1. J Bacteriol. 2018 Mar 12;200(7). pii: e00698-17. doi: 10.1128/JB.00698-17. Print 
2018 Apr 1.

Genome-Wide Identification by Transposon Insertion Sequencing of Escherichia coli
K1 Genes Essential for In Vitro Growth, Gastrointestinal Colonizing Capacity, and
Survival in Serum.

McCarthy AJ(1), Stabler RA(2), Taylor PW(3).

Author information: 
(1)School of Pharmacy, University College London, London, United Kingdom.
(2)London School of Hygiene and Tropical Medicine, London, United Kingdom.
(3)School of Pharmacy, University College London, London, United Kingdom
peter.taylor@ucl.ac.uk.

Escherichia coli K1 strains are major causative agents of invasive disease of
newborn infants. The age dependency of infection can be reproduced in neonatal
rats. Colonization of the small intestine following oral administration of K1
bacteria leads rapidly to invasion of the blood circulation; bacteria that avoid 
capture by the mesenteric lymphatic system and evade antibacterial mechanisms in 
the blood may disseminate to cause organ-specific infections such as meningitis. 
Some E. coli K1 surface constituents, in particular the polysialic acid capsule, 
are known to contribute to invasive potential, but a comprehensive picture of the
factors that determine the fully virulent phenotype has not emerged so far. We
constructed a library and constituent sublibraries of ∼775,000 Tn5 transposon
mutants of E. coli K1 strain A192PP and employed transposon-directed insertion
site sequencing (TraDIS) to identify genes required for fitness for infection of 
2-day-old rats. Transposon insertions were lacking in 357 genes following
recovery on selective agar; these genes were considered essential for growth in
nutrient-replete medium. Colonization of the midsection of the small intestine
was facilitated by 167 E. coli K1 gene products. Restricted bacterial
translocation across epithelial barriers precluded TraDIS analysis of
gut-to-blood and blood-to-brain transits; 97 genes were required for survival in 
human serum. This study revealed that a large number of bacterial genes, many of 
which were not previously associated with systemic E. coli K1 infection, are
required to realize full invasive potential.IMPORTANCEEscherichia coli K1 strains
cause life-threatening infections in newborn infants. They are acquired from the 
mother at birth and colonize the small intestine, from where they invade the
blood and central nervous system. It is difficult to obtain information from
acutely ill patients that sheds light on physiological and bacterial factors
determining invasive disease. Key aspects of naturally occurring age-dependent
human infection can be reproduced in neonatal rats. Here, we employ
transposon-directed insertion site sequencing to identify genes essential for the
in vitro growth of E. coli K1 and genes that contribute to the colonization of
susceptible rats. The presence of bottlenecks to invasion of the blood and
cerebrospinal compartments precluded insertion site sequencing analysis, but we
identified genes for survival in serum.

Copyright © 2018 McCarthy et al.

DOI: 10.1128/JB.00698-17 
PMID: 29339415 


2. J Antimicrob Chemother. 2017 Oct 1;72(10):2729-2736. doi: 10.1093/jac/dkx204.

Modifications in the pmrB gene are the primary mechanism for the development of
chromosomally encoded resistance to polymyxins in uropathogenic Escherichia coli.

Phan MD(1)(2), Nhu NTK(1)(2), Achard MES(1)(2), Forde BM(1)(2)(3), Hong KW(4),
Chong TM(4), Yin WF(4), Chan KG(4), West NP(1)(2), Walker MJ(1)(2), Paterson
DL(2)(5), Beatson SA(1)(2)(3), Schembri MA(1)(2).

Author information: 
(1)School of Chemistry & Molecular Biosciences, The University of Queensland,
Brisbane, Queensland, Australia.
(2)Australian Infectious Diseases Research Centre, The University of Queensland, 
Brisbane, Queensland, Australia.
(3)Australian Centre for Ecogenomics, The University of Queensland, Brisbane,
Queensland, Australia.
(4)Division of Genetics and Molecular Biology, Institute of Biological Sciences, 
Faculty of Science, University of Malaya, 50603 Kuala, Lumpur, Malaysia.
(5)The University of Queensland Centre for Clinical Research, The University of
Queensland, Brisbane, Queensland, Australia.

Objectives: Polymyxins remain one of the last-resort drugs to treat infections
caused by MDR Gram-negative pathogens. Here, we determined the mechanisms by
which chromosomally encoded resistance to colistin and polymyxin B can arise in
the MDR uropathogenic Escherichia coli ST131 reference strain EC958.
Methods: Two complementary approaches, saturated transposon mutagenesis and
spontaneous mutation induction with high concentrations of colistin and polymyxin
B, were employed to select for mutations associated with resistance to
polymyxins. Mutants were identified using transposon-directed insertion-site
sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and
further investigated using RT-PCR. Competitive growth assays were used to measure
fitness cost.
Results: A transposon insertion at nucleotide 41 of the pmrB gene
(EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold
increased MIC of colistin and polymyxin B, respectively. Three spontaneous
mutations, also located within the pmrB gene, conferred resistance to both
colistin and polymyxin B with a corresponding increase in transcription of the
pmrCAB genes. All three mutations incurred a fitness cost in the absence of
colistin and polymyxin B.
Conclusions: This study identified the pmrB gene as the main chromosomal target
for induction of colistin and polymyxin B resistance in E. coli.

© The Author 2017. Published by Oxford University Press on behalf of the British 
Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions,
please email: journals.permissions@oup.com.

DOI: 10.1093/jac/dkx204 
PMID: 29091192 


3. MBio. 2017 Oct 24;8(5). pii: e01558-17. doi: 10.1128/mBio.01558-17.

Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic
Escherichia coli.

Goh KGK(1)(2), Phan MD(1)(2), Forde BM(1)(2)(3), Chong TM(4), Yin WF(4), Chan
KG(4), Ulett GC(5), Sweet MJ(2)(6), Beatson SA(1)(2)(3), Schembri MA(7)(2).

Author information: 
(1)School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, Queensland, Australia.
(2)Australian Infectious Diseases Research Centre, University of Queensland,
Brisbane, Queensland, Australia.
(3)Australian Centre for Ecogenomics, University of Queensland, Brisbane,
Queensland, Australia.
(4)Division of Genetics and Molecular Biology, Institute of Biological Sciences, 
Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
(5)School of Medical Science and Menzies Health Institute Queensland, Griffith
University, Gold Coast, Australia.
(6)Institute for Molecular Bioscience, University of Queensland, Brisbane,
Queensland, Australia.
(7)School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, Queensland, Australia m.schembri@uq.edu.au.

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and
bloodstream infections and possesses an array of virulence factors for
colonization, survival, and persistence. One such factor is the polysaccharide K 
capsule. Among the different K capsule types, the K1 serotype is strongly
associated with UPEC infection. In this study, we completely sequenced the K1
UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1
capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput
transposon-directed insertion site sequencing (TraDIS) to identify the complement
of genes required for capsule production. Our analysis identified known genes
involved in capsule biosynthesis, as well as two additional regulatory genes
(mprA and lrhA) that we characterized at the molecular level. Mutation of mprA
resulted in protection against K1 phage-mediated killing, a phenotype restored by
complementation. We also identified a significantly increased unidirectional Tn5 
insertion frequency upstream of the lrhA gene and showed that strong expression
of LrhA induced by a constitutive Pcl promoter led to loss of capsule production.
Further analysis revealed loss of MprA or overexpression of LrhA affected the
transcription of capsule biosynthesis genes in PA45B and increased sensitivity to
killing in whole blood. Similar phenotypes were also observed in UPEC strains
UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain
nor capsule type specific. Overall, this study defined the genome of a UPEC
urosepsis isolate and identified and characterized two new regulatory factors
that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs)
are among the most common bacterial infections in humans and are primarily caused
by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a
polysaccharide K capsule that provides protection against host innate immune
factors and contributes to survival and persistence during infection. The K1
serotype is one example of a polysaccharide capsule type and is strongly
associated with UPEC strains that cause UTIs, bloodstream infections, and
meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily
increasing, highlighting the need to better understand factors (e.g., the
capsule) that contribute to UPEC pathogenesis. This study describes the original 
and novel application of lytic capsule-specific phage killing, saturated Tn5
transposon mutagenesis, and high-throughput transposon-directed insertion site
sequencing to define the entire complement of genes required for capsule
production in UPEC. Our comprehensive approach uncovered new genes involved in
the regulation of this key virulence determinant.

Copyright © 2017 Goh et al.

DOI: 10.1128/mBio.01558-17 
PMCID: PMC5654933
PMID: 29066548 


4. Nucleic Acids Res. 2017 Sep 6;45(15):e145. doi: 10.1093/nar/gkx594.

Data exploration, quality control and statistical analysis of ChIP-exo/nexus
experiments.

Welch R(1), Chung D(2), Grass J(3)(4), Landick R(3)(4)(5), Keles S(1)(6).

Author information: 
(1)Department of Statistics, University of Wisconsin-Madison, Madison, WI 53706, 
USA.
(2)Department of Public Health Sciences, Medical University of South Carolina, SC
29425, USA.
(3)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison,
Madison, WI 53726, USA.
(4)Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
53706, USA.
(5)Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
53706, USA.
(6)Department of Biostatistics and Medical Informatics, University of
Wisconsin-Madison, Madison, WI 53792, USA.

ChIP-exo/nexus experiments rely on innovative modifications of the commonly used 
ChIP-seq protocol for high resolution mapping of transcription factor binding
sites. Although many aspects of the ChIP-exo data analysis are similar to those
of ChIP-seq, these high throughput experiments pose a number of unique quality
control and analysis challenges. We develop a novel statistical quality control
pipeline and accompanying R/Bioconductor package, ChIPexoQual, to enable
exploration and analysis of ChIP-exo and related experiments. ChIPexoQual
evaluates a number of key issues including strand imbalance, library complexity, 
and signal enrichment of data. Assessment of these features are facilitated
through diagnostic plots and summary statistics computed over regions of the
genome with varying levels of coverage. We evaluated our QC pipeline with both
large collections of public ChIP-exo/nexus data and multiple, new ChIP-exo
datasets from Escherichia coli. ChIPexoQual analysis of these datasets resulted
in guidelines for using these QC metrics across a wide range of sequencing depths
and provided further insights for modelling ChIP-exo data.

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

DOI: 10.1093/nar/gkx594 
PMCID: PMC5587812
PMID: 28911122  [Indexed for MEDLINE]


5. Int J Genomics. 2017;2017:6489383. doi: 10.1155/2017/6489383. Epub 2017 Jul 16.

Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella
tularensis-Exposed hPBMCs Reveal Potential Biomarkers.

Cer RZ(1)(2), Herrera-Galeano JE(1)(2)(3), Frey KG(1)(2), Schully KL(1)(2), Luu
TV(1)(2), Pesce J(1)(2)(4), Mokashi VP(1)(5), Keane-Myers AM(1)(2)(6),
Bishop-Lilly KA(1)(2).

Author information: 
(1)Genomics and Bioinformatics Department, Biological Defense Research
Directorate, Naval Medical Research Center, Frederick, MD, USA.
(2)Henry M. Jackson Foundation for the Advancement of Military Medicine,
Bethesda, MD, USA.
(3)KCE Services and Consulting LLC, Columbia, MD, USA.
(4)Division of Microbiology and Infectious Diseases, National Institute of
Allergy and Infectious Diseases, Bethesda, MD, USA.
(5)Navy Drug Screening Laboratory, Jacksonville, FL, USA.
(6)Immunology, National Institute of Health, Bethesda, MD, USA.

Increasing evidence that microRNAs (miRNAs) play important roles in the immune
response against infectious agents suggests that miRNA might be exploitable as
signatures of exposure to specific infectious agents. In order to identify
potential early miRNA biomarkers of bacterial infections, human peripheral blood 
mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia
pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the
nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three 
early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq
analyses identified 87 miRNAs to be differentially expressed (DE) in a linear
fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay.
Through RNAseq identification and qPCR validation, we identified differentially
expressed miRNA species that may be involved in the early response to bacterial
infections. Based upon its upregulation at early time points postexposure in two 
different individuals, hsa-mir-30c-5p is a miRNA species that could be studied
further as a potential biomarker for exposure to these gram-negative
intracellular pathogens. Gene ontology functional analyses demonstrated that
programmed cell death is the first ranking biological process associated with
miRNAs that are upregulated in F. tularensis-exposed hPBMCs.

DOI: 10.1155/2017/6489383 
PMCID: PMC5534298
PMID: 28791299 


6. NPJ Syst Biol Appl. 2017 Jun 22;3:17. doi: 10.1038/s41540-017-0019-y. eCollection
2017.

Reverse engineering highlights potential principles of large gene regulatory
network design and learning.

Carré C(1)(2), Mas A(1), Krouk G(2).

Author information: 
(1)Institut Montpelliérain Alexander Grothendieck, Université de Montpellier,
Montpellier, France.
(2)Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de
Biologie Intégrative des Plantes 'Claude Grignon', UMR5004 CNRS, INRA, SupAgro,
UM, Place Pierre Viala, Montpellier, 34060 France.

