Copyright 2003 Wiley-Liss, Inc.

DOI: 10.1002/gcc.10155 
PMID: 12619160  [Indexed for MEDLINE]


1288. Genome Res. 2003 Apr;13(4):644-53. Epub 2003 Mar 12.

Chromosomal deletion formation system based on Tn5 double transposition: use for 
making minimal genomes and essential gene analysis.

Goryshin IY(1), Naumann TA, Apodaca J, Reznikoff WS.

Author information: 
(1)Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706,
USA.

In this communication, we describe the use of specialized transposons (Tn5
derivatives) to create deletions in the Escherichia coli K-12 chromosome. These
transposons are essentially rearranged composite transposons that have been
assembled to promote the use of the internal transposon ends, resulting in
intramolecular transposition events. Two similar transposons were developed. The 
first deletion transposon was utilized to create a consecutive set of deletions
in the E. coli chromosome. The deletion procedure has been repeated 20 serial
times to reduce the genome an average of 200 kb (averaging 10 kb per deletion).
The second deletion transposon contains a conditional origin of replication that 
allows deleted chromosomal DNA to be captured as a complementary plasmid. By
plating cells on media that do not support plasmid replication, the deleted
chromosomal material is lost and if it is essential, the cells do not survive.
This methodology was used to analyze 15 chromosomal regions and more than 100
open reading frames (ORFs). This provides a robust technology for identifying
essential and dispensable genes.

DOI: 10.1101/gr.611403 
PMCID: PMC430159
PMID: 12654720  [Indexed for MEDLINE]


1289. J Bacteriol. 2003 Apr;185(8):2667-72.

Global analysis of genes regulated by EvgA of the two-component regulatory system
in Escherichia coli.

Nishino K(1), Inazumi Y, Yamaguchi A.

Author information: 
(1)Department of Cell Membrane Biology, Institute of Scientific and Industrial
Research, Osaka University, Ibaraki-shi, Japan.

The response regulator EvgA controls expression of multiple genes conferring
antibiotic resistance in Escherichia coli (K. Nishino and A. Yamaguchi, J.
Bacteriol. 184:2319-2323, 2002). To understand the whole picture of EvgA
regulation, DNA macroarray analysis of the effect of EvgA overproduction was
performed. EvgA activated genes related to acid resistance, osmotic adaptation,
and drug resistance.


PMCID: PMC152604
PMID: 12670992  [Indexed for MEDLINE]


1290. J Biol Chem. 2003 Mar 14;278(11):9227-34. Epub 2003 Jan 3.

Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs
based on complementation of a bacterial mutant.

Leal NA(1), Park SD, Kima PE, Bobik TA.

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida 32611, USA.

In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) 
lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns.
Such deficiencies can result from inborn errors in the MCM structural gene or
from mutations that impair the assimilation of dietary cobalamins into coenzyme
B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin
adenosyltransferase (ATR) catalyzes the terminal step in the conversion of
cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in
this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has
not been identified. Here we report the identification of the bovine and human
ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression
library was screened for clones that complemented an ATR-deficient bacterial
strain for color formation on aldehyde indicator medium, and four positive clones
were isolated. The DNA sequences of two clones were determined and found to be
identical. Sequence similarity searching was then used to identify a homologous
human cDNA (89% identity) and its corresponding gene that is located on
chromosome XII. The bovine and human cDNAs were independently cloned and
expressed in Escherichia coli. Enzyme assays showed that expression strains
produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific
activities are in line with values reported previously for bacterial ATR enzymes.
Subsequent studies showed that the human cDNA clone complemented an ATR-deficient
bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This
demonstrated that the human ATR is active under physiological conditions albeit
in a heterologous host. In addition, Western blots were used to show that ATR
expression is altered in cell lines derived from cblB methylmalonyl aciduria
patients compared with cell lines from normal individuals. We propose that inborn
errors in the human ATR gene identified here result in methylmalonyl aciduria.
The identification of genes involved in this disorder will allow improvements in 
the diagnosis and treatment of this serious disease.

DOI: 10.1074/jbc.M212739200 
PMID: 12514191  [Indexed for MEDLINE]


1291. Antimicrob Agents Chemother. 2003 Mar;47(3):1101-11.

Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer
membrane proteins.

Jo JT(1), Brinkman FS, Hancock RE.

Author information: 
(1)Department of Microbiology and Immunology, University of British Columbia,
Vancouver, Canada V6T 1Z3.

The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in
Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of
antimicrobials, including beta-lactams, chloramphenicol, macrolides, quinolones, 
and tetracycline. The MexX-MexY linker-pump combination has been shown to be
involved in intrinsic resistance to aminoglycosides, but the identity of the
cognate outer membrane channel component remains under debate. Fourteen
uncharacterized OprM homologs identified in the genome of P. aeruginosa were
examined as candidates for this role by assessing the minimum inhibitory
concentrations (MICs) of aminoglycosides in P. aeruginosa strain PAK knockout
mutants lacking the corresponding genes. Insertional inactivation of OpmG, OpmI, 
and OpmH resulted in decreases of various degrees in the MICs of streptomycin,
kanamycin, and gentamicin. When reintroduced into P. aeruginosa on multicopy
plasmids, OpmG was able to complement the susceptibility of an opmG::miniTn5
mutant; however, cloned opmH, the proposed ortholog of Escherichia coli tolC
according to our phylogenetic analysis, was able to only partially complement the
opmH::miniTn5 mutant. Mini-microarray hybridization analysis demonstrated that
opmG disruption does not affect expression of OpmI or OpmH (ruling out such
indirect effects on aminoglycoside resistance); however, opmH disruption did have
possible effects on expression of OpmG and OpmI. Based on the data, we propose
that OpmG is a major outer membrane efflux channel involved in aminoglycoside
efflux in P. aeruginosa PAK and that OpmI, its most related paralog, may share an
overlapping function.


PMCID: PMC149301
PMID: 12604548  [Indexed for MEDLINE]


1292. Appl Environ Microbiol. 2003 Mar;69(3):1759-74.

DNA microarray analyses of the long-term adaptive response of Escherichia coli to
acetate and propionate.

Polen T(1), Rittmann D, Wendisch VF, Sahm H.

Author information: 
(1)Forschungszentrum Jülich, Institut für Biotechnologie I, 52425 Jülich,
Germany.

In its natural environment, Escherichia coli is exposed to short-chain fatty
acids, such as acetic acid or propionic acid, which can be utilized as carbon
sources but which inhibit growth at higher concentrations. DNA microarray
experiments revealed expression changes during exponential growth on complex
medium due to the presence of sodium acetate or sodium propionate at a neutral
external pH. The adaptive responses to acetate and propionate were similar and
involved genes in three categories. First, the RNA levels for chemotaxis and
flagellum genes increased. Accordingly, the expression of chromosomal fliC'-'lacZ
and flhDC'-'lacZ fusions and swimming motility increased after adaptation to
acetate or propionate. Second, the expression of many genes that are involved in 
the uptake and utilization of carbon sources decreased, indicating some kind of
catabolite repression by acetate and propionate. Third, the expression of some
genes of the general stress response increased, but the increases were more
pronounced after short-term exposure for this response than for the adaptive
response. Adaptation to propionate but not to acetate involved increased
expression of threonine and isoleucine biosynthetic genes. The gene expression
changes after adaptation to acetate or propionate were not caused solely by
uncoupling or osmotic effects but represented specific characteristics of the
long-term response of E. coli to either compound.


PMCID: PMC150104
PMID: 12620868  [Indexed for MEDLINE]


1293. Biotechnol Prog. 2003 Mar-Apr;19(2):612-23.

Gene array-based identification of changes that contribute to ethanol tolerance
in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant
mutant).

Gonzalez R(1), Tao H, Purvis JE, York SW, Shanmugam KT, Ingram LO.

Author information: 
(1)Department of Chemical Engineering and Department of Food Science and Human
Nutrition, Iowa State University, Ames, Iowa 50011, USA.

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the
production of ethanol. Gene arrays were used to identify expression changes that 
occurred in the mutant, LY01, during directed evolution to improve ethanol
tolerance (defined as extent of growth in the presence of added ethanol).
Expression levels for 205 (5%) of the ORFs were found to differ significantly (p 
< 0.10) between KO11 and LY01 under each of six different growth conditions (p < 
0.000001). Statistical evaluation of differentially expressed genes according to 
various classification schemes identified physiological areas of importance. A
large fraction of differentially expressed ORFs were globally regulated, leading 
to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a 
putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) 
in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the
absence of ethanol. Other differences in gene expression provided additional
clues that permitted experimentation. Tolerance appears to involve increased
metabolism of glycine (higher expression of gcv genes) and increased production
of betaine (higher expression of betIBA and betT encoding betaine synthesis from 
choline and choline uptake, respectively). Addition of glycine (10 mM) increased 
ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition
of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and
KO11 but had no effect on growth in the absence of ethanol. Both glycine and
betaine can serve as protective osmolytes, and this may be the basis of their
beneficial action. In addition, the marAB genes encoding multiple antibiotic
resistance proteins were expressed at higher levels in LY01 as compared to KO11. 
Interestingly, overexpression of marAB in KO11 made this strain more
ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol
tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine
deamination (sdaB) also appear beneficial for LY01. Addition of serine increased 
the growth of LY01 in the presence and absence of ethanol but had no effect on
KO11. Changes in the expression of several genes concerned with the synthesis of 
the cell envelope components were also noted, which may contribute to increased
ethanol tolerance.

DOI: 10.1021/bp025658q 
PMID: 12675606  [Indexed for MEDLINE]


1294. J Bacteriol. 2003 Mar;185(6):2017-21.

Definition of the Escherichia coli MC4100 genome by use of a DNA array.

Peters JE(1), Thate TE, Craig NL.

Author information: 
(1)Howard Hughes Medical Institute, Department of Molecular Biology and Genetics,
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

We have used an Escherichia coli K-12 whole-genome array based on the DNA
sequence of strain MG1655 as a tool to identify deletions in another E. coli K-12
strain, MC4100, by probing the array with labeled chromosomal DNA. Despite the
continued widespread use of MC4100 as an experimental system, the specific
genetic relationship of this strain to the sequenced K-12 derivative MG1655 has
not been resolved. MC4100 was found to contain four deletions, ranging from 1 to 
97 kb in size. The exact nature of three of the deletions was previously
unresolved, and the fourth deletion was altogether unknown.


PMCID: PMC150127
PMID: 12618467  [Indexed for MEDLINE]


1295. J Bacteriol. 2003 Mar;185(6):1831-40.

Analysis of genome plasticity in pathogenic and commensal Escherichia coli
isolates by use of DNA arrays.

Dobrindt U(1), Agerer F, Michaelis K, Janka A, Buchrieser C, Samuelson M,
Svanborg C, Gottschalk G, Karch H, Hacker J.

Author information: 
(1)Institut für Molekulare Infektionsbiologie der Universität Würzburg, 97070
Würzburg, Germany. ulrich.dobrindt@mail.uni-wuerzburg.de

Genomes of prokaryotes differ significantly in size and DNA composition.
Escherichia coli is considered a model organism to analyze the processes involved
in bacterial genome evolution, as the species comprises numerous pathogenic and
commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the
presence and absence of additional DNA elements contributing to specific
virulence traits and also in the presence and absence of additional genetic
information. To analyze the genetic diversity of pathogenic and commensal E. coli
isolates, a whole-genome approach was applied. Using DNA arrays, the presence of 
all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain 
MG1655 was investigated in 26 E. coli isolates, including various extraintestinal
and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion
mutants, and commensal and laboratory strains. Additionally, the presence of
virulence-associated genes of E. coli was determined using a DNA "pathoarray"
developed in our laboratory. The frequency and distributional pattern of genomic 
variations vary widely in different E. coli strains. Up to 10% of the E. coli
K-12-specific ORFs were not detectable in the genomes of the different strains.
DNA sequences described for extraintestinal or intestinal pathogenic E. coli are 
more frequently detectable in isolates of the same origin than in other
pathotypes. Several genes coding for virulence or fitness factors are also
present in commensal E. coli isolates. Based on these results, the conserved E.
coli core genome is estimated to consist of at least 3,100 translatable ORFs. The
absence of K-12-specific ORFs was detectable in all chromosomal regions. These
data demonstrate the great genome heterogeneity and genetic diversity among E.
coli strains and underline the fact that both the acquisition and deletion of DNA
elements are important processes involved in the evolution of prokaryotes.


PMCID: PMC150128
PMID: 12618447  [Indexed for MEDLINE]


1296. Microbiol Mol Biol Rev. 2003 Mar;67(1):86-156, table of contents.

Bacteriophage T4 genome.

Miller ES(1), Kutter E, Mosig G, Arisaka F, Kunisawa T, Rüger W.

Author information: 
(1)Department of Microbiology, North Carolina State University, Raleigh, North
Carolina 27695-7615, USA. eric_miller@ncsu.edu

Phage T4 has provided countless contributions to the paradigms of genetics and
biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene
products. T4 biology and its genomic sequence provide the best-understood model
for modern functional genomics and proteomics. Variations on gene expression,
including overlapping genes, internal translation initiation, spliced genes,
translational bypassing, and RNA processing, alert us to the caveats of purely
computational methods. The T4 transcriptional pattern reflects its dependence on 
the host RNA polymerase and the use of phage-encoded proteins that sequentially
modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, 
anti-sigma, and sigma decoy proteins also act to specify early, middle, and late 
promoter recognition. Posttranscriptional controls by T4 provide excellent
systems for the study of RNA-dependent processes, particularly at the structural 
level. The redundancy of DNA replication and recombination systems of T4 reveals 
how phage and other genomes are stably replicated and repaired in different
environments, providing insight into genome evolution and adaptations to new
hosts and growth environments. Moreover, genomic sequence analysis has provided
new insights into tail fiber variation, lysis, gene duplications, and membrane
localization of proteins, while high-resolution structural determination of the
"cell-puncturing device," combined with the three-dimensional image
reconstruction of the baseplate, has revealed the mechanism of penetration during
infection. Despite these advances, nearly 130 potential T4 genes remain
uncharacterized. Current phage-sequencing initiatives are now revealing the
similarities and differences among members of the T4 family, including those that
infect bacteria other than Escherichia coli. T4 functional genomics will aid in
the interpretation of these newly sequenced T4-related genomes and in broadening 
our understanding of the complex evolution and ecology of phages-the most
abundant and among the most ancient biological entities on Earth.


PMCID: PMC150520
PMID: 12626685  [Indexed for MEDLINE]


1297. Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):1072-7. Epub 2003 Jan 21.

Parallel changes in gene expression after 20,000 generations of evolution in
Escherichiacoli.

Cooper TF(1), Rozen DE, Lenski RE.

Author information: 
(1)Center for Microbial Ecology, Michigan State University, East Lansing, MI
48824, USA. cooperti@msu.edu

Twelve populations of Escherichia coli, derived from a common ancestor, evolved
in a glucose-limited medium for 20,000 generations. Here we use DNA expression
arrays to examine whether gene-expression profiles in two populations evolved in 
parallel, which would indicate adaptation, and to gain insight into the
mechanisms underlying their adaptation. We compared the expression profile of the
ancestor to that of clones sampled from both populations after 20,000
generations. The expression of 59 genes had changed significantly in both
populations. Remarkably, all 59 were changed in the same direction relative to
the ancestor. Many of these genes were members of the cAMP-cAMP receptor protein 
(CRP) and guanosine tetraphosphate (ppGpp) regulons. Sequencing of several genes 
controlling the effectors of these regulons found a nonsynonymous mutation in
spoT in one population. Moving this mutation into the ancestral background showed
that it increased fitness and produced many of the expression changes manifest
after 20,000 generations. The same mutation had no effect on fitness when
introduced into the other evolved population, indicating that a mutation of
similar effect was present already. Our study demonstrates the utility of
expression arrays for addressing evolutionary issues including the quantitative
measurement of parallel evolution in independent lineages and the identification 
of beneficial mutations.

DOI: 10.1073/pnas.0334340100 
PMCID: PMC298728
PMID: 12538876  [Indexed for MEDLINE]


1298. Genome Res. 2003 Feb;13(2):206-15.

A microarray-based antibiotic screen identifies a regulatory role for
supercoiling in the osmotic stress response of Escherichia coli.

Cheung KJ(1), Badarinarayana V, Selinger DW, Janse D, Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,
USA.

Changes in DNA supercoiling are induced by a wide range of environmental stresses
in Escherichia coli, but the physiological significance of these responses
remains unclear. We now demonstrate that an increase in negative supercoiling is 
necessary for transcriptional activation of a large subset of osmotic
stress-response genes. Using a microarray-based approach, we have characterized
supercoiling-dependent gene transcription by expression profiling under
conditions of high salt, in conjunction with the microbial antibiotics
novobiocin, pefloxacin, and chloramphenicol. Algorithmic clustering and
statistical measures for gauging cellular function show that this subset is
enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven
stationary phase adaptation. Transcription factor binding site analysis also
supports regulation by the global stress sigma factor rpoS. In addition, these
studies implicate 60 uncharacterized genes in the osmotic stress regulon, and
offer evidence for a broader role for supercoiling in the control of
stress-induced transcription.

DOI: 10.1101/gr.401003 
PMCID: PMC420364
PMID: 12566398  [Indexed for MEDLINE]


1299. J Bacteriol. 2003 Feb;185(3):1071-81.

Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa.

Firoved AM(1), Deretic V.

Author information: 
(1)Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109-0620, USA.

Erratum in
    J Bacteriol. 2003 Dec;185(23):7029.

Pseudomonas aeruginosa is the dominant pathogen causing chronic respiratory
infections in cystic fibrosis (CF). After an initial phase characterized by
intermittent infections, a chronic colonization is established in CF upon the
conversion of P. aeruginosa to the mucoid, exopolysaccharide
alginate-overproducing phenotype. The emergence of mucoid P. aeruginosa in CF is 
associated with respiratory decline and poor prognosis. The switch to mucoidy in 
most CF isolates is caused by mutations in the mucA gene encoding an anti-sigma
factor. The mutations in mucA result in the activation of the alternative sigma
factor AlgU, the P. aeruginosa ortholog of Escherichia coli extreme stress sigma 
factor sigma(E). Because of the global nature of the regulators of mucoidy, we
have hypothesized that other genes, in addition to those specific for alginate
production, must be induced upon conversion to mucoidy, and their production may 
contribute to the pathogenesis in CF. Here we applied microarray analysis to
identify on the whole-genome scale those genes that are coinduced with the AlgU
sigmulon upon conversion to mucoidy. Gene expression profiles of AlgU-dependent
conversion to mucoidy revealed coinduction of a specific subset of known
virulence determinants (the major protease elastase gene, alkaline
metalloproteinase gene aprA, and the protease secretion factor genes aprE and
aprF) or toxic factors (cyanide synthase) that may have implications for disease 
in CF. Analysis of promoter regions of the most highly induced genes (>40-fold, P
< or = 10(-4)) revealed a previously unrecognized, putative AlgU promoter
upstream of the osmotically inducible gene osmE. This newly identified
AlgU-dependent promoter of osmE was confirmed by mapping the mRNA 5' end by
primer extension. The recognition of genes induced in mucoid P. aeruginosa, other
than those associated with alginate biosynthesis, reported here revealed the
identity of previously unappreciated factors potentially contributing to the
morbidity and mortality caused by mucoid P. aeruginosa in CF.


PMCID: PMC142812
PMID: 12533483  [Indexed for MEDLINE]


1300. Bioinformatics. 2003;19 Suppl 1:i34-43.

Predicting bacterial transcription units using sequence and expression data.

Bockhorst J(1), Qiu Y, Glasner J, Liu M, Blattner F, Craven M.

Author information: 
(1)Department of Computer Sciences, University of Wisconsin, Madison, Wisconsin
53706, USA. joebock@biostat.wisc.edu

MOTIVATION: A key aspect of elucidating gene regulation in bacterial genomes is
identifying the basic units of transcription. We present a method, based on
probabilistic language models, that we apply to predict operons, promoters and
terminators in the genome of Escherichia coli K-12. Our approach has two key
properties: (i) it provides a coherent set of predictions for related regulatory 
elements of various types and (ii) it takes advantage of both DNA sequence and
gene expression data, including expression measurements from inter-genic probes.
RESULTS: Our experimental results show that we are able to predict operons and
localize promoters and terminators with high accuracy. Moreover, our models that 
use both sequence and expression data are more accurate than those that use only 
one of these two data sources.


PMID: 12855435  [Indexed for MEDLINE]


1301. Environ Monit Assess. 2003 Jan-Feb;81(1-3):327-36.

Characterization of microbial communities from coastal waters using microarrays.

Stin OC(1), Carnahan A, Singh R, Powell J, Furuno JP, Dorsey A, Silbergeld E,
Williams HN, Morris JG.

Author information: 
(1)Department of Epidemiology and Preventive Medicine, School of Medicine,
University of Maryland Baltimore, Baltimore, Maryland, USA.
ostin001@umaryland.edu

Molecular methods, including DNA probes, were used to identify and enumerate
pathogenic Vibrio species in the Chesapeake Bay; our data indicated that Vibrio
vulnificus exhibits seasonal fluctuations in number. Our work included a
characterization of total microbial communities from the Bay; development of
microarrays that identify and quantify the diversity of those communities; and
observation of temporal changes in those communities. To identify members of the 
microbial community, we amplified the 16S rDNA gene from community DNA isolated
from a biofilm sample collected from the Chesapeake Bay in February, 2000. The
resultant 75 sequences were 95% or more similar to 7 species including two
recently described Shewanella species, baltica and frigidimarina, that have not
been previously isolated from the Chesapeake. When the genera of bacteria from
biofilm after culturing are compared to those detected by subcloning amplified
16S fragments from community DNA, the cultured sample exhibited a strong bias. In
oysters collected in February, the most common bacteria were previously unknown. 
Based on our 16S findings, we are developing microarrays to detect these and
other microbial species in these estuarine communities. The microarrays will
detect each species using four distinct loci, with the multiple loci serving as
an internal control. The accuracy of the microarray will be measured using
sentinel species such as Aeromonas species, Escherichia coli, and Vibrio
vulnificus. Using microarrays, it should be possible to determine the annual
fluctuations of bacterial species (culturable and non-culturable, pathogenic and 
non-pathogenic). The data may be applied to understanding patterns of
environmental change; assessing the "health" of the Bay; and evaluating the risk 
of human illness associated with exposure to and ingestion of water and
shellfish.


PMID: 12620025  [Indexed for MEDLINE]


1302. Genome Inform. 2003;14:94-103.

Construction of genetic network using evolutionary algorithm and combined fitness
function.

Shin A(1), Iba H.

Author information: 
(1)Dept. of Electronics, School of Engineering, University of Tokyo, Japan.
ando@iba.k.u-tokyo.ac.jp

This paper proposes a method to capture the dynamics in gene expression data
using S-system formalism and construct genetic network models. Our purposed
method exploits the probabilistic heuristic search and divide-and-conquer
approach to estimate the network structure. In evaluating the network structure, 
we attempt a primitive integration of other knowledge to the statistical
criterion. The Z-score is used to analyze the robust and significant parameters
from stochastic search results. We evaluated the proposed method on artificially 
generated data and E.coli mRNA expression data.


PMID: 15706524  [Indexed for MEDLINE]


1303. J Bacteriol. 2003 Jan;185(2):534-43.

FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff
pathway through induction of the methyl-accepting chemotaxis protein Aer.

Prüss BM(1), Campbell JW, Van Dyk TK, Zhu C, Kogan Y, Matsumura P.

Author information: 
(1)Department of Microbiology and Immunology, University of Illinois at Chicago, 
60612-7344, USA. pruess@UIC.EDU

The regulation by two transcriptional activators of flagellar expression (FlhD
and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass 
slide DNA microarrays. An flhD::Kan insertion and an aer deletion were
independently introduced into two Escherichia coli K-12 strains, and the effects 
upon gene regulation were investigated. Altogether, the flhD::Kan insertion
altered the expression of 29 operons of known function. Among them was Aer, which
in turn regulated a subset of these operons, namely, the ones involved in
anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC
repressed enzymes involved in aerobic respiration and regulated many other
metabolic enzymes and transporters in an Aer-independent manner. Expression of 12
genes of uncharacterized function was also affected. FlhD increased gltBD,
gcvTHP, and ompT expression. The regulation of half of these genes was
subsequently confirmed with reporter gene fusions, enzyme assays, and real-time
PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype
MicroArrays and correlated with gene expression.


PMCID: PMC145316
PMID: 12511500  [Indexed for MEDLINE]


1304. J Mol Microbiol Biotechnol. 2003;5(2):105-22.

Comparison of the changes in global gene expression of Escherichia coli induced
by four bactericidal agents.

Shaw KJ(1), Miller N, Liu X, Lerner D, Wan J, Bittner A, Morrow BJ.

Author information: 
(1)Johnson & Johnson Pharmaceutical Research & Development, La Jolla, CA 92121,
USA. kshaw@prdus.jnj.com

DNA microarrays provide a global view of the physiological state of the cell by
parallel analysis of the expression levels of all the genes in an organism. The
effects of four bactericidal agents on the expression pattern of Escherichia coli
MG1655 were assessed. Compounds were chosen on the basis of their different
mechanisms of action and included inhibitors of DNA replication and
recombination, translation, transcription and cell wall biosynthesis. The
addition of rifampin resulted in increased expression of the target, rpoB, as
well as several genes involved in nucleotide salvage and purine biosynthesis. The
addition of ampicillin resulted in overall changes in gene expression that showed
some similarity to changes induced by rifampin. The addition of the antibiotics
kanamycin or norfloxacin resulted in the induction of unique gene expression
signatures: a heat shock response to kanamycin and an SOS response to
norfloxacin. Several genes of unknown function showed expression profiles similar
to the genes associated with the SOS or the heat shock response. Thus, these
profiles define families of genes with similar expression phenotypes that can be 
tested for related function.

Copyright 2003 S. Karger AG, Basel

DOI: 10.1159/000069981 
PMID: 12736533  [Indexed for MEDLINE]


1305. J Mol Microbiol Biotechnol. 2003;5(1):37-45.

Escherichia coli response to exogenous pyrophosphate and analogs.

Biville F(1), Oshima T, Mori H, Kawagoe Y, Bouvet O, Rager MN, Perrotte-Piquemal 
M, Danchin A.