Inferring transcriptional gene regulatory networks from transcriptomic datasets
is a key challenge of systems biology, with potential impacts ranging from
medicine to agronomy. There are several techniques used presently to
experimentally assay transcription factors to target relationships, defining
important information about real gene regulatory networks connections. These
techniques include classical ChIP-seq, yeast one-hybrid, or more recently,
DAP-seq or target technologies. These techniques are usually used to validate
algorithm predictions. Here, we developed a reverse engineering approach based on
mathematical and computer simulation to evaluate the impact that this prior
knowledge on gene regulatory networks may have on training machine learning
algorithms. First, we developed a gene regulatory networks-simulating engine
called FRANK (Fast Randomizing Algorithm for Network Knowledge) that is able to
simulate large gene regulatory networks (containing 104 genes) with
characteristics of gene regulatory networks observed in vivo. FRANK also
generates stable or oscillatory gene expression directly produced by the
simulated gene regulatory networks. The development of FRANK leads to important
general conclusions concerning the design of large and stable gene regulatory
networks harboring scale free properties (built ex nihilo). In combination with
supervised (accepting prior knowledge) support vector machine algorithm we (i)
address biologically oriented questions concerning our capacity to accurately
reconstruct gene regulatory networks and in particular we demonstrate that
prior-knowledge structure is crucial for accurate learning, and (ii) draw
conclusions to inform experimental design to performed learning able to solve
gene regulatory networks in the future. By demonstrating that our predictions
concerning the influence of the prior-knowledge structure on support vector
machine learning capacity holds true on real data (Escherichia coli K14 network
reconstruction using network and transcriptomic data), we show that the formalism
used to build FRANK can to some extent be a reasonable model for gene regulatory 
networks in real cells.

DOI: 10.1038/s41540-017-0019-y 
PMCID: PMC5481436
PMID: 28649444 


7. PLoS One. 2017 Jun 14;12(6):e0178966. doi: 10.1371/journal.pone.0178966.
eCollection 2017.

Intestinal organoids model human responses to infection by commensal and Shiga
toxin producing Escherichia coli.

Karve SS(1), Pradhan S(1), Ward DV(2), Weiss AA(1).

Author information: 
(1)Department of Molecular Genetics, Biochemistry, and Microbiology, University
of Cincinnati, Cincinnati, Ohio, United States of America.
(2)Center for Microbiome Research and Department of Microbiology and
Physiological Systems, University of Massachusetts Medical School, Worcester,
Massachusetts, United States of America.

Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the
potentially fatal complication hemolytic uremic syndrome, and currently only
supportive therapy is available. Lack of suitable animal models has hindered
study of this disease. Induced human intestinal organoids (iHIOs), generated by
in vitro differentiation of pluripotent stem cells, represent differentiated
human intestinal tissue. We show that iHIOs with addition of human neutrophils
can model E. coli intestinal infection and innate cellular responses. Commensal
and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high
numbers. Commensal E. coli did not cause damage, and were completely contained
within the lumen, suggesting defenses, such as mucus production, can constrain
non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. 
Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew
as filaments, consistent with activation of the bacterial SOS stress response.
SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased 
ROS production. Transcriptional profiling (RNAseq) demonstrated that both
commensal and O157:H7 upregulated genes associated with gastrointestinal
maturation, while infection with O157:H7 upregulated inflammatory responses,
including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment,
and infection with O157:H7 resulted in recruitment of human neutrophils into the 
iHIO tissue.

DOI: 10.1371/journal.pone.0178966 
PMCID: PMC5470682
PMID: 28614372  [Indexed for MEDLINE]


8. J Bacteriol. 2017 Jun 13;199(13). pii: e00086-17. doi: 10.1128/JB.00086-17. Print
2017 Jul 1.

Genome-Wide Analysis of ResD, NsrR, and Fur Binding in Bacillus subtilis during
Anaerobic Fermentative Growth by In Vivo Footprinting.

Chumsakul O(1), Anantsri DP(2), Quirke T(3), Oshima T(1), Nakamura K(4), Ishikawa
S(5)(6), Nakano MM(7).

Author information: 
(1)Graduate School of Biological Sciences, Nara Institute of Science and
Technology, Nara, Japan.
(2)Institute of Environmental Health, Oregon Health & Science University,
Portland, Oregon, USA.
(3)Sam Barlow High School, Gresham, Oregon, USA.
(4)Department of Life Science and Informatics, Maebashi Institute of Technology, 
Gunma, Japan.
(5)Graduate School of Biological Sciences, Nara Institute of Science and
Technology, Nara, Japan shu@people.kobe-u.ac.jp nakanom@ohsu.edu.
(6)Graduate School of Science, Technology & Innovation, Kobe University, Kobe,
Japan.
(7)Institute of Environmental Health, Oregon Health & Science University,
Portland, Oregon, USA shu@people.kobe-u.ac.jp nakanom@ohsu.edu.

Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate 
ResD response regulator play primary roles in the transcriptional activation of
genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR 
repressor controls transcription to support nitrate respiration. In addition, the
ferric uptake repressor (Fur) can modulate transcription under anaerobic
conditions. However, whether these controls are direct or indirect has been
investigated only in a gene-specific manner. To gain a genomic view of anaerobic 
gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR,
and Fur transcription factors (TFs) using in situ DNase I footprinting combined
with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting
by high-throughput sequencing [GeF-seq]). A significant number of sites were
targets of ResD and NsrR, and a majority of them were also bound by Fur. The
binding of multiple TFs to overlapping targets affected each individual TF's
binding, which led to combinatorial transcriptional control. ResD bound to both
the promoters and the coding regions of genes under its positive control. Other
genes showing enrichment of ResD at only the promoter regions are targets of
direct ResD-dependent repression or antirepression. The results support previous 
findings of ResD as an RNA polymerase (RNAP)-binding protein and indicated that
ResD can associate with the transcription elongation complex. The data set
allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and
uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by 
an A- and T-rich sequence are often found at sites where all three TFs
competitively bind.IMPORTANCE Bacteria encounter oxygen fluctuation in their
natural environment as well as in host organisms. Hence, understanding how
bacteria respond to oxygen limitation will impact environmental and human health.
ResD, NsrR, and Fur control transcription under anaerobic conditions. This work
using in situ DNase I footprinting uncovered the genome-wide binding profile of
the three transcription factors (TFs). Binding of the TFs is often competitive or
cooperative depending on the promoters and the presence of other TFs, indicating 
that transcriptional regulation by multiple TFs is much more complex than we
originally thought. The results from this study provide a more complete picture
of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to
our further understanding of anaerobic physiology.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/JB.00086-17 
PMCID: PMC5472814
PMID: 28439033  [Indexed for MEDLINE]


9. BMC Genet. 2017 Mar 7;18(1):21. doi: 10.1186/s12863-017-0488-4.

Deciphering alternative splicing and nonsense-mediated decay modulate expression 
in primary lymphoid tissues of birds infected with avian pathogenic E. coli
(APEC).

Sun H(1).

Author information: 
(1)College of Animal Science and Technology, Yangzhou University, Yangzhou,
Jiangsu, 225009, China. hongyans2392@163.com.

BACKGROUND: Avian pathogenic E. coli (APEC) can lead to a loss in millions of
dollars in poultry annually because of mortality and produce contamination.
Studies have verified that many immune-related genes undergo changes in
alternative splicing (AS), along with nonsense mediated decay (NMD), to regulate 
the immune system under different conditions. Therefore, the splicing profiles of
primary lymphoid tissues with systemic APEC infection need to be comprehensively 
examined.
RESULTS: Gene expression in RNAseq data were obtained for three different immune 
tissues (bone marrow, thymus, and bursa) from three phenotype birds
(non-challenged, resistant, and susceptible birds) at two time points.
Alternative 5' splice sites and exon skipping/inclusion were identified as the
major alternative splicing events in avian primary immune organs under systemic
APEC infection. In this study, we detected hundreds of
differentially-expressed-transcript-containing genes (DETs) between different
phenotype birds at 5 days post-infection (dpi). DETs, PSAP and STT3A, with NMD
have important functions under systemic APEC infection. DETs, CDC45, CDK1, RAG2, 
POLR1B, PSAP, and DNASE1L3, from the same transcription start sites (TSS)
indicate that cell death, cell cycle, cellular function, and maintenance were
predominant in host under systemic APEC.
CONCLUSIONS: With the use of RNAseq technology and bioinformatics tools, this
study provides a portrait of the AS event and NMD in primary lymphoid tissues,
which play critical roles in host homeostasis under systemic APEC infection.
According to this study, AS plays a pivotal regulatory role in the immune
response in chicken under systemic APEC infection via either NMD or alternative
TSSs. This study elucidates the regulatory role of AS for the immune complex
under systemic APEC infection.

DOI: 10.1186/s12863-017-0488-4 
PMCID: PMC5341183
PMID: 28270101  [Indexed for MEDLINE]


10. PLoS Negl Trop Dis. 2017 Jan 6;11(1):e0005273. doi: 10.1371/journal.pntd.0005273.
eCollection 2017 Jan.

Transcriptome Sequencing Reveals Large-Scale Changes in Axenic Aedes aegypti
Larvae.

Vogel KJ(1), Valzania L(1), Coon KL(1), Brown MR(1), Strand MR(1).

Author information: 
(1)Department of Entomology, The University of Georgia, Athens, Georgia, United
States of America.

Mosquitoes host communities of microbes in their digestive tract that consist
primarily of bacteria. We previously reported that Aedes aegypti larvae colonized
by a native community of bacteria and gnotobiotic larvae colonized by only
Escherichia coli develop very similarly into adults, whereas axenic larvae never 
molt and die as first instars. In this study, we extended these findings by first
comparing the growth and abundance of bacteria in conventional, gnotobiotic, and 
axenic larvae during the first instar. Results showed that conventional and
gnotobiotic larvae exhibited no differences in growth, timing of molting, or
number of bacteria in their digestive tract. Axenic larvae in contrast grew
minimally and never achieved the critical size associated with molting by
conventional and gnotobiotic larvae. In the second part of the study we compared 
patterns of gene expression in conventional, gnotobiotic and axenic larvae by
conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h
post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially
expressed in axenic versus conventional or gnotobiotic larvae. However, this
profile consisted primarily of transcripts in seven categories that included the 
down-regulation of select peptidases in the gut and up-regulation of several
genes in the gut and carcass with roles in amino acid transport, hormonal
signaling, and metabolism. Overall, our results indicate that axenic larvae
exhibit alterations in gene expression consistent with defects in acquisition and
assimilation of nutrients required for growth.

DOI: 10.1371/journal.pntd.0005273 
PMCID: PMC5245907
PMID: 28060822  [Indexed for MEDLINE]

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


11. Appl Environ Microbiol. 2017 Feb 15;83(5). pii: e03028-16. doi:
10.1128/AEM.03028-16. Print 2017 Mar 1.

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions
Identified by Transposon Insertion Sequencing.

de Moraes MH(1), Desai P(2), Porwollik S(2), Canals R(2), Perez DR(3), Chu W(2), 
McClelland M(2), Teplitski M(3).

Author information: 
(1)Soil and Water Science Department, Genetics Institute, University of
Florida-IFAS, Gainesville, Florida, USA mhdmoraes@gmail.com.
(2)Department of Microbiology and Molecular Genetics, University of California,
Irvine, Irvine, California, USA.
(3)Soil and Water Science Department, Genetics Institute, University of
Florida-IFAS, Gainesville, Florida, USA.

Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia
coli, are increasingly recognized as causes of gastroenteritis outbreaks
associated with the consumption of fruits and vegetables. Persistence in plants
represents an important part of the life cycle of these pathogens. The
identification of the full complement of Salmonella genes involved in the
colonization of the model plant (tomato) was carried out using transposon
insertion sequencing analysis. With this approach, 230,000 transposon insertions 
were screened in tomato pericarps to identify loci with reduction in fitness,
followed by validation of the screen results using competition assays of the
isogenic mutants against the wild type. A comparison with studies in animals
revealed a distinct plant-associated set of genes, which only partially overlaps 
with the genes required to elicit disease in animals. De novo biosynthesis of
amino acids was critical to persistence within tomatoes, while amino acid
scavenging was prevalent in animal infections. Fitness reduction of the
Salmonella amino acid synthesis mutants was generally more severe in the tomato
rin mutant, which hyperaccumulates certain amino acids, suggesting that these
nutrients remain unavailable to Salmonella spp. within plants. Salmonella
lipopolysaccharide (LPS) was required for persistence in both animals and plants,
exemplifying some shared pathogenesis-related mechanisms in animal and plant
hosts. Similarly to phytopathogens, Salmonella spp. required biosynthesis of
amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it
appears that while Salmonella shares some strategies with phytopathogens and taps
into its animal virulence-related functions, colonization of tomatoes represents 
a distinct strategy, highlighting this pathogen's flexible metabolism.IMPORTANCE 
Outbreaks of gastroenteritis caused by human pathogens have been increasingly
associated with foods of plant origin, with tomatoes being one of the common
culprits. Recent studies also suggest that these human pathogens can use plants
as alternate hosts as a part of their life cycle. While dual (animal/plant)
lifestyles of other members of the Enterobacteriaceae family are well known, the 
strategies with which Salmonella colonizes plants are only partially understood. 
Therefore, we undertook a high-throughput characterization of the functions
required for Salmonella persistence within tomatoes. The results of this study
were compared with what is known about genes required for Salmonella virulence in
animals and interactions of plant pathogens with their hosts to determine whether
Salmonella repurposes its virulence repertoire inside plants or whether it
behaves more as a phytopathogen during plant colonization. Even though Salmonella
utilized some of its virulence-related genes in tomatoes, plant colonization
required a distinct set of functions.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/AEM.03028-16 
PMCID: PMC5311394
PMID: 28039131  [Indexed for MEDLINE]


12. Antimicrob Agents Chemother. 2017 Jan 24;61(2). pii: e01740-16. doi:
10.1128/AAC.01740-16. Print 2017 Feb.

Identification of IncA/C Plasmid Replication and Maintenance Genes and
Development of a Plasmid Multilocus Sequence Typing Scheme.

Hancock SJ(1)(2), Phan MD(3)(2), Peters KM(1)(2), Forde BM(1)(2), Chong TM(4),
Yin WF(4), Chan KG(4), Paterson DL(5), Walsh TR(6), Beatson SA(1)(2), Schembri
MA(3)(2).

Author information: 
(1)Australian Infectious Diseases Research Centre, University of Queensland,
Brisbane, Australia.
(2)School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, Australia.
(3)Australian Infectious Diseases Research Centre, University of Queensland,
Brisbane, Australia m.phan1@uq.edu.au m.schembri@uq.edu.au.
(4)Faculty of Science, Division of Genetics and Molecular Biology, Institute of
Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia.
(5)University of Queensland Centre for Clinical Research, Brisbane, Australia.
(6)Department of Medical Microbiology and Infectious Disease, Cardiff University,
Cardiff, United Kingdom.

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly
prevalent within pathogenic Enterobacteriaceae They are associated with the
dissemination of multiple clinically relevant resistance genes, including blaCMY 
and blaNDM Current typing methods for IncA/C plasmids offer limited resolution.
In this study, we present the complete sequence of a blaNDM-1-positive IncA/C
plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia 
coli strain. Hypersaturated transposon mutagenesis, coupled with
transposon-directed insertion site sequencing (TraDIS), was employed to identify 
conserved genetic elements required for replication and maintenance of pMS6198A. 
Our analysis of TraDIS data identified roles for the replicon, including repA, a 
toxin-antitoxin system; two putative partitioning genes, parAB; and a putative
gene, 053 Construction of mini-IncA/C plasmids and examination of their stability
within E. coli confirmed that the region encompassing 053 contributes to the
stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance
genes (repA, parAB, and 053) were used to construct a new plasmid multilocus
sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to
a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with 
two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) 
fall into ST1, suggesting acquisition and subsequent expansion of this
blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a
standardized tool to identify, track, and analyze the dissemination of important 
IncA/C plasmid lineages, particularly in the context of epidemiological studies.

Copyright © 2017 American Society for Microbiology.

DOI: 10.1128/AAC.01740-16 
PMCID: PMC5278728
PMID: 27872077  [Indexed for MEDLINE]


13. EMBO J. 2017 Feb 1;36(3):374-387. doi: 10.15252/embj.201694639. Epub 2016 Nov 11.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of
RNase E.

Waters SA(1), McAteer SP(2), Kudla G(3), Pang I(1)(4), Deshpande NP(1)(4), Amos
TG(1), Leong KW(5), Wilkins MR(1)(4), Strugnell R(5), Gally DL(2), Tollervey
D(6), Tree JJ(7).

Author information: 
(1)School of Biotechnology and Biomolecular Sciences, University of New South
Wales, Sydney, NSW, Australia.
(2)The Roslin Institute, University of Edinburgh, Edinburgh, UK.
(3)MRC Human Genetic Unit, University of Edinburgh, Edinburgh, UK.
(4)Systems Biology Initiative, University of New South Wales, Sydney, NSW,
Australia.
(5)Peter Doherty Institute, University of Melbourne, Melbourne, Victoria,
Australia.
(6)Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK
j.tree@unsw.edu.au d.tollervey@ed.ac.uk.
(7)School of Biotechnology and Biomolecular Sciences, University of New South
Wales, Sydney, NSW, Australia j.tree@unsw.edu.au d.tollervey@ed.ac.uk.

RNA sequencing studies have identified hundreds of non-coding RNAs in bacteria,
including regulatory small RNA (sRNA). However, our understanding of sRNA
function has lagged behind their identification due to a lack of tools for the
high-throughput analysis of RNA-RNA interactions in bacteria. Here we demonstrate
that in vivo sRNA-mRNA duplexes can be recovered using UV-crosslinking, ligation 
and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase
E, to facilitate processing of mRNAs. We were able to recover base-paired
sRNA-mRNA duplexes in association with RNase E, allowing proximity-dependent
ligation and sequencing of cognate sRNA-mRNA pairs as chimeric reads. We verified
that this approach captures bona fide sRNA-mRNA interactions. Clustering analyses
identified novel sRNA seed regions and sets of potentially co-regulated target
mRNAs. We identified multiple mRNA targets for the pathotype-specific sRNA Esr41,
which was shown to regulate colicin sensitivity and iron transport in E. coli
Numerous sRNA interactions were also identified with non-coding RNAs, including
sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

© 2016 The Authors. Published under the terms of the CC BY 4.0 license.

DOI: 10.15252/embj.201694639 
PMCID: PMC5286369
PMID: 27836995  [Indexed for MEDLINE]


14. Nucleic Acids Res. 2016 Dec 1;44(21):10117-10131. Epub 2016 Aug 4.

Silencing of cryptic prophages in Corynebacterium glutamicum.

Pfeifer E(1), Hünnefeld M(1), Popa O(2), Polen T(1), Kohlheyer D(1), Baumgart
M(1), Frunzke J(3).

Author information: 
(1)Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum
Jülich, 52425 Jülich, Germany.
(2)Quantitative and Theoretical Biology, Heinrich-Heine-Universität Düsseldorf,
40225, Düsseldorf, Germany.
(3)Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum
Jülich, 52425 Jülich, Germany j.frunzke@fz-juelich.de.

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its
integration into host regulatory circuits is a pivotal driver of microbial
evolution but requires the stringent regulation of phage gene activity. In this
study, we describe the nucleoid-associated protein CgpS, which represents an
essential protein functioning as a xenogeneic silencer in the Gram-positive
Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C.
glutamicum strain ATCC 13032 and was first identified by DNA affinity
chromatography using an early phage promoter of CGP3. Genome-wide profiling of
CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq)
revealed its association with AT-rich DNA elements, including the entire CGP3
prophage region (187 kbp), as well as several other elements acquired by
horizontal gene transfer. Countersilencing of CgpS resulted in a significantly
increased induction frequency of the CGP3 prophage. In contrast, a strain lacking
the CGP3 prophage was not affected and displayed stable growth. In a
bioinformatics approach, cgpS orthologs were identified primarily in
actinobacterial genomes as well as several phage and prophage genomes. Sequence
analysis of 618 orthologous proteins revealed a strong conservation of the
secondary structure, supporting an ancient function of these xenogeneic silencers
in phage-host interaction.

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

DOI: 10.1093/nar/gkw692 
PMCID: PMC5137423
PMID: 27492287  [Indexed for MEDLINE]


15. Poult Sci. 2016 Dec 1;95(12):2803-2814. Epub 2016 Jul 27.

Thymus transcriptome reveals novel pathways in response to avian pathogenic
Escherichia coli infection.

Sun H(1)(2), Liu P(3), Nolan LK(4), Lamont SJ(5).

Author information: 
(1)College of Animal Science and Technology, Yangzhou University, Yangzhou,
Jiangsu, China, 225009.
(2)Department of Animal Science, Iowa State University, Ames 50011.
(3)Department of Statistics, Iowa State University, Ames 50011.
(4)Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames 50011.
(5)Department of Animal Science, Iowa State University, Ames 50011
sjlamont@iastate.edu.

Avian pathogenic Escherichia coli (APEC) can cause significant morbidity in
chickens. The thymus provides the essential environment for T cell development;
however, the thymus transcriptome has not been examined for gene expression in
response to APEC infection. An improved understanding of the host genomic
response to APEC infection could inform future breeding programs for disease
resistance and APEC control. We therefore analyzed the transcriptome of the
thymus of birds challenged with APEC, contrasting susceptible and resistant
phenotypes. Thousands of genes were differentially expressed in birds of the
5-day post infection (dpi) challenged-susceptible group vs. 5 dpi non-challenged,
in 5 dpi challenged-susceptible vs. 5 dpi challenged-resistant birds, as well as 
in 5 dpi vs. one dpi challenged-susceptible birds. The Toll-like receptor
signaling pathway was the major innate immune response for birds to respond to
APEC infection. Moreover, lysosome and cell adhesion molecules pathways were
common mechanisms for chicken response to APEC infection. The T-cell receptor
signaling pathway, cell cycle, and p53 signaling pathways were significantly
activated in resistant birds to resist APEC infection. These results provide a
comprehensive assessment of global gene networks and biological functionalities
of differentially expressed genes in the thymus under APEC infection. These
findings provide novel insights into key molecular genetic mechanisms that
differentiate host resistance from susceptibility in this primary lymphoid
tissue, the thymus.

© The Author 2016. Published by Oxford University Press on behalf of Poultry
Science Association.

DOI: 10.3382/ps/pew202 
PMCID: PMC5144662
PMID: 27466434  [Indexed for MEDLINE]


16. Sci Rep. 2016 Jul 18;6:30025. doi: 10.1038/srep30025.

Systematic analysis of an evolved Thermobifida fusca muC producing malic acid on 
organic and inorganic nitrogen sources.

Deng Y(1)(2), Lin J(3), Mao Y(1)(2), Zhang X(4).

Author information: 
(1)National Engineering Laboratory for Cereal Fermentation Technology (NELCF),
Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
(2)The Key Laboratory of Industrial Biotechnology, Ministry of Education,
Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
(3)College of Life Science, North China University of Science and Technology,
Tangshan 063000, China.
(4)School of pharmaceutical science, Jiangnan University, 1800 Lihu Road, Wuxi,
Jiangsu 214122, China.

Thermobifida fusca is a thermophilic actinobacterium. T. fusca muC obtained by
adaptive evolution preferred yeast extract to ammonium sulfate for accumulating
malic acid and ammonium sulfate for cell growth. We did transcriptome analysis of
T. fusca muC on Avicel and cellobiose with addition of ammonium sulfate or yeast 
extract, respectively by RNAseq. The transcriptional results indicate that
ammonium sulfate induced the transcriptions of the genes related to carbohydrate 
metabolisms significantly more than yeast extract. Importantly, Tfu_2487,
encoding histidine-containing protein (HPr), didn't transcribe on yeast extract
at all, while it transcribed highly on ammonium sulfate. In order to understand
the impact of HPr on malate production and cell growth of the muC strain, we
deleted Tfu_2487 to get a mutant strain: muCΔ2487, which had
1.33 mole/mole-glucose equivalent malate yield, much higher than that on yeast
extract. We then developed an E. coli-T. fusca shuttle plasmid for
over-expressing HPr in muCΔ2487, a strain without HPr background, forming the
muCΔ2487S strain. The muCΔ2487S strain had a much lower malate yield but faster
cell growth than the muC strain. The results of both mutant strains confirmed
that HPr was the key regulatory protein for T. fusca's metabolisms on nitrogen
sources.

DOI: 10.1038/srep30025 
PMCID: PMC4948018
PMID: 27424527 


17. PLoS One. 2016 Jun 23;11(6):e0157480. doi: 10.1371/journal.pone.0157480.
eCollection 2016.

The Impact of Intramammary Escherichia coli Challenge on Liver and Mammary
Transcriptome and Cross-Talk in Dairy Cows during Early Lactation Using RNAseq.

Moyes KM(1), Sørensen P(2), Bionaz M(3).

Author information: 
(1)Department of Animal and Avian Sciences, University of Maryland, College Park,
Maryland, United States of America.
(2)Center for Quantitative Genetics and Genomics, Department of Molecular Biology
and Genetics, Aarhus University, 8830 Tjele, Denmark.
(3)Department of Animal and Rangeland Sciences, Oregon State University,
Corvallis, Oregon, United States of America.

Our objective was to identify the biological response and the cross-talk between 
liver and mammary tissue after intramammary infection (IMI) with Escherichia coli
(E. coli) using RNAseq technology. Sixteen cows were inoculated with live E. coli
into one mammary quarter at ~4-6 weeks in lactation. For all cows, biopsies were 
performed at -144, 12 and 24 h relative to IMI in liver and at 24 h post-IMI in
infected and non-infected (control) mammary quarters. For a subset of cows (n =
6), RNA was extracted from both liver and mammary tissue and sequenced using a
100 bp paired-end approach. Ingenuity Pathway Analysis and the Dynamic Impact
Approach analysis of differentially expressed genes (overall effect False
Discovery Rate≤0.05) indicated that IMI induced an overall activation of
inflammation at 12 h post-IMI and a strong inhibition of metabolism, especially
related to lipid, glucose, and xenobiotics at 24 h post-IMI in liver. The data
indicated in mammary tissue an overall induction of inflammatory response with
little effect on metabolism at 24 h post-IMI. We identified a large number of
up-stream regulators potentially involved in the response to IMI in both tissues 
but a relatively small core network of transcription factors controlling the
response to IMI for liver whereas a large network in mammary tissue.
Transcriptomic results in liver and mammary tissue were supported by changes in
inflammatory and metabolic mediators in blood and milk. The analysis of potential
cross-talk between the two tissues during IMI uncovered a large communication
from the mammary tissue to the liver to coordinate the inflammatory response but 
a relatively small communication from the liver to the mammary tissue. Our
results indicate a strong induction of the inflammatory response in mammary
tissue and impairment of liver metabolism 24h post-IMI partly driven by the
signaling from infected mammary tissue.

DOI: 10.1371/journal.pone.0157480 
PMCID: PMC4919052
PMID: 27336699  [Indexed for MEDLINE]


18. Genes Dev. 2016 Jun 1;30(11):1327-38. doi: 10.1101/gad.280834.116.

S1-DRIP-seq identifies high expression and polyA tracts as major contributors to 
R-loop formation.

Wahba L(1), Costantino L(1), Tan FJ(2), Zimmer A(1), Koshland D(1).

Author information: 
(1)Department of Cell and Molecular Biology, University of California at
Berkeley, Berkeley, California 94720, USA;
(2)Department of Embryology, Carnegie Institution for Science, Baltimore,
Maryland 21218, USA.