Author information: 
(1)Département de Biochimie et Génétique moléculaire, Institut Pasteur, Paris,
France. fbiville@pasteur.fr

The addition of exogenous pyrophosphate increases the growth yield and cAMP
synthesis in stationary phase when Escherichia coli is grown in minimal medium.
Pyrophosphate increases the yield by altering the enterobactin uptake system. We 
studied the physiological effects and examined how the E. coli transcriptome was 
modified when two structural analogs of pyrophosphate were added to the growth
medium. Methylenediphosphonic acid or a high concentration of iron had the same
positive effects as pyrophosphate on growth yield, cAMP synthesis and the
repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect
these cellular processes significantly. The transcriptome modifications generated
by pyrophosphate or methylenediphosphonic acid were more similar than those
generated by imidodiphosphate or excess iron. The transcriptome data also
indicated that processes other than iron uptake might be involved in the cellular
response to exogenous pyrophosphate or methylenediphosphonic acid.

Copyright 2003 S. Karger AG, Basel

DOI: 10.1159/000068722 
PMID: 12673060  [Indexed for MEDLINE]


1306. Methods Enzymol. 2003;370:278-80.

Analysis of microarray data for the marA, soxS, and rob regulons of Escherichia
coli.

Martin RG(1), Rosner JL.

Author information: 
(1)Laboratory of Molecular Biology, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892-0560, USA.

DOI: 10.1016/S0076-6879(03)70024-X 
PMID: 14712652  [Indexed for MEDLINE]


1307. Methods Mol Biol. 2003;224:61-78.

Escherichia coli spotted double-strand DNA microarrays: RNA extraction, labeling,
hybridization, quality control, and data management.

Khodursky AB(1), Bernstein JA, Peter BJ, Rhodius V, Wendisch VF, Zimmer DP.

Author information: 
(1)Department of Biochemistry, Biophysics and Molecular Biology, University of
Minnesota, St. Paul, USA.

DOI: 10.1385/1-59259-364-X:61 
PMID: 12710666  [Indexed for MEDLINE]


1308. Mol Microbiol. 2003 Jan;47(2):383-96.

Proteome analysis of Escherichia coli K-12 by two-dimensional native-state
chromatography and MALDI-MS.

Champion MM(1), Campbell CS, Siegele DA, Russell DH, Hu JC.

Author information: 
(1)Department of Biochemistry, Texas A & M University, College Station TX
77843-2128, USA.

To identify proteins expressed in Escherichia coli K-12 MG1655 during exponential
growth in defined medium, we separated soluble proteins of E. coli over two
dimensions of native-state high-performance liquid chromatography, and examined
the components of the protein mixtures in each of 380 fractions by peptide mass
fingerprinting. To date, we have identified the products of 310 genes covering a 
wide range of cellular functions. Validation of protein assignments was made by
comparing the assignments of proteins to specific first-dimension fractions to
proteins visualized by two-dimensional gel electrophoresis. Co-fractionation of
proteins suggests the possible identities of components of multiprotein
complexes. This approach yields high-throughput gel-independent identification of
proteins. It can also be used to assign identities to spots visualized by
two-dimensional gels, and should be useful to evaluate differences in expressed
proteome content and protein complexes among strains or between different
physiological states.


PMID: 12519190  [Indexed for MEDLINE]


1309. Pac Symp Biocomput. 2003:41-52.

MOPAC: motif finding by preprocessing and agglomerative clustering from
microarrays.

Ganesh R(1), Siegele DA, Ioerger TR.

Author information: 
(1)Department of Computer Science, Texas A&M University, College Station, TX
77840, USA.

We propose a novel strategy for discovering motifs from gene expression data. The
gene expression data in our experiments comes from DNA Microarray analysis of the
bacterium E. coli in response to recovery from nutrient starvation. We have
annotated the data and identified the upregulated genes. Our interest is to find 
common regulatory motifs that are responsible for the upregulation of these
specific genes. We assume that a common motif that a regulatory protein can bind 
to will be present in the upstream region of the upregulated genes and will not
be present in the upstream regions of genes that showed a constant level of
expression over time. Our objective is to find the common motifs that are present
in at least some of the upstream sequences of upregulated genes and not present
in the control set, which is the set of genes whose expression remained the same.
Because it is possible that there could be several subsets of co-regulated genes 
under different control mechanisms among the co-expressed genes, we do not want
to require motifs to be present in all upregulated sequences. Therefore, we
propose a new algorithm for finding such motifs through stages of pre-processing,
denoising, agglomerative clustering and consensus checking. Through this process,
we have found some motifs that are good candidates for further validation.


PMID: 12603016  [Indexed for MEDLINE]


1310. Nucleic Acids Res. 2002 Dec 15;30(24):5360-8.

Interaction of C5 protein with RNA aptamers selected by SELEX.

Lee JH(1), Kim H, Ko J, Lee Y.

Author information: 
(1)Department of Chemistry, Center for Molecular Design and Synthesis, Korea
Advanced Institute of Science and Technology, Daejeon 305-701, South Korea.

RNA aptamers binding to C5 protein, the protein component of Escherichia coli
RNase P, were selected and characterized as an initial step in elucidating the
mechanism of action of C5 protein as an RNA-binding protein. Sequence analyses of
the RNA aptamers suggest that C5 protein binds various RNA molecules with
dissociation constants comparable to that of M1 RNA, the RNA component of RNase
P. The dominant sequence, W2, was chosen for further study. Interactions between 
W2 and C5 protein were independent of Mg2+, in contrast to the Mg2+ dependency of
M1 RNA-C5 protein interactions. The affinity of W2 for C5 protein increased with 
increasing concentration of monovalent NH4+, suggesting interactions via
hydrophobic attraction. W2 forms a fairly stable complex with C5 protein,
although the stability of this complex is lower than that of the complex of M1
RNA with C5 protein. The core RNA motif essential for interaction with C5 protein
was identified as a stem-loop structure, comprising a 5 bp stem and a 20 nt loop.
Our results strongly imply that C5 protein is an interacting partner protein of
some cellular RNA species apart from M1 RNA.


PMCID: PMC140078
PMID: 12490703  [Indexed for MEDLINE]


1311. J Biol Chem. 2002 Dec 13;277(50):48558-64. Epub 2002 Sep 24.

RNA binding properties of the AU-rich element-binding recombinant
Nup475/TIS11/tristetraprolin protein.

Worthington MT(1), Pelo JW, Sachedina MA, Applegate JL, Arseneau KO, Pizarro TT.

Author information: 
(1)Digestive Health Center of Excellence, University of Virginia Health Sciences 
Center, Charlottesville 22908, USA. mtw3p@virginia.edu

Regulation of messenger RNA stability by AU-rich elements is an important means
of regulating genes induced by growth factors and cytokines. Nup475 (also known
as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding
Cys(3)His motif proteins required for proper regulation of tumor necrosis factor 
mRNA stability in macrophages. We developed an Escherichia coli expression system
to produce soluble Nup475 protein in quantity to study its RNA binding
properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a
broad range of pH and salt concentrations by RNA gel shift. This binding was
inhibited by excess zinc metal, providing a potential mechanism for previous
reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs.
Immobilized Nup475 protein was used to select its optimal binding site by RNA
SELEX and revealed a strong preference for the extended sequence UUAUUUAUU,
rather than a simple AUUUA motif. These findings were confirmed by site-directed 
mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos,
interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity
toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially
disrupt the Nup475-tumor necrosis factor AU-rich element complex.

DOI: 10.1074/jbc.M206505200 
PMID: 12324455  [Indexed for MEDLINE]


1312. Ann Periodontol. 2002 Dec;7(1):8-16.

Bacterial diversity in necrotizing ulcerative periodontitis in HIV-positive
subjects.

Paster BJ(1), Russell MK, Alpagot T, Lee AM, Boches SK, Galvin JL, Dewhirst FE.

Author information: 
(1)Department of Molecular Genetics, The Forsyth Institute, Boston, Massachusetts
02115, USA. bpaster@forsyth.org

BACKGROUND: Necrotizing ulcerative periodontitis (NUP) is a painful and
potentially debilitating affliction that affects about 2% to 6% of HIV-positive
subjects. NUP may be caused by specific microorganisms that are presently unknown
or by microbial species not usually thought to cause periodontal infections. The 
purpose of this study was to define the bacterial species associated with NUP in 
HIV-positive patients.
METHODS: 16S rRNA bacterial genes of DNA isolated from subgingival plaque of 8
HIV-positive subjects with NUP were amplified by polymerase chain reaction (PCR) 
and cloned into Escherichia coli. The sequences of cloned inserts were used to
determine species identity or closest relatives by comparison with known
sequences. The microbial profiles in subgingival plaque of subjects with NUP,
chronic periodontitis, and periodontal health were compared using a battery of
over 200 oligonucleotide probes in a PCR-based, reverse-capture, checkerboard
DNA-DNA hybridization assay.
RESULTS: Sequence analysis of over 400 clones revealed 108 species; 65 were
"uncultivable" phylotypes, of which 26 were novel to NUP subjects. Species or
phylotypes most commonly detected were Bulleidia extructa, Dialister,
Fusobacterium, Selenomonas, Peptostreptococcus, Veillonella, and the phylum TM7. 
Based on sequence analysis and checkerboard analysis, NUP did not possess the
classical periodontal pathogens such as Porphyromonas gingivalis. Otherwise, the 
microbial profiles of NUP and periodontitis had many similarities. The microbial 
profiles of subgingival plaque from periodontally healthy subjects were different
and less complex in comparison to the profiles of both disease groups.
CONCLUSIONS: Certain species appear to be associated with health and periodontal 
diseases. The putative pathogens associated with periodontal disease in
HIV-negative subjects are not associated with NUP in HIV-positive subjects.

DOI: 10.1902/annals.2002.7.1.8 
PMID: 16013212  [Indexed for MEDLINE]


1313. J Bacteriol. 2002 Dec;184(23):6725-9.

DNA microarray analysis of the expression profile of Escherichia coli in response
to treatment with 4,5-dihydroxy-2-cyclopenten-1-one.

Phadtare S(1), Kato I, Inouye M.

Author information: 
(1)Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway,
New Jersey 08854, USA.

We carried out DNA microarray-based global transcript profiling of Escherichia
coli in response to 4,5-dihydroxy-2-cyclopenten-1-one to explore the
manifestation of its antibacterial activity. We show that it has widespread
effects in E. coli affecting genes encoding proteins involved in cell metabolism 
and membrane synthesis and functions. Genes belonging to the regulon involved in 
synthesis of Cys are upregulated. In addition, rpoS and RpoS-regulated genes
responding to various stresses and a number of genes responding to oxidative
stress are upregulated.


PMCID: PMC135408
PMID: 12426362  [Indexed for MEDLINE]


1314. J Bacteriol. 2002 Dec;184(23):6551-8.

Gene expression profiling of the pH response in Escherichia coli.

Tucker DL(1), Tucker N, Conway T.

Author information: 
(1)Advanced Center for Genome Technology, The University of Oklahoma, Norman,
Oklahoma 73069-0245, USA.

Escherichia coli MG1655 acid-inducible genes were identified by whole-genome
expression profiling. Cultures were grown to the mid-logarithmic phase on
acidified glucose minimal medium, conditions that induce glutamate-dependent acid
resistance (AR), while the other AR systems are either repressed or not induced. 
A total of 28 genes were induced in at least two of three experiments in which
the gene expression profiles of cells grown in acid (pH 5.5 or 4.5) were compared
to those of cells grown at pH 7.4. As expected, the genes encoding glutamate
decarboxylase, gadA and gadB, were significantly induced. Interestingly, two
acid-inducible genes code for small basic proteins with pIs of >10.5, and six
code for small acidic proteins with pIs ranging from 5.7 to 4.0; the roles of
these small basic and acidic proteins in acid resistance are unknown. The
acid-induced genes represented only five functional grouping categories,
including eight genes involved in metabolism, nine associated with cell envelope 
structures or modifications, two encoding chaperones, six regulatory genes, and
six unknown genes. It is unlikely that all of these genes are involved in the
glutamate-dependent AR. However, nine acid-inducible genes are clustered in the
gadA region, including hdeA, which encodes a putative periplasmic chaperone, and 
four putative regulatory genes. One of these putative regulators, yhiE, was shown
to significantly increase acid resistance when overexpressed in cells that had
not been preinduced by growth at pH 5.5, and mutation of yhiE decreased acid
resistance; yhiE could therefore encode an activator of AR genes. Thus, the
acid-inducible genes clustered in the gadA region appear to be involved in
glutatmate-dependent acid resistance, although their specific roles remain to be 
elucidated.


PMCID: PMC135413
PMID: 12426343  [Indexed for MEDLINE]


1315. Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14646-51. Epub 2002 Nov 4.

S-layer-streptavidin fusion proteins as template for nanopatterned molecular
arrays.

Moll D(1), Huber C, Schlegel B, Pum D, Sleytr UB, Sára M.

Author information: 
(1)Center for Ultrastructure Research and Ludwig Boltzmann Institute for
Molecular Nanotechnology, University of Agricultural Sciences, A-1180 Vienna,
Austria.

Biomolecular self-assembly can be used as a powerful tool for nanoscale
engineering. In this paper, we describe the development of building blocks for
nanobiotechnology, which are based on the fusion of streptavidin to a crystalline
bacterial cell surface layer (S-layer) protein with the inherent ability to
self-assemble into a monomolecular protein lattice. The fusion proteins and
streptavidin were produced independently in Escherichia coli, isolated, and mixed
to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled
chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers,
and on accessory cell wall polymer containing cell wall fragments. The
two-dimensional protein crystals displayed streptavidin in defined repetitive
spacing, and they were capable of binding d-biotin and biotinylated proteins.
Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned
molecular affinity matrix to arrange biotinylated compounds on a surface. In
addition, it has application potential as a functional coat of liposomes.

DOI: 10.1073/pnas.232299399 
PMCID: PMC137473
PMID: 12417763  [Indexed for MEDLINE]


1316. Allergy. 2002 Nov;57(11):1053-8.

Severe IgE-mediated anaphylaxis following consumption of fried frog legs:
definition of alpha-parvalbumin as the allergen in cause.

Hilger C(1), Grigioni F, Thill L, Mertens L, Hentges F.

Author information: 
(1)Unit of Immunology and Allergology, Centre Hospitalier de Luxembourg, 4 rue
Barblé, L-1210 Luxembourg. hilger.christiane@chl.lu

BACKGROUND: IgE-mediated allergic reactions to bullfrog and edible frog have been
reported. The implicated allergens have not been defined so far. The frog
material and the patient's serum from a case of severe food-induced anaphylaxis
were used to define the implicated allergen at the protein and DNA level.
METHODS: Immunoblotting techniques and N-terminal protein microsequencing were
used to define the allergen recognized by the patient's serum. Back translation
from the identified protein sequence was used to design degenerated primers to
amplify the allergen's cDNA by polymerase chain reaction (PCR). We defined the
nucleotide sequence of the allergen from the frog of Indonesian origin that was
consumed by the patient, and the homologous cDNA from Rana esculenta.
RESULTS: Protein microsequencing revealed that the implicated frog allergen
belonged to the parvalbumin family. cDNAs coding for alpha- and beta-parvalbumin 
of R. esculenta and Rana species were cloned. Recombinant proteins were expressed
in Escherichia coli. The patient's serum IgE antibodies recognized parvalbumin
prepared from frog muscle and recombinant alpha-parvalbumin from R. species but
not from R. esculenta. Recombinant beta-parvalbumin was not recognized by the IgE
antibodies.
CONCLUSION: This work defines at the protein and DNA levels alpha-parvalbumin as 
the allergen implicated in a case of IgE-mediated anaphylaxis to frog muscle. It 
also shows that a protein belonging to the parvalbumin family is implicated in
type I allergies outside the fish species.


PMID: 12359003  [Indexed for MEDLINE]


1317. Environ Microbiol. 2002 Nov;4(11):735-43.

Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays
using surface plasmon resonance imaging.

Nelson BP(1), Liles MR, Frederick KB, Corn RM, Goodman RM.

Author information: 
(1)Department of Chemistry, University of Wisconsin-Madison, 1101 University
Avenue, Madison, WI 53706-1396, USA.

In this paper, we describe the detection of bacterial cell-extracted 16S
ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance
(SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of
molecular interactions on surfaces in response to changes in the index of
refraction, therefore eliminating the need for a fluorescent or radioactive
label. A variation of the more common SPR techniques, SPR imaging enables
detection from multiple probes in a reusable array format. The arrays developed
here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA
gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly
conserved sequence found in rRNAs from most members of the domain Bacteria. We
report species-specific hybridization of cell-extracted total RNA and in vitro
transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple 
probe sequences for each species, and found that success or failure of
hybridization was dependent upon probe position in the 16S rRNA molecule. It was 
also determined that one of the probes intended to bind 16S rRNA also bound an
unknown protein. The amount of binding to these probes was quantified with SPR
imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E.
coli total cellular RNA under the experimental conditions used. These results
indicate the feasibility of using SPR imaging for 16S rRNA identification and
encourage further development of this method for direct detection of other RNA
molecules.


PMID: 12460281  [Indexed for MEDLINE]


1318. J Bacteriol. 2002 Nov;184(22):6225-34.

Escherichia coli gene expression responsive to levels of the response regulator
EvgA.

Masuda N(1), Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,
USA.

To investigate the function of the EvgA response regulator, we compared the
genome-wide transcription profile of EvgA-overexpressing and EvgA-lacking
Escherichia coli strains by oligonucleotide microarrays. The microarray
measurements allowed the identification of at least 37 EvgA-activated genes,
including acid resistance-related genes gadABC and hdeAB, efflux pump genes yhiUV
and emrK, and 21 genes with unknown function. EvgA overexpression conferred acid 
resistance to exponentially growing cells. This acid resistance was abolished by 
deletion of ydeP, ydeO, or yhiE, which was induced by EvgA overexpression. These 
results suggest that ydeP, ydeO, and yhiE are novel genes related to acid
resistance and that EvgA regulates several acid resistance genes. Furthermore,
the deletion of yhiE completely abolished acid resistance in stationary-phase
cells, suggesting that YhiE plays a critical role in stationary-phase acid
resistance. The multidrug resistance in an acrB deletion mutant caused by EvgA
overexpression was completely abolished by deletion of yhiUV, while the emrKY
deletion had no effect on the increase in resistance by EvgA overexpression. In
addition, EvgA overexpression did not confer resistance in a tolC-deficient
strain. These results suggest that YhiUV induced by EvgA overexpression is
functionally associated with TolC and contributes to multidrug resistance.


PMCID: PMC151933
PMID: 12399493  [Indexed for MEDLINE]


1319. J Biol Chem. 2002 Oct 25;277(43):40309-23. Epub 2002 Jul 18.

Global gene expression profiling in Escherichia coli K12. The effects of
leucine-responsive regulatory protein.

Hung SP(1), Baldi P, Hatfield GW.

Author information: 
(1)Department of Microbiology and Molecular Genetics, College of Medicine,
University of California, Irvine, CA 92697, USA.

Leucine-responsive regulatory protein (Lrp) is a global regulatory protein that
affects the expression of multiple genes and operons in bacteria. Although the
physiological purpose of Lrp-mediated gene regulation remains unclear, it has
been suggested that it functions to coordinate cellular metabolism with the
nutritional state of the environment. The results of gene expression profiles
between otherwise isogenic lrp(+) and lrp(-) strains of Escherichia coli support 
this suggestion. The newly discovered Lrp-regulated genes reported here are
involved either in small molecule or macromolecule synthesis or degradation, or
in small molecule transport and environmental stress responses. Although many of 
these regulatory effects are direct, others are indirect consequences of
Lrp-mediated changes in the expression levels of other global regulatory
proteins. Because computational methods to analyze and interpret high dimensional
DNA microarray data are still an early stage, much of the emphasis of this work
is directed toward the development of methods to identify differentially
expressed genes with a high level of confidence. In particular, we describe a
Bayesian statistical framework for a posterior estimate of the standard deviation
of gene measurements based on a limited number of replications. We also describe 
an algorithm to compute a posterior estimate of differential expression for each 
gene based on the experiment-wide global false positive and false negative level 
for a DNA microarray data set. This allows the experimenter to compute posterior 
probabilities of differential expression for each individual differential gene
expression measurement.

DOI: 10.1074/jbc.M204044200 
PMID: 12130640  [Indexed for MEDLINE]


1320. Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13471-6. Epub 2002 Oct 8.

Adaptation to famine: a family of stationary-phase genes revealed by microarray
analysis.

Tani TH(1), Khodursky A, Blumenthal RM, Brown PO, Matthews RG.

Author information: 
(1)Biophysics Research Division and Department of Biological Chemistry,
University of Michigan, Ann Arbor, MI 48109, USA.

Bacterial adaptation to nutrient limitation and increased population densities is
central to survival and virulence. Surprisingly, <3% of Escherichia coli genes
are known to play roles specific to the stationary phase. There is evidence that 
the leucine-responsive regulatory protein (Lrp) may play an important role in
stationary phase, so this study used microarrays representing >98% of E. coli
genes to more comprehensively identify those controlled by Lrp. The primary
analysis compared isogenic Lrp(+) and Lrp(-) strains in cells growing in steady
state in glucose minimal medium, either in the presence or absence of leucine.
More than 400 genes were significantly Lrp-responsive under the conditions used. 
Transcription of 147 genes was lower in Lrp(+) than in Lrp(-) cells whether or
not leucine was present; most of these genes were tightly coregulated under
several conditions, including a burst of synthesis on transition to stationary
phase. This cluster includes 56 of 115 genes already known to play roles in
stationary phase. Our results suggest that the actual number of genes induced on 
entrance into stationary phase is closer to 200 and that Lrp affects nearly
three-quarters of them, including genes involved in response to nutrient
limitation, high concentrations of organic acids, and osmotic stress.

DOI: 10.1073/pnas.212510999 
PMCID: PMC129697
PMID: 12374860  [Indexed for MEDLINE]


1321. J Bacteriol. 2002 Oct;184(19):5502-7.

Profiling early osmostress-dependent gene expression in Escherichia coli using
DNA macroarrays.

Weber A(1), Jung K.

Author information: 
(1)Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück,
D-49069 Osnabrück, Germany.

DNA macroarray technology was used to monitor early transcriptional alterations
of Escherichia coli in response to an osmotic upshift imposed by the addition of 
0.4 M NaCl. Altered mRNA levels of 152 genes were detected; 45 genes showed
increased expression while the expression of the remaining 107 genes was reduced.
Northern blot analysis of several selected genes differing in their relative
expression values confirmed the results obtained by the array technology.


PMCID: PMC135335
PMID: 12218039  [Indexed for MEDLINE]


1322. Mol Cell Probes. 2002 Oct;16(5):371-8.

Virulence typing of Escherichia coli using microarrays.

van Ijperen C(1), Kuhnert P, Frey J, Clewley JP.

Author information: 
(1)Molecular Biology Unit, Central Public Health Laboratory, 61 Colindale Avenue,
London, NW9 5HT, UK.

We describe a microarray based broad-range screening technique for Escherichia
coli virulence typing. Gene probes were amplified by PCR from a plasmid bank of
characterised E. coli virulence genes and were spotted onto a glass slide to form
an array of capture probes. Genomic DNA from E. coli strains which were to be
tested for the presence of these virulence gene sequences was labelled with
fluorescent cyanine dyes by random amplification and then hybridised against the 
array of probes. The hybridisation, washing and data analysis conditions were
optimised for glass slides, and the applicability of the method for identifying
the presence of the virulence genes was determined using reference strains and
clinical isolates. It was found to be a sensitive screening method for detecting 
virulence genes, and a powerful tool for determining the pathotype of E. coli. It
will be possible to expand and automate this microarray technique to make it
suitable for rapid and reliable diagnostic screening of bacterial isolates.


PMID: 12477441  [Indexed for MEDLINE]


1323. Mol Cell Probes. 2002 Oct;16(5):341-50.

Development of a membrane-array method for the detection of human intestinal
bacteria in fecal samples.

Wang RF(1), Kim SJ, Robertson LH, Cerniglia CE.

Author information: 
(1)Division of Microbiology, National Center for Toxicological Research, Food and
Drug Administration, 3900 NCTR Rd. Jefferson, AR 72079, USA. rwang@nctr.fda.gov

A membrane-array method was developed for the detection of human intestinal
bacteria in fecal samples without using the expensive microarray-arrayer and
laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal
bacterial species were used to design oligonucleotide probes. Three 40-mer
oligonucleotides specific for each bacterial species (total 60 probes) were
synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled
16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal
samples or pure cultured bacteria using two universal primers, and were
hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP
color development. The 20 intestinal bacterial species tested were Bacteroides
thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium
clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus
productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium 
longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens,
Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two
universal primers were able to amplify full size 16S rDNA from all of the 20
bacterial species tested. The hybridization results indicated that the
membrane-array method is a reliable technique for the detection of predominant
human intestinal bacteria in the fecal samples. The result was also confirmed by 
using specific PCR methods for these bacteria.


PMID: 12477438  [Indexed for MEDLINE]


1324. Mol Microbiol. 2002 Oct;46(1):281-91.

Transcriptome analysis of all two-component regulatory system mutants of
Escherichia coli K-12.

Oshima T(1), Aiba H, Masuda Y, Kanaya S, Sugiura M, Wanner BL, Mori H, Mizuno T.

Author information: 
(1)Research and Education Center for Genetic Information, Nara Institute of
Science and Technology, Ikoma 630-0101, Japan.

We have systematically examined the mRNA profiles of 36 two-component deletion
mutants, which include all two-component regulatory systems of Escherichia coli, 
under a single growth condition. DNA microarray results revealed that the mutants
belong to one of three groups based on their gene expression profiles in
Luria-Bertani broth under aerobic conditions: (i) those with no or little change;
(ii) those with significant changes; and (iii) those with drastic changes. Under 
these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive
EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes.
Cellular functions such as flagellar synthesis and expression of the RpoS regulon
were affected by multiple two-component systems. A high correlation coefficient
of expression profile was found between several two-component mutants. Together, 
these results support the view that a network of functional interactions, such as
cross-regulation, exists between different two-component systems. The compiled
data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/
2_components).


PMID: 12366850  [Indexed for MEDLINE]


1325. Plant Cell. 2002 Oct;14(10):2325-38.

Rose scent: genomics approach to discovering novel floral fragrance-related
genes.

Guterman I(1), Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G, Bar E,
Davydov O, Ovadis M, Emanuel M, Wang J, Adam Z, Pichersky E, Lewinsohn E, Zamir
D, Vainstein A, Weiss D.

Author information: 
(1)Institute of Plant Sciences and Genetics in Agriculture, Faculty of
Agricultural, Food, and Environmental Quality Sciences, Hebrew University of
Jerusalem, P.O. Box 12, Rehovot 76100, Israel.

Erratum in
    Plant Cell. 2002 Nov;14(11):2975..

Comment in
    Plant Cell. 2002 Oct;14(10):2315-7.

For centuries, rose has been the most important crop in the floriculture
industry; its economic importance also lies in the use of its petals as a source 
of natural fragrances. Here, we used genomics approaches to identify novel
scent-related genes, using rose flowers from tetraploid scented and nonscented
cultivars. An annotated petal EST database of approximately 2100 unique genes
from both cultivars was created, and DNA chips were prepared and used for
expression analyses of selected clones. Detailed chemical analysis of volatile
composition in the two cultivars, together with the identification of secondary
metabolism-related genes whose expression coincides with scent production, led to
the discovery of several novel flower scent-related candidate genes. The function
of some of these genes, including a germacrene D synthase, was biochemically
determined using an Escherichia coli expression system. This work demonstrates
the advantages of using the high-throughput approaches of genomics to detail
traits of interest expressed in a cultivar-specific manner in nonmodel plants.
EST sequences were submitted to the GenBank database (accession numbers BQ 103855
to BQ 106728).


PMCID: PMC151220
PMID: 12368489  [Indexed for MEDLINE]


1326. Bioinformatics. 2002 Sep;18(9):1153-61.

Genome structures embossed by oligonucleotide-stickiness.

Nishigaki K(1), Saito A.

Author information: 
(1)Department of Functional Materials Science, Saitama University, 255
Shimo-Okubo, Saitama, Saitama 338-8570, Japan. koichi@fms.saitama-u.ac.jp

MOTIVATION: An unmanageably large amount of data on genome sequences is
accumulating, prompting researchers to develop new methods to analyze them. We
have devised a novel method designated oligostickiness, a measure roughly
proportional to the binding affinity of an oligonucleotide to a DNA of interest, 
in order to analyze genome sequences as a whole.
RESULTS: Fifteen representative genomes such as Bacillus subtilis, Escherichia
coli, Saccharomyces cerevisiae, Caenorhabditis elegans, H. sapiens and others
were analyzed by this method using more than 50 probe dodecanucleotides, offering
the following findings: (i) Genome sequences can be specifically featured by way 
of oligostickiness maps. (ii) Oligostickiness analysis, which is similar to but
more informative than (G + C) content or repetitive sequence analysis, can reveal
intra-genomic structures such as mosaic structures (E. coli and B. subtilis) and 
highly sticky/non-sticky regions of biological meanings. (iii) Some probe
oligonucleotides such as dC(12) and dT(12) can be used for classifying genomes,
clearly discriminating prokaryotes and eukaryotes. (iv) Based on global
oligostickiness, which is the average value of the local oligostickinesses, the
features of a genome could be visualized in spider web mode. The pattern of a
spider web as well as a set of oligostickiness maps is highly characteristic to
each genome or chromosome. Thus, we called it as chromosome texture, leading to a
finding that all the chromosomes contained in a cell, so far investigated, have a
common texture.
AVAILABILITY: Oligostickinesses maps used in this work are available at
http://gp.fms.saitama-u.ac.jp/
CONTACT: koichi@fms.saitama-u.ac.jp


PMID: 12217906  [Indexed for MEDLINE]


1327. J Bacteriol. 2002 Sep;184(17):4846-56.

Identification of a DtxR-regulated operon that is essential for
siderophore-dependent iron uptake in Corynebacterium diphtheriae.

Qian Y(1), Lee JH, Holmes RK.

Author information: 
(1)Department of Microbiology, University of Colorado Health Sciences Center,
4200 East Ninth Avenue, Denver, CO 80262, USA.

The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits
transcription from iron-regulated promoters (IRPs) in Corynebacterium
diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a
SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of
appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia
coli system was iron dependent. The open reading frames (ORFs) downstream from
IRP6 and previously described promoter IRP1 were found to encode proteins
homologous to components of ATP-binding cassette (ABC) transport systems involved
in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed
under high-iron conditions in wild-type C. diphtheriae C7(beta), but they were
expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which
were shown previously to be defective in corynebactin-dependent iron uptake. A
clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive
corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a 
clone of the wild-type irp1 operon failed to complement any of these strains.
Complementation by subclones of pCM6ABC demonstrated that mutant alleles of
irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5,
respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single 
amino acid substitutions in their predicted protein products, and the irp6C
allele in HC4 caused premature chain termination of its predicted protein
product. Strain HC3 was found to have a chain-terminating mutation in dtxR in
addition to a missense mutation in its irp6B allele. These findings demonstrated 
that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that
specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in
corynebactin-dependent iron uptake, and that complementation of these mutant
alleles restores repression of corynebactin production under high-iron growth
conditions, most likely as a consequence of restoring siderophore-dependent iron 
uptake mediated by the irp6 operon.


PMCID: PMC135300
PMID: 12169610  [Indexed for MEDLINE]


1328. Mol Microbiol. 2002 Sep;45(6):1575-88.

Amplification of Hot DNA segments in Escherichia coli.

Kodama K(1), Kobayashi T, Niki H, Hiraga S, Oshima T, Mori H, Horiuchi T.

Author information: 
(1)National Institute for Basic Biology, Myodaiji, Okazaki, Aichi, Japan.

In Escherichia coli, a replication fork blocking event at a DNA replication
terminus (Ter) enhances homologous recombination at the nearby sister chromosomal
region, converting the region into a recombination hotspot, Hot, site. Using a
RNaseH negative (rnhA-) mutant, we identified eight kinds of Hot DNAs (HotA-H).
Among these, enhanced recombination of three kinds of Hot DNAs (HotA-C) was
dependent on fork blocking events at Ter sites. In the present study, we examined
whether HotA DNAs are amplified when circular DNA (HotA plus a drug-resistance
DNA) is inserted into the homologous region on the chromosome of a rnhA- mutant. 
The resulting HotA DNA transformants were analysed using pulsed-field gel
electrophoresis, fluorescence in situ hybridization and DNA microarray technique.
The following results were obtained: (i) HotA DNA is amplified by about 40-fold
on average; (ii) whereas 90% of the cells contain about 6-10 copies of HotA DNA, 
the remaining 10% of cells have as many as several hundred HotA copies; and (iii)
amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs
are amplified to the same level as HotA. Furthermore, HotL DNA, which is
activated by blocking the clockwise oriC-starting replication fork at the
artificially inserted TerL site in the fork-blocked strain with a rnhA+
background, is also amplified, but is not amplified in the non-blocked strain.
From these data, we propose a model that can explain production of three distinct
forms of Hot DNA molecules by the following three recombination pathways: (i)
unequal intersister recombination; (ii) intrasister recombination, followed by
rolling-circle replication; and (iii) intrasister recombination, producing
circular DNA molecules.


PMID: 12354226  [Indexed for MEDLINE]


1329. Nucleic Acids Res. 2002 Sep 1;30(17):3732-8.

Transcriptome analysis of Escherichia coli using high-density oligonucleotide
probe arrays.

Tjaden B(1), Saxena RM, Stolyar S, Haynor DR, Kolker E, Rosenow C.

Author information: 
(1)Department of Computer Science, University of Washington, Seattle, WA 98195,
USA.

Microarrays traditionally have been used to analyze the expression behavior of
large numbers of coding transcripts. Here we present a comprehensive approach for
high-throughput transcript discovery in Escherichia coli focused mainly on
intergenic regions which, together with analysis of coding transcripts, provides 
us with a more complete insight into the organism's transcriptome. Using a whole 
genome array, we detected expression for 4052 coding transcripts and identified
1102 additional transcripts in the intergenic regions of the E.coli genome.
Further classification reveals 317 novel transcripts with unknown function. Our
results show that, despite sophisticated approaches to genome annotation, many
cellular transcripts remain unidentified. Through the experimental identification
of all RNAs expressed under a specific condition, we gain a more thorough
understanding of all cellular processes.


PMCID: PMC137427
PMID: 12202758  [Indexed for MEDLINE]


1330. Ren Fail. 2002 Sep;24(5):567-75.

Cytokine expression in the renal tubular epithelial cells stimulated by Shiga
toxin 2 of Escherichia coli O157:H7.

Lee JE(1), Kim JS, Choi IH, Tagawa M, Kohsaka T, Jin DK.

Author information: 
(1)Department of Clinical Research Center, Samsung Biomedical Research Institute,
Seoul, Korea.

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure
in children worldwide. Shiga toxin (Stx) associated HUS, the most common type, is
now known to be caused by Escherichia coli O157:7, which produces Stxl or the
more potent, Stx2. Since the renal tubule is the major tissue affected in the
course of HUS and Stx2 is known to be toxic to the renal tubular cells (RTC), we 
attempted to elucidate the mechanism of renal injury in HUS by studying the
alteration of cytokines in the RTC evoked by Stx2. cDNA-array is a powerful tool 
for evaluating changes in the expression of a group of critical genes and also
gives insights on the overview of the gene activation. In this study, we purified
Stx2 from the E. coli O157:7, which was isolated from a typical
diarrhea-associated HUS patient and then tried to compare the cytokine gene
expression between the stimulated RTC and un-stimulated RTC using cDNA-array. Our
results showed that one third of the examined cytokine genes were up regulated at
least twice by the addition of Vtx2. These up-regulated genes represented the
chemokines (macrophage related cytokines), fibrosis-related cytokine (TNF, PDGF) 
and leukemia inhibitory factors. However, the expression of IL-6, one of the
pleiotropic cytokines, was significantly decreased and this finding was confirmed
by northern analysis. Our results suggest that VT2 up-regulates the
pro-inflammatory cytokines and fibrosis prone growth factors in RTC and that the 
inhibition of the activation of these cytokines may ameliorate the renal tubular 
injury in the HUS caused by E. coli O157:7.


PMID: 12380901  [Indexed for MEDLINE]


1331. FEMS Microbiol Lett. 2002 Aug 6;213(2):175-82.

Design and evaluation of oligonucleotide-microarray method for the detection of
human intestinal bacteria in fecal samples.

Wang RF(1), Beggs ML, Robertson LH, Cerniglia CE.

Author information: 
(1)Microbiology Division, National Center for Toxicological Research, US-FDA,
Jefferson, AR 72079, USA. rwang@nctr.fda.gov

An oligonucleotide-microarray method was developed for the detection of
intestinal bacteria in fecal samples collected from human subjects. The 16S rDNA 
sequences of 20 predominant human intestinal bacterial species were used to
design oligonucleotide probes. Three 40-mer oligonucleotides specific for each
bacterial species (total 60 probes) were synthesized and applied to glass slides.
Cyanine5 (CY5)-labeled 16S rDNAs were amplified by polymerase chain reaction
(PCR) from human fecal samples or bacterial DNA using two universal primers and
were hybridized to the oligo-microarray. The 20 intestinal bacterial species
tested were Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides
fragilis, Bacteroides distasonis, Clostridium clostridiiforme, Clostridium
leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus
obeum, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus albus,
Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis,
Eubacterium biforme, Eubacterium aerofaciens, Lactobacillus acidophilus,
Escherichia coli, and Enterococcus faecium. The two universal primers were able
to amplify full size 16S rDNA from all of the 20 bacterial species tested. The
hybridization results indicated that the oligo-microarray method developed in
this study is a reliable method for the detection of predominant human intestinal
bacteria in the fecal samples.


PMID: 12167534  [Indexed for MEDLINE]


1332. Antonie Van Leeuwenhoek. 2002 Aug;81(1-4):15-25.

Gene expression analysis of the response by Escherichia coli to seawater.

Rozen Y(1), Larossa RA, Templeton LJ, Smulski DR, Belkin S.

Author information: 
(1)Fredy and Nadine Herrmann Graduate School of Applied Science, Hebrew
University, Jerusalem, Israel.

Gene expression of Escherichia coli cells exposed to seawater for 20 h was
compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA
microarray technology. The expression of most (ca. 3,000) of the 4,228 open
reading frames on the microarray remained unchanged; the relative expression of
about 320 genes decreased in seawater, whereas that of ca. one fourth (937)
increased. Clearly coherent expression patterns were observed for several
functional gene groups. Induced genes were numerous in groups specifying the
degradation of small molecules (carbon compounds, amino acids and fatty acids),
energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and 
TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures
and phage related functions. Repressed genes were clustered in two groups, cell
division and nucleotides biosynthesis, indicating a cessation of growth.


PMID: 12448701  [Indexed for MEDLINE]


1333. Mol Genet Genomics. 2002 Aug;267(6):721-9. Epub 2002 Jun 21.

DNA microarray analysis of fim mutations in Escherichia coli.

Schembri MA(1), Ussery DW, Workman C, Hasman H, Klemm P.

Author information: 
(1)Microbial Adhesion Group, Section of Molecular Microbiology, Bldg. 301,
BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark.

Bacterial adhesion is often mediated by complex polymeric surface structures
referred to as fimbriae. Type 1 fimbriae of Escherichia coli represent the
archetypical and best characterised fimbrial system. These adhesive organelles
mediate binding to D-mannose and are directly associated with virulence in the
urinary tract. A typical type 1 fimbriated bacterium has up to 500 fimbriae on
its surface, with each fimbria consisting of approximately 1000 individual
subunits. This equates to approximately 8% of the total cellular protein and is
potentially a significant resource drain for the cell. Here we have used DNA
microarray analysis to examine the molecular events involved in response to
fimbrial gene expression in E. coli K-12. Observed differential expression levels
of the fim genes were in good agreement with our current knowledge of the
stoichiometry of type 1 fimbriae. Changes in fim expression correlated directly
with alterations in colony morphology. Deletion of the entire fim gene cluster
resulted in the converse expression of another surface protein Antigen 43 (Ag43).
Specific deletion of the fimH gene did not affect expression of other fim genes
or Ag43, but did dramatically reduce the number of fimbriae expressed on the cell
surface. The use of high-resolution oligonucleotide arrays for defining points of
transcription initiation and termination is also demonstrated.

DOI: 10.1007/s00438-002-0705-2 
PMID: 12207220  [Indexed for MEDLINE]


1334. Mol Microbiol. 2002 Aug;45(3):673-95.

Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene
expression in Escherichia coli.

Oshima T(1), Wada C, Kawagoe Y, Ara T, Maeda M, Masuda Y, Hiraga S, Mori H.

Author information: 
(1)CREST, JST (Japan Science and Technology).

Deoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in
5'-GATC-3' sequences and is important in many cellular processes in Escherichia
coli. We performed a computational analysis of the entire E. coli genome and
confirmed that GATC sequences are distributed unevenly in regulatory regions,
which suggests that Dam might regulate gene transcription. To test this, a
high-density DNA microarray of 4097 E. coli genes was constructed and used to
assess the gene expression profiles of the wild type and the dam-16::kam mutant
strain grown under four different conditions. We also used two-dimensional
electrophoretic analysis of the proteome to assess the protein profiles. The
expression of a large number of genes was affected by the dam deficiency. Genes
involved in aerobic respiration, stress and SOS responses, amino acid metabolism 
and nucleotide metabolism were expressed at higher levels in the mutant cells,
especially in aerobic conditions. In contrast, transcription of genes
participating in anaerobic respiration, flagella biosynthesis, chemotaxis and
motility was decreased in the dam mutant strain under both aerobic and low
aerobic conditions. Thus, Dam-controlled genes are involved in adjusting the
metabolic and respiratory pathways and bacterial motility to suit particular
environmental conditions. The promoters of most of these Dam-controlled genes
were also found to contain GATC sequences that overlap with recognition sites for
two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator 
protein (CRP). We propose that Dam-mediated methylation plays an important role
in the global regulation of genes, particularly those with Fnr and CRP binding
sites.


PMID: 12139615  [Indexed for MEDLINE]


1335. Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9697-702. Epub 2002 Jul 15.

Global analysis of mRNA decay and abundance in Escherichia coli at single-gene
resolution using two-color fluorescent DNA microarrays.

Bernstein JA(1), Khodursky AB, Lin PH, Lin-Chao S, Cohen SN.

Author information: 
(1)Department of Genetics, Stanford University School of Medicine, Stanford, CA
94305, USA.

Much of the information available about factors that affect mRNA decay in
Escherichia coli, and by inference in other bacteria, has been gleaned from study
of less than 25 of the approximately 4,300 predicted E. coli messages. To
investigate these factors more broadly, we examined the half-lives and
steady-state abundance of known and predicted E. coli mRNAs at single-gene
resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy
for normalization of microarray data was developed to permit quantitation of mRNA
decay after transcriptional arrest by rifampicin. We found that globally, mRNA
half-lives were similar in nutrient-rich media and defined media in which the
generation time was approximately tripled. A wide range of stabilities was
observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs
had half-lives between 3 and 8 min. Genes having biologically related metabolic
functions were commonly observed to have similar stabilities. Whereas the
half-lives of a limited number of mRNAs correlated positively with their
abundance, we found that overall, increased mRNA stability is not predictive of
increased abundance. Neither the density of putative sites of cleavage by RNase
E, which is believed to initiate mRNA decay in E. coli, nor the free energy of
folding of 5' or 3' untranslated region sequences was predictive of mRNA
half-life. Our results identify previously unsuspected features of mRNA decay at 
a global level and also indicate that generalizations about decay derived from
the study of individual gene transcripts may have limited applicability.

DOI: 10.1073/pnas.112318199 
PMCID: PMC124983
PMID: 12119387  [Indexed for MEDLINE]


1336. J Bacteriol. 2002 Jul;184(13):3759-64.

The enigmatic Escherichia coli fadE gene is yafH.

Campbell JW(1), Cronan JE Jr.

Author information: 
(1)Department of Microbiology, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801, USA.

The identity of the gene encoding acyl coenzyme A dehydrogenase is a major
remaining mystery of the Escherichia coli fatty acid degradation (fad) regulon.
Our prior genome array analyses showed that transcription of the yafH gene is
controlled by the FadR regulatory protein. We now report direct experimental
proof that yafH and fadE are the same gene.


PMCID: PMC135136
PMID: 12057976  [Indexed for MEDLINE]


1337. J Mol Microbiol Biotechnol. 2002 Jul;4(4):379-88.

Specific growth inhibition by acetate of an Escherichia coli strain expressing
Era-dE, a dominant negative Era mutant.

Inoue K(1), Chen J, Kato I, Inouye M.

Author information: 
(1)Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ
08854, USA.

Escherichia coli Era is a GTP binding protein and essential for cell growth. We
have previously reported that an Era mutant, designated Era-dE, causes a dominant
negative effect on the growth and the loss of the ability to utilize TCA cycle
metabolites as carbon source when overproduced. To investigate the role of Era,
the gene expression in the cells overproducing Era-dE was examined by DNA
microarray analysis. The expression of lipA and nadAB, which are involved in
lipoic acid synthesis and NAD synthesis, respectively, was found to be reduced in
the cells overproducing Era-dE. Lipoic acid and NAD are essential cofactors for
the activities of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase
complex and glycine cleavage enzyme complex. The expression of numerous genes
involved in dissimilatory carbon metabolism and carbon source transport was
increased. This set of genes partially overlaps with the set of genes controlled 
by cAMP-CAP in E coli. Moreover, the growth defect of Era-dE overproduction was
specifically enhanced by acetate but not by TCA cycle metabolites both in rich
and synthetic media. Intracellular serine pool in Era-dE overproducing cells was 
found to be increased significantly compared to that of the cells overproducing
wild-type Era. It was further found that even the wild-type E. coli cells not
overproducing Era-dE became sensitive to acetate in the presence of serine in a
medium. We propose that when Era-dE is overproduced, carbon fluxes to the TCA
cycle and to C1 units become impaired, resulting in a higher cellular serine
concentration. We demonstrated that such cells with a high serine concentration
became sensitive to acetate, however the reason for this acetate sensitivity is
not known at the present.


PMID: 12125819  [Indexed for MEDLINE]


1338. Mol Microbiol. 2002 Jul;45(2):521-32.

LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis 
genes in Escherichia coli.

Lehnen D(1), Blumer C, Polen T, Wackwitz B, Wendisch VF, Unden G.

Author information: 
(1)Institut für Mikrobiologie and Weinforschung, Johannes Gutenberg-Universität
Mainz, Becherweg 15, 55099 Mainz, Germany.

The function of the LysR-type regulator LrhA of Escherichia coli was defined by
comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA 
mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes
involved in flagellation, motility and chemotaxis showed relative mRNA abundances
increased by factors between 3 and 80. When a representative set of five
flagellar, motility and chemotaxis genes was tested in lacZ reporter gene
fusions, similar factors for derepression were found in the lrhA mutant. In gel
retardation experiments, the LrhA protein bound specifically to flhD and lrhA
promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, 
fliA and trg were not bound by LrhA. The expression of flhDC (encoding
FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2)
is known as the master regulator for the expression of flagellar and chemotaxis
genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA
promoters were identified. The lrhA gene was under positive autoregulation by
LrhA as shown by gel retardation and lrhA expression studies. It is suggested
that LrhA is a key regulator controlling the transcription of flagellar, motility
and chemotaxis genes by regulating the synthesis and concentration of
FlhD(2)C(2).


PMID: 12123461  [Indexed for MEDLINE]


1339. Mol Plant Microbe Interact. 2002 Jul;15(7):619-29.

Microarray profiling of Erwinia chrysanthemi 3937 genes that are regulated during
plant infection.

Okinaka Y(1), Yang CH, Perna NT, Keen NT.

Author information: 
(1)Department of Plant Pathology and Center for Plant Cell Biology, University of
California, Riverside 92521, USA. okinaka@mail.ecr.edu

Microarray technology was used to identify genes in Erwinia chrysanthemi 3937
that are specifically up- or down-regulated in a plant host compared with growth 
in laboratory culture medium. Several genes were plant down-regulated, and almost
all of them were homologues of well-known housekeeping genes, such as those
encoding metabolic functions, oxidative phosphorylation components, and
transcription or translation processes. On the other hand, almost all of the
plant up-regulated genes were involved with specialized functions, including
already known or new putative virulence factors, anaerobiosis, iron uptake,
transporters or permeases, xenobiotic resistance, chemotaxis, and stress
responses to reactive oxygen species and heat. A substantial number of the plant 
up-regulated genes do not appear to be directly involved in damaging the host,
but are probably important in adapting the pathogen to the host environment. We
constructed insertion mutations in several of the plant up-regulated E.
chrysanthemi 3937 genes. Among these, mutations of Bacillus subtilis pps1,
Escherichia coli purU, and Pseudomonas aeruginosa pheC homologues reduced
virulence on African violet leaves. Thus, new insights were obtained into genes
important in bacterial virulence.

DOI: 10.1094/MPMI.2002.15.7.619 
PMID: 12118877  [Indexed for MEDLINE]


1340. Nucleic Acids Res. 2002 Jul 1;30(13):2886-93.

Co-expression pattern from DNA microarray experiments as a tool for operon
prediction.

Sabatti C(1), Rohlin L, Oh MK, Liao JC.

Author information: 
(1)Department of Human Genetics and Statistics and Department of Chemical
Engineering, University of California, Los Angeles, CA 90095, USA.

The prediction of operons, the smallest unit of transcription in prokaryotes, is 
the first step towards reconstruction of a regulatory network at the whole genome
level. Sequence information, in particular the distance between open reading
frames, has been used to predict if adjacent Escherichia coli genes are in an
operon. While appreciably successful, these predictions need to be validated and 
refined experimentally. As a growing number of gene expression array experiments 
on E.coli became available, we investigated to what extent they could be used to 
improve and validate these predictions. To this end, we examined a large
collection of published microarry data. The correlation between expression ratios
of adjacent genes was used in a Bayesian classification scheme to predict whether
the genes are in an operon or not. We found that for the genes whose expression
levels change significantly across the experiments in the data set, the currently
available gene expression data allowed a significant refinement of the
sequenced-based predictions. We report these co-expression correlations in an
E.coli genomic map. For a significant portion of gene pairs, however, the set of 
array experiments considered did not contain sufficient information to determine 
whether they are in the same transcriptional unit. This is not due to
unreliability of the array data per se, but to the design of the experiments
analyzed. In general, experiments that perturb a large number of genes offer more
information for operon prediction than confined perturbations. These results
provide a rationale for conducting expression studies comparing conditions that
cause global changes in gene expression.


PMCID: PMC117043
PMID: 12087173  [Indexed for MEDLINE]


1341. J Biol Chem. 2002 Jun 14;277(24):21657-65. Epub 2002 Apr 8.

Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback
repressor pathway regulating Wnt signaling.

Leung JY(1), Kolligs FT, Wu R, Zhai Y, Kuick R, Hanash S, Cho KR, Fearon ER.

Author information: 
(1)Department of Internal Medicine, University of Michigan Medical School, Ann
Arbor, Michigan 48109-0638, USA.

The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in
the pathway play a key role in many cancers. Although Wnts act to stabilize
beta-catenin levels in the cytosol and nucleus, a multiprotein complex containing
adenomatous polyposis coli, glycogen synthase kinase 3beta, and Axin1 or its
homolog Axin2/Axil/conductin promotes beta-catenin phosphorylation and subsequent
proteasomal degradation. We found that the rat Axil gene was strongly induced
upon neoplastic transformation of RK3E cells by mutant beta-catenin or
gamma-catenin or after ligand-induced activation of a beta-catenin-estrogen
receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells
activated the conductin gene, and human cancers with defective beta-catenin
regulation had elevated AXIN2 gene and protein expression. Expression of
AXIN2/Axil was strongly repressed in cancer cells by restoration of wild type
adenomatous polyposis coli function or expression of a dominant negative form of 
T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role 
in the ability of beta-catenin to activate AXIN2 transcription. In contrast to
AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by
beta-catenin-TCF. Because Axin2 can inhibit beta-catenin abundance and function, 
the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling.
Additionally, although Axin1 and Axin2 have been thought to have comparable
functions, the observation that Wnt pathway activation elevates AXIN2 but not
AXIN1 expression suggests that there may be potentially significant functional
differences between the two proteins.