R loops form when transcripts hybridize to homologous DNA on chromosomes,
yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the
genome of many organisms, regulating chromosome stability, gene expression, and
DNA repair. Understanding the parameters dictating R-loop formation in vivo has
been hampered by the limited quantitative and spatial resolution of current
genomic strategies for mapping R loops. We report a novel whole-genome method,
S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for
mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this
methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome.
Given the pervasive transcription of the yeast genome, this result suggests that 
R-loop formation is dictated by characteristics of the DNA, RNA, and/or
chromatin. We successfully identified two features highly predictive of hybrid
formation: high transcription and long homopolymeric dA:dT tracts. These
accounted for >60% of the hybrid regions found in the genome. We demonstrated
that these two factors play a causal role in hybrid formation by genetic
manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the
identification of the first global genomic features causal for R-loop formation
in yeast.

© 2016 Wahba et al.; Published by Cold Spring Harbor Laboratory Press.

DOI: 10.1101/gad.280834.116 
PMCID: PMC4911931
PMID: 27298336  [Indexed for MEDLINE]


19. BMC Genomics. 2016 Mar 22;17:253. doi: 10.1186/s12864-016-2387-x.

FabR regulates Salmonella biofilm formation via its direct target FabB.

Hermans K(1), Roberfroid S(1), Thijs IM(1), Kint G(1), De Coster D(1), Marchal
K(1), Vanderleyden J(1), De Keersmaecker SC(1)(2), Steenackers HP(3).

Author information: 
(1)Department of Microbial and Molecular Systems, Centre of Microbial and Plant
Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001, Leuven, 
Belgium.
(2)Platform Biotechnology and Molecular Biology, Scientific Institute of Public
Health (WIV-ISP), Brussels, Belgium.
(3)Department of Microbial and Molecular Systems, Centre of Microbial and Plant
Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001, Leuven, 
Belgium. Hans.Steenackers@biw.kuleuven.be.

BACKGROUND: Biofilm formation is an important survival strategy of Salmonella in 
all environments. By mutant screening, we showed a knock-out mutant of fabR,
encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have
impaired biofilm formation. In order to unravel how this regulator impinges on
Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR
regulon. Hereto, we applied a combinatorial high-throughput approach, combining
ChIP-chip with transcriptomics.
RESULTS: All the previously identified E. coli FabR transcriptional target genes 
(fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as
well. As we found a fabB overexpressing strain to partly mimic the biofilm defect
of the fabR mutant, the effect of FabR on biofilms can be attributed at least
partly to FabB, which plays a key role in UFA biosynthesis. Additionally,
ChIP-chip identified a number of novel direct FabR targets (the intergenic
regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a.
genes involved in tRNA metabolism, ribosome synthesis and translation). Next to
UFA biosynthesis, a number of these direct targets and other indirect targets
identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB,
osmC, sseI), could possibly contribute to the effect of FabR on biofilm
formation.
CONCLUSION: Overall, our results point at the importance of FabR and UFA
biosynthesis in Salmonella biofilm formation and their role as potential targets 
for biofilm inhibitory strategies.

DOI: 10.1186/s12864-016-2387-x 
PMCID: PMC4804515
PMID: 27004424  [Indexed for MEDLINE]


20. J Vis Exp. 2016 Jan 27;(107):e53620. doi: 10.3791/53620.

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput
Sequencing of Adenine-methylated DNA Fragments.

Wu F(1), Olson BG(1), Yao J(2).

Author information: 
(1)Department of Cell Biology, Yale University School of Medicine.
(2)Department of Cell Biology, Yale University School of Medicine;
yao.j@yale.edu.

The DNA adenine methyltransferase identification (DamID) assay is a powerful
method to detect protein-DNA interactions both locally and genome-wide. It is an 
alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion
protein consisting of the protein of interest and the E. coli DNA adenine
methyltransferase can methylate the adenine base in GATC motifs near the sites of
protein-DNA interactions. Adenine-methylated DNA fragments can then be
specifically amplified and detected. The original DamID assay detects the genomic
locations of methylated DNA fragments by hybridization to DNA microarrays, which 
is limited by the availability of microarrays and the density of predetermined
probes. In this paper, we report the detailed protocol of integrating high
throughput DNA sequencing into DamID (DamID-seq). The large number of short reads
generated from DamID-seq enables detecting and localizing protein-DNA
interactions genome-wide with high precision and sensitivity. We have used the
DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian
cells, and have noticed that DamID-seq provides a high resolution and a wide
dynamic range in detecting genome-NL interactions. The DamID-seq approach enables
probing NL associations within gene structures and allows comparing genome-NL
interaction maps with other functional genomic data, such as ChIP-seq and
RNA-seq.

DOI: 10.3791/53620 
PMCID: PMC4781701
PMID: 26862720  [Indexed for MEDLINE]


21. Semin Cell Dev Biol. 2016 May;53:2-9. doi: 10.1016/j.semcdb.2015.11.012. Epub
2015 Dec 17.

Connecting the dots of the bacterial cell cycle: Coordinating chromosome
replication and segregation with cell division.

Hajduk IV(1), Rodrigues CD(2), Harry EJ(3).

Author information: 
(1)The ithree institute, University of Technology Sydney, Sydney 2007, NSW,
Australia.
(2)Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 
MA 02115, USA.
(3)The ithree institute, University of Technology Sydney, Sydney 2007, NSW,
Australia. Electronic address: liz.harry@uts.edu.au.

Proper division site selection is crucial for the survival of all organisms. What
still eludes us is how bacteria position their division site with high precision,
and in tight coordination with chromosome replication and segregation. Until
recently, the general belief, at least in the model organisms Bacillus subtilis
and Escherichia coli, was that spatial regulation of division comes about by the 
combined negative regulatory mechanisms of the Min system and nucleoid occlusion.
However, as we review here, these two systems cannot be solely responsible for
division site selection and we highlight additional regulatory mechanisms that
are at play. In this review, we put forward evidence of how chromosome
replication and segregation may have direct links with cell division in these
bacteria and the benefit of recent advances in chromosome conformation capture
techniques in providing important information about how these three processes
mechanistically work together to achieve accurate generation of progenitor cells.

Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.semcdb.2015.11.012 
PMID: 26706151  [Indexed for MEDLINE]


22. BMC Genomics. 2015 Nov 4;16:895. doi: 10.1186/s12864-015-2162-4.

Analysis of the FnrL regulon in Rhodobacter capsulatus reveals limited regulon
overlap with orthologues from Rhodobacter sphaeroides and Escherichia coli.

Kumka JE(1), Bauer CE(2).

Author information: 
(1)Molecular and Cellular Biochemistry Department, Indiana University, Simon Hall
MSB, 212 S. Hawthorne Dr, Bloomington, IN, 47405-7003, USA.
(2)Molecular and Cellular Biochemistry Department, Indiana University, Simon Hall
MSB, 212 S. Hawthorne Dr, Bloomington, IN, 47405-7003, USA. bauer@indiana.edu.

BACKGROUND: FNR homologues constitute an important class of transcription factors
that control a wide range of anaerobic physiological functions in a number of
bacterial species. Since FNR homologues are some of the most pervasive
transcription factors, an understanding of their involvement in regulating
anaerobic gene expression in different species sheds light on evolutionary
similarity and differences. To address this question, we used a combination of
high throughput RNA-Seq and ChIP-Seq analysis to define the extent of the FnrL
regulon in Rhodobacter capsulatus and related our results to that of FnrL in
Rhodobacter sphaeroides and FNR in Escherichia coli.
RESULTS: Our RNA-seq results show that FnrL affects the expression of 807 genes, 
which accounts for over 20 % of the Rba. capsulatus genome. ChIP-seq results
indicate that 42 of these genes are directly regulated by FnrL. Importantly, this
includes genes involved in the synthesis of the anoxygenic photosystem.
Similarly, FnrL in Rba. sphaeroides affects 24 % of its genome, however, only 171
genes are differentially expressed in common between two Rhodobacter species,
suggesting significant divergence in regulation.
CONCLUSIONS: We show that FnrL in Rba. capsulatus activates photosynthesis while 
in Rba. sphaeroides FnrL regulation reported to involve repression of the
photosystem. This analysis highlights important differences in transcriptional
control of photosynthetic events and other metabolic processes controlled by FnrL
orthologues in closely related Rhodobacter species. Furthermore, we also show
that the E. coli FNR regulon has limited transcriptional overlap with the FnrL
regulons from either Rhodobacter species.

DOI: 10.1186/s12864-015-2162-4 
PMCID: PMC4634722
PMID: 26537891  [Indexed for MEDLINE]


23. J Bacteriol. 2015 Oct 19;198(1):187-200. doi: 10.1128/JB.00658-15. Print 2016 Jan
1.

Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation
Response of Caulobacter crescentus.

Lubin EA(1), Henry JT(2), Fiebig A(2), Crosson S(2), Laub MT(3).

Author information: 
(1)Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts, USA.
(2)Department of Biochemistry and Molecular Biology, University of Chicago,
Chicago, Illinois, USA.
(3)Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts, USA Howard Hughes Medical Institute, Massachusetts Institute of
Technology, Cambridge, Massachusetts, USA laub@mit.edu.

An ability to sense and respond to changes in extracellular phosphate is critical
for the survival of most bacteria. For Caulobacter crescentus, which typically
lives in phosphate-limited environments, this process is especially crucial. Like
many bacteria, Caulobacter responds to phosphate limitation through a conserved
two-component signaling pathway called PhoR-PhoB, but the direct regulon of PhoB 
in this organism is unknown. Here we used chromatin immunoprecipitation-DNA
sequencing (ChIP-Seq) to map the global binding patterns of the
phosphate-responsive transcriptional regulator PhoB under phosphate-limited and
-replete conditions. Combined with genome-wide expression profiling, our work
demonstrates that PhoB is induced to regulate nearly 50 genes under
phosphate-starved conditions. The PhoB regulon is comprised primarily of genes
known or predicted to help Caulobacter scavenge for and import inorganic
phosphate, including 15 different membrane transporters. We also investigated the
regulatory role of PhoU, a widely conserved protein proposed to coordinate
phosphate import with expression of the PhoB regulon by directly modulating the
histidine kinase PhoR. However, our studies show that it likely does not play
such a role in Caulobacter, as PhoU depletion has no significant effect on
PhoB-dependent gene expression. Instead, cells lacking PhoU exhibit striking
accumulation of large polyphosphate granules, suggesting that PhoU participates
in controlling intracellular phosphate metabolism.IMPORTANCE: The transcription
factor PhoB is widely conserved throughout the bacterial kingdom, where it helps 
organisms respond to phosphate limitation by driving the expression of a battery 
of genes. Most of what is known about PhoB and its target genes is derived from
studies of Escherichia coli. Our work documents the PhoB regulon in Caulobacter
crescentus, and comparison to the regulon in E. coli reveals significant
differences, highlighting the evolutionary plasticity of transcriptional
responses driven by highly conserved transcription factors. We also demonstrated 
that the conserved protein PhoU, which is implicated in bacterial persistence,
does not regulate PhoB activity, as previously suggested. Instead, our results
favor a model in which PhoU affects intracellular phosphate accumulation,
possibly through the high-affinity phosphate transporter.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.00658-15 
PMCID: PMC4686198
PMID: 26483520  [Indexed for MEDLINE]


24. Microb Genom. 2015 Oct 13;1(4):e000031. doi: 10.1099/mgen.0.000031. eCollection
2015 Oct.

Genome-wide analysis of the response to nitric oxide in uropathogenic Escherichia
coli CFT073.

Mehta HH(1), Liu Y(2), Zhang MQ(2), Spiro S(1).

Author information: 
(1)Department of Biological Sciences, University of Texas at Dallas, 800 W
Campbell Road, Richardson, TX 75080, USA.
(2)Department of Biological Sciences, University of Texas at Dallas, 800 W
Campbell Road, Richardson, TX 75080, USA; Center for Systems Biology, University 
of Texas at Dallas, 800 W Campbell Road, Richardson, TX 75080, USA.

Uropathogenic Escherchia coli (UPEC) is the causative agent of urinary tract
infections. Nitric oxide (NO) is a toxic water-soluble gas that is encountered by
UPEC in the urinary tract. Therefore, UPEC probably requires mechanisms to
detoxify NO in the host environment. Thus far, flavohaemoglobin (Hmp), an NO
denitrosylase, is the only demonstrated NO detoxification system in UPEC. Here we
show that, in E. coli strain CFT073, the NADH-dependent NO reductase
flavorubredoxin (FlRd) also plays a major role in NO scavenging. We generated a
mutant that lacks all known and candidate NO detoxification pathways (Hmp, FlRd
and the respiratory nitrite reductase, NrfA). When grown and assayed
anaerobically, this mutant expresses an NO-inducible NO scavenging activity,
pointing to the existence of a novel detoxification mechanism. Expression of this
activity is inducible by both NO and nitrate, and the enzyme is
membrane-associated. Genome-wide transcriptional profiling of UPEC grown under
anaerobic conditions in the presence of nitrate (as a source of NO) highlighted
various aspects of the response of the pathogen to nitrate and NO. Several
virulence-associated genes are upregulated, suggesting that host-derived NO is a 
potential regulator of UPEC virulence. Chromatin immunoprecipitation and
sequencing was used to evaluate the NsrR regulon in CFT073. We identified 49 NsrR
binding sites in promoter regions in the CFT073 genome, 29 of which were not
previously identified in E. coli K-12. NsrR may regulate some CFT073 genes that
do not have homologues in E. coli K-12.

DOI: 10.1099/mgen.0.000031 
PMCID: PMC5320621
PMID: 28348816 


25. PLoS One. 2015 Sep 21;10(9):e0138466. doi: 10.1371/journal.pone.0138466.
eCollection 2015.

ChIP-Seq Analysis of the σE Regulon of Salmonella enterica Serovar Typhimurium
Reveals New Genes Implicated in Heat Shock and Oxidative Stress Response.