DOI: 10.1074/jbc.M200139200 
PMID: 11940574  [Indexed for MEDLINE]


1342. Mol Microbiol. 2002 Jun;44(6):1611-24.

Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of
directly activated promoters by application of molecular genetics and informatics
to microarray data.

Martin RG(1), Rosner JL.

Author information: 
(1)Laboratory of Molecular Biology, National Institute of Diabetes and Digestive 
and Kidney Diseases/NIH, Bethesda, MD 20892-0560, USA. rgmartin@helix.nih.gov

Microarray analyses are providing a plethora of data concerning transcriptional
responses to specific gene regulators and their inducers but do not distinguish
between direct and indirect responses. Here, we identify directly activated
promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by
applying informatics, genomics and molecular genetics to microarray data obtained
by others. Those studies found that overexpression of marA, or the treatment of
cells with salicylate to derepress marA, or treatment with paraquat to induce
soxS, resulted in elevated transcription of 153 genes. However, only 27 out of
the promoters showed increased transcription under at least two of the
aforementioned conditions and eight of those were previously known to be directly
activated. A computer algorithm was used to identify potential activator binding 
sites located upstream of the remaining 19 promoters of this subset, and
conventional genetic and biochemical approaches were applied to test whether
these sites are critical for activation by the homologous MarA, SoxS and Rob
transcriptional activators. Only seven out of the 19 promoters were found to be
activated when fused to lacZ and tested as single lysogens. All seven contained
an essential activator binding site. The remaining promoters were insensitive to 
stimulation by the inducers suggesting that the great majority of elevated
microarray transcripts either were misidentified or resulted from indirect
effects requiring sequences outside of the promoter region. We estimate that the 
total number of directly activated promoters in the regulon is less than 40.


PMID: 12067348  [Indexed for MEDLINE]


1343. Proc Natl Acad Sci U S A. 2002 May 14;99(10):7033-8. Epub 2002 May 7.

Genome-wide screening for trait conferring genes using DNA microarrays.

Gill RT(1), Wildt S, Yang YT, Ziesman S, Stephanopoulos G.

Author information: 
(1)Department of Chemical Engineering, Massachusetts Institute of Technology, 77 
Massachusetts Avenue, Cambridge, MA 02139, USA.

We report a DNA microarray-based method for genome-wide monitoring of
competitively grown transformants to identify genes whose overexpression confers 
a specific cellular phenotype. Whereas transcriptional profiling identifies
differentially expressed genes that are correlated with particular aspects of the
cellular phenotype, this functional genomics approach determines genes that
result in a specific physiology. This parallel gene-trait mapping method consists
of transforming a strain with a genomic library, enriching the cell population in
transformants containing the trait conferring gene(s), and finally using DNA
microarrays to simultaneously isolate and identify the enriched gene inserts.
Various methods of enrichment can be used; here, genes conferring low-level
antibiotic resistance were identified by growth in selective media. We
demonstrated the method by transforming Escherichia coli cells with a genomic E. 
coli library and selecting for transformants exhibiting a growth advantage in the
presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol
tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on
DNA microarrays containing 1,160 E. coli gene probes, extra-chromosomal DNA
isolated from transformed cells grown in the presence of various levels of
Pine-Sol. Results were further validated by plating and sequencing of individual 
colonies, and also by assessing the Pine-Sol resistance of cells transformed with
enriched plasmid library or individual resistance genes identified by the
microarrays. Applications of this method beyond antibiotic resistance include
identification of genes resulting in resistance to chemotherapeutic agents, genes
yielding resistance to toxic products (recombinant proteins, chemical feedstocks)
in industrial fermentations, genes providing enhanced growth in cell culture or
high cell density fermentations, genes facilitating growth on unconventional
substrates, and others.

DOI: 10.1073/pnas.102154799 
PMCID: PMC124523
PMID: 11997466  [Indexed for MEDLINE]


1344. Gut. 2002 May;50(5):658-64.

Characteristics of the peroxisome proliferator activated receptor gamma
(PPARgamma) ligand induced apoptosis in colon cancer cells.

Shimada T(1), Kojima K, Yoshiura K, Hiraishi H, Terano A.

Author information: 
(1)Department of Gastroenterology, Dokkyo University School of Medicine, Mibu,
Tochigi 321-0293, Japan. tshimada@dokkyomed.ac.jp

Comment in
    Z Gastroenterol. 2003 Oct;41(10):1035-8.

BACKGROUND: Involvement of peroxisome proliferator activated receptor gamma
(PPARgamma) in the growth response of colon cancer cells has been suggested.
AIMS: To investigate the characteristics of PPARgamma induced apoptosis in colon 
cancer cells.
METHODS: The effects of ligands for each of the PPAR subtypes (alpha, delta, and 
gamma) on DNA synthesis and cell viability were examined in HT-29 colon cancer
cells. Modulation of apoptosis related gene expression by PPARgamma ligands was
screened with cDNA arrays, and the results were confirmed by quantitative reverse
transcription-polymerase chain reaction (RT-PCR) analysis.
RESULTS: PPARalpha, PPARdelta, and PPARgamma were all expressed in HT-29 cells.
PPARgamma ligands, 15-deoxy-delta(12,)(14)-prostaglandin J2 (15d-PGJ2) and
troglitazone (TGZ), suppressed DNA synthesis of HT-29 cells whereas ligands for
PPARalpha and PPARdelta had no significant effects. Both 15d-PGJ2 and TGZ induced
HT-29 cell death in a dose dependent manner which was associated with an increase
in fragmented DNA and was sensitive to a caspase inhibitor. Among several genes
selected by cDNA array screening, quantitative RT-PCR analysis confirmed
downregulation of c-myc expression and upregulation of c-jun and gadd153
expression by 15d-PGJ2 and TGZ. PPARgamma induced apoptosis was antagonised by
the presence of serum in the culture medium, and interaction between PPARgamma
signalling and cell survival signalling through the phosphatidylinositol 3-kinase
pathway was suggested.
CONCLUSIONS: As c-myc is an important target gene of the adenomatous polyposis
coli (APC)/beta-catenin and/or APC/gamma-catenin pathway, activation of PPARgamma
signalling appears to compensate for deregulated c-myc expression caused by
mutated APC. The present results suggest the potential usefulness of PPARgamma
ligands for chemoprevention and treatment of colon cancers.


PMCID: PMC1773196
PMID: 11950812  [Indexed for MEDLINE]


1345. Nat Biotechnol. 2002 May;20(5):478-83.

An array of target-specific screening strains for antibacterial discovery.

DeVito JA(1), Mills JA, Liu VG, Agarwal A, Sizemore CF, Yao Z, Stoughton DM,
Cappiello MG, Barbosa MD, Foster LA, Pompliano DL.

Author information: 
(1)Department of Antimicrobial Research, Bristol-Myers Squibb Company,
Wilmington, DE 19880, USA.

As the global threat of drug- and antibiotic-resistant bacteria continues to
rise, new strategies are required to advance the drug discovery process. This
work describes the construction of an array of Escherichia coli strains for use
in whole-cell screens to identify new antimicrobial compounds. We used the
recombination systems from bacteriophages lambda and P1 to engineer each strain
in the array for low-level expression of a single, essential gene product, thus
making each strain hypersusceptible to specific inhibitors of that gene target.
Screening of nine strains from the array in parallel against a large chemical
library permitted identification of new inhibitors of bacterial growth. As an
example of the target specificity of the approach, compounds identified in the
whole-cell screen for MurA inhibitors were also found to block the biochemical
function of the target when tested in vitro.

DOI: 10.1038/nbt0502-478 
PMID: 11981561  [Indexed for MEDLINE]


1346. J Biol Chem. 2002 Apr 12;277(15):13175-83. Epub 2002 Jan 28.

Global expression profiling of acetate-grown Escherichia coli.

Oh MK(1), Rohlin L, Kao KC, Liao JC.

Author information: 
(1)Department of Chemical Engineering, UCLA, Los Angeles, California 90095, USA.

This study characterized the transcript profile of Escherichia coli in acetate
cultures using DNA microarray on glass slides. Glucose-grown cultures were used
as a reference. At the 95% confidence level, 354 genes were up-regulated in
acetate, while 370 genes were down-regulated compared with the glucose-grown
culture. Generally, more metabolic genes were up-regulated in acetate than other 
gene groups, while genes involved in cell replication, transcription, and
translation machinery tended to be down-regulated. It appears that E. coli
commits more resources to metabolism at the expense of growth when cultured in
the poor carbon source. The expression profile confirms many known features in
acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic
acid cycle, and gluconeogenic genes. It also provided many previously unknown
features, including induction of malic enzymes, ppsA, and the glycolate pathway
and repression of glycolytic and glucose phosphotransferase genes in acetate. The
carbon flux delivered from the malic enzymes and PpsA in acetate was further
confirmed by deletion mutations. In general, the gene expression profiles
qualitatively agree with the metabolic flux changes and may serve as a predictor 
for gene function and metabolic flux distribution.

DOI: 10.1074/jbc.M110809200 
PMID: 11815613  [Indexed for MEDLINE]


1347. Nature. 2002 Apr 11;416(6881):644-8. Epub 2002 Mar 24.

Functional genomic analysis of phagocytosis and identification of a Drosophila
receptor for E. coli.

Rämet M(1), Manfruelli P, Pearson A, Mathey-Prevot B, Ezekowitz RA.

Author information: 
(1)Laboratory of Developmental Immunology, Massachusetts General Hospital for
Children, and Department of Pediatrics, Harvard Medical School, 55 Fruit Street, 
Boston, Massachusetts 02114, USA. mramet@partners.org

The recognition and phagocytosis of microbes by macrophages is a principal aspect
of innate immunity that is conserved from insects to humans. Drosophila
melanogaster has circulating macrophages that phagocytose microbes similarly to
mammalian macrophages, suggesting that insect macrophages can be used as a model 
to study cell-mediated innate immunity. We devised a double-stranded RNA
interference-based screen in macrophage-like Drosophila S2 cells, and have
defined 34 gene products involved in phagocytosis. These include proteins that
participate in haemocyte development, vesicle transport, actin cytoskeleton
regulation and a cell surface receptor. This receptor, Peptidoglycan recognition 
protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not
Gram-positive bacteria. Drosophila humoral immunity also distinguishes between
Gram-negative and Gram-positive bacteria through the Imd and Toll pathways,
respectively; however, a receptor for the Imd pathway has not been identified.
Here we show that PGRP-LC is important for antibacterial peptide synthesis
induced by Escherichia coli both in vitro and in vivo. Furthermore, totem
mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E.
coli), but not Gram-positive, bacterial infection. Our results demonstrate that
PGRP-LC is an essential component for recognition and signalling of Gram-negative
bacteria. Furthermore, this functional genomic approach is likely to have
applications beyond phagocytosis.

DOI: 10.1038/nature735 
PMID: 11912489  [Indexed for MEDLINE]


1348. Mol Microbiol. 2002 Apr;44(1):89-105.

Over 1000 genes are involved in the DNA damage response of Escherichia coli.

Khil PP(1), Camerini-Otero RD.

Author information: 
(1)Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, 10
Center Drive, Bethesda, MD 20892, USA.

Changes in gene expression after treatment of Escherichia coli cultures with
mitomycin C were assessed using gene array technology. Unexpectedly, a large
number of genes (nearly 30% of all genes) displayed significant changes in their 
expression level. Analysis and classification of expression profiles of the
corresponding genes allowed us to assign this large number of genes into one or
two dozen small clusters of genes with similar expression profiles. This
assignment allowed us to describe systematically the changes in the level of gene
expression in response to DNA damage. Among the damage-induced genes, more than
100 are novel. From those genes involved in DNA metabolism that have not
previously been shown to be induced by DNA damage, the mutS gene involved in
mismatch repair is especially noteworthy. In addition to the SOS response, we
observed the induction of other stress response pathways, such as those of
oxidative stress and osmotic protection. Among the genes that are downregulated
in response to DNA damage are numerous protein biosynthesis genes. Analysis of
the gene expression data highlighted the essential involvement of
sigma(s)-regulated genes and the general stress response network in the response 
to DNA damage.


PMID: 11967071  [Indexed for MEDLINE]


1349. Mutat Res. 2002 Mar 20;500(1-2):125-34.

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a
base excision repair-deficient Escherichia coli strain.

Salmelin C(1), Vilpo J.

Author information: 
(1)Leukemia Research Laboratory, Department of Clinical Chemistry, Tampere
University Hospital and Tampere University Medical School, P.O. Box 2000,
FIN-33521 Tampere, Finland.

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a
bifunctional alkylating agent widely used as an anticancer drug and also as an
immunosuppressant. Its chemical structure and clinical experience indicate that
CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to
induce mutations and gene expression changes in the wild-type (WT) Escherichia
coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E.
coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA
array method. The results showed that CLB is a potent mutagen in MV1932 cells
compared with the E. coli WT strain AB1157, emphasizing the role of
3-methyladenine DNA glycosylases I and II in protecting the cells from
CLB-induced DNA damage and subsequent mutations. Global gene expression profiling
revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of
4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these
MV1932 genes were downregulated, while 22% were upregulated in WT cells. The
downregulated genes in MV1932 represented most (19/23) functional categories, and
unexpectedly, many of them code for proteins responsible for genomic integrity.
These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance 
to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of
RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC
(genetic recombination). Our results strongly suggest that in addition to the DNA
damage itself, the downregulation of central protecting genes is responsible for 
the decreased cell survival (demonstrated in a previous work) and the increased
mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.


PMID: 11890942  [Indexed for MEDLINE]


1350. FEBS Lett. 2002 Mar 6;514(1):90-5.

RNA aptamers directed against release factor 1 from Thermus thermophilus.

Szkaradkiewicz K(1), Nanninga M, Nesper-Brock M, Gerrits M, Erdmann VA, Sprinzl
M.

Author information: 
(1)Laboratorium für Biochemie, Universität Bayreuth, Universitätsstrasse 30,
D-95440, Bayreuth, Germany.

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers
that specifically bind Thermus thermophilus release factor 1 (RF1). From 31
isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 
5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure
indicate that in both groups the invariable sequences are located in
single-stranded regions of hairpin structures. Complex formations between RF1 and
aptamers of both groups were identified by electrophoretic shift assay and
chemical footprinting. Deletion of the invariable sequences did not effect the
secondary structure of the aptamers but abolished their binding to RF1. RNA
motifs matching the invariable sequences of the aptamers are present as consensus
sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1
recognizes UAG stop codons in an Escherichia coli in vitro translation system.
Aptamers from both groups inhibited this RF1 activity.


PMID: 11904188  [Indexed for MEDLINE]


1351. Appl Environ Microbiol. 2002 Mar;68(3):1425-30.

Quantitative detection of microbial genes by using DNA microarrays.

Cho JC(1), Tiedje JM.

Author information: 
(1)Center for Microbial Ecology, Plant and Soil Sciences Bldg., Michigan State
University, East Lansing, MI 48824, USA.

To quantify target genes in biological samples using DNA microarrays, we employed
reference DNA to normalize variations in spot size and hybridization. This method
was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and
Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA 
as the reference DNA. We observed a good linearity between the log signal ratio
and log DNA concentration ratio at DNA concentrations above the method's
detection limit, which was approximately 10 pg. This approach for designing
quantitative microarrays and the inferred equation from this study provide a
simple and convenient way to estimate the target gene concentration from the
hybridization signal ratio.


PMCID: PMC123775
PMID: 11872496  [Indexed for MEDLINE]


1352. Int J Oncol. 2002 Mar;20(3):441-51.

Expression analysis of pediatric solid tumor cell lines using oligonucleotide
microarrays.

Wai DH(1), Schaefer KL, Schramm A, Korsching E, Van Valen F, Ozaki T, Boecker W, 
Schweigerer L, Dockhorn-Dworniczak B, Poremba C.

Author information: 
(1)Gerhard-Domagk-Institute of Pathology, University of Muenster, D-48149
Muenster, Germany.

We identified patterns of differentially-expressed genes in cell lines derived
from several pediatric solid tumors. Affymetrix Human Cancer G110 Arrays,
carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell lines 
originating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of
soft parts. Hierarchical clustering clearly differentiated these 3 entities and
revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific
up-regulation in these cell lines, respectively. Whereas ET lines demonstrated
increased expression of microtubule-associated protein tau (MAPT), protein
phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene
a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples
exhibited high expression of wingless-type mouse mammary tumor virus integration 
site family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and
adenomatous polyposis coli (APC) which are involved in regulating free
beta-catenin levels. These genes likely maintain tumor-specific characteristics
and participate in key downstream regulatory mechanisms. We also correlated the
expression levels of up-regulated genes in ETs with their chromosomal
localization and compared these data to the comparative genomic hybridization
profiles of the cell lines. We demonstrate that gains of genetic material
contribute essentially to differential gene expression.


PMID: 11836553  [Indexed for MEDLINE]


1353. J Biol Chem. 2002 Mar 1;277(9):7412-9. Epub 2001 Dec 13.

Molecular regulation of urothelial renewal and host defenses during infection
with uropathogenic Escherichia coli.

Mysorekar IU(1), Mulvey MA, Hultgren SJ, Gordon JI.

Author information: 
(1)Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, 660 S Euclid Avenue, St. Louis, MO 63110, USA.

Uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract
infection in women, attaches to the superficial facet cell layer of the bladder
epithelium (urothelium) via its FimH adhesin. Attachment triggers exfoliation of 
bacteria-laden superficial facet cells, followed by rapid reconstitution of the
urothelium through differentiation of underlying basal and intermediate cells. We
have used DNA microarrays to define the molecular regulators of urothelial
renewal and host defense expressed in adult C57Bl/6 female mice during the early 
phases of infection with isogenic virulent (FimH+) or avirulent (FimH-) UPEC
strains. The temporal evolution and cellular origins of selected responses were
then characterized by real time quantitative reverse transcriptase-PCR, in situ
hybridization, and immunohistochemical analyses. Well before exfoliation is
evident, FimH-mediated attachment suppresses transforming growth factor-beta
(Bmp4) and Wnt5a/Ca(2+) signaling to promote subsequent differentiation of
basal/intermediate cells. The early transcriptional responses to attachment also 
include induction of regulators of proliferation (e.g. epidermal growth factor
family members), induction of the ETS transcription factor Elf3, which
transactivates genes involved in epithelial differentiation and host defense
(inducible nitric-oxide synthase), induction of modulators, and mediators of
pro-inflammatory responses (e.g. Socs3, Cebp/delta, Bcl3, and CC/CXC chemokines),
induction of modulators of apoptotic responses (A20), and induction of
intermediate cell tight junction components (claudin-4). Both early and late
phases of the host response exhibit remarkable specificity for the FimH+ strain
and provide new insights about the molecular cascade mobilized to combat
UPEC-associated urinary tract infection.

DOI: 10.1074/jbc.M110560200 
PMID: 11744708  [Indexed for MEDLINE]


1354. Mol Microbiol. 2002 Mar;43(6):1445-56.

RNase G complementation of rne null mutation identifies functional
interrelationships with RNase E in Escherichia coli.

Lee K(1), Bernstein JA, Cohen SN.

Author information: 
(1)Department of Genetics, Stanford University School of Medicine, CA 94305-5120,
USA.

Erratum in
    Mol Microbiol 2002 Oct;46(1):295.

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have
sequence similarity and broadly similar sequence specificity. Whereas the absence
of Rne normally is lethal, we show here that E. coli bacteria that lack the rne
gene can be made viable by overexpression of Rng. Rng-complemented cells
accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase 
P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was 
not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated
from precursors by RNase G cleavage in vitro. Using DNA microarrays containing
4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose
steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of
RNase E. Most transcript species affected by RNase E deficiency were also
elevated in an rne deletion mutant complemented by Rng. However, approximately
100 mRNAs that accumulated in Rne-deficient cells were decreased by
rng-complemention, thus identifying targets whose processing or degradation may
be the basis for RNase E essentiality. Remarkably prominent in this group were
mRNAs implicated in energy-generating pathways or in the synthesis or degradation
of macromolecules.


PMID: 11952897  [Indexed for MEDLINE]


1355. J Biol Chem. 2002 Feb 15;277(7):5476-83. Epub 2001 Dec 5.

Blocking the secretion of hepatic very low density lipoproteins renders the liver
more susceptible to toxin-induced injury.

Björkegren J(1), Beigneux A, Bergo MO, Maher JJ, Young SG.

Author information: 
(1)Gladstone Institute of Cardiovascular Disease, Cardiovascular Research
Institute, University of California, San Francisco 94110, USA.

Recently, we generated mice lacking microsomal triglyceride transfer protein
(MTP) in the liver (Mttp(Delta/Delta)) and demonstrated that very low density
lipoprotein secretion from hepatocytes was almost completely blocked. The
blockade in lipoprotein production was accompanied by mild to moderate hepatic
steatosis, but the mice appeared healthy. Although hepatic MTP deficiency
appeared to be innocuous, we hypothesized that a blockade in very low density
lipoprotein secretion and the accompanying steatosis might increase the
sensitivity of Mttp(Delta/Delta) livers to additional hepatic insults. To address
this issue, we compared the susceptibility of Mttp(Delta/Delta) mice and
Mttp(flox/flox) controls to hepatic injury from Escherichia coli
lipopolysaccharides, concanavalin A, and Pseudomonas aeruginosa exotoxin A. At
baseline, neither the Mttp(Delta/Delta) nor the Mttp(flox/flox) mice had elevated
serum transaminases or histologic evidence of hepatic inflammation. After the
administration of the toxins, however, the Mttp(Delta/Delta) mice manifested
higher levels of transaminases and, unlike the Mttp(flox/flox) mice, developed
histologic evidence of hepatic inflammation. The toxic challenge induced tumor
necrosis factor-alpha to a similar extent in Mttp(Delta/Delta) and
Mttp(flox/flox) mice, but other parameters of injury (e.g. chemokine transcript
levels and lipid peroxides) were disproportionately increased in the
Mttp(Delta/Delta) mice. Our results suggest that blocking lipoprotein secretion
in the liver may increase the susceptibility of the liver to certain toxic
challenges.

DOI: 10.1074/jbc.M108514200 
PMID: 11739387  [Indexed for MEDLINE]


1356. Biochem Biophys Res Commun. 2002 Feb 1;290(4):1328-35.

A rapid method to capture and screen for transcription factors by SELDI mass
spectrometry.

Forde CE(1), Gonzales AD, Smessaert JM, Murphy GA, Shields SJ, Fitch JP,
McCutchen-Maloney SL.

Author information: 
(1)Biology and Biotechnology Research Program, Lawrence Livermore National
Laboratory, Livermore, California 94550, USA.

A novel method to screen for transcription factors binding to promoter DNA
sequences has been developed using DNA chip surfaces and mass spectrometry. This 
technique was demonstrated with Escherichia coli lac repressor, LacI. The
consensus promoter binding sequence for LacI and a scrambled version of the same 
DNA sequence were prepared on two affinity chip surfaces. Total E. coli protein
lysate was applied to the two surfaces. A 38.2 kDa protein, as detected by
SELDI-MS, was captured on the chip surface containing the binding sequence for
LacI but not on the surface containing the scrambled sequence. The protein was
identified following one-step, small-scale affinity capture and peptide mapping. 
Subsequent database searches identified the 38.2 kDa protein as the lac repressor
of E. coli. We discuss application of DNA chip affinity capture to characterize
transcription factors and to screen for differences in cellular regulatory
networks.

©2002 Elsevier Science (USA).

DOI: 10.1006/bbrc.2002.6352 
PMID: 11812009  [Indexed for MEDLINE]


1357. Protein Eng. 2002 Feb;15(2):153-60.

Gene expression response to misfolded protein as a screen for soluble recombinant
protein.

Lesley SA(1), Graziano J, Cho CY, Knuth MW, Klock HE.

Author information: 
(1)Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row,
San Diego, CA 92121, USA. lesley@gnf.org

Proper protein folding is key to producing recombinant proteins for structure
determination. We have examined the effect of misfolded recombinant protein on
gene expression in Escherichia coli. Comparison of expression patterns indicates 
a unique set of genes responding to translational misfolding. The response is in 
part analogous to heat shock and suggests a translational component to the
regulation. We have further utilized the expression information to generate
reporters responsive to protein misfolding. These reporters were used to identify
properly folded recombinant proteins and to create soluble domains of insoluble
proteins for structural studies.


PMID: 11917152  [Indexed for MEDLINE]


1358. Annu Rev Microbiol. 2002;56:599-624. Epub 2002 Jan 30.

Impact of genomic technologies on studies of bacterial gene expression.

Rhodius V(1), Van Dyk TK, Gross C, LaRossa RA.

Author information: 
(1)Department of Stomatology, University of California, San Francisco, 94143,
USA. rhodius@itsa.ucsf.edu

The ability to simultaneously monitor expression of all genes in any bacterium
whose genome has been sequenced has only recently become available. This requires
not only careful experimentation but also that voluminous data be organized and
interpreted. Here we review the emerging technologies that are impacting the
study of bacterial global regulatory mechanisms with a view toward discussing
both perceived best practices and the current state of the art. To do this, we
concentrate upon examples using Escherichia coli and Bacillus subtilis because
prior work in these organisms provides a sound basis for comparison.

DOI: 10.1146/annurev.micro.56.012302.160925 
PMID: 12142487  [Indexed for MEDLINE]


1359. Bioinformatics. 2002;18 Suppl 1:S337-44.

Identifying operons and untranslated regions of transcripts using Escherichia
coli RNA expression analysis.

Tjaden B(1), Haynor DR, Stolyar S, Rosenow C, Kolker E.

Author information: 
(1)Department of Computer Science and Engineering, University of Washington, Box 
352350, Seattle, WA 98195, USA. tjaden@cs.washington.edu

Microarrays traditionally have been used to assay the transcript expression of
coding regions of genes. Here, we use Escherichia coli oligonucleotide
microarrays to assay transcript expression of both open reading frames (ORFs) and
intergenic regions. We then use hidden Markov models to analyse this expression
data and estimate transcription boundaries of genes. This approach allows us to
identify 5' untranslated regions (5' UTRs) of transcripts as well as genes that
are likely to be operon members. The operon elements we identify correspond to
documented operons with 99% specificity and 63% sensitivity. Similarly we find
that our 5' UTR results accurately coincide with experimentally verified promoter
regions for most genes.