Li J(1), Overall CC(2), Johnson RC(1), Jones MB(3), McDermott JE(2), Heffron
F(1), Adkins JN(2), Cambronne ED(1).

Author information: 
(1)Department of Molecular Microbiology and Immunology, Oregon Health & Science
University, Portland, Oregon, United States of America.
(2)Biological Sciences Division, Pacific Northwest National Laboratory, Richland,
Washington, United States of America.
(3)Department of Infectious Diseases, J. Craig Venter Institute, Rockville,
Maryland, United States of America.

The alternative sigma factor σE functions to maintain bacterial homeostasis and
membrane integrity in response to extracytoplasmic stress by regulating thousands
of genes both directly and indirectly. The transcriptional regulatory network
governed by σE in Salmonella and E. coli has been examined using microarray,
however a genome-wide analysis of σE-binding sites in Salmonella has not yet been
reported. We infected macrophages with Salmonella Typhimurium over a select time 
course. Using chromatin immunoprecipitation followed by high-throughput DNA
sequencing (ChIP-seq), 31 σE-binding sites were identified. Seventeen sites were 
new, which included outer membrane proteins, a quorum-sensing protein, a cell
division factor, and a signal transduction modulator. The consensus sequence
identified for σE in vivo binding was similar to the one previously reported,
except for a conserved G and A between the -35 and -10 regions. One third of the 
σE-binding sites did not contain the consensus sequence, suggesting there may be 
alternative mechanisms by which σE modulates transcription. By dissecting direct 
and indirect modes of σE-mediated regulation, we found that σE activates gene
expression through recognition of both canonical and reversed consensus sequence.
New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in
heat shock and oxidative stress responses.

DOI: 10.1371/journal.pone.0138466 
PMCID: PMC4577112
PMID: 26389830  [Indexed for MEDLINE]


26. Pathogens. 2015 Jun 26;4(3):422-30. doi: 10.3390/pathogens4030422.

Molecular Characterization of the Multidrug Resistant Escherichia coli ST131
Clone.

Schembri MA(1)(2), Zakour NL(3)(4), Phan MD(5)(6), Forde BM(7)(8), Stanton-Cook
M(9)(10), Beatson SA(11)(12).

Author information: 
(1)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. m.schembri@uq.edu.au.
(2)Australian Infectious Diseases Research Centre, the University of Queensland, 
Brisbane, Queensland 4072, Australia. m.schembri@uq.edu.au.
(3)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. n.benzakour@uq.edu.au.
(4)Australian Infectious Diseases Research Centre, the University of Queensland, 
Brisbane, Queensland 4072, Australia. n.benzakour@uq.edu.au.
(5)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. m.phan1@uq.edu.au.
(6)Australian Infectious Diseases Research Centre, the University of Queensland, 
Brisbane, Queensland 4072, Australia. m.phan1@uq.edu.au.
(7)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. b.forde@uq.edu.au.
(8)Australian Infectious Diseases Research Centre, the University of Queensland, 
Brisbane, Queensland 4072, Australia. b.forde@uq.edu.au.
(9)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. m.stantoncook@uq.edu.au.
(10)Australian Infectious Diseases Research Centre, the University of Queensland,
Brisbane, Queensland 4072, Australia. m.stantoncook@uq.edu.au.
(11)School of Chemistry & Molecular Biosciences, the University of Queensland,
Brisbane, Queensland 4072, Australia. s.beatson@uq.edu.au.
(12)Australian Infectious Diseases Research Centre, the University of Queensland,
Brisbane, Queensland 4072, Australia. s.beatson@uq.edu.au.

Escherichia coli ST131 is a recently emerged and globally disseminated multidrug 
resistant clone associated with urinary tract and bloodstream infections in both 
community and clinical settings. The most common group of ST131 strains are
defined by resistance to fluoroquinolones and possession of the type 1 fimbriae
fimH30 allele. Here we provide an update on our recent work describing the
globally epidemiology of ST131. We review the phylogeny of ST131 based on whole
genome sequence data and highlight the important role of recombination in the
evolution of this clonal lineage. We also summarize our findings on the virulence
of the ST131 reference strain EC958, and highlight the use of transposon directed
insertion-site sequencing to define genes associated with serum resistance and
essential features of its large antibiotic resistance plasmid pEC958.

DOI: 10.3390/pathogens4030422 
PMCID: PMC4584265
PMID: 26131613 


27. PLoS Genet. 2015 Jun 12;11(6):e1005289. doi: 10.1371/journal.pgen.1005289.
eCollection 2015 Jun.

The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through
Transcription Units.

Merrikh CN(1), Brewer BJ(2), Merrikh H(3).

Author information: 
(1)Department of Microbiology, University of Washington, Seattle, Washington,
United States of America.
(2)Department of Genome Sciences, University of Washington, Seattle, Washington, 
United States of America.
(3)Department of Microbiology, University of Washington, Seattle, Washington,
United States of America; Department of Genome Sciences, University of
Washington, Seattle, Washington, United States of America.

In bacteria the concurrence of DNA replication and transcription leads to
potentially deleterious encounters between the two machineries, which can occur
in either the head-on (lagging strand genes) or co-directional (leading strand
genes) orientations. These conflicts lead to replication fork stalling and can
destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution 
factors that reduce the severity of these encounters. Though Escherichia coli
accessory helicases have been implicated in the mitigation of head-on conflicts, 
direct evidence of these proteins mitigating co-directional conflicts is lacking.
Furthermore, the endogenous chromosomal regions where these helicases act, and
the mechanism of recruitment, have not been identified. We show that the
essential Bacillus subtilis accessory helicase PcrA aids replication progression 
through protein coding genes of both head-on and co-directional orientations, as 
well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional
conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are 
major targets of PcrA activity on the chromosome. Partial depletion of PcrA
renders cells extremely sensitive to head-on conflicts, linking the essential
function of PcrA to conflict resolution. Furthermore, ablating PcrA's
ATPase/helicase activity simultaneously increases its association with conflict
regions, while incapacitating its ability to mitigate conflicts, and leads to
cell death. In contrast, disruption of PcrA's C-terminal RNA polymerase
interaction domain does not impact its ability to mitigate conflicts between
replication and transcription, its association with conflict regions, or cell
survival. Altogether, this work establishes PcrA as an essential factor involved 
in mitigating transcription-replication conflicts and identifies chromosomal
regions where it routinely acts. As both conflicts and accessory helicases are
found in all domains of life, these results are broadly relevant.

DOI: 10.1371/journal.pgen.1005289 
PMCID: PMC4466434
PMID: 26070154  [Indexed for MEDLINE]


28. PLoS One. 2015 Apr 15;10(4):e0122369. doi: 10.1371/journal.pone.0122369.
eCollection 2015.

Molecular characterization of a multidrug resistance IncF plasmid from the
globally disseminated Escherichia coli ST131 clone.

Phan MD(1), Forde BM(1), Peters KM(1), Sarkar S(1), Hancock S(1), Stanton-Cook
M(1), Ben Zakour NL(1), Upton M(2), Beatson SA(1), Schembri MA(1).

Author information: 
(1)Australian Infectious Diseases Research Centre, School of Chemistry and
Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072,
Australia.
(2)Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth,
United Kingdom.

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and
globally disseminated multidrug resistant clone associated with urinary tract and
bloodstream infections. Plasmids represent a major vehicle for the carriage of
antibiotic resistance genes in E. coli ST131. In this study, we determined the
complete sequence and performed a comprehensive annotation of pEC958, an IncF
plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb
in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic
resistance genes (including the blaCTX-M-15 gene). We also carried out
hyper-saturated transposon mutagenesis and multiplexed transposon directed
insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS
data showed that while only the RepFII replicon was required for pEC958
replication, the RepFIA replicon contains genes essential for its partitioning.
Thus, our data provides direct evidence that the RepFIA and RepFII replicons in
pEC958 cooperate to ensure their stable inheritance. The gene encoding the
antitoxin component (ccdA) of the post-segregational killing system CcdAB was
also protected from mutagenesis, demonstrating this system is active. Sequence
comparison with a global collection of ST131 strains suggest that IncF represents
the most common type of plasmid in this clone, and underscores the need to
understand its evolution and contribution to the spread of antibiotic resistance 
genes in E. coli ST131.

DOI: 10.1371/journal.pone.0122369 
PMCID: PMC4398462
PMID: 25875675  [Indexed for MEDLINE]


29. Nucleic Acids Res. 2015 Jul 27;43(13):e87. doi: 10.1093/nar/gkv300. Epub 2015 Apr
14.

cMonkey2: Automated, systematic, integrated detection of co-regulated gene
modules for any organism.

Reiss DJ(1), Plaisier CL(2), Wu WJ(2), Baliga NS(3).

Author information: 
(1)Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109, USA
dreiss@systemsbiology.org.
(2)Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109, USA.
(3)Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109, USA
Department of Microbiology, University of Washington, Seattle, WA 98103, USA
nbaliga@systemsbiology.org.

The cMonkey integrated biclustering algorithm identifies conditionally
co-regulated modules of genes (biclusters). cMonkey integrates various orthogonal
pieces of information which support evidence of gene co-regulation, and optimizes
biclusters to be supported simultaneously by one or more of these prior
constraints. The algorithm served as the cornerstone for constructing the first
global, predictive Environmental Gene Regulatory Influence Network (EGRIN) model 
for a free-living cell, and has now been applied to many more organisms. However,
due to its computational inefficiencies, long run-time and complexity of various 
input data types, cMonkey was not readily usable by the wider community. To
address these primary concerns, we have significantly updated the cMonkey
algorithm and refactored its implementation, improving its usability and
extendibility. These improvements provide a fully functioning and user-friendly
platform for building co-regulated gene modules and the tools necessary for their
exploration and interpretation. We show, via three separate analyses of data for 
E. coli, M. tuberculosis and H. sapiens, that the updated algorithm and inclusion
of novel scoring functions for new data types (e.g. ChIP-seq and transcription
factor over-expression [TFOE]) improve discovery of biologically informative
co-regulated modules. The complete cMonkey2 software package, including source
code, is available at https://github.com/baliga-lab/cmonkey2.

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

DOI: 10.1093/nar/gkv300 
PMCID: PMC4513845
PMID: 25873626  [Indexed for MEDLINE]


30. Methods Mol Biol. 2015;1284:3-25. doi: 10.1007/978-1-4939-2444-8_1.

Epigenome profiling of specific plant cell types using a streamlined INTACT
protocol and ChIP-seq.

Wang D(1), Deal RB.

Author information: 
(1)Department of Biology, O. Wayne Rollins Research Center, Emory University,
1510 Clifton Road NE, Atlanta, GA, 30322, USA.

Plants consist of many functionally specialized cell types, each with its own
unique epigenome, transcriptome, and proteome. Characterization of these cell
type-specific properties is essential to understanding cell fate specification
and the responses of individual cell types to the environment. In this chapter we
describe an approach to map chromatin features in specific cell types of
Arabidopsis thaliana using nuclei purification from individual cell types with
the INTACT method (isolation of nuclei tagged in specific cell types) followed by
chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). The
INTACT system employs two transgenes to generate affinity-labeled nuclei in the
cell type of interest, and these tagged nuclei can then be selectively purified
from tissue homogenates. The primary transgene encodes the nuclear tagging fusion
protein (NTF), which consists of a nuclear envelope-targeting domain, the green
fluorescent protein, and a biotin ligase recognition peptide, while the second
transgene encodes the E. coli biotin ligase (BirA), which selectively
biotinylates NTF. Expression of NTF and BirA in a specific cell type thus yields 
nuclei that are coated with biotin and can be purified by virtue of their
affinity for streptavidin-coated magnetic beads. Compared with the original
INTACT nuclei purification protocol, the procedure presented here is greatly
simplified and shortened. After nuclei purification, we provide detailed
instructions for chromatin isolation, shearing, and immunoprecipitation. Finally,
we present a low input ChIP-seq library preparation protocol based on the
nano-ChIP-seq method of Adli and Bernstein, and we describe multiplex Illumina
sequencing of these libraries to produce high quality, cell type-specific
epigenome profiles at a relatively low cost. The procedures given here are
optimized for Arabidopsis but should be easily adaptable to other plant species.

DOI: 10.1007/978-1-4939-2444-8_1 
PMID: 25757765  [Indexed for MEDLINE]


31. Genom Data. 2014 Dec;2:110-113.

Genome-Wide Mapping of the Distribution of CarD, RNAP σA, and RNAP β on the
Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation
Sequencing.

Landick R(1), Krek A(2), Glickman MS(3), Socci ND(2), Stallings CL(4).

Author information: 
(1)Departments of Biochemistry and Bacteriology, University of Wisconsin,
Madison, WI, 53706, USA.
(2)Bioinformatics Core, Memorial Sloan-Kettering Cancer Center, New York, NY,
10065, USA.
(3)Immunology Program, Sloan-Kettering Institute, and Division of Infectious
Diseases, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA.
(4)Department of Molecular Microbiology, Washington University School of
Medicine, St. Louis, MO 63110, USA.

CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP)
and affects the transcriptional profile of Mycobacterium smegmatis and
Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating
RNAP function but our prior experiments had not determined at what stage of
transcription CarD was functioning and at which genes CarD interacted with the
RNAP. To begin to address these open questions, we performed Chromatin
Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD
throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and
σA were also profiled. We expected that RNAP β would be present throughout
transcribed regions and RNAP σA would be predominantly enriched at promoters
based on work in Escherichia coli (3), however this had yet to be determined in
mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the
genome in the absence of RNAP, was primarily associated with promoter regions,
and was highly correlated with the distribution of RNAP σA. The colocalization of
σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation 
complexes at most promoters and is therefore a global regulator of transcription 
initiation. Here we describe in detail the data from the ChIP-seq experiments
associated with the study published by Srivastava and colleagues in the
Proceedings of the National Academy of Science in 2013 (5) as well as discuss the
findings from this dataset in relation to both CarD and mycobacterial
transcription as a whole. The ChIP-seq data have been deposited in the Gene
Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.
GSE48164).