PMID: 12169564  [Indexed for MEDLINE]


1360. Biotechniques. 2002 Jan;32(1):110, 112-4, 116, 118-9.

DNA array analysis in a Microsoft Windows environment.

Conway T(1), Kraus B, Tucker DL, Smalley DJ, Dorman AF, McKibben L.

Author information: 
(1)Department of Botany and Microbiology, The University of Oklahoma, Norman
73069-0245, USA. tconway@ou.edu

Microsoft Windows-based computers have evolved to the point that they provide
sufficient computational and visualization power for robust analysis of DNA array
data. In fact, smaller laboratories might prefer to carry out some or all of
their analyses and visualization in a Windows environment, rather than
alternative platforms such as UNIX. We have developed a series of manually
executed macros written in Visual Basic for Microsoft Excel spreadsheets, that
allows for rapid and comprehensive gene expression data analysis. The first macro
assigns gene names to spots on the DNA array and normalizes individual
hybridizations by expressing the signal intensity for each gene as a percentage
of the sum of all gene intensities. The second macro streamlines statistical
consideration of the confidence in individual gene measurements for sets of
experimental replicates by calculating probability values with the Student's t
test. The third macro introduces a threshold value, calculates expression ratios 
between experimental conditions, and calculates the standard deviation of the
mean of the log ratio values. Selected columns of data are copied by a fourth
macro to create a processed data set suitable for entry into a Microsoft Access
database. An Access database structure is described that allows simple queries
across multiple experiments and export of data into third-party data
visualization software packages. These analysis tools can be used in their
present form by others working with commercial E. coli membrane arrays, or they
may be adapted for use with other systems. The Excel spreadsheets with embedded
Visual Basic macros and detailed instructions for their use are available at
http://www.ou.edu/microarray.


PMID: 11808684  [Indexed for MEDLINE]


1361. Methods Enzymol. 2002;358:177-88.

Transcript profiling of Escherichia coli using high-density DNA microarrays.

Picataggio SK(1), Templeton LJ, Smulski DR, LaRossa RA.

Author information: 
(1)DuPont Central Research and Development, Wilmington, Delaware 19880, USA.


PMID: 12474387  [Indexed for MEDLINE]


1362. Mol Microbiol. 2002 Jan;43(2):307-22.

Functional characterization of the antagonistic flagellar late regulators FliA
and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome.

Josenhans C(1), Niehus E, Amersbach S, Hörster A, Betz C, Drescher B, Hughes KT, 
Suerbaum S.

Author information: 
(1)Institute for Hygiene and Microbiology, University of Wuerzburg, Germany.
cjosenhans@hygiene.uni-wuerzburg.de

Helicobacter pylori is thought to regulate gene expression with a very small set 
of regulatory genes. We identified a previously unannotated open reading frame
(ORF) in the H. pylori 26695 genome (HP1122) as a putative H. pylori flgM gene
(sigma28 factor antagonist) by a motif-based bioinformatic approach. Deletion of 
HP1122 resulted in a fourfold increase in transcription of the sigma28-dependent 
major flagellin gene flaA, supporting the function of HP1122 as H. pylori FlgM.
Helicobacter pylori FlgM lacks a conserved 20-amino-acid N-terminal domain of
enterobacterial FlgM proteins, but was able to interact with the Salmonella
typhimurium sigma28 (FliA) and inhibit the expression of FliA-dependent genes in 
Salmonella. Helicobacter pylori FlgM inhibited FliA to the same extent in a
Salmonella strain with an intact flagellar export system and in an
export-deficient strain. Helicobacter pylori FliA was able to drive transcription
of FliA-dependent genes in Salmonella. The effects of mutations in the H. pylori 
flgM and fliA genes on the H. pylori transcriptome were analysed using whole
genome DNA microarrays. The antagonistic roles of FlgM and FliA in controlling
the transcription of the major flagellin gene flaA were confirmed, and two
additional FliA/FlgM dependent operons (HP472 and HP1051/HP1052) were identified.
None of the three genes contained in these operons has a known function in
flagellar biogenesis in other bacteria. Like other motile bacteria, H. pylori has
a FliA/FlgM pair of sigma and anti-sigma factors, but the genes controlled by
these differ markedly from the Salmonella/Escherichia coli paradigm.


PMID: 11985711  [Indexed for MEDLINE]


1363. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14973-8.

Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta
-catenin signaling is activated in human colon tumors.

Yan D(1), Wiesmann M, Rohan M, Chan V, Jefferson AB, Guo L, Sakamoto D, Caothien 
RH, Fuller JH, Reinhard C, Garcia PD, Randazzo FM, Escobedo J, Fantl WJ, Williams
LT.

Author information: 
(1)Chiron Corporation, 4560 Horton Street, Q-407B, Emeryville, CA 94608, USA.

Genetic studies have identified mutations in key regulators of the
Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon
cancers. However, whether the pathway is activated in clinical cancer samples is 
not easily determined, and therefore it is useful to find markers that could be
surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression
profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin
levels are stabilized as a consequence of truncated adenomatous polyposis coli
and were compared with profiles of the same cells transfected with antisense
oligodeoxynucleotides. Treatment of cells with beta-catenin antisense
oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human
naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these 
mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines
derived from different origins, axin2 and hnkd were expressed only in human colon
cancer cell lines that are known to have activating mutations in the
Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also
found to be elevated in about 65% of laser microdissected cells from human colon 
tumors compared with laser microdissected cells of normal morphology from the
same patient samples. The increased expression of axin2 and hnkd correlated with 
truncations in adenomatous polyposis coli in the same patient samples. These
results reveal that it is possible to detect activation of a carcinogenic pathway
in human cancer samples with specific markers.

DOI: 10.1073/pnas.261574498 
PMCID: PMC64968
PMID: 11752446  [Indexed for MEDLINE]


1364. Biosens Bioelectron. 2001 Dec;16(9-12):695-9.

The extremely rapid oligonucleotide hybridization and high throughput detection
of microbial gene sequences using fluorescence polarization.

Tsuruoka M(1), Murano S, Okada M, Ohiso I, Fujii T.

Author information: 
(1)Advanced Science and Technology Laboratory, Hiroshima City, Ozuka 151-366
Hiroshima-shi, Hiroshima 731-3162, Japan. tsuruoka@sentan.city.hiroshima.jp

The hybridization of oligonucleotide sequences complementary to the genes of
Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli
(EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence
polarization under the reaction condition of high salt concentration (0.8 M
NaCl), which was optimized to obtain a higher rate of hybridization. The time
courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the
amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR 
and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA
in HCV, respectively. Probe DNA sequences were designed which hybridized
extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and
that of HCV. In the cases using the three different DNA probes, the hybridization
was 90% complete in about 1 min, considerably faster than that of the 3 min
reported previously. The rapidity of this hybridization could not be explained by
the melting temperature or the G+C content of the probe sequences but its
relationship with high order structure of the single stranded DNA or RNA of the
amplicons in the solution was strongly suggested.


PMID: 11679246  [Indexed for MEDLINE]


1365. Clin Cancer Res. 2001 Dec;7(12):4013-20.

Tissue microarray analysis of beta-catenin in colorectal cancer shows nuclear
phospho-beta-catenin is associated with a better prognosis.

Chung GG(1), Provost E, Kielhorn EP, Charette LA, Smith BL, Rimm DL.

Author information: 
(1)Department of Internal Medicine, Section Medical Oncology, Yale University
School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA.

PURPOSE: Beta-catenin is involved in homotypic cell-cell adhesion and the wnt
signaling pathway. Deregulation of beta-catenin levels, caused in part by
mutations of the adenomatous polyposis coli gene, is thought to play a role in
the development of colorectal and other cancers. To further elucidate their
roles, the expression pattern of beta-catenin and phosphospecific beta-catenin
was correlated with clinical outcome in a series of patients with colorectal
cancer.
EXPERIMENTAL DESIGN: Immunohistochemical analysis of a tissue microarray with 650
colorectal cancer specimens was performed to study the expression and subcellular
localization of beta-catenin and phosphospecific beta-catenin. These results were
correlated with other clinicopathological factors and with overall survival.
RESULTS: The majority of cancers retained some degree of beta-catenin membranous 
staining, whereas cytoplasmic or nuclear expression was seen in 42.5% and 20.4%
of specimens, respectively. Phospho-beta-catenin showed nuclear staining in 9.5% 
of specimens, and there was no apparent membranous or cytoplasmic staining. There
was no significant association between beta-catenin or phospho-beta-catenin and
grade or stage. However, there was a positive correlation between beta-catenin
and phospho-beta-catenin (P = 0.039), with phospho-beta-catenin representing a
subset of nuclear beta-catenin. Patients with nuclear expression of beta-catenin 
did not have an altered survival compared with those that did not (P = 0.5611).
Nuclear expression of phospho-beta-catenin, however, was associated with an
improved survival (P = 0.0006). In multivariate analysis, only stage and
phospho-beta-catenin were independently predictive of overall survival (P < 0.001
and P = 0.0034, respectively).
CONCLUSIONS: These findings support a role for beta-catenin overexpression in
colorectal tumorigenesis and provide initial evidence that phospho-beta-catenin
may be a marker for improved overall survival independent of stage and grade.


PMID: 11751495  [Indexed for MEDLINE]


1366. Nucleic Acids Res. 2001 Nov 15;29(22):E112.

Prokaryotic RNA preparation methods useful for high density array analysis:
comparison of two approaches.

Rosenow C(1), Saxena RM, Durst M, Gingeras TR.

Author information: 
(1)Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA.
carsten_rosenow@affymetrix.com

High density oligonucleotide arrays have been used extensively for expression
studies of eukaryotic organisms. We have designed a prokaryotic high density
oligonucleotide array using the complete Escherichia coli genome sequence to
monitor expression levels of all genes and intergenic regions in the genome.
Because previously described methods for preparing labeled target nucleic acids
are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment
and direct labeling protocol was developed together with a cDNA synthesis
protocol. The reproducibility of each labeling method was determined using high
density oligonucleotide probe arrays as a read-out methodology and the expression
results from direct labeling were compared to the expression results from the
cDNA synthesis. About 50% of all annotated E.coli open reading frames are
observed to be transcribed, as measured by both protocols, when the cells were
grown in rich LB medium. Each labeling method individually showed a high degree
of concordance in replica experiments (95 and 99%, respectively), but when each
sample preparation method was compared to the other, approximately 32% of the
genes observed to be expressed were discordant. However, both labeling methods
can detect the same relative gene expression changes when RNA from IPTG-induced
cells was labeled and compared to RNA from uninduced E.coli cells.


PMCID: PMC92579
PMID: 11713332  [Indexed for MEDLINE]


1367. Am J Physiol Cell Physiol. 2001 Nov;281(5):C1587-95.

Human endothelial cell response to gram-negative lipopolysaccharide assessed with
cDNA microarrays.

Zhao B(1), Bowden RA, Stavchansky SA, Bowman PD.

Author information: 
(1)United States Army Institute of Surgical Research and Clinical Investigation, 
Brook Army Medical Center, San Antonio, Texas 78234, USA.

To assess the feasibility of using cDNA microarrays to understand the response of
endothelial cells to lipopolysaccharide (LPS) and to evaluate potentially
beneficial agents in treatment of septic shock, human umbilical vein endothelial 
cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h. Total RNA
was isolated and reverse-transcribed into (33)P-labeled cDNA probes that were
hybridized to human GeneFilter microarrays containing approximately 4,000 genes. 
The mRNA levels of several genes known to respond to LPS changed after
stimulation. In addition, a number of genes not previously implicated in the
response of endothelial cells to LPS also appeared to be altered in expression.
Nuclear factor-kappaB (NF-kappaB) was shown to play an important role in
regulating genes identified from the microarray studies. Pretreatment of
endothelial cells with a specific NF-kappaB translocation inhibitor eliminated
most of the alterations in gene expression. Quantitative RT-PCR results
independently confirmed the microarray results for monocyte chemotactic protein-1
and interleukin-8, and enzyme-linked immunosorbent assays demonstrated that
augmented transcription was followed by translation and secretion.

DOI: 10.1152/ajpcell.2001.281.5.C1587 
PMID: 11600422  [Indexed for MEDLINE]


1368. Mutat Res. 2001 Nov 1;483(1-2):1-11.

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes.

Porwollik S(1), Wong RM, Sims SH, Schaaper RM, DeMarini DM, McClelland M.

Author information: 
(1)Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121, USA.

Erratum in
    Mutat Res 2001 Dec 12;484(1-2):107-10.

The DeltauvrB mutations present in strains of Salmonella enterica Typhimurium
used commonly in the Salmonella (Ames) mutagenicity assay were isolated
independently for at least five different his mutants. These deletions all
involved the galactose operon, biotin operon, nucleotide-excision-repair uvrB
gene, and chlorate-resistance genes. Beyond this, the size of the deletions and
the number and type of genes deleted have remained unknown for nearly 30 years.
Here, we have used genomic hybridization to a Typhimurium microarray to
characterize these five DeltauvrB deletions. The number of genes (and amount of
DNA) deleted due to the DeltauvrB mutations are 15 (16kb) each in TA97 and TA104,
47 (50kb) in TA100, 87 (96kb) in TA1537, and 119 (125kb) in TA98, accounting for 
0.3, 0.3, 1.0, 1.9, and 2.6% of the genome, respectively. In addition, TA97 and
TA104 contain an identical three-gene deletion elsewhere in their genomes, and,
most remarkably, TA104 contains a 282-gene amplification, representing 7% of the 
genome. Missing genes include mfdA and mdaA, encoding a multi-drug translocase
and a major nitroreductase, respectively, both absent in TA98; dps, encoding a
DNA-binding protein absent in TA1537 and TA98; and dinG, encoding a
lexA-regulated repair enzyme, absent in three DeltauvrB lineages. Genes involved 
in molybdenum cofactor biosynthesis and a number of ORFs of unknown functions are
missing in all DeltauvrB strains investigated. Studies in DeltauvrB strains of
Escherichia coli have found that the enhanced mutagenesis of some base analogues 
was due to the deletion of genes involved in molybdenum cofactor biosynthesis
rather than to deletion of uvrB. These discoveries do not diminish the value of
the data generated in the Ames strains. However, absence of genes other than uvrB
may account for the enhanced mutagenicity of some compounds in DeltauvrB Ames
strains. In general, microarrays will be useful for characterizing the extent and
nature of deletion and amplification mutations.


PMID: 11600126  [Indexed for MEDLINE]


1369. Nat Biotechnol. 2001 Nov;19(11):1060-5.

Selection analyses of insertional mutants using subgenic-resolution arrays.

Badarinarayana V(1), Estep PW 3rd, Shendure J, Edwards J, Tavazoie S, Lam F,
Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

We describe a method of genome-wide analysis of quantitative growth phenotypes
using insertional mutagenesis and DNA microarrays. We applied the method to
assess the fitness contributions of Escherichia coli gene domains under specific 
growth conditions. A transposon library was subjected to competitive growth
selection in Luria-Bertani (LB) and in glucose minimal media.
Transposon-containing genomic DNA fragments from the selected libraries were
compared with the initial unselected transposon insertion library on DNA
microarrays to identify insertions that affect fitness. Genes involved in the
biosynthesis of nutrients not provided in the growth medium were found to be
significantly enriched in the set of genes containing negatively selected
insertions. The data also identify fitness contributions of several
uncharacterized genes, including putative transcriptional regulators and enzymes.
The applicability of this high-resolution array selection in other species is
discussed.

DOI: 10.1038/nbt1101-1060 
PMID: 11689852  [Indexed for MEDLINE]


1370. Science. 2001 Oct 26;294(5543):870-5.

The plasticity of dendritic cell responses to pathogens and their components.

Huang Q(1), Liu D, Majewski P, Schulte LC, Korn JM, Young RA, Lander ES, Hacohen 
N.

Author information: 
(1)Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

Comment in
    Science. 2001 Oct 26;294(5543):799-801.
    Science. 2001 Nov 9;294(5545):1279.

Dendritic cells are involved in the initiation of both innate and adaptive
immunity. To systematically explore how dendritic cells modulate the immune
system in response to different pathogens, we used oligonucleotide microarrays to
measure gene expression profiles of dendritic cells in response to Escherichia
coli, Candida albicans, and influenza virus as well as to their molecular
components. Both a shared core response and pathogen-specific programs of gene
expression were observed upon exposure to each of these pathogens. These results 
reveal that dendritic cells sense diverse pathogens and elicit tailored
pathogen-specific immune responses.

DOI: 10.1126/science.294.5543.870 
PMID: 11679675  [Indexed for MEDLINE]


1371. Int J Food Microbiol. 2001 Oct 22;70(1-2):143-54.

Automated immunomagnetic separation and microarray detection of E. coli O157:H7
from poultry carcass rinse.

Chandler DP(1), Brown J, Call DR, Wunschel S, Grate JW, Holman DA, Olson L,
Stottlemyre MS, Bruckner-Lea CJ.

Author information: 
(1)Analytical Microbiology Group, Pacific Northwest National Laboratory,
Richland, WA 99352, USA. dp.chandler@pnl.gov

We describe the development and application of an electromagnetic flow cell and
fluidics system for automated immunomagnetic separation (IMS) of Escherichia coli
O157:H7 directly from poultry carcass rinse. We further describe the biochemical 
coupling of automated sample preparation with nucleic acid microarrays. Both the 
cell concentration system and microarray detection method did not require cell
growth or enrichment from the poultry carcass rinse prior to IMS. Highly porous
Ni foam was used to enhance the magnetic field gradient within the flow path,
providing a mechanism for immobilizing immunomagnetic particles throughout the
fluid rather than the tubing wall. A maximum of 32% recovery efficiency of
non-pathogenic E. coli was achieved within the automated system with 6 s cell
contact times using commercially available antibodies targeted against the O and 
K antigens. A 15-min protocol (from sample injection though elution) provided a
cell recovery efficiency that was statistically similar to > I h batch captures. 
O157:H7 cells were reproducibly isolated directly from poultry carcass rinse with
39% recovery efficiency at 10(3) CFU ml(-1) inoculum. Direct plating of washed
beads showed positive recovery of O157:H7 directly from poultry carcass rinse at 
an inoculum of 10 CFU ml(-1). Recovered beads were used for direct polymerase
chain reaction (PCR) amplification and microarray detection, with a process-level
detection limit (automated cell concentration though microarray detection) of <
10(3)CFU ml(-1) in poultry carcass rinse.


PMID: 11759752  [Indexed for MEDLINE]


1372. Infect Immun. 2001 Oct;69(10):6140-7.

Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine
mRNAs in intestinal epithelial cells, resulting in increased chemokine
expression.

Thorpe CM(1), Smith WE, Hurley BP, Acheson DW.

Author information: 
(1)Division of Geographic Medicine and Infectious Diseases, Department of
Medicine, Tufts University School of Medicine, New England Medical Center,
Boston, Massachusetts, USA. cthorpe@lifespan.org

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic
syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive
enteric pathogen, the extent to which Stxs can cross the host intestinal
epithelium may affect the risk of developing HUS. We have previously shown that
Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial
cells (IECs) in vitro via a ribotoxic stress response. We used cytokine
expression arrays to determine the effect of Stx1 on various C-X-C chemokine
genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the
mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and
ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and
superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein
in a dose-response fashion, despite an overall inhibition in host cell protein
synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude
that Stxs are able to increase mRNA and protein levels of multiple C-X-C
chemokines in IECs, with increased mRNA stability at least one mechanism
involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can
alter host signal transduction in IECs, resulting in the production of multiple
chemokine mRNAs, leading to increased expression of specific proteins. Taken
together, these data suggest that exposing IECs to Stxs may stimulate a
proinflammatory response, resulting in influx of acute inflammatory cells and
thus contributing to the intestinal tissue damage seen in STEC infection.

DOI: 10.1128/IAI.69.10.6140-6147.2001 
PMCID: PMC98744
PMID: 11553553  [Indexed for MEDLINE]


1373. J Bacteriol. 2001 Oct;183(19):5496-505.

LuxArray, a high-density, genomewide transcription analysis of Escherichia coli
using bioluminescent reporter strains.

Van Dyk TK(1), DeRose EJ, Gonye GE.

Author information: 
(1)Central Research and Development Department, DuPont Company, Wilmington,
Delaware 19880-0173, USA. Tina.K.Van-Dyk@usa.dupont.com

A sequenced collection of plasmid-borne random fusions of Escherichia coli DNA to
a Photorhabdus luminescens luxCDABE reporter was used as a starting point to
select a set of 689 nonredundant functional gene fusions. This group, called
LuxArray 1.0, represented 27% of the predicted transcriptional units in E. coli. 
High-density printing of the LuxArray 1.0 reporter strains to membranes on agar
plates was used for simultaneous reporter gene assays of gene expression. The
cellular response to nalidixic acid perturbation was analyzed using this format. 
As expected, fusions to promoters of LexA-controlled SOS-responsive genes dinG,
dinB, uvrA, and ydjM were found to be upregulated in the presence of nalidixic
acid. In addition, six fusions to genes not previously known to be induced by
nalidixic acid were also reproducibly upregulated. The responses of two of these,
fusions to oraA and yigN, were induced in a LexA-dependent manner by both
nalidixic acid and mitomycin C, identifying these as members of the LexA regulon.
The responses of the other four were neither induced by mitomycin C nor dependent
on lexA function. Thus, the promoters of ycgH, intG, rihC, and a putative operon 
consisting of lpxA, lpxB, rnhB, and dnaE were not generally DNA damage responsive
and represent a more specific response to nalidixic acid. These results
demonstrate that cellular arrays of reporter gene fusions are an important
alternative to DNA arrays for genomewide transcriptional analyses.

DOI: 10.1128/JB.183.19.5496-5505.2001 
PMCID: PMC95439
PMID: 11544210  [Indexed for MEDLINE]


1374. Am J Obstet Gynecol. 2001 Sep;185(3):716-24.

A high-throughput study of gene expression in preterm labor with a subtractive
microarray approach.

Muhle RA(1), Pavlidis P, Grundy WN, Hirsch E.

Author information: 
(1)Department of Obstetrics and Gynecology, Columbia University College of
Physicians and Surgeons, New York, NY, USA.

OBJECTIVE: We propose that elucidation of the pathophysiology of preterm labor
can be achieved with genome-scale analyses of differential gene expression.
STUDY DESIGN: CD-1 mice on day 14.5 of a 19- to 20-day gestation were assigned to
one of 4 treatment groups modeling different clinical conditions (n = 5 per
group): group A, infection with labor (intrauterine injection of 10(10)
heat-killed Escherichia coli, which causes delivery within an average of 20
hours); group B, infection without labor (intrauterine injection of 10(7)
heat-killed E coli, which leads to normal delivery at term); group C, labor
without infection (ovariectomy, which causes delivery within an average of 27
hours); and group D, no infection and no labor (intrauterine injection of
vehicle). Total pooled myometrial RNA was prepared 3.5 hours after surgery for
groups A, B, and D and 5 hours after surgery for group C. The relative expression
of 4963 genes was assayed in these pools by using DNA microarrays. Transcripts
specifically involved in infection-induced labor were identified by subtracting
from the list of differentially regulated genes in group A those with common
expression in groups B and C.
RESULTS: In group A 68 differentially expressed transcripts (>or=2-fold
upregulation or downregulation) were identified. Among these are 39 characterized
genes. Fourteen (45%) are involved in inflammatory responses, 7 (18%) are
involved in growth-differentiation-oncogenesis, and 3 (8%) are involved in
apoptosis. Subtraction identified 13 gene products most likely to be important
for bacterially induced labor, as opposed to labor without infection or bacterial
exposure without labor.
CONCLUSION: This study demonstrates the potential of the subtractive DNA
microarray technique to identify transcripts important specifically for
bacterially induced preterm labor.

DOI: 10.1067/mob.2001.117183 
PMID: 11568803  [Indexed for MEDLINE]


1375. J Bacteriol. 2001 Sep;183(18):5239-47.

DNA microarray-based identification of genes controlled by autoinducer
2-stimulated quorum sensing in Escherichia coli.

DeLisa MP(1), Wu CF, Wang L, Valdes JJ, Bentley WE.

Author information: 
(1)Center for Agricultural Biotechnology, University of Maryland Biotechnology
Institute, University of Maryland, College Park, Maryland 20742, USA.

Bacterial cell-to-cell communication facilitates coordinated expression of
specific genes in a growth rate-II and cell density-dependent manner, a process
known as quorum sensing. While the discovery of a diffusible Escherichia coli
signaling pheromone, termed autoinducer 2 (AI-2), has been made along with
several quorum sensing genes, the overall number and coordination of genes
controlled by quorum sensing through the AI-2 signal has not been studied
systematically. We investigated global changes in mRNA abundance elicited by the 
AI-2 signaling molecule through the use of a luxS mutant that was unable to
synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the E. coli
genome, exhibited significant transcriptional changes (either induction or
repression) in response to a 300-fold AI-2 signaling differential, with many of
the identified genes displaying high induction levels (more than fivefold).
Significant induction of ygeV, a putative sigma(54)-dependent transcriptional
activator, and yhbH, a sigma(54) modulating protein, suggests sigma(54) may be
involved in E. coli quorum sensing.


PMCID: PMC95404
PMID: 11514505  [Indexed for MEDLINE]


1376. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9853-8. Epub 2001 Aug 7.

DNA/DNA hybridization to microarrays reveals gene-specific differences between
closely related microbial genomes.

Murray AE(1), Lies D, Li G, Nealson K, Zhou J, Tiedje JM.