DOI: 10.1016/j.gdata.2014.05.012 
PMCID: PMC4115788
PMID: 25089258 


32. BMC Microbiol. 2014 Aug 1;14:206. doi: 10.1186/s12866-014-0206-6.

Characterization and analysis of the Burkholderia pseudomallei BsaN virulence
regulon.

Chen Y, Schröder I, French CT, Jaroszewicz A, Yee XJ, Teh BE, Toesca IJ, Miller
JF, Gan YH(1).

Author information: 
(1)Department of Biochemistry, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore 117597, Singapore. yunn_hwen_gan@nuhs.edu.sg.

BACKGROUND: Burkholderia pseudomallei is a facultative intracellular pathogen and
the causative agent of melioidosis. A conserved type III secretion system (T3SS3)
and type VI secretion system (T6SS1) are critical for intracellular survival and 
growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated
by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR
regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci.
RESULTS: To elucidate the scope of the BsaN regulon, we used RNAseq analysis to
compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion
mutant. The 60 genes positively-regulated by BsaN include those that we had
previously identified in addition to a polyketide biosynthesis locus and genes
involved in amino acid biosynthesis. BsaN was also found to repress the
transcription of 51 genes including flagellar motility loci and those encoding
components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli,
we show that BsaN together with the chaperone BicA directly control the
expression of the T3SS3 translocon, effector and associated regulatory genes that
are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis
approach, a consensus regulatory motif in the promoter regions of BsaN-regulated 
genes was shown to be essential for transcriptional activation.
CONCLUSIONS: BsaN/BicA functions as a central regulator of key virulence clusters
in B. pseudomallei within a more extensive network of genetic regulation. We
propose that BsaN/BicA controls a gene expression program that facilitates the
adaption and intracellular survival of the pathogen within eukaryotic hosts.

DOI: 10.1186/s12866-014-0206-6 
PMCID: PMC4236580
PMID: 25085508  [Indexed for MEDLINE]


33. PLoS Genet. 2014 Apr 17;10(4):e1004288. doi: 10.1371/journal.pgen.1004288.
eCollection 2014 Apr.

Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

Chan YA(1), Aristizabal MJ(2), Lu PY(2), Luo Z(3), Hamza A(1), Kobor MS(4),
Stirling PC(5), Hieter P(6).

Author information: 
(1)Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.
(2)Centre for Molecular Medicine and Therapeutics, Child and Family Research
Institute, Vancouver, Canada.
(3)Wine Research Centre, University of British Columbia, Vancouver, Canada.
(4)Centre for Molecular Medicine and Therapeutics, Child and Family Research
Institute, Vancouver, Canada; Department of Medical Genetics, University of
British Columbia, Vancouver, Canada.
(5)Department of Medical Genetics, University of British Columbia, Vancouver,
Canada; Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
(6)Michael Smith Laboratories, University of British Columbia, Vancouver, Canada;
Department of Medical Genetics, University of British Columbia, Vancouver,
Canada.

DNA:RNA hybrid formation is emerging as a significant cause of genome instability
in biological systems ranging from bacteria to mammals. Here we describe the
genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae
by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling
microarray. These profiles show that DNA:RNA hybrids preferentially accumulated
at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open
reading frames (ORFs). The latter are generally highly transcribed and have high 
GC content. Interestingly, significant DNA:RNA hybrid enrichment was also
detected at genes associated with antisense transcripts. The expression of
antisense-associated genes was also significantly altered upon overexpression of 
RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific
differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion
mutant and Hpr1 THO complex mutant compared to wild type, suggesting different
roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA
hybrid prone loci provide a resource for understanding the properties of
hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating
enzymes, and contribute to models of antisense-mediated gene regulation. A
summary of this paper was presented at the 26th International Conference on Yeast
Genetics and Molecular Biology, August 2013.

DOI: 10.1371/journal.pgen.1004288 
PMCID: PMC3990523
PMID: 24743342  [Indexed for MEDLINE]


34. Methods. 2014 Jun 1;67(3):294-303. doi: 10.1016/j.ymeth.2014.03.006. Epub 2014
Mar 17.

Inferring gene regulatory networks by integrating ChIP-seq/chip and transcriptome
data via LASSO-type regularization methods.

Qin J(1), Hu Y(2), Xu F(1), Yalamanchili HK(1), Wang J(3).

Author information: 
(1)Department of Biochemistry, LKS Faculty of Medicine, The University of Hong
Kong, Hong Kong Special Administrative Region, China; Shenzhen Institute of
Research & Innovation, The University of Hong Kong, Shenzhen, China.
(2)Department of Mathematics, Zhejiang University, Hangzhou, China; Department of
Applied Mathematics, The Hong Kong Polytechnic University, Hung Hom, Kowloon,
Hong Kong Special Administrative Region, China.
(3)Department of Biochemistry, LKS Faculty of Medicine, The University of Hong
Kong, Hong Kong Special Administrative Region, China; Shenzhen Institute of
Research & Innovation, The University of Hong Kong, Shenzhen, China; Centre for
Genomic Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong
Special Administrative Region, China. Electronic address: junwen@hku.hk.

Inferring gene regulatory networks from gene expression data at whole genome
level is still an arduous challenge, especially in higher organisms where the
number of genes is large but the number of experimental samples is small. It is
reported that the accuracy of current methods at genome scale significantly drops
from Escherichia coli to Saccharomyces cerevisiae due to the increase in number
of genes. This limits the applicability of current methods to more complex
genomes, like human and mouse. Least absolute shrinkage and selection operator
(LASSO) is widely used for gene regulatory network inference from gene expression
profiles. However, the accuracy of LASSO on large genomes is not satisfactory. In
this study, we apply two extended models of LASSO, L0 and L1/2 regularization
models to infer gene regulatory network from both high-throughput gene expression
data and transcription factor binding data in mouse embryonic stem cells (mESCs).
We find that both the L0 and L1/2 regularization models significantly outperform 
LASSO in network inference. Incorporating interactions between transcription
factors and their targets remarkably improved the prediction accuracy. Current
study demonstrates the efficiency and applicability of these two models for gene 
regulatory network inference from integrative omics data in large genomes. The
applications of the two models will facilitate biologists to study the gene
regulation of higher model organisms in a genome-wide scale.

Copyright © 2014 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ymeth.2014.03.006 
PMID: 24650566  [Indexed for MEDLINE]


35. BMC Syst Biol. 2013;7 Suppl 6:S13. doi: 10.1186/1752-0509-7-S6-S13. Epub 2013 Dec
13.

Inferring functional transcription factor-gene binding pairs by integrating
transcription factor binding data with transcription factor knockout data.

Yang TH, Wu WS.

BACKGROUND: Chromatin immunoprecipitation (ChIP) experiments are now the most
comprehensive experimental approaches for mapping the binding of transcription
factors (TFs) to their target genes. However, ChIP data alone is insufficient for
identifying functional binding target genes of TFs for two reasons. First, there 
is an inherent high false positive/negative rate in ChIP-chip or ChIP-seq
experiments. Second, binding signals in the ChIP data do not necessarily imply
functionality.
METHODS: It is known that ChIP-chip data and TF knockout (TFKO) data reveal
complementary information on gene regulation. While ChIP-chip data can provide
TF-gene binding pairs, TFKO data can provide TF-gene regulation pairs. Therefore,
we propose a novel network approach for identifying functional TF-gene binding
pairs by integrating the ChIP-chip data with the TFKO data. In our method, a
TF-gene binding pair from the ChIP-chip data is regarded to be functional if it
also has high confident curated TFKO TF-gene regulatory relation or deduced
hypostatic TF-gene regulatory relation.
RESULTS AND CONCLUSIONS: We first validated our method on a gathered ground truth
set. Then we applied our method to the ChIP-chip data to identify functional
TF-gene binding pairs. The biological significance of our identified functional
TF-gene binding pairs was shown by assessing their functional enrichment, the
prevalence of protein-protein interaction, and expression coherence. Our results 
outperformed the results of three existing methods across all measures. And our
identified functional targets of TFs also showed statistical significance over
the randomly assigned TF-gene pairs. We also showed that our method is dataset
independent and can apply to ChIP-seq data and the E. coli genome. Finally, we
provided an example showing the biological applicability of our notion.

DOI: 10.1186/1752-0509-7-S6-S13 
PMCID: PMC4029220
PMID: 24565265  [Indexed for MEDLINE]


36. PLoS Genet. 2013;9(10):e1003834. doi: 10.1371/journal.pgen.1003834. Epub 2013 Oct
3.

The serum resistome of a globally disseminated multidrug resistant uropathogenic 
Escherichia coli clone.

Phan MD(1), Peters KM, Sarkar S, Lukowski SW, Allsopp LP, Gomes Moriel D, Achard 
ME, Totsika M, Marshall VM, Upton M, Beatson SA, Schembri MA.

Author information: 
(1)Australian Infectious Diseases Research Centre, The University of Queensland, 
Brisbane, Queensland, Australia ; School of Chemistry and Molecular Biosciences, 
The University of Queensland, Brisbane, Queensland, Australia.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone
responsible for a high proportion of urinary tract and bloodstream infections.
The rapid emergence and successful spread of E. coli ST131 is strongly associated
with antibiotic resistance; however, this phenotype alone is unlikely to explain 
its dominance amongst multidrug resistant uropathogens circulating worldwide in
hospitals and the community. Thus, a greater understanding of the molecular
mechanisms that underpin the fitness of E. coli ST131 is required. In this study,
we employed hyper-saturated transposon mutagenesis in combination with
multiplexed transposon directed insertion-site sequencing to define the essential
genes required for in vitro growth and the serum resistome (i.e. genes required
for resistance to human serum) of E. coli EC958, a representative of the
predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E.
coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum
resistome comprised 56 genes, the majority of which encode membrane proteins or
factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis
confirmed a role in serum resistance for 46 (82%) of these genes. The murein
lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, 
were most strongly associated with serum resistance. While LPS was the main
resistance mechanism defined for E. coli EC958 in serum, the enterobacterial
common antigen and colanic acid also impacted on this phenotype. Our analysis
also identified a novel function for two genes, hyxA and hyxR, as minor
regulators of O-antigen chain length. This study offers novel insight into the
genetic make-up of E. coli ST131, and provides a framework for future research on
E. coli and other Gram-negative pathogens to define their essential gene
repertoire and to dissect the molecular mechanisms that enable them to survive in
the bloodstream and cause disease.

DOI: 10.1371/journal.pgen.1003834 
PMCID: PMC3789825
PMID: 24098145  [Indexed for MEDLINE]

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


37. BMC Genomics. 2013 Sep 22;14:638. doi: 10.1186/1471-2164-14-638.

"Non-canonical protein-DNA interactions identified by ChIP are not artifacts":
response.

Schindler D(1), Waldminghaus T.

Author information: 
(1)LOEWE-Center for Synthetic Microbiology (SYNMIKRO), Philipps-Universität
Marburg, Hans-Meerwein-Str, 6, D-35043, Marburg, Germany.
Torsten.Waldminghaus@synmikro.uni-marburg.de.

BACKGROUND: Studies of protein association with DNA on a genome wide scale are
possible through methods like ChIP-Chip or ChIP-Seq. Massive problems with false 
positive signals in our own experiments motivated us to revise the standard
ChIP-Chip protocol. Analysis of chromosome wide binding of the alternative sigma 
factor σ³² in Escherichia coli with this new protocol resulted in detection of
only a subset of binding sites found in a previous study by Wade and colleagues. 
We suggested that the remainder of binding sites detected in the previous study
are likely to be false positives. In a recent article the Wade group claimed that
our conclusion is wrong and that the disputed sites are genuine σ³² binding
sites. They further claimed that the non-detection of these sites in our study
was due to low data quality.
RESULTS/DISCUSSION: We respond to the criticism of Wade and colleagues and
discuss some general questions of ChIP-based studies. We outline why the quality 
of our data is sufficient to derive meaningful results. Specific points are: (i) 
the modifications we introduced into the standard ChIP-Chip protocol do not
necessarily result in a low dynamic range, (ii) correlation between ChIP-Chip
replicates should not be calculated based on the whole data set as done in
transcript analysis, (iii) control experiments are essential for identifying
false positives. Suggestions are made how ChIP-based methods could be further
optimized and which alternative approaches can be used to strengthen conclusions.
CONCLUSION: We appreciate the ongoing discussion about the ChIP-Chip method and
hope that it helps other scientist to analyze and interpret their results. The
modifications we introduced into the ChIP-Chip protocol are a first step towards 
reducing false positive signals but there is certainly potential for further
optimization. The discussion about the σ³² binding sites in question highlights
the need for alternative approaches and further investigation of appropriate
methods for verification.

DOI: 10.1186/1471-2164-14-638 
PMCID: PMC3870955
PMID: 24053571  [Indexed for MEDLINE]


38. BMC Bioinformatics. 2013 Jul 17;14:227. doi: 10.1186/1471-2105-14-227.

A fast weak motif-finding algorithm based on community detection in graphs.

Jia C(1), Carson MB, Yu J.