Author information: 
(1)Earth and Ecosystem Sciences, Desert Research Institute, Reno, NV 89512, USA. 
alison@dri.edu

DNA microarrays constructed with full length ORFs from Shewanella oneidensis,
MR-1, were hybridized with genomic DNA from nine other Shewanella species and
Escherichia coli K-12. This approach enabled visualization of relationships
between organisms by comparing individual ORF hybridizations to 164 genes and is 
further amenable to high-density high-throughput analyses of complete microbial
genomes. Conserved genes (arcA and ATP synthase) were identified among all
species investigated. The mtr operon, which is involved in iron reduction, was
poorly conserved among other known metal-reducing Shewanella species. Results
were most informative for closely related organisms with small subunit rRNA
sequence similarities greater than 93% and gyrB sequence similarities greater
than 80%. At this level of relatedness, the similarity between hybridization
profiles was strongly correlated with sequence divergence in the gyrB gene.
Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly
identical, with only 3% of the ORFs hybridizing poorly, in contrast to
hybridizations with Shewanella putrefaciens, formerly considered to be the same
species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below
-0.75). Genomic hybridizations showed that genes in operons had consistent levels
of hybridization across an operon in comparison to a randomly sampled data set,
suggesting that similar applications will be informative for identification of
horizontally acquired genes. The full value of microbial genomic hybridizations
lies in providing the ability to understand and display specific differences
between closely related organisms providing a window into understanding
microheterogeneity, bacterial speciation, and taxonomic relationships.

DOI: 10.1073/pnas.171178898 
PMCID: PMC55542
PMID: 11493693  [Indexed for MEDLINE]


1377. J Bacteriol. 2001 Aug;183(15):4562-70.

DNA microarray-mediated transcriptional profiling of the Escherichia coli
response to hydrogen peroxide.

Zheng M(1), Wang X, Templeton LJ, Smulski DR, LaRossa RA, Storz G.

Author information: 
(1)Cell Biology and Metabolism Branch, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

The genome-wide transcription profile of Escherichia coli cells treated with
hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli
open reading frames. By measuring gene expression in isogenic wild-type and oxyR 
deletion strains, we confirmed that the peroxide response regulator OxyR
activates most of the highly hydrogen peroxide-inducible genes. The DNA
microarray measurements allowed the identification of several new OxyR-activated 
genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which 
may participate in Fe-S cluster assembly or repair; and four genes of unknown
function. We also identified several genes, including uxuA, encoding mannonate
hydrolase, whose expression might be repressed by OxyR, since their expression
was elevated in the DeltaoxyR mutant strain. In addition, the induction of some
genes was found to be OxyR independent, indicating the existence of other
peroxide sensors and regulators in E. coli. For example, the isc operon, which
specifies Fe-S cluster formation and repair activities, was induced by hydrogen
peroxide in strains lacking either OxyR or the superoxide response regulators
SoxRS. These results expand our understanding of the oxidative stress response
and raise interesting questions regarding the nature of other regulators that
modulate gene expression in response to hydrogen peroxide.

DOI: 10.1128/JB.183.15.4562-4570.2001 
PMCID: PMC95351
PMID: 11443091  [Indexed for MEDLINE]


1378. Int J Food Microbiol. 2001 Jul 20;67(1-2):71-80.

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and
nucleic acid microarrays.

Call DR(1), Brockman FJ, Chandler DP.

Author information: 
(1)Environmental Microbiology, Pacific Northwest National Laboratory, Richland,
WA 99352, USA. drcall@wsu.edu

Rapid detection and characterization of food borne pathogens such as Escherichia 
coli O157:H7 is crucial for epidemiological investigations and food safety
surveillance. As an alternative to conventional technologies, we examined the
sensitivity and specificity of nucleic acid microarrays for detecting and
genotyping E. coli O157:H7. The array was composed of oligonucleotide probes
(25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I
and II, and hemolysin A). Target DNA was amplified from whole cells or from
purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR
products were hybridized to the array without further modification or
purification. The array was 32-fold more sensitive than gel electrophoresis and
capable of detecting amplification products from < 1 cell equivalent of genomic
DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used
to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of
pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for
which genotypic data were available, were unambiguously genotyped with this
array. Glass-based microarrays are relatively simple to construct and provide a
rapid and sensitive means to detect multiplexed PCR products; the system is
amenable to automation.


PMID: 11482571  [Indexed for MEDLINE]


1379. Appl Environ Microbiol. 2001 Jul;67(7):3258-63.

Microarray analysis of microbial virulence factors.

Chizhikov V(1), Rasooly A, Chumakov K, Levy DD.

Author information: 
(1)Food and Drug Administration Center for Biologics Evaluation and Research,
Rockville, Maryland, USA.

Hybridization with oligonucleotide microchips (microarrays) was used for
discrimination among strains of Escherichia coli and other pathogenic enteric
bacteria harboring various virulence factors. Oligonucleotide microchips are
miniature arrays of gene-specific oligonucleotide probes immobilized on a glass
surface. The combination of this technique with the amplification of genetic
material by PCR is a powerful tool for the detection of and simultaneous
discrimination among food-borne human pathogens. The presence of six genes (eaeA,
slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants
and virulence factors of bacterial strains was monitored by multiplex PCR
followed by hybridization of the denatured PCR product to the gene-specific
oligonucleotides on the microchip. The assay was able to detect these virulence
factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip 
analysis were confirmed by hybridization of radiolabeled gene-specific probes to 
genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of
the multiplex PCR products used for the microarray analysis produced ambiguous
results due to the presence of unexpected and uncharacterized bands. Our results 
suggest that microarray analysis of microbial virulence factors might be very
useful for automated identification and characterization of bacterial pathogens.

DOI: 10.1128/AEM.67.7.3258-3263.2001 
PMCID: PMC93008
PMID: 11425749  [Indexed for MEDLINE]


1380. Bioorg Khim. 2001 Jul-Aug;27(4):282-90.

[Extensive complementarity of the Shine-Dalgarno region and 3'-terminal sequence 
of 16S ribosomal RNA is inefficient for translation in vivo].

[Article in Russian]

Komarova AV(1), Chufistova LS, Supina EV, Boni IV.

Author information: 
(1)Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia.

Translation initiation in Escherichia coli involves as a rule complementary
interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation
codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD). The
translation efficiency is believed to be directly affected by the affinity of the
ribosome to the mRNA initiation region. Earlier, high-affinity RNA ligands to E. 
coli ribosomes were selected by the SELEX approach, with the ligands containing
an extended SD-sequence well represented. In this work, we examined the ability
of artificial ribosome binding sites (RBSs) containing such an extended (10-nt)
SD-sequence (superSD) to drive translation in vivo, as well as its ability to
form the translation initiation complex in vitro. Toe print experiments showed
the formation of a ternary initiation complex on mRNA comprising superSD.
Moreover, they proved the formation of an extended SD-duplex in the binary
30S-mRNA complex. Nevertheless, the superSD appeared to be inefficient in
translation in vivo. We believe that the initiation complex involving a
superSD-element is too stable to be functional; it may impede the transition from
initiation to elongation, thus disrupting the transcription-translation coupling 
and inhibiting the formation of polysomes.


PMID: 11558262  [Indexed for MEDLINE]


1381. Genes Dev. 2001 Jul 1;15(13):1637-51.

Identification of novel small RNAs using comparative genomics and microarrays.

Wassarman KM(1), Repoila F, Rosenow C, Storz G, Gottesman S.

Author information: 
(1)Cell Biology and Metabolism Branch, National Institute of Child Health & Human
Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

A burgeoning list of small RNAs with a variety of regulatory functions has been
identified in both prokaryotic and eukaryotic cells. However, it remains
difficult to identify small RNAs by sequence inspection. We used the high
conservation of small RNAs among closely related bacterial species, as well as
analysis of transcripts detected by high-density oligonucleotide probe arrays, to
predict the presence of novel small RNA genes in the intergenic regions of the
Escherichia coli genome. The existence of 23 distinct new RNA species was
confirmed by Northern analysis. Of these, six are predicted to encode short ORFs,
whereas 17 are likely to be novel functional small RNAs. We discovered that many 
of these small RNAs interact with the RNA-binding protein Hfq, pointing to a
global role of the Hfq protein in facilitating small RNA function. The approaches
used here should allow identification of small RNAs in other organisms.

DOI: 10.1101/gad.901001 
PMCID: PMC312727
PMID: 11445539  [Indexed for MEDLINE]


1382. Genome Res. 2001 Jul;11(7):1246-55.

Phenotype microarrays for high-throughput phenotypic testing and assay of gene
function.

Bochner BR(1), Gadzinski P, Panomitros E.

Author information: 
(1)Biolog, Inc., Hayward, California 94545, USA. bbochner@biolog.com

The bacterium Escherichia coli is used as a model cellular system to test and
validate a new technology called Phenotype MicroArrays (PMs). PM technology is a 
high-throughput technology for simultaneous testing of a large number of cellular
phenotypes. It consists of preconfigured well arrays in which each well tests a
different cellular phenotype and an automated instrument that continuously
monitors and records the response of the cells in all wells of the arrays. For
example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a
cell suspension into seven microplate arrays. PMs can be used to directly assay
the effects of genetic changes on cells, especially gene knock-outs. Here, we
provide data on phenotypic analysis of six strains and show that we can detect
expected phenotypes as well as, in some cases, unexpected phenotypes.

DOI: 10.1101/gr.186501 
PMCID: PMC311101
PMID: 11435407  [Indexed for MEDLINE]


1383. Mol Microbiol. 2001 Jul;41(1):59-71.

Alkaline shock induces the Bacillus subtilis sigma(W) regulon.

Wiegert T(1), Homuth G, Versteeg S, Schumann W.

Author information: 
(1)Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany.
thomas.wiegert@uni-bayreuth.de

When confronted with a stress factor, bacteria react with a specific stress
response, a genetically encoded programme resulting in the transiently enhanced
expression of a subset of genes. One of these stress factors is a sudden increase
in the external pH. As a first step to understand the response of Bacillus
subtilis cells towards an alkali shock at the transcriptional level, we attempted
to identify alkali-inducible genes using the DNA macroarray technique. To define 
the appropriate challenging conditions, we used the ydjF gene, the orthologue of 
the Escherichia coli pspA, as a model gene for an alkali-inducible gene.
Hybridization of 33P-labelled cDNA to a DNA macroarray revealed induction of more
than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9. It
was discovered that a large subset of these genes belong to the recently
described sigmaW regulon, which was confirmed by the analysis of a sigW knockout.
A comparison of B. subtilis wild type with the congenic sigW knockout also led to
the discovery of new members of the sigmaW regulon. In addition, we found several
genes clearly not belonging to that regulon. This analysis represents the first
report of an extracellular stimulus inducing the sigmaW regulon.


PMID: 11454200  [Indexed for MEDLINE]


1384. Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7546-51. Epub 2001 Jun 12.

A novel application of gene arrays: Escherichia coli array provides insight into 
the biology of the obligate endosymbiont of tsetse flies.

Akman L(1), Aksoy S.

Author information: 
(1)Department of Epidemiology and Public Health, Section of Vector Biology, Yale 
University School of Medicine, New Haven, CT 06510, USA.

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the 
functional roles of obligate symbionts have been difficult to study because it
has not been possible to cultivate these organisms in vitro. The medically
important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont, 
Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related
to Escherichia coli, for fertility and possibly nutrition. We show here that the 
intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an
attempt to understand the composition of its genome, we used the gene arrays
developed for E. coli. We were able to identify 650 orthologous genes in
Wigglesworthia corresponding to approximately 85% of its genome. The arrays were 
also applied for expression analysis using Wigglesworthia cDNA and 61 gene
products were detected, presumably coding for some of its most abundant products.
Overall, genes involved in cell processes, DNA replication, transcription, and
translation were found largely retained in the small genome of Wigglesworthia. In
addition, genes coding for transport proteins, chaperones, biosynthesis of
cofactors, and some amino acids were found to comprise a significant portion,
suggesting an important role for these proteins in its symbiotic life. Based on
its expression profile, we predict that Wigglesworthia may be a facultative
anaerobic organism that utilizes ammonia as its major source of nitrogen. We
present an application of E. coli gene arrays to obtain broad genome information 
for a closely related organism in the absence of complete genome sequence data.

DOI: 10.1073/pnas.131057498 
PMCID: PMC34705
PMID: 11404467  [Indexed for MEDLINE]


1385. Nucleic Acids Res. 2001 Jun 15;29(12):2549-57.

Issues in cDNA microarray analysis: quality filtering, channel normalization,
models of variations and assessment of gene effects.

Tseng GC(1), Oh MK, Rohlin L, Liao JC, Wong WH.

Author information: 
(1)Department of Biostatistics, Harvard School of Public Health, 655 Huntington
Avenue, Boston, MA 02115, USA.

We consider the problem of comparing the gene expression levels of cells grown
under two different conditions using cDNA microarray data. We use a quality
index, computed from duplicate spots on the same slide, to filter out outlying
spots, poor quality genes and problematical slides. We also perform calibration
experiments to show that normalization between fluorescent labels is needed and
that the normalization is slide dependent and non-linear. A rank invariant method
is suggested to select non-differentially expressed genes and to construct
normalization curves in comparative experiments. After normalization the
residuals from the calibration data are used to provide prior information on
variance components in the analysis of comparative experiments. Based on a
hierarchical model that incorporates several levels of variations, a method for
assessing the significance of gene effects in comparative experiments is
presented. The analysis is demonstrated via two groups of experiments with 125
and 4129 genes, respectively, in Escherichia coli grown in glucose and acetate.


PMCID: PMC55725
PMID: 11410663  [Indexed for MEDLINE]


1386. J Biol Chem. 2001 Jun 8;276(23):19937-44. Epub 2001 Mar 20.

Improved statistical inference from DNA microarray data using analysis of
variance and a Bayesian statistical framework. Analysis of global gene expression
in Escherichia coli K12.

Long AD(1), Mangalam HJ, Chan BY, Tolleri L, Hatfield GW, Baldi P.

Author information: 
(1)Department of Ecology, School of Biological Sciences, University of
California, Irvine, California 92697, USA.

We describe statistical methods based on the t test that can be conveniently used
on high density array data to test for statistically significant differences
between treatments. These t tests employ either the observed variance among
replicates within treatments or a Bayesian estimate of the variance among
replicates within treatments based on a prior estimate obtained from a local
estimate of the standard deviation. The Bayesian prior allows statistical
inference to be made from microarray data even when experiments are only
replicated at nominal levels. We apply these new statistical tests to a data set 
that examined differential gene expression patterns in IHF(+) and IHF(-)
Escherichia coli cells (Arfin, S. M., Long, A. D., Ito, E. T., Tolleri, L.,
Riehle, M. M., Paegle, E. S., and Hatfield, G. W. (2000) J. Biol. Chem. 275,
29672-29684). These analyses identify a more biologically reasonable set of
candidate genes than those identified using statistical tests not incorporating a
Bayesian prior. We also show that statistical tests based on analysis of variance
and a Bayesian prior identify genes that are up- or down-regulated following an
experimental manipulation more reliably than approaches based only on a t test or
fold change. All the described tests are implemented in a simple-to-use web
interface called Cyber-T that is located on the University of California at
Irvine genomics web site.

DOI: 10.1074/jbc.M010192200 
PMID: 11259426  [Indexed for MEDLINE]


1387. Genetics. 2001 May;158(1):41-64.

Comparative gene expression profiles following UV exposure in wild-type and
SOS-deficient Escherichia coli.

Courcelle J(1), Khodursky A, Peter B, Brown PO, Hanawalt PC.

Author information: 
(1)Department of Biochemistry, Howard Hughes Medical Institute, Stanford
University, Stanford, CA 94305, USA. jcourcelle@biology.msstate.edu

The SOS response in UV-irradiated Escherichia coli includes the upregulation of
several dozen genes that are negatively regulated by the LexA repressor. Using
DNA microarrays containing amplified DNA fragments from 95.5% of all open reading
frames identified on the E. coli chromosome, we have examined the changes in gene
expression following UV exposure in both wild-type cells and lexA1 mutants, which
are unable to induce genes under LexA control. We report here the time courses of
expression of the genes surrounding the 26 documented lexA-regulated regions on
the E. coli chromosome. We observed 17 additional sites that responded in a
lexA-dependent manner and a large number of genes that were upregulated in a
lexA-independent manner although upregulation in this manner was generally not
more than twofold. In addition, several transcripts were either downregulated or 
degraded following UV irradiation. These newly identified UV-responsive genes are
discussed with respect to their possible roles in cellular recovery following
exposure to UV irradiation.


PMCID: PMC1461638
PMID: 11333217  [Indexed for MEDLINE]


1388. J Bacteriol. 2001 May;183(10):2979-88.

Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux
and levels of expression of glycolytic genes during xylose fermentation.

Tao H(1), Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, Shanmugam
KT.

Author information: 
(1)Institute of Food and Agricultural Sciences, Department of Microbiology and
Cell Science, University of Florida, Gainesville, FL 32611, USA.

Replacement of the native fermentation pathway in Escherichia coli B with a
homo-ethanol pathway from Zymomonas mobilis (pdc and adhB genes) resulted in a 30
to 50% increase in growth rate and glycolytic flux during the anaerobic
fermentation of xylose. Gene array analysis was used as a tool to investigate
differences in expression levels for the 30 genes involved in xylose catabolism
in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total
open reading frames, only 8% were expressed at a significantly higher level in
KO11 (P < 0.05). In contrast, over half of the 30 genes involved in the
catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher
levels in KO11. For 14 of the 30 genes, higher expression was statistically
significant at the 95% confidence level (xylAB, xylE, xylFG, xylR, rpiA, rpiB,
pfkA, fbaA, tpiA, gapA, pgk, and pykA) during active fermentation (6, 12, and 24 
h). Values at single time points for only four of these genes (eno, fbaA, fbaB,
and talA) were higher in strain B than in KO11. The relationship between changes 
in mRNA (cDNA) levels and changes in specific activities was verified for two
genes (xylA and xylB) with good agreement. In KO11, expression levels and
activities were threefold higher than in strain B for xylose isomerase (xylA) and
twofold higher for xylulokinase (xylB). Increased expression of genes involved in
xylose catabolism is proposed as the basis for the increase in growth rate and
glycolytic flux in ethanologenic KO11.

DOI: 10.1128/JB.183.10.2979-2988.2001 
PMCID: PMC95196
PMID: 11325924  [Indexed for MEDLINE]


1389. Appl Environ Microbiol. 2001 Apr;67(4):1911-21.

Genomic interspecies microarray hybridization: rapid discovery of three thousand 
genes in the maize endophyte, Klebsiella pneumoniae 342, by microarray
hybridization with Escherichia coli K-12 open reading frames.

Dong Y(1), Glasner JD, Blattner FR, Triplett EW.

Author information: 
(1)Department of Agronomy, Wisconsin Gene Expression Center, University of
Wisconsin-Madison, Madison, WI 53706, USA.

In an effort to efficiently discover genes in the diazotrophic endophyte of
maize, Klebsiella pneumoniae 342, DNA from strain 342 was hybridized to a
microarray containing 96% (n = 4,098) of the annotated open reading frames from
Escherichia coli K-12. Using a criterion of 55% identity or greater, 3,000 (70%) 
of the E. coli K-12 open reading frames were also found to be present in strain
342. Approximately 24% (n = 1,030) of the E. coli K-12 open reading frames are
absent in strain 342. For 1.6% (n = 68) of the open reading frames, the signal
was too low to make a determination regarding the presence or absence of the
gene. Genes with high identity between the two organisms are those involved in
energy metabolism, amino acid metabolism, fatty acid metabolism, cofactor
synthesis, cell division, DNA replication, transcription, translation, transport,
and regulatory proteins. Functions that were less highly conserved included
carbon compound metabolism, membrane proteins, structural proteins, putative
transport proteins, cell processes such as adaptation and protection, and central
intermediary metabolism. Open reading frames of E. coli K-12 with little or no
identity in strain 342 included putative regulatory proteins, putative
chaperones, surface structure proteins, mobility proteins, putative enzymes,
hypothetical proteins, and proteins of unknown function, as well as genes
presumed to have been acquired by lateral transfer from sources such as phage,
plasmids, or transposons. The results were in agreement with the physiological
properties of the two strains. Whole genome comparisons by genomic interspecies
microarray hybridization are shown to rapidly identify thousands of genes in a
previously uncharacterized bacterial genome provided that the genome of a close
relative has been fully sequenced. This approach will become increasingly more
useful as more full genome sequences become available.

DOI: 10.1128/AEM.67.4.1911-1921.2001 
PMCID: PMC92813
PMID: 11282649  [Indexed for MEDLINE]


1390. FEMS Microbiol Lett. 2001 Apr 1;197(1):91-7.

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

Prüss BM(1), Liu X, Hendrickson W, Matsumura P.

Author information: 
(1)Department of Microbiology and Immunology (M/C 790), College of Medicine,
University of Illinois at Chicago, E-603 Medical Sciences Building, 835 S.
Wolcott, Chicago, IL 60612-7344, USA. pruess@uic.edu

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially
identified as a flagella-specific activator, is a global regulator involved in
many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, 
eight new targets of FlhD/FlhC were recognized. These are the transporter for
galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate
dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol
3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC;
nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the
modulator for hydrogenases, HydNHypF).


PMID: 11287152  [Indexed for MEDLINE]


1391. Nat Biotechnol. 2001 Apr;19(4):336-41.

Immobilized RNA switches for the analysis of complex chemical and biological
mixtures.

Seetharaman S(1), Zivarts M, Sudarsan N, Breaker RR.

Author information: 
(1)Department of Molecular, Cellular and Developmental Biology, Yale University, 
New Haven, CT 06520-8103, USA.

Comment in
    Nat Biotechnol. 2001 Apr;19(4):313-4.

A prototype biosensor array has been assembled from engineered RNA molecular
switches that undergo ribozyme-mediated self-cleavage when triggered by specific 
effectors. Each type of switch is prepared with a 5'-thiotriphosphate moiety that
permits immobilization on gold to form individually addressable pixels. The
ribozymes comprising each pixel become active only when presented with their
corresponding effector, such that each type of switch serves as a specific
analyte sensor. An addressed array created with seven different RNA switches was 
used to report the status of targets in complex mixtures containing metal ion,
enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was
used to determine the phenotypes of Escherichia coli strains for adenylate
cyclase function by detecting naturally produced 3',5'- cyclic adenosine
monophosphate (cAMP) in bacterial culture media.

DOI: 10.1038/86723 
PMID: 11283591  [Indexed for MEDLINE]


1392. Anal Biochem. 2001 Mar;290(2):205-13.

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and
total RNA on DNA microarrays.

Wendisch VF(1), Zimmer DP, Khodursky A, Peter B, Cozzarelli N, Kustu S.

Author information: 
(1)Department of Plant and Microbial Biology, University of California, Berkeley,
California 94720, USA.

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of 
Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini.
We have developed a method to isolate Escherichia coli mRNA by polyadenylating it
in crude cell extracts with E. coli poly(A) polymerase I and purifying it by
oligo(dT) chromatography. Differences in lacZRNA levels were similar with
purified mRNA and total RNA in dot blot hydridizations for cultures grown with or
without gratuitous induction of the lactose operon. More broadly, changes in gene
expression upon induction were similar when cDNAs primed from mRNA or total RNA
with random hexanucleotides were hydridized to DNA microarrays for the E. coli
genome. Comparable signal intensities were obtained with only 1% as much
oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears 
to be selective for mRNA. These and additional studies of genome-wide expression 
with DNA microarrays provide evidence that in vitro poly(A) tailing works
universally for E. coli mRNAs.

Copyright 2001 Academic Press.

DOI: 10.1006/abio.2000.4982 
PMID: 11237321  [Indexed for MEDLINE]


1393. J Clin Microbiol. 2001 Mar;39(3):1097-104.

Antimicrobial resistance and bacterial identification utilizing a microelectronic
chip array.

Westin L(1), Miller C, Vollmer D, Canter D, Radtkey R, Nerenberg M, O'Connell JP.

Author information: 
(1)Department of Advanced Research, Nanogen, Inc., 10398 Pacific Center Ct., San 
Diego, CA 92121, USA.

Species-specific bacterial identification of clinical specimens is often limited 
to a few species due to the difficulty of performing multiplex reactions. In
addition, discrimination of amplicons is time-consuming and laborious, consisting
of gel electrophoresis, probe hybridization, or sequencing technology. In order
to simplify the process of bacterial identification, we combined anchored in situ
amplification on a microelectronic chip array with discrimination and detection
on the same platform. Here, we describe the simultaneous amplification and
discrimination of six gene sequences which are representative of different
bacterial identification assays: Escherichia coli gyrA, Salmonella gyrA,
Campylobacter gyrA, E. coli parC, Staphylococcus mecA, and Chlamydia cryptic
plasmid. The assay can detect both plasmid and transposon genes and can also
discriminate strains carrying antibiotic resistance single-nucleotide
polymorphism mutations. Finally, the assay is similarly capable of discriminating
between bacterial species through reporter-specific discrimination and
allele-specific amplification. Anchored strand displacement amplification allows 
multiplex amplification and complex genotype discrimination on the same platform.
This assay simplifies the bacterial identification process greatly, allowing
molecular biology techniques to be performed with minimal processing of samples
and practical experience.

DOI: 10.1128/JCM.39.3.1097-1104.2001 
PMCID: PMC87879
PMID: 11230433  [Indexed for MEDLINE]


1394. Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2555-60. Epub 2001 Feb 13.

A genomic approach to gene fusion technology.

Van Dyk TK(1), Wei Y, Hanafey MK, Dolan M, Reeve MJ, Rafalski JA, Rothman-Denes
LB, LaRossa RA.

Author information: 
(1)DuPont Company, Biochemical Sciences and Engineering, Wilmington, DE 19880,
USA. Tina.K.Van-Dyk@usa.dupont.com

Gene expression profiling provides powerful analyses of transcriptional responses
to cellular perturbation. In contrast to DNA array-based methods, reporter gene
technology has been underused for this application. Here we describe a
genomewide, genome-registered collection of Escherichia coli bioluminescent
reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E.
coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed
precise mapping of each fusion. The utility of this collection covering about 30%
of the transcriptional units was tested by analyzing individual fusions
representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive
regulons. Each fusion strain responded as anticipated to environmental conditions
known to activate the corresponding regulatory circuit. Thus, the collection
mirrors E. coli's transcriptional wiring diagram. This genomewide collection of
gene fusions provides an independent test of results from other gene expression
analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. 
coli indicated elevated expression of expected and unanticipated genes. Selected 
luxCDABE fusions corresponding to these up-regulated genes were used to confirm
or contradict the DNA microarray results. The power of partnering gene fusion and
DNA microarray technology to discover promoters and define operons was
demonstrated when data from both suggested that a cluster of 20 genes encoding
production of type I extracellular polysaccharide in E. coli form a single
operon.