Author information: 
(1)School of Computer and Information Technology, Beijing Jiaotong University,
Beijing 100044, China. cyjia@bjtu.edu.cn

BACKGROUND: Identification of transcription factor binding sites (also called
'motif discovery') in DNA sequences is a basic step in understanding genetic
regulation. Although many successful programs have been developed, the problem is
far from being solved on account of diversity in gene expression/regulation and
the low specificity of binding sites. State-of-the-art algorithms have their own 
constraints (e.g., high time or space complexity for finding long motifs, low
precision in identification of weak motifs, or the OOPS constraint: one
occurrence of the motif instance per sequence) which limit their scope of
application.
RESULTS: In this paper, we present a novel and fast algorithm we call TFBSGroup. 
It is based on community detection from a graph and is used to discover long and 
weak (l,d) motifs under the ZOMOPS constraint (zero, one or multiple
occurrence(s) of the motif instance(s) per sequence), where l is the length of a 
motif and d is the maximum number of mutations between a motif instance and the
motif itself. Firstly, TFBSGroup transforms the (l, d) motif search in sequences 
to focus on the discovery of dense subgraphs within a graph. It identifies these 
subgraphs using a fast community detection method for obtaining coarse-grained
candidate motifs. Next, it greedily refines these candidate motifs towards the
true motif within their own communities. Empirical studies on synthetic (l, d)
samples have shown that TFBSGroup is very efficient (e.g., it can find true (18, 
6), (24, 8) motifs within 30 seconds). More importantly, the algorithm has
succeeded in rapidly identifying motifs in a large data set of prokaryotic
promoters generated from the Escherichia coli database RegulonDB. The algorithm
has also accurately identified motifs in ChIP-seq data sets for 12 mouse
transcription factors involved in ES cell pluripotency and self-renewal.
CONCLUSIONS: Our novel heuristic algorithm, TFBSGroup, is able to quickly
identify nearly exact matches for long and weak (l, d) motifs in DNA sequences
under the ZOMOPS constraint. It is also capable of finding motifs in real
applications. The source code for TFBSGroup can be obtained from
http://bioinformatics.bioengr.uic.edu/TFBSGroup/.

DOI: 10.1186/1471-2105-14-227 
PMCID: PMC3726413
PMID: 23865838  [Indexed for MEDLINE]


39. PLoS One. 2013 May 23;8(5):e64688. doi: 10.1371/journal.pone.0064688. Print 2013.

Genome-wide analysis of the salmonella Fis regulon and its regulatory mechanism
on pathogenicity islands.

Wang H(1), Liu B, Wang Q, Wang L.

Author information: 
(1)TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA,
Tianjin, PR China.

Fis, one of the most important nucleoid-associated proteins, functions as a
global regulator of transcription in bacteria that has been comprehensively
studied in Escherichia coli K12. Fis also influences the virulence of Salmonella 
enterica and pathogenic E. coli by regulating their virulence genes, however, the
relevant mechanism is unclear. In this report, using combined RNA-seq and
chromatin immunoprecipitation (ChIP)-seq technologies, we first identified 1646
Fis-regulated genes and 885 Fis-binding targets in the S. enterica serovar
Typhimurium, and found a Fis regulon different from that in E. coli. Fis has been
reported to contribute to the invasion ability of S. enterica. By using cell
infection assays, we found it also enhances the intracellular replication ability
of S. enterica within macrophage cell, which is of central importance for the
pathogenesis of infections. Salmonella pathogenicity islands (SPI)-1 and SPI-2
are crucial for the invasion and survival of S. enterica in host cells. Using
mutation and overexpression experiments, real-time PCR analysis, and
electrophoretic mobility shift assays, we demonstrated that Fis regulates 63 of
the 94 Salmonella pathogenicity island (SPI)-1 and SPI-2 genes, by three
regulatory modes: i) binds to SPI regulators in the gene body or in upstream
regions; ii) binds to SPI genes directly to mediate transcriptional activation of
themselves and downstream genes; iii) binds to gene encoding OmpR which affects
SPI gene expression by controlling SPI regulators SsrA and HilD. Our results
provide new insights into the impact of Fis on SPI genes and the pathogenicity of
S. enterica.

DOI: 10.1371/journal.pone.0064688 
PMCID: PMC3662779
PMID: 23717649  [Indexed for MEDLINE]


40. DNA Res. 2013 Aug;20(4):325-38. doi: 10.1093/dnares/dst013. Epub 2013 Apr 11.

High-resolution mapping of in vivo genomic transcription factor binding sites
using in situ DNase I footprinting and ChIP-seq.

Chumsakul O(1), Nakamura K, Kurata T, Sakamoto T, Hobman JL, Ogasawara N, Oshima 
T, Ishikawa S.

Author information: 
(1)Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

Accurate identification of the DNA-binding sites of transcription factors and
other DNA-binding proteins on the genome is crucial to understanding their
molecular interactions with DNA. Here, we describe a new method: Genome
Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo
DNase I digestion of genomic DNA with ChIP coupled with high-throughput
sequencing. We have determined the in vivo binding sites of a Bacillus subtilis
global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding
sequences, which were protected from in vivo DNase I digestion, were resolved at 
a comparable resolution to that achieved by in vitro DNase I footprinting, and
this was simply attained without the necessity of prediction by peak-calling
programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the
closely positioned AbrB-binding sites, which had previously appeared as one peak 
in ChAP-chip and ChAP-seq experiments. The high-resolution determination of
AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in
96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with
very low-binding intensities, single TGGNA motifs were also identified. Thus,
GeF-seq is a powerful method to elucidate the molecular mechanism of target
protein binding to its cognate DNA sequences.

DOI: 10.1093/dnares/dst013 
PMCID: PMC3738160
PMID: 23580539  [Indexed for MEDLINE]


41. Integr Biol (Camb). 2013 May;5(5):796-806. doi: 10.1039/c3ib20221f.

Efficient transcription initiation in bacteria: an interplay of protein-DNA
interaction parameters.

Djordjevic M(1).

Author information: 
(1)Institute of Physiology and Biochemistry, Faculty of Biology, University of
Belgrade, Studentski trg 16, 11000 Belgrade, Serbia. dmarko@bio.bg.ac.rs

As the first, and usually rate-limiting, step of transcription initiation,
bacterial RNA polymerase (RNAP) binds to double stranded DNA (dsDNA) and
subsequently opens the two strands of DNA (the open complex formation). The rate 
determining step in the open complex formation is opening of a short (6 bp) DNA
called the -10 region, which interacts with RNAP in both dsDNA and single
stranded (ssDNA) forms. Accordingly, formation of the open complex depends on
(physically independent) domains of RNAP that interact with ssDNA and dsDNA, as
well as on parameters of DNA melting and sequences of -10 regions. We here aim to
understand how these different interactions are mutually related to ensure
efficient open complex formation. To achieve this, we use a recently developed
biophysical model of transcription initiation, which allows the calculation of
the kinetic parameters of transcription initiation on the scale of whole genome. 
We consequently investigate kinetic properties of sequences derived from all E.
coli intergenic regions, and from more than 300 experimentally confirmed E. coli 
σ(70) promoters. We find that interaction specificities of σ(70) DNA binding
domains reduce the number of sequences where RNAP binds strongly, but forms the
open complex too slowly to achieve functional transcription (so-called poised
promoters). However, we find that, despite this reduction, there is still a
significant number of such poised promoters in the intergenic regions, which may 
provide a major source of false positives in genome-wide searches of
transcription start sites. Furthermore, we surprisingly find that sequences of
-10 regions of the functional promoters increase the extent of RNAP poising,
which we interpret in terms of an extension of a recently proposed model of
promoter recognition ('mix-and-match model') to kinetic parameters. Overall, our 
results allow better understanding of the design of σ(70) DNA binding domains and
promoter sequences, and place a fundamental limit on accuracy of methods for
promoter detection that are based on strong RNAP binding (e.g. ChIP-chip).

DOI: 10.1039/c3ib20221f 
PMID: 23511241  [Indexed for MEDLINE]


42. Nucleic Acids Res. 2013 Apr;41(8):4549-64. doi: 10.1093/nar/gkt148. Epub 2013 Mar
6.

A comparison of dense transposon insertion libraries in the Salmonella serovars
Typhi and Typhimurium.

Barquist L(1), Langridge GC, Turner DJ, Phan MD, Turner AK, Bateman A, Parkhill
J, Wain J, Gardner PP.

Author information: 
(1)Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
lb14@sanger.ac.uk

Salmonella Typhi and Typhimurium diverged only ∼50 000 years ago, yet have very
different host ranges and pathogenicity. Despite the availability of multiple
whole-genome sequences, the genetic differences that have driven these changes in
phenotype are only beginning to be understood. In this study, we use
transposon-directed insertion-site sequencing to probe differences in gene
requirements for competitive growth in rich media between these two closely
related serovars. We identify a conserved core of 281 genes that are required for
growth in both serovars, 228 of which are essential in Escherichia coli. We are
able to identify active prophage elements through the requirement for their
repressors. We also find distinct differences in requirements for genes involved 
in cell surface structure biogenesis and iron utilization. Finally, we
demonstrate that transposon-directed insertion-site sequencing is not only
applicable to the protein-coding content of the cell but also has sufficient
resolution to generate hypotheses regarding the functions of non-coding RNAs
(ncRNAs) as well. We are able to assign probable functions to a number of
cis-regulatory ncRNA elements, as well as to infer likely differences in
trans-acting ncRNA regulatory networks.

DOI: 10.1093/nar/gkt148 
PMCID: PMC3632133
PMID: 23470992  [Indexed for MEDLINE]


43. Nucleic Acids Res. 2013 Mar 1;41(5):e59. doi: 10.1093/nar/gks1342. Epub 2012 Dec 
28.

Nanobody(R)-based chromatin immunoprecipitation/micro-array analysis for
genome-wide identification of transcription factor DNA binding sites.

Nguyen-Duc T(1), Peeters E, Muyldermans S, Charlier D, Hassanzadeh-Ghassabeh G.

Author information: 
(1)Research group of Cellular and Molecular Immunology, Vrije Universiteit
Brussel, Pleinlaan 2, B-1050 Brussel, Belgium.

Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain
antibodies. Because of their small size, straightforward production in
Escherichia coli, easy tailoring, high affinity, specificity, stability and
solubility, nanobodies® have been exploited in various biotechnological
applications. A major challenge in the post-genomics and post-proteomics era is
the identification of regulatory networks involving nucleic acid-protein and
protein-protein interactions. Here, we apply a nanobody® in chromatin
immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for
genome-wide identification of DNA-protein interactions. The Lrp-like regulator
Ss-LrpB, arguably one of the best-studied specific transcription factors of the
hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this
proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific
nanobodies®, each interacting with a different epitope, were generated for ChIP. 
Genome-wide ChIP-chip with one of these nanobodies® identified the
well-established Ss-LrpB binding sites and revealed several unknown target
sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator
sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a 
novel class of affinity reagents for ChIP. Taking into account the unique
characteristics of nanobodies®, in particular, their short generation time,
nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq
experiments, especially in organisms with no (or limited) possibility of genetic 
manipulation.

DOI: 10.1093/nar/gks1342 
PMCID: PMC3597646
PMID: 23275538  [Indexed for MEDLINE]


44. MBio. 2012 Dec 11;3(6). pii: e00407-12. doi: 10.1128/mBio.00407-12.

Crp is a global regulator of antibiotic production in streptomyces.

Gao C(1), Hindra, Mulder D, Yin C, Elliot MA.

Author information: 
(1)Department of Biology and Institute for Infectious Disease Research, McMaster 
University, Hamilton, Ontario, Canada.

Cyclic AMP receptor protein (Crp) is a transcription regulator controlling
diverse cellular processes in many bacteria. In Streptomyces coelicolor, it is
well established that Crp plays a critical role in spore germination and colony
development. Here, we demonstrate that Crp is a key regulator of secondary
metabolism and antibiotic production in S. coelicolor and show that it may
additionally coordinate precursor flux from primary to secondary metabolism. We
found that crp deletion adversely affected the synthesis of three
well-characterized antibiotics in S. coelicolor: actinorhodin (Act),
undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA). Using chromatin
immunoprecipitation-microarray (ChIP-chip) assays, we determined that eight (out 
of 22) secondary metabolic clusters encoded by S. coelicolor contained
Crp-associated sites. We followed the effect of Crp induction using transcription
profiling analyses and found secondary metabolic genes to be significantly
affected: included in this Crp-dependent group were genes from six of the
clusters identified in the ChIP-chip experiments. Overexpressing Crp in a panel
of Streptomyces species led to enhanced antibiotic synthesis and new metabolite
production, suggesting that Crp control over secondary metabolism is broadly
conserved in the streptomycetes and that Crp overexpression could serve as a
powerful tool for unlocking the chemical potential of these organisms. IMPORTANCE
Streptomyces produces a remarkably diverse array of secondary metabolites,
including many antibiotics. In recent years, genome sequencing has revealed that 
these products represent only a small proportion of the total secondary
metabolite potential of Streptomyces. There is, therefore, considerable interest 
in discovering ways to stimulate the production of new metabolites. Here, we show
that Crp (the classical regulator of carbon catabolite repression in Escherichia 
coli) is a master regulator of secondary metabolism in Streptomyces. It binds to 
eight of 22 secondary metabolic gene clusters in the Streptomyces coelicolor
genome and directly affects the expression of six of these. Deletion of crp in S.
coelicolor leads to dramatic reductions in antibiotic levels, while Crp
overexpression enhances antibiotic production. We find that the
antibiotic-stimulatory capacity of Crp extends to other streptomycetes, where its
overexpression activates the production of "cryptic" metabolites that are not
otherwise seen in the corresponding wild-type strain.

DOI: 10.1128/mBio.00407-12 
PMCID: PMC3520106
PMID: 23232715  [Indexed for MEDLINE]


45. Mol Microbiol. 2013 Feb;87(3):526-38. doi: 10.1111/mmi.12111. Epub 2012 Dec 19.

ChIP-seq and transcriptome analysis of the OmpR regulon of Salmonella enterica
serovars Typhi and Typhimurium reveals accessory genes implicated in host
colonization.