DOI: 10.1073/pnas.041620498 
PMCID: PMC30176
PMID: 11226277  [Indexed for MEDLINE]


1395. Appl Environ Microbiol. 2001 Feb;67(2):922-8.

Portable system for microbial sample preparation and oligonucleotide microarray
analysis.

Bavykin SG(1), Akowski JP, Zakhariev VM, Barsky VE, Perov AN, Mirzabekov AD.

Author information: 
(1)BioChip Technology Center, Argonne National Laboratory, Argonne, Illinois
60439, USA.

We have developed a three-component system for microbial identification that
consists of (i) a universal syringe-operated silica minicolumn for successive DNA
and RNA isolation, fractionation, fragmentation, fluorescent labeling, and
removal of excess free label and short oligonucleotides; (ii) microarrays of
immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a
portable battery-powered device for imaging the hybridization of fluorescently
labeled RNA fragments with the arrays. The minicolumn combines a guanidine
thiocyanate method of nucleic acid isolation with a newly developed hydroxyl
radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA
can also be fractionated through differential binding of double- and
single-stranded forms of nucleic acids to the silica. The procedure involves
sequential washing of the column with different solutions. No vacuum filtration
steps, phenol extraction, or centrifugation is required. After hybridization, the
overall fluorescence pattern is captured as a digital image or as a Polaroid
photo. This three-component system was used to discriminate Escherichia coli,
Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is
rapid: beginning with whole cells, it takes approximately 25 min to obtain
labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the
microarray image using a stationary image analysis system or the portable imager.

DOI: 10.1128/AEM.67.2.922-928.2001 
PMCID: PMC92667
PMID: 11157263  [Indexed for MEDLINE]


1396. Biochimie. 2001 Feb;83(2):235-41.

H-NS and H-NS-like proteins in Gram-negative bacteria and their multiple role in 
the regulation of bacterial metabolism.

Bertin P(1), Hommais F, Krin E, Soutourina O, Tendeng C, Derzelle S, Danchin A.

Author information: 
(1)Unité de Régulation de l' Expression Génétique, Institut Pasteur, 28, rue du
Dr.-Roux, 75724 Paris cedex 15, France. phbertin@pasteur.fr

In Escherichia coli, the H-NS protein plays an important role in the structure
and the functioning of bacterial chromosome. A homologous protein has also been
identified in several enteric bacteria and in closely related organisms such as
Haemophilus influenzae. To get information on their structure and their function,
we identified H-NS-like proteins in various microorganisms by different
procedures. In silico analysis of their amino acid sequence and/or in vivo
experiments provide evidence that more than 20 proteins belong to the same class 
of regulatory proteins. Moreover, large scale technologies demonstrate that, at
least in E. coli, the loss of motility in hns mutants results from a lack of
flagellin biosynthesis, due to the in vivo repression of flagellar gene
expression. In contrast, several genes involved in adaptation to low pH are
strongly induced in a H-NS deficient strain, resulting in an increased resistance
to acidic stress. Finally, expression profiling and phenotypic analysis suggest
that, unlike H-NS, its paralogous protein StpA does not play any role in these
processes.


PMID: 11278074  [Indexed for MEDLINE]


1397. Biochimie. 2001 Feb;83(2):201-12.

Genome organisation and chromatin structure in Escherichia coli.

Ussery D(1), Larsen TS, Wilkes KT, Friis C, Worning P, Krogh A, Brunak S.

Author information: 
(1)Center for Biological Sequence Analysis, Department of Biotechnology, Building
208, The Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
dave@cbs.dtu.dk

We have analysed the complete sequence of the Escherichia coli K12 isolate MG1655
genome for chromatin-associated protein binding sites, and compared the predicted
location of predicted sites with experimental expression data from 'DNA chip'
experiments. Of the dozen proteins associated with chromatin in E. coli, only
three have been shown to have significant binding preferences: integration host
factor (IHF) has the strongest binding site preference, and FIS sites show a weak
consensus, and there is no clear consensus site for binding of the H-NS protein. 
Using hidden Markov models (HMMs), we predict the location of 608 IHF sites,
scattered throughout the genome. A subset of the IHF sites associated with
repeats tends to be clustered around the origin of replication. We estimate there
could be roughly 6000 FIS sites in E. coli, and the sites tend to be localised in
two regions flanking the replication termini. We also show that the regions
upstream of genes regulated by H-NS are more curved and have a higher AT content 
than regions upstream of other genes. These regions in general would also be
localised near the replication terminus.


PMID: 11278070  [Indexed for MEDLINE]


1398. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):525-30. Epub 2001 Jan 9.

Genetic architecture of thermal adaptation in Escherichia coli.

Riehle MM(1), Bennett AF, Long AD.

Author information: 
(1)Department of Ecology and Evolutionary Biology, University of California,
Irvine, CA 92697-2525, USA. mriehle@uci.edu

Elucidating the genetic basis of adaptation on a genomewide scale has evaded
biologists, but complete genome sequences and DNA high-density array technology
make genomewide surveys more tractable. Six lines of Escherichia coli adapted for
2,000 generations to a stressful high temperature of 41.5 degrees C were examined
on a genomewide scale for duplication/deletion events by using DNA high-density
arrays. A total of five duplication and deletion events were detected. These five
events occurred in three of the six lines, whereas the remaining three lines
contained no detectable events. Three of the duplications were at 2.85 Mb of the 
E. coli chromosome, providing evidence for the replicability of the adaptation to
high temperature. Four candidate genes previously shown to play roles in stress
and starvation survival were identified in the region of common duplication.
Expression of the two candidate genes examined is elevated over expression levels
in the ancestral lines or the lines without the duplication. In the two cases
where the duplication at 2.85 Mb has been further characterized, the timing of
the genome reorganization is coincident with significant increases in relative
fitness. In both of these cases, the model for the origin of the duplication is a
complex recombination event involving insertion sequences and repeat sequences.
These results provide additional evidence for the idea that gene duplication
plays an integral role in adaptation, specifically as a means for gene
amplification.

DOI: 10.1073/pnas.021448998 
PMCID: PMC14620
PMID: 11149947  [Indexed for MEDLINE]


1399. Biotechnol Bioeng. 2001 Jan 5;72(1):85-95.

Genomic analysis of high-cell-density recombinant Escherichia coli fermentation
and "cell conditioning" for improved recombinant protein yield.

Gill RT(1), DeLisa MP, Valdes JJ, Bentley WE.

Author information: 
(1)Department of Chemical Engineering, University of Maryland, College Park,
Maryland 20742, USA.

The Escherichia coli stress gene transcription profile and response to
recombinant protein overexpression were substantially altered at high cell
density when compared with low cell density. Reverse trascription-polymerase
chain reaction RT-PCR-amplified mRNA from low (4 g[DCW]/L) and high-cell-density 
(43.5 g [DCW]/L) conditions were hybridized with a DNA microarray of Kohara
clones encompassing 16% of the E. coli genome, and differentially displayed genes
were identified. Transcript-specific RNA dot blots indicated that molecular
chaperones (groEL, ibpA, degP), proteases (degP, ftsH), the lysis gene mltB, and 
DNA damage/bacteriophage-associated gene transcript levels (ftsH, recA, alpA,
uvrB) increased 10- to 43-fold at high cell density. In addition, overexpression 
of recombinant green fluorescent protein (GFP(uv))/chloramphenicol
acetyltransferase (CAT) fusion protein did not change the rates of cell growth or
cell lysis. The stress gene transcription profile at high cell density was used
to evaluate "cell conditioning" strategies to alter the levels of chaperones,
proteases, and other intracellular proteins prior to recombinant protein
overexpression. Interestingly, the addition of 1 g/L dithiothreitol (DTT) 20 min 
prior to induction of a GFP(uv)/CAT fusion protein resulted in a twofold increase
in CAT activity when compared with the unconditioned controls. In addition, RNA
dot blots of five stress genes confirmed that cell conditioning strategies
significantly altered the dynamic stress gene response to foreign protein
overexpression.

Copyright 2001 John Wiley & Sons, Inc.


PMID: 11084598  [Indexed for MEDLINE]


1400. Anal Chem. 2001 Jan 1;73(1):1-7.

Surface plasmon resonance imaging measurements of DNA and RNA hybridization
adsorption onto DNA microarrays.

Nelson BP(1), Grimsrud TE, Liles MR, Goodman RM, Corn RM.

Author information: 
(1)Department of Chemistry, University of Wisconsin, Madison 53706-1396, USA.

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic
technique for measuring interactions between unlabeled biological molecules with 
arrays of surface-bound species. In this paper, SPR imaging is used to
quantitatively detect the hybridization adsorption of short (18-base) unlabeled
DNA oligonucleotides at low concentration, as well as, for the first time, the
hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S
ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array.
For the hybridization adsorption of both DNA and RNA oligonucleotides, a
detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, 
concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA 
probes to the gold surface leads to high surface probe density (10(12)
molecules/cm2) and excellent probe stability that enables more than 25 cycles of 
hybridization and denaturing without loss in signal or specificity. Fresnel
calculations are used to show that changes in percent reflectivity as measured by
SPR imaging are linear with respect to surface coverage of adsorbed DNA
oligonucleotides. Data from SPR imaging is used to construct a quantitative
adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA
18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir
isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).


PMID: 11195491  [Indexed for MEDLINE]


1401. Genome Inform. 2001;12:44-53.

Extraction of correlated gene clusters by multiple graph comparison.

Nakaya A(1), Goto S, Kanehisa M.

Author information: 
(1)Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji,
Kyoto 611-0011, Japan. nakaya@kuicr.kyoto-u.ac.jp

This paper presents a new method to extract a set of correlated genes with
respect to multiple biological features. Relationships among genes on a specific 
feature are encoded as a graph structure whose nodes correspond to genes. For
example, the genome is a graph representing positional correlations of genes on
the chromosome, the pathway is a graph representing functional correlations of
gene products, and the expression profile is a graph representing gene expression
similarities. When a set of genes are localized in a single graph, such as a gene
cluster on the chromosome, an enzyme cluster in the metabolic pathway, or a set
of coexpressed genes in the microarray gene expression profile, this may suggest 
a functional link among those genes. The functional link would become stronger
when the clusters are correlated; namely, when a set of corresponding genes form 
clusters in multiple graphs. The newly introduced heuristic algorithm extracts
such correlated gene clusters as isomorphic subgraphs in multiple graphs by using
inter-graph links that are defined based on biological relevance. Using the
method, we found E.coli correlated gene clusters in which genes are related with 
respect to the positions in the genome and the metabolic pathway, as well as the 
3D structural similarity. We also analyzed protein-protein interaction data by
two-hybrid experiments and gene coexpression data by microarrays in S.cerevisiae,
and estimated the possibility of utilizing our method for screening the datasets 
that are likely to contain many false positive relations.


PMID: 11791223  [Indexed for MEDLINE]


1402. J Bacteriol. 2001 Jan;183(2):545-56.

High-density microarray-mediated gene expression profiling of Escherichia coli.

Wei Y(1), Lee JM, Richmond C, Blattner FR, Rafalski JA, LaRossa RA.

Author information: 
(1)Central Research and Development, DuPont Company, Wilmington, Delaware
19880-0173, USA.

A nearly complete collection of 4,290 Escherichia coli open reading frames was
amplified and arrayed in high density on glass slides. To exploit this reagent,
conditions for RNA isolation from E. coli cells, cDNA production with attendant
fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation
have been established. A brief isopropyl-beta-D-thiogalactopyranoside (IPTG)
treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in
contrast, most other transcript titers remained unchanged. Distinct RNA
expression patterns between E. coli cultures in the exponential and transitional 
phases of growth were catalogued, as were differences associated with culturing
in minimal and rich media. The relative abundance of each transcript was
estimated by using hybridization of a genomic DNA-derived, fluorescently labeled 
probe as a correction factor. This inventory provided a quantitative view of the 
steady-state level of each mRNA species. Genes the expression of which was
detected by this method were enumerated, and results were compared with the
current understanding of E. coli physiology.

DOI: 10.1128/JB.183.2.545-556.2001 
PMCID: PMC94910
PMID: 11133948  [Indexed for MEDLINE]


1403. J Comput Biol. 2001;8(1):37-52.

On differential variability of expression ratios: improving statistical inference
about gene expression changes from microarray data.

Newton MA(1), Kendziorski CM, Richmond CS, Blattner FR, Tsui KW.

Author information: 
(1)Department of Statistics, University of Wisconsin, Madison, WI 53792, USA.
newton@stat.wisc.edu

We consider the problem of inferring fold changes in gene expression from cDNA
microarray data. Standard procedures focus on the ratio of measured fluorescent
intensities at each spot on the microarray, but to do so is to ignore the fact
that the variation of such ratios is not constant. Estimates of gene expression
changes are derived within a simple hierarchical model that accounts for
measurement error and fluctuations in absolute gene expression levels.
Significant gene expression changes are identified by deriving the posterior odds
of change within a similar model. The methods are tested via simulation and are
applied to a panel of Escherichia coli microarrays.

DOI: 10.1089/106652701300099074 
PMID: 11339905  [Indexed for MEDLINE]


1404. Pac Symp Biocomput. 2001:127-38.

BioProspector: discovering conserved DNA motifs in upstream regulatory regions of
co-expressed genes.

Liu X(1), Brutlag DL, Liu JS.

Author information: 
(1)Stanford Medical Informatics, 251 Campus Dr. X215, Stanford University,
Stanford, CA 94305-5479, USA. xliu@smi.stanford.edu

The development of genome sequencing and DNA microarray analysis of gene
expression gives rise to the demand for data-mining tools. BioProspector, a C
program using a Gibbs sampling strategy, examines the upstream region of genes in
the same gene expression pattern group and looks for regulatory sequence motifs. 
BioProspector uses zero to third-order Markov background models whose parameters 
are either given by the user or estimated from a specified sequence file. The
significance of each motif found is judged based on a motif score distribution
estimated by a Monte Carlo method. In addition, BioProspector modifies the motif 
model used in the earlier Gibbs samplers to allow for the modeling of gapped
motifs and motifs with palindromic patterns. All these modifications greatly
improve the performance of the program. Although testing and development are
still in progress, the program has shown preliminary success in finding the
binding motifs for Saccharomyces cerevisiae RAP1, Bacillus subtilis RNA
polymerase, and Escherichia coli CRP. We are currently working on combining
BioProspector with a clustering program to explore gene expression networks and
regulatory mechanisms.


PMID: 11262934  [Indexed for MEDLINE]


1405. Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14674-9.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as
a defense against nitrogen limitation.

Zimmer DP(1), Soupene E, Lee HL, Wendisch VF, Khodursky AB, Peter BJ, Bender RA, 
Kustu S.

Author information: 
(1)Departments of Plant and Microbial Biology and Molecular and Cell Biology,
University of California, Berkeley, CA 94720, USA.

Comment in
    Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14044-5.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription 
of genes/operons whose products minimize the slowing of growth under
nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we
compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes
[glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA
microarrays. Both strains could be grown under conditions of nitrogen excess.
Thus, we could avoid differences in gene expression caused by slow growth or
nitrogen limitation per se. Rearranging the spot images from microarrays in
genome order allowed us to detect all of the operons known to be under NtrC
control and facilitated detection of a number of new ones. Many of these operons 
encode transport systems for nitrogen-containing compounds, including compounds
recycled during cell-wall synthesis, and hence scavenging appears to be a primary
response to nitrogen limitation. In all, approximately 2% of the E. coli genome
appears to be under NtrC control, although transcription of some operons depends 
on the nitrogen assimilation control protein, which serves as an adapter between 
NtrC and final sigma(70)-dependent promoters.

DOI: 10.1073/pnas.97.26.14674 
PMCID: PMC18977
PMID: 11121068  [Indexed for MEDLINE]


1406. FEMS Microbiol Lett. 2000 Dec 1;193(1):99-103.

Rapid cloning of metK encoding methionine adenosyltransferase from
Corynebacterium glutamicum by screening a genomic library on a high density
colony-array.

Grossmann K(1), Herbster K, Mack M.

Author information: 
(1)BASF-LYNX Bioscience AG, Im Neuenheimer Feld 515, 69120, Heidelberg, Germany.

The genes SAM1 and SAM2 encoding the two different methionine
adenosyltransferases (EC 2.5.1.6) in Saccharomyces cerevisiae were used as
templates to generate specific DNA-probes. This heterologous mixture of
DNA-probes was hybridized under low stringency hybridization conditions to a
Corynebacterium glutamicum colony-array representing the complete genome.
Subsequently, one genomic fragment was isolated which contained the C. glutamicum
methionine adenosyltransferase gene metK (1.224 kb). When overproduced in
Escherichia coli, MetK (44.2 kDa) of C. glutamicum had methionine
adenosyltransferase activity. In addition, overexpression of metK in C.
glutamicum led to an increased intracellular S-adenosylmethionine concentration. 
The metK transcript was detected by reverse transcription PCR in C. glutamicum
cells in the exponential growth phase but not in the stationary phase.


PMID: 11094286  [Indexed for MEDLINE]


1407. Nat Biotechnol. 2000 Dec;18(12):1262-8.

RNA expression analysis using a 30 base pair resolution Escherichia coli genome
array.

Selinger DW(1), Cheung KJ, Mei R, Johansson EM, Richmond CS, Blattner FR,
Lockhart DJ, Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, 200 Longwood Avenue Boston, MA
02115, USA.

Comment in
    Nat Biotechnol. 2000 Dec;18(12):1241-2.

We have developed a high-resolution "genome array" for the study of gene
expression and regulation in Escherichia coli. This array contains on average one
25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one
every 6 bases for the intergenic regions and every 60 bases for the 4,290 open
reading frames (ORFs). Twofold concentration differences can be detected at
levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be
seen over a dynamic range of three orders of magnitude. In rich medium we
detected transcripts for 97% and 87% of the ORFs in stationary and log phases,
respectively. We found that 1, 529 transcripts were differentially expressed
under these conditions. As expected, genes involved in translation were expressed
at higher levels in log phase, whereas many genes known to be involved in the
starvation response were expressed at higher levels in stationary phase. Many
previously unrecognized growth phase-regulated genes were identified, such as a
putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of
which are highly upregulated in stationary phase. Transcription of between 3,000 
and 4,000 predicted ORFs was observed from the antisense strand, indicating that 
most of the genome is transcribed at a detectable level. Examples are also
presented for high-resolution array analysis of transcript start and stop sites
and RNA secondary structure.

DOI: 10.1038/82367 
PMID: 11101804  [Indexed for MEDLINE]


1408. Nucleic Acids Res. 2000 Nov 1;28(21):E93.

Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic
SELEX.

Shtatland T(1), Gill SC, Javornik BE, Johansson HE, Singer BS, Uhlenbeck OC,
Zichi DA, Gold L.

Author information: 
(1)Department of Molecular, University of Colorado, Boulder, CO 80309-0347, USA.

Genomic SELEX is a method for studying the network of nucleic acid-protein
interactions within any organism. Here we report the discovery of several
interesting and potentially biologically important interactions using genomic
SELEX. We have found that bacteriophage MS2 coat protein binds several
Escherichia coli mRNA fragments more tightly than it binds the natural,
well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG
(involved in formation of lipopolysaccharide in the bacterial outer membrane),
ebgR (lactose utilization repressor), as well as from several other genes.
Genomic SELEX may yield experimentally induced artifacts, such as molecules in
which the fixed sequences participate in binding. We describe several methods
(annealing of oligonucleotides complementary to fixed sequences or switching
fixed sequences) to eliminate some, or almost all, of these artifacts. Such
methods may be useful tools for both randomized sequence SELEX and genomic SELEX.


PMCID: PMC113162
PMID: 11058143  [Indexed for MEDLINE]


1409. Microbiology. 2000 Sep;146 ( Pt 9):2277-82.

Metal-ion tolerance in Escherichia coli: analysis of transcriptional profiles by 
gene-array technology.

Brocklehurst KR(1), Morby AP.

Author information: 
(1)School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US,
UK.

Escherichia coli was adapted to grow in medium containing substantially elevated 
concentrations of either Zn(II), Cd(II), Co(II) or Ni(II). Whole-genome
transcriptional profiles were generated from adapted strains and analysed for
significant alteration in transcript abundance with reference to a wild-type
strain. Similar alterations in specific message levels were observed for strains 
adapted to the four metal ions. One unexpected trend was the increase in
transcript level of genes involved in transposition of IS elements, particularly 
insA. Subsequent expression of insA-7 from a heterologous promoter in E. coli
conferred tolerance to Zn(II).

DOI: 10.1099/00221287-146-9-2277 
PMID: 10974115  [Indexed for MEDLINE]


1410. Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9419-24.

Analysis of topoisomerase function in bacterial replication fork movement: use of
DNA microarrays.

Khodursky AB(1), Peter BJ, Schmid MB, DeRisi J, Botstein D, Brown PO, Cozzarelli 
NR.

Author information: 
(1)Department of Molecular and Cell Biology, University of California, Berkeley
94720, USA.

We used DNA microarrays of the Escherichia coli genome to trace the progression
of chromosomal replication forks in synchronized cells. We found that both DNA
gyrase and topoisomerase IV (topo IV) promote replication fork progression. When 
both enzymes were inhibited, the replication fork stopped rapidly. The elongation
rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended
these results. Inactivation of gyrase alone caused a slow stop of replication.
Topo IV activity was sufficient to prevent accumulation of (+) supercoils in
plasmid DNA in vivo, suggesting that topo IV can promote replication by removing 
(+) supercoils in front of the chromosomal fork.


PMCID: PMC16879
PMID: 10944214  [Indexed for MEDLINE]


1411. Metab Eng. 2000 Jul;2(3):201-9.

DNA microarray detection of metabolic responses to protein overproduction in
Escherichia coli.

Oh MK(1), Liao JC.

Author information: 
(1)Department of Chemical Engineering, University of California, Los Angeles,
California 90095, USA.

It has been commonly observed that gratuitous overexpression of proteins in
Escherichia coli causes growth retardation. However, the molecular events
involved in the metabolic response to the over-expression of proteins are still
unclear. Here we used DNA microarray technology to characterize the changes in
transcriptional patterns of selected host genes during protein overexpression. A 
nontoxic, soluble protein, LuxA (coded by luxA), which is the alpha-subunit of
the luciferase heterodimer, was overexpressed for this purpose. A total of 132 E.
coli genes, including those in the central metabolism, key biosynthetic pathways,
and selected regulatory functions, were used as probes for detecting the level of
mRNA transcripts in E. coli strains JM109, MC4100, and VJS676A during protein
overexpression. Upon induction, these strains shared several common responses,
such as the upregulation of glk and the heat shock genes as well as the
downregulation of fba, ppc, atpA, and gnd. In addition, the biosynthesis genes
glnA, glyA, and leuA were downregulated in all three strains. Media-dependent
responses were also observed in our study. For example, many respiratory genes
that were upregulated in defined media showed an opposite effect in complex media
under protein-overproducing conditions. These results demonstrate that gratuitous
overexpression of proteins triggers a complex global response that involves
several metabolic and regulatory systems. Explanations based on either existing
knowledge of global regulations such as the heat shock response and the stringent
response or stoichiometric analysis without regulatory considerations cannot
account for the response induced by protein overexpression.

Copyright 2000 Academic Press.

DOI: 10.1006/mben.2000.0149 
PMID: 11056062  [Indexed for MEDLINE]


1412. J Bacteriol. 2000 Jun;182(12):3467-74.

Differential expression of over 60 chromosomal genes in Escherichia coli by
constitutive expression of MarA.

Barbosa TM(1), Levy SB.

Author information: 
(1)Center for Adaptation Genetics and Drug Resistance and the Departments of
Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, 
Massachusetts 02111, USA.

In Escherichia coli, the MarA protein controls expression of multiple chromosomal
genes affecting resistance to antibiotics and other environmental hazards. For a 
more-complete characterization of the mar regulon, duplicate macroarrays
containing 4,290 open reading frames of the E. coli genome were hybridized to
radiolabeled cDNA populations derived from mar-deleted and mar-expressing E.
coli. Strains constitutively expressing MarA showed altered expression of more
than 60 chromosomal genes: 76% showed increased expression and 24% showed
decreased expression. Although some of the genes were already known to be MarA
regulated, the majority were newly determined and belonged to a variety of
functional groups. Some of the genes identified have been associated with iron
transport and metabolism; other genes were previously known to be part of the
soxRS regulon. Northern blot analysis of selected genes confirmed the results
obtained with the macroarrays. The findings reveal that the mar locus mediates a 
global stress response involving one of the largest networks of genes described.


PMCID: PMC101932
PMID: 10852879  [Indexed for MEDLINE]


1413. Biotechnol Prog. 2000 Mar-Apr;16(2):278-86.

Gene expression profiling by DNA microarrays and metabolic fluxes in Escherichia 
coli.

Oh MK(1), Liao JC.

Author information: 
(1)Department of Chemical Engineering, University of California, Los Angeles,
California 90095, USA.

DNA microarray technology was applied to detect differential transcription
profiles of a subset of the Escherichia coli genome. A total of 111 E. coli
genes, including those in central metabolism, key biosyntheses, and some
regulatory functions, were cloned, amplified, and used as probes for detecting
the level of transcripts. An E. coli strain was grown in glucose, acetate, and
glycerol media, and the transcript levels of the selected genes were detected.
Despite extensive studies on E. coli physiology, many new features were found in 
the regulation of these genes. For example, several genes were unexpectedly
up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and
asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate.
These genes were regulated with no apparent reasons by unknown mechanisms.
Meanwhile, many genes were regulated for apparent purposes but by unknown
mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both
acetate and glycerol media were down-regulated, and the ppc, glycolytic, and
biosynthetic genes in acetate were also down-regulated because of the reduced
fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore,
a group of genes were regulated by known mechanisms, but the physiological roles 
of such regulation remain unclear. This group includes pckA and aspA, which are
up-regulated in glycerol, and gnd and aspA, which are down- and up-regulated,
respectively, in acetate. The DNA microarray technology demonstrated here is a
powerful yet economical tool for characterizing gene regulation and will prove to
be useful for strain improvement and bioprocess development.

DOI: 10.1021/bp000002n 
PMID: 10753455  [Indexed for MEDLINE]


1414. Xenobiotica. 2000 Feb;30(2):155-77.

DNA arrays: technology, options and toxicological applications.

Rockett JC(1), Dix DJ.

Author information: 
(1)Reproductive Toxicology Division, National Health and Environmental Effects
Research Laboratory, US Environmental Protection Agency, NC 27711, USA.
rockett.john@epa.gov

The human genome contains an estimated 3 billion bases of DNA making up some
100000 genes, and the variation within this genome accounts for human diversity
and, in many cases, disease. Defining and understanding the expression profile of
given genotypes is essential to understanding adverse effects from acute or
chronic exposure to environmental toxicants or other stimuli. DNA array
technology could help researchers understand how organisms function in response
to exposure by elucidating the molecular mechanisms that underlie them. DNA
arrays have been developed and refined over the past 5 years and matured into a
relatively accessible and affordable technology. They vary in design from
membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens
of thousands of genetic elements. Mammalian DNA arrays will soon allow expression
analysis on a genome-wide scale, similar to that already accomplished in some
lower organisms (e.g. S. cerevisiae, E. coli). These whole-genome arrays will be 
powerful tools for identifying and characterizing toxicants in environmental and 
pharmaceutical science. This review discusses the technology behind the
production of DNA arrays, the options available to those interested in applying
them to their own research, and the possible toxicological applications of this
exciting new technology.

DOI: 10.1080/004982500237758	 
PMID: 10718123  [Indexed for MEDLINE]


1415. Cytogenet Cell Genet. 2000;91(1-4):39-43.

Cell lines from the same cervical carcinoma but with different radiosensitivities
exhibit different cDNA microarray patterns of gene expression.

Achary MP(1), Jaggernauth W, Gross E, Alfieri A, Klinger HP, Vikram B.

Author information: 
(1)Department of Radiation Oncology, Albert Einstein College of Medicine of
Yeshiva University, and Montefiore Medical Center, Bronx, NY, USA.
achary@aecom.yu.edu

Combining chemotherapy with radiotherapy has improved the cure rate among
patients with cancers of the cervix. Although one-half to two-thirds of the
patients can be cured by radiation alone, such patients cannot be identified at
present and must therefore suffer the burden of chemotherapy. Our long-range goal
is to identify those cervical cancers that are radiosensitive and could be cured 
by radiotherapy alone. The advent of methods that permit the simultaneous
analysis of expression patterns of thousands of genes, make it feasible to
attempt to identify the molecular events related to radiosensitivity and the
associated regulatory pathways. We hypothesize that the sensitivity of tumor
cells to ionizing radiation (IR) is determined by the level of expression of
specific genes that may be identified with the aid of cDNA microarrays. As the
first step in testing this hypothesis, we determined the gene expression
differences between two cell lines exhibiting different degrees of
radiosensitivity. These were derived from the same tumor prior to treatment from 
a patient with squamous cell carcinoma of the cervix. The mRNA from these cells
was subjected to cDNA analysis on a microarray of 5,776 known genes and ESTs. The
expression of 52 genes of the total of 5,776 was elevated (maximum 4.1 fold) in
the radioresistant cells as compared to the radiosensitive cells. Ten of the 52
sequences are known genes while 42 are ESTs. Conversely, the expression of 18
genes was elevated in the sensitive cells as compared to the resistant cells.
Seven of these 18 are known genes while eleven are ESTs. Among the genes
expressed differentially between the resistant and sensitive cells were several
known to be associated with response to IR and many more genes and ESTs that had 
not previously been reported to be related to radiosensitivity. The genes that
showed the greatest overexpression in the radioresistant cell line were
metal-regulatory transcription factor-1, cytochrome P450 CYP1B1, adenomatosis
polyposis coli, translation elongation factor-1, cytochrome-c oxidase, whereas in
the sensitive cell line, transcription factor NF-kappa-B, metalloproteinase
inhibitor-1 precursor, superoxide dismutase-2, insulin-like growth factor binding
protein-3, guanine nucleotide-binding protein and transforming growth factor
beta-induced protein were overexpressed.

Copyright 2001 S. Karger AG, Basel

DOI: 10.1159/000056815 
PMID: 11173827  [Indexed for MEDLINE]


1416. J Biol Chem. 1999 Oct 29;274(44):31236-44.

Highly specific recognition of primer RNA structures for 2'-OH priming reaction
by bacterial reverse transcriptases.

Inouye S(1), Hsu MY, Xu A, Inouye M.

Author information: 
(1)Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway,
New Jersey 08854, USA.

A minor population of Escherichia coli contains retro-elements called retrons,
which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs
called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA
structures in their individual primer-template RNAs to initiate cDNA synthesis
from the 2'-OH group of a specific internal G residue (branching G residue). The 
resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly
any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific 
RNA structure in its primer-template RNA. On the basis of structural comparison
with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86
and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71
identical residues (22%). From the analysis of 10 such constructs, the C-terminal
91-residue sequence of RT-Ec86 was found to be essential for the recognition of
the unique stem-loop structure and the branching G residue in the primer-template
RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by
exponential enrichment) method with RT-Ec86 and primer RNAs containing random
sequences, the identical stem-loop structure (including the 3-U loop) to that
found in the retron-Ec86 primer-template RNA was enriched. In addition, the
highly conserved 4-base sequence (UAGC), including the branching G residue, was
also enriched. These results indicate that the highly diverse C-terminal region
recognizes specific stem-loop structures and the branching G residue located
upstream of the stem-loop structure. The present results with seemingly primitive
RNA-dependent DNA polymerases provide insight into the mechanisms for specific
protein RNA recognition.


PMID: 10531319  [Indexed for MEDLINE]


1417. Biochimie. 1999 Oct;81(10):995-1002.

Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by
optical biosensor technique.

Karlsson M(1), Pavlov MY, Malmqvist M, Persson B, Ehrenberg M.

Author information: 
(1)Department of Cell and Molecular Biology, BMC, Box 596, 75124 Uppsala, Sweden.

The optical biosensor technique, based on the surface plasmon resonance (SPR)
phenomenon, has been used to study the initiation of protein synthesis by E. coli
ribosomes on surface coupled mRNA. mRNA was first periodate oxidized and then
hydrazide coupled to the surface of a CM5 sensor chip. The formation of
initiation complexes on the surface coupled mRNA was monitored in real-time with 
a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes
were assembled on mRNA by sequential introduction of the 30S and 50S subunits
supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that
the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds
efficiently only in the presence of tRNA. Moreover, 70S*mRNA*fMet-tRNA(Met)
complexes formed with fMet-tRNA(Met) are more stable than similar complexes
formed with deacylated tRNAs. The efficient formation and slow dissociation of
mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by 
the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met).
This work demonstrates the feasibility of the BIACORE technique for studying the 
initiation of protein synthesis.


PMID: 10575353  [Indexed for MEDLINE]


1418. J Bacteriol. 1999 Oct;181(20):6425-40.

Functional genomics: expression analysis of Escherichia coli growing on minimal
and rich media.

Tao H(1), Bausch C, Richmond C, Blattner FR, Conway T.

Author information: 
(1)Department of Microbiology, The Ohio State University, Columbus, Ohio
43210-1292, USA.

DNA arrays of the entire set of Escherichia coli genes were used to measure the
genomic expression patterns of cells growing in late logarithmic phase on minimal
glucose medium and on Luria broth containing glucose. Ratios of the transcript
levels for all 4,290 E. coli protein-encoding genes (cds) were obtained, and
analysis of the expression ratio data indicated that the physiological state of
the cells under the two growth conditions could be ascertained. The cells in the 
rich medium grew faster, and expression of the majority of the translation
apparatus genes was significantly elevated under this growth condition,
consistent with known patterns of growth rate-dependent regulation and increased 
rate of protein synthesis in rapidly growing cells. The cells grown on minimal
medium showed significantly elevated expression of many genes involved in
biosynthesis of building blocks, most notably the amino acid biosynthetic
pathways. Nearly half of the known RpoS-dependent genes were expressed at
significantly higher levels in minimal medium than in rich medium, and rpoS
expression was similarly elevated. The role of RpoS regulation in these
logarithmic phase cells was suggested by the functions of the RpoS dependent
genes that were induced. The hallmark features of E. coli cells growing on
glucose minimal medium appeared to be the formation and excretion of acetate,
metabolism of the acetate, and protection of the cells from acid stress. A
hypothesis invoking RpoS and UspA (universal stress protein, also significantly
elevated in minimal glucose medium) as playing a role in coordinating these
various aspects and consequences of glucose and acetate metabolism was generated.
This experiment demonstrates that genomic expression assays can be applied in a
meaningful way to the study of whole-bacterial-cell physiology for the generation
of hypotheses and as a guide for more detailed studies of particular genes of
interest.


PMCID: PMC103779
PMID: 10515934  [Indexed for MEDLINE]


1419. Nucleic Acids Res. 1999 Oct 1;27(19):3821-35.

Genome-wide expression profiling in Escherichia coli K-12.

Richmond CS(1), Glasner JD, Mau R, Jin H, Blattner FR.

Author information: 
(1)Laboratory of Genetics, University of Wisconsin, Madison, WI 53706, USA.
craig@genetics.wisc.edu

We have established high resolution methods for global monitoring of gene
expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots 
on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to
glass microarrays for efficiency and reproducibility. A complete set of PCR
primers was created for all 4290 annotated open reading frames (ORFs) from the
complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays
of PCR products were prepared and used to assess global changes in gene
expression. Full-length coding sequences for array printing were generated by
two-step PCR amplification. In this study we measured changes in RNA levels after
exposure to heat shock and following treatment with
isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and
fluorescence-based methods showed comparable results. Treatment with IPTG
resulted in high level induction of the lacZYA and melAB operons. Following heat 
shock treatment 119 genes were shown to have significantly altered expression
levels, including 35 previously uncharacterized ORFs and most genes of the heat
shock stimulon. Analysis of spot intensities from hybridization to replicate
arrays identified sets of genes with signals consistently above background
suggesting that at least 25% of genes were expressed at detectable levels during 
growth in rich media.


PMCID: PMC148645
PMID: 10481021  [Indexed for MEDLINE]


1420. Mol Microbiol. 1999 Sep;33(5):1004-14.

Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the
CspA family of Escherichia coli.

Phadtare S(1), Inouye M.

Author information: 
(1)Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, NJ 08854, USA.

The CspA family of Escherichia coli comprises nine homologous proteins, CspA to
CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity
and low binding affinity. This is considered to be important for its proposed
function as an RNA chaperone to prevent the formation of secondary structures in 
RNA molecules, thus facilitating translation at low temperature. The cellular
functions of other Csp proteins are yet to be fully elucidated, and their
sequence specific binding capabilities have not been identified. As a step
towards identification of the target genes of Csp proteins, we investigated the
RNA binding specificities of CspB, CspC and CspE by an in vitro selection
approach (SELEX). In the present study, we show that these proteins are able to
bind preferentially to specific RNA/single-stranded DNA sequences. The consensus 
sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT-rich
regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the
range 0.23-0.9 x 10(-6) M, while CspC has 10-fold lower binding affinity.
Consistent with our recent findings of transcriptional regulation of cspA by
CspE, we have identified a motif identical to the CspE consensus. This motif is
the putative CspE-mediated transcription pause recognition site in a
5'-untranslated region of the cspA mRNA.


PMID: 10476034  [Indexed for MEDLINE]


1421. RNA. 1999 Sep;5(9):1180-90.

In vitro selection of RNA aptamers that bind special elongation factor SelB, a
protein with multiple RNA-binding sites, reveals one major interaction domain at 
the carboxyl terminus.

Klug SJ(1), Hüttenhofer A, Famulok M.

Author information: 
(1)Institut für Biochemie der Universität München, Germany.

Erratum in
    RNA. 2014 Jun;20(6):956.

The SelB protein of Escherichia coli is a special elongation factor required for 
the cotranslational incorporation of the uncommon amino acid selenocysteine into 
proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously
to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of
formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA)
codon. The protein is also thought to contain binding sites allowing its
interaction with ribosomal proteins and/or rRNA. SelB thus includes specific
binding sites for a variety of different RNA molecules. We used an in vitro
selection approach with a pool completely randomized at 40 nt to isolate new
high-affinity SelB-binding RNA motifs. Our main objective was to investigate
which of the various RNA-binding domains in SelB would turn out to be prime
targets for aptamer interaction. The resulting sequences were compared with those
from a previous SELEX experiment using a degenerate pool of the wild-type
formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc.
Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of 
tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers
were different in their primary sequence and most bore no recognizable consensus 
to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite
the different RNA-binding sites in the protein, the vast majority of aptamers
bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin
binding.


PMCID: PMC1369841
PMID: 10496219  [Indexed for MEDLINE]


1422. J Mol Biol. 1998 Nov 27;284(2):241-54.

A comprehensive library of DNA-binding site matrices for 55 proteins applied to
the complete Escherichia coli K-12 genome.

Robison K(1), McGuire AM, Church GM.

Author information: 
(1)Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

A major mode of gene regulation occurs via the binding of specific proteins to
specific DNA sequences. The availability of complete bacterial genome sequences
offers an unprecedented opportunity to describe networks of such interactions by 
correlating existing experimental data with computational predictions. Of the 240
candidate Escherichia coli DNA-binding proteins, about 55 have DNA-binding sites 
identified by DNA footprinting. We used these sites to construct recognition
matrices, which we used to search for additional binding sites in the E. coli
genomic sequence. Many of these matrices show a strong preference for non-coding 
DNA. Discrepancies are identified between matrices derived from natural sites and
those derived from SELEX (Systematic Evolution of Ligands by Exponential
enrichment) experiments. We have constructed a database of these proteins and
binding sites, called DPInteract (available at
http://arep.med.harvard.edu/dpinteract).

Copyright 1998 Academic Press.

DOI: 10.1006/jmbi.1998.2160 
PMID: 9813115  [Indexed for MEDLINE]


1423. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9761-6.

Identification of an UP element consensus sequence for bacterial promoters.

Estrem ST(1), Gaal T, Ross W, Gourse RL.

Author information: 
(1)Department of Bacteriology, University of Wisconsin, 1550 Linden Drive,
Madison, WI 53706, USA.

The UP element, a component of bacterial promoters located upstream of the -35
hexamer, increases transcription by interacting with the RNA polymerase
alpha-subunit. By using a modification of the SELEX procedure for identification 
of protein-binding sites, we selected in vitro and subsequently screened in vivo 
for sequences that greatly increased promoter activity when situated upstream of 
the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream
sequences increased transcription from 136- to 326-fold in vivo, considerably
more than the natural rrnB P1 UP element, and was used to derive a consensus
sequence: -59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38. The most active selected
sequence contained the derived consensus, displayed all of the properties of an
UP element, and the interaction of this sequence with the alpha C-terminal domain
was similar to that of previously characterized UP elements. The identification
of the UP element consensus should facilitate a detailed understanding of the
alpha-DNA interaction. Based on the evolutionary conservation of the residues in 
alpha responsible for interaction with UP elements, we suggest that the UP
element consensus sequence should be applicable throughout eubacteria, should
generally facilitate promoter prediction, and may be of use for biotechnological 
applications.


PMCID: PMC21410
PMID: 9707549  [Indexed for MEDLINE]


1424. Bioinformatics. 1998;14(3):271-8.

Systematic genomic screening and analysis of mRNA in untranslated regions and
mRNA precursors: combining experimental and computational approaches.

Dandekar T(1), Beyer K, Bork P, Kenealy MR, Pantopoulos K, Hentze M, Sonntag-Buck
V, Flouriot G, Gannon F, Schreiber S.

Author information: 
(1)European Molecular Biology Laboratory, Postfach 102209, D-69012 Heidelberg,
Germany. dandekar@embl-heidelberg.de

MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and
downstream (3'UTR) of the open reading frame, as well as the mRNA precursor,
carry important regulatory sequences. To reveal unidentified regulatory signals, 
we combine information from experiments with computational approaches. Depending 
on available knowledge, three different strategies are employed.
RESULTS: Searching with a consensus template, new RNAs with regulatory RNA
elements can be identified in genomic screens. By this approach, we identify new 
candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of 
HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet
defined, it may be analyzed by combining results from SELEX (selective enrichment
of ligands by exponential amplification) and a search of databases from RNA or
genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the 
polyadenylation site in the mRNA precursor serves as a test example.
Alternatively, the regulatory RNA element may be found by studying different RNA 
foldings and their correlation with simple experimental tests. We delineate a
novel instability element in the 3'UTR of the estrogen receptor mRNA in this way.
AVAILABILITY: Strategy, methods and programs are available on request from
T.Dandekar.
CONTACT: dandekar@embl-heidelberg.de


PMID: 9614270  [Indexed for MEDLINE]


1425. RNA. 1997 Mar;3(3):255-68.

The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and
hydroxyl radical probing studies.

Moine H(1), Cachia C, Westhof E, Ehresmann B, Ehresmann C.

Author information: 
(1)UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, 
France. moine@ibmc.u-strasbg.fr.

The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a
small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and
thus represents a model system for understanding RNA/protein interaction rules.
The S8 binding site on 16S rRNA was suspected to contain noncanonical features
difficult to prove with classical genetical or biochemical means. We performed in
vitro iterative selection of RNA aptamers that bind S8. For the different
aptamers, the interactions with the protein were probed with hydroxyl radicals.
Aptamers that were recognized according to the same structural rules as wild-type
RNA, but with variations not found in nature, were identified. These aptamers
revealed features in the S8 binding site that had been concealed during previous 
characterizations by the high base conservation throughout evolution. Our data
demonstrate that the core structure of the S8 binding site is composed of three
interdependent bases (nt 597/641/643), with an essential intervening adenine
nucleotide (position 642). The other elements important for the binding site are 
a base pair (598/640) above the three interdependent bases and a bulged base at
position 595, the identity of which is not important. Possible implications on
the geometry of the S8 binding site are discussed with the help of a
three-dimensional model.


PMCID: PMC1369478
PMID: 9056763  [Indexed for MEDLINE]


1426. Nucleic Acids Res. 1997 Feb 15;25(4):781-6.

Libraries for genomic SELEX.

Singer BS(1), Shtatland T, Brown D, Gold L.

Author information: 
(1)Department of Molecular Biology, University of Colorado, Boulder 80309-0347,
USA.

Erratum in
    Nucleic Acids Res 1997 Nov 1;25(21):4430.

An increasing number of proteins are being identified that regulate gene
expression by binding specific nucleic acidsin vivo. A method termed genomic
SELEX facilitates the rapid identification of networks of protein-nucleic acid
interactions by identifying within the genomic sequences of an organism the
highest affinity sites for any protein of the organism. As with its progenitor,
SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding,
partitioning, and amplification of nucleic acids. The two methods differ in that 
the variable region of the nucleic acid library for genomic SELEX is derived from
the genome of an organism. We have used a quick and simple method to construct
Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries
that can be transcribed with T7 RNA polymerase. We present evidence that the
libraries contain overlapping inserts starting at most of the positions within
the genome, making these libraries suitable for genomic SELEX.


PMCID: PMC146522
PMID: 9016629  [Indexed for MEDLINE]


1427. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4409-14.

Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle
selection of iron-regulated genes.

Ochsner UA(1), Vasil ML.

Author information: 
(1)Department of Microbiology, University of Colorado Health Science Center,
Denver 80262, USA.

The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is
under direct control of the "ferric uptake regulator" (Fur). Novel targets of the
Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by
exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur
interaction, binding to anti-Fur antibody, purification on protein G, and PCR
amplification. DNA fragments obtained after at least three exponential enrichment
cycles were cloned and subjected to DNA mobility-shift assays and DNase I
footprint analyses to verify the specific interaction with the Fur protein in
vitro. Iron-dependent expression of the corresponding genes in vivo was monitored
by RNase protection analysis. In total, 20 different DNA fragments were
identified which represent actual Pseudomonas iron-regulated genes (PIGs). While 
four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC,
respectively), 16 PIGs represent previously unknown genes. Homology studies of
the putative proteins encoded by the PIGs allowed us to speculate about their
possible function. Two PIG products were highly similar to siderophore receptors 
from various species, and three PIG products were significantly homologous to
alternative sigma factors. Furthermore, homologs of the Escherichia coli
ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component
regulatory system similar to the Pseudomonas syringae LemA sensor kinase were
identified. The putative gene products of seven additional PIGs did not show
significant homologies to any known proteins. The PIGs were mapped on the
P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of 
P. aeruginosa is discussed.


PMCID: PMC39551
PMID: 8633080  [Indexed for MEDLINE]


1428. Biochemistry. 1996 Feb 20;35(7):2349-56.

In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB
endonuclease.

Jayasena VK(1), Brown D, Shtatland T, Gold L.

Author information: 
(1)Department of Molecular, Cellular and Developmental Biology, University of
Colorado, Boulder 80309-0347, USA.

T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4
messages, rendering them nonfunctional. Not all -GGAG- sites are processed
equally by RegB; those found at the Shine-Dalgarno sequences and in
intercistronic regions are processed with higher efficiency than the -GGAG- sites
located in coding regions. The low activity of RegB observed in vitro is enhanced
by 1-2 orders of magnitude by the Escherichia coli ribosomal protein S1. We have 
used SELEX (systematic evolution of ligands by exponential enrichment) on a
combinatorial RNA library to obtain molecules that are specifically cleaved by T4
RegB endonuclease in the presence of S1. The sequences obtained contain the
required -GGAG- tetranucleotide and were unusually enriched in adenosine and
cytosine nucleotides. No consensus structure or sequence motif other than -GGAG- 
was conserved among the selected molecules. The majority of the RNAs are entirely
dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be 
S1 independent but are cleaved by RegB with much lower rates.

DOI: 10.1021/bi951879b 
PMID: 8652576  [Indexed for MEDLINE]


1429. J Mol Biol. 1996 Jan 12;255(1):55-66.

In vitro evolution of the DNA binding sites of Escherichia coli methionine
repressor, MetJ.

He YY(1), Stockley PG, Gold L.

Author information: 
(1)Department of Molecular Cellular and Developmental Biology, University of
Colorado at Boulder 80309, USA.

The SELEX procedure was used to study the recognition between the E. coli
methionine repressor (MetJ) and its DNA binding sites. DNA ligands with high
affinity for either the holo-repressor or apo-repressor were isolated from a pool
of molecules randomized over 20 base-pairs. Among 90 DNA ligands selected by
holo-repressor binding, roughly 90% contain variations of two tandem, perfect
eight base-pair Met-boxes, which are the consensus deduced from natural met
operators. Base-pairs that are important, for specific interactions with the
protein are highly conserved. The data also reveal the importance of the
non-contacted operator base-pairs in facilitating the conformational changes in
the operator which must occur for repressor binding. There are also effects due
to the sequences of the base-pairs immediately flanking the operator site. DNA
ligands selected by apo-repressor share a very similar, but not identical,
consensus with that selected by holo-repressor, suggesting that the corepressor
does not greatly alter the specificity of repressor binding.

DOI: 10.1006/jmbi.1996.0006 
PMID: 8568875  [Indexed for MEDLINE]


1430. J Bacteriol. 1995 Sep;177(17):4872-80.

A consensus sequence for binding of Lrp to DNA.

Cui Y(1), Wang Q, Stormo GD, Calvo JM.

Author information: 
(1)Section of Biochemistry, Molecular and Cell Biology, Cornell University,
Ithaca, New York 14853, USA.

Lrp (leucine-responsive regulatory protein) is a major regulatory protein
involved in the expression of numerous operons in Escherichia coli. For ilvIH,
one of the operons positively regulated by Lrp, Lrp binds to multiple sites
upstream of the transcriptional start site and activates transcription. An
alignment of 12 Lrp binding sites within ilvIH DNA from two different organisms
revealed a tentative consensus sequence AGAAT TTTATTCT (Q. Wang, M. Sacco, E.
Ricca, C.T. Lago, M. DeFelice, and J.M. Calvo, Mol. Microbiol. 7:883-891, 1993). 
To further characterize the binding specificity of Lrp, we used a variation of
the Selex procedure of C. Tuerk and L. Gold (Science 249:505-510, 1990) to
identify sequences that bound Lrp out of a pool of 10(12) different DNA
molecules. We identified 63 related DNA sequences that bound Lrp and estimated
their relative binding affinities for Lrp. A consensus sequence derived from
analysis of these sequences, YAGHAWATTWT DCTR, where Y = C or T, H = not G, W = A
or T, D = not C, and R = A or G, contains clear dyad symmetry and is very similar
to the one defined earlier. To test the idea that Lrp in the presence of leucine 
might bind to a different subset of DNA sequences, we carried out a second
selection experiment with leucine present during the binding reactions. DNA
sequences selected in the presence or absence of leucine were similar, and
leucine did not stimulate binding to any of the sequences that were selected in
the presence of leucine. Therefore, it is unlikely that leucine changes the
specificity of Lrp binding.


PMCID: PMC177260
PMID: 7665463  [Indexed for MEDLINE]


1431. FASEB J. 1993 Jan;7(1):201-7.

Selective enrichment of RNA species for tight binding to Escherichia coli rho
factor.

Schneider D(1), Gold L, Platt T.

Author information: 
(1)Department of Molecular, Cellular, and Developmental Biology, University of
Colorado, Boulder 80309.

We have applied the SELEX procedure (systematic evolution of ligands by
exponential enrichment) to obtain RNA molecules that bind tightly to the
Escherichia coli transcription termination factor rho. The starting pool was a
population of RNA molecules 77 nucleotides (nt) long, in which was embedded a
cassette of 30 nt of randomized sequence. The apparent dissociation constant of
this RNA pool for hexameric rho factor was about 1 microM. After eight rounds of 
selection by filter binding, with RNA in either 10-fold or 40 to 100-fold excess 
at each step, the dissociation constant of the selected RNA had dropped by more
than 500-fold to about 1 nM. Analysis of 29 clonal isolates from the population
revealed that five had KDs substantially weaker than 10 nM (presumably background
carryover), 40% were C-rich (as might have been predicted from rho's known
substrate binding), and 40% had a strikingly preserved potential hairpin, in most
cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The
rho-dependent trp t' terminator region includes a related potential hairpin
structure; however, it is energetically unfavorable. The implications of the
sequence findings for elucidating both static and dynamic aspects of rho factor
recognition and response to its RNA target site are discussed.


PMID: 7678562  [Indexed for MEDLINE]