Perkins TT(1), Davies MR, Klemm EJ, Rowley G, Wileman T, James K, Keane T,
Maskell D, Hinton JC, Dougan G, Kingsley RA.

Author information: 
(1)The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus,
Hinxton, Cambridge CB10 1SA, UK.

OmpR is a multifunctional DNA binding regulator with orthologues in many enteric 
bacteria that exhibits classical regulator activity as well as
nucleoid-associated protein-like characteristics. In the enteric pathogen
Salmonella enterica, using chromatin immunoprecipitation of OmpR:FLAG and
nucleotide sequencing, 43 putative OmpR binding sites were identified in
S. enterica serovar Typhi, 22 of which were associated with OmpR-regulated genes.
Mutation of a sequence motif (TGTWACAW) that was associated with the putative
OmpR binding sites abrogated binding of OmpR:6×His to the tviA upstream region. A
core set of 31 orthologous genes were found to exhibit OmpR-dependent expression 
in both S. Typhi and S. Typhimurium. S. Typhimurium-encoded orthologues of two
divergently transcribed OmpR-regulated operons (SL1068-71 and SL1066-67) had a
putative OmpR binding site in the inter-operon region in S. Typhi, and were
characterized using in vitro and in vivo assays. These operons are widely
distributed within S. enterica but absent from the closely related Escherichia
coli. SL1066 and SL1067 were required for growth on N-acetylmuramic acid as a
sole carbon source. SL1068-71 exhibited sequence similarity to sialic acid uptake
systems and contributed to colonization of the ileum and caecum in the
streptomycin-pretreated mouse model of colitis.

© 2012 Blackwell Publishing Ltd.

DOI: 10.1111/mmi.12111 
PMCID: PMC3586657
PMID: 23190111  [Indexed for MEDLINE]


46. Nucleic Acids Res. 2012 Dec;40(22):e175. doi: 10.1093/nar/gks771. Epub 2012 Aug
25.

Improved predictions of transcription factor binding sites using physicochemical 
features of DNA.

Maienschein-Cline M(1), Dinner AR, Hlavacek WS, Mu F.

Author information: 
(1)Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.

Typical approaches for predicting transcription factor binding sites (TFBSs)
involve use of a position-specific weight matrix (PWM) to statistically
characterize the sequences of the known sites. Recently, an alternative
physicochemical approach, called SiteSleuth, was proposed. In this approach, a
linear support vector machine (SVM) classifier is trained to distinguish TFBSs
from background sequences based on local chemical and structural features of DNA.
SiteSleuth appears to generally perform better than PWM-based methods. Here, we
improve the SiteSleuth approach by considering both new physicochemical features 
and algorithmic modifications. New features are derived from Gibbs energies of
amino acid-DNA interactions and hydroxyl radical cleavage profiles of DNA.
Algorithmic modifications consist of inclusion of a feature selection step, use
of a nonlinear kernel in the SVM classifier, and use of a consensus-based
post-processing step for predictions. We also considered SVM classification based
on letter features alone to distinguish performance gains from use of SVM-based
models versus use of physicochemical features. The accuracy of each of the
variant methods considered was assessed by cross validation using data available 
in the RegulonDB database for 54 Escherichia coli TFs, as well as by experimental
validation using published ChIP-chip data available for Fis and Lrp.

DOI: 10.1093/nar/gks771 
PMCID: PMC3526315
PMID: 22923524  [Indexed for MEDLINE]


47. Gene. 2012 Oct 25;508(2):221-8. doi: 10.1016/j.gene.2012.07.064. Epub 2012 Aug 4.

Under-representation of intrinsic terminators across bacterial genomic islands:
Rho as a principal regulator of xenogenic DNA expression.

Mitra A(1), Nagaraja V.

Author information: 
(1)Department of Microbiology and Cell Biology, Indian Institute of Science,
Bangalore, India.

Two transcription termination mechanisms - intrinsic and Rho-dependent - have
evolved in bacteria. The Rho factor occurs in most bacterial lineages, and has
been hypothesized to play a global regulatory role. Genome-wide studies using
microarray, 2D-gel electrophoresis and ChIP-chip provided evidence that Rho
serves to silence transcription from horizontally acquired genes and prophages in
Escherichia coli K-12, implicating the factor to be a part of the "cellular
immune mechanism" protecting against deleterious phages and aberrant gene
expression from acquired xenogenic DNA. We have investigated this model by
adopting an alternate in silico approach and have extended the study to other
species. Our analysis shows that several genomic islands across diverse phyla
have under-representation of intrinsic terminators, similar to that
experimentally observed in E. coli K-12. This implies that Rho-dependent
termination is the predominant process operational in these islands and that
silencing of foreign DNA is a conserved function of Rho. From the present
analysis, it is evident that horizontally acquired islands have lost intrinsic
terminators to facilitate Rho-dependent termination. These results underscore the
importance of Rho as a conserved, genome-wide sentinel that regulates potentially
toxic xenogenic DNA.

Copyright © 2012 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.gene.2012.07.064 
PMID: 22890136  [Indexed for MEDLINE]


48. Gut Microbes. 2012 Mar-Apr;3(2):93-103. doi: 10.4161/gmic.19578. Epub 2012 Mar 1.

Signature tagged mutagenesis in the functional genetic analysis of
gastrointestinal pathogens.

Cummins J(1), Gahan CG.

Author information: 
(1)Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

Signature tagged mutagenesis is a genetic approach that was developed to identify
novel bacterial virulence factors. It is a negative selection method in which
unique identification tags allow analysis of pools of mutants in mixed
populations. The approach is particularly well suited to functional genetic
analysis of the gastrointestinal phase of infection in foodborne pathogens and
has the capacity to guide the development of novel vaccines and therapeutics. In 
this review we outline the technical principles underpinning signature-tagged
mutagenesis as well as novel sequencing-based approaches for transposon mutant
identification such as TraDIS (transposon directed insertion-site sequencing). We
also provide an analysis of screens that have been performed in gastrointestinal 
pathogens which are a global health concern (Escherichia coli, Listeria
monocytogenes, Helicobacter pylori, Vibrio cholerae and Salmonella enterica). The
identification of key virulence loci through the use of signature tagged
mutagenesis in mice and relevant larger animal models is discussed.

DOI: 10.4161/gmic.19578 
PMCID: PMC3370953
PMID: 22555467  [Indexed for MEDLINE]


49. Mol Endocrinol. 2012 Feb;26(2):349-57. doi: 10.1210/me.2011-1080. Epub 2011 Dec
29.

Research resource: The estrogen receptor α cistrome defined by DamIP.

Xiao R(1), Sun D, Ayers S, Xi Y, Li W, Baxter JD, Moore DD.

Author information: 
(1)Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, Texas 77030, USA.

Gene expression is tightly regulated by transcription factors and cofactors that 
function by directly or indirectly interacting with DNA of the genome.
Understanding how and where these proteins bind provides essential information to
uncover genetic regulatory mechanisms. We have developed a new method to study
DNA-protein interaction in vivo called DNA adenine methyltransferase (Dam)IP,
which is based on fusing a protein of interest to a mutant form of Dam from
Escherichia coli. We showed previously that DamIP can efficiently identify in
vivo binding sites of Dam-tethered human estrogen receptor (hER)α. In current
study, we present the cistrome of hERα determined by DamIP and high throughput
sequencing (DamIP-seq). The DamIP-seq-defined hERα cistrome identifies many new
binding regions and overlaps with those determined by chromatin
immunoprecipitation (ChIP)-chip or ChIP-seq. Elements uniquely identified by
DamIP-seq include a unique class of elements that show low, but persistent, hERα 
binding when reexamined by conventional ChIP. In contrast, DamIP-seq fails to
detect some elements with very transient hERα binding. The methyl-adenine
modifications introduced by Dam are stable and do not decrease over 12 d. In
summary, the current study provides both an alternate view of the hERα cistrome
to further understand the mechanism of hERα-mediated transcription and a new tool
to explore other transcriptional factors and cofactors that is very different
from conventional ChIP.

DOI: 10.1210/me.2011-1080 
PMCID: PMC3384088
PMID: 22207717  [Indexed for MEDLINE]


50. J Bacteriol. 2011 Jun;193(12):3090-9. doi: 10.1128/JB.00086-11. Epub 2011 Apr 22.

Transcription factor GreA contributes to resolving promoter-proximal pausing of
RNA polymerase in Bacillus subtilis cells.

Kusuya Y(1), Kurokawa K, Ishikawa S, Ogasawara N, Oshima T.

Author information: 
(1)Graduate School of Information Science, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan. taku@bs.naist.jp

Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate
intrinsic cleavage of the nascent transcript at the active site of RNAP.
Biochemical and genetic studies to date have shown that Escherichia coli Gre
factors prevent transcriptional arrest during elongation and enhance
transcription fidelity. Furthermore, Gre factors participate in the stimulation
of promoter escape and the suppression of promoter-proximal pausing during the
beginning of RNA synthesis in E. coli. Although Gre factors are conserved in
general bacteria, limited functional studies have been performed in bacteria
other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity
precipitation coupled with DNA microarray) was conducted to visualize the
distribution of Bacillus subtilis GreA on the chromosome and to determine the
effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is
uniformly distributed in the transcribed region from the promoter to coding
region with core RNAP, and its inactivation induces RNAP accumulation at many
promoter or promoter-proximal regions. Based on these findings, we propose that
GreA would constantly associate with core RNAP during transcriptional initiation 
and elongation and resolves its stalling at promoter or promoter-proximal
regions, thus contributing to the even distribution of RNAP along the promoter
and coding regions in B. subtilis cells.

DOI: 10.1128/JB.00086-11 
PMCID: PMC3133182
PMID: 21515770  [Indexed for MEDLINE]


51. J Bacteriol. 2011 Apr;193(7):1771-6. doi: 10.1128/JB.01292-10. Epub 2011 Jan 28.

Retrospective application of transposon-directed insertion site sequencing to a
library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7
screened in cattle.

Eckert SE(1), Dziva F, Chaudhuri RR, Langridge GC, Turner DJ, Pickard DJ, Maskell
DJ, Thomson NR, Stevens MP.

Author information: 
(1)Roslin Institute and Royal (Dick) School of Veterinary Studies, University of 
Edinburgh, Bush Farm Road, Roslin, Midlothian EH25 9RG, United Kingdom.

Massively parallel sequencing of transposon-flanking regions assigned the
genotype and fitness score to 91% of Escherichia coli O157:H7 mutants previously 
screened in cattle by signature-tagged mutagenesis (STM). The method obviates the
limitations of STM and markedly extended the functional annotation of the
prototype E. coli O157:H7 genome without further animal use.

DOI: 10.1128/JB.01292-10 
PMCID: PMC3067669
PMID: 21278291  [Indexed for MEDLINE]


52. PLoS Comput Biol. 2010 Nov 18;6(11):e1001007. doi: 10.1371/journal.pcbi.1001007.

Using sequence-specific chemical and structural properties of DNA to predict
transcription factor binding sites.

Bauer AL(1), Hlavacek WS, Unkefer PJ, Mu F.

Author information: 
(1)Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos
National Laboratory, Los Alamos, New Mexico, United States of America.

An important step in understanding gene regulation is to identify the DNA binding
sites recognized by each transcription factor (TF). Conventional approaches to
prediction of TF binding sites involve the definition of consensus sequences or
position-specific weight matrices and rely on statistical analysis of DNA
sequences of known binding sites. Here, we present a method called SiteSleuth in 
which DNA structure prediction, computational chemistry, and machine learning are
applied to develop models for TF binding sites. In this approach, binary
classifiers are trained to discriminate between true and false binding sites
based on the sequence-specific chemical and structural features of DNA. These
features are determined via molecular dynamics calculations in which we consider 
each base in different local neighborhoods. For each of 54 TFs in Escherichia
coli, for which at least five DNA binding sites are documented in RegulonDB, the 
TF binding sites and portions of the non-coding genome sequence are mapped to
feature vectors and used in training. According to cross-validation analysis and 
a comparison of computational predictions against ChIP-chip data available for
the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX
SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms
QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. 
The main advantage of SiteSleuth is a lower false positive rate.

DOI: 10.1371/journal.pcbi.1001007 
PMCID: PMC2987836
PMID: 21124945  [Indexed for MEDLINE]


53. Nucleic Acids Res. 2011 Mar;39(5):1656-65. doi: 10.1093/nar/gkq848. Epub 2010 Nov
4.

Sensitive and accurate identification of protein-DNA binding events in ChIP-chip 
assays using higher order derivative analysis.

Barrett CL(1), Cho BK, Palsson BO.

Author information: 
(1)Department of Bioengineering, University of California, San Diego, La Jolla,
CA 92093, USA. cbarrett@ucsd.edu

Immuno-precipitation of protein-DNA complexes followed by microarray
hybridization is a powerful and cost-effective technology for discovering
protein-DNA binding events at the genome scale. It is still an unresolved
challenge to comprehensively, accurately and sensitively extract binding event
information from the produced data. We have developed a novel strategy composed
of an information-preserving signal-smoothing procedure, higher order derivative 
analysis and application of the principle of maximum entropy to address this
challenge. Importantly, our method does not require any input parameters to be
specified by the user. Using genome-scale binding data of two Escherichia coli
global transcription regulators for which a relatively large number of
experimentally supported sites are known, we show that ∼90% of known sites were
resolved to within four probes, or ∼88 bp. Over half of the sites were resolved
to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy
delivers significant quantitative and qualitative performance gains over
available methods. Such accurate and sensitive binding site resolution has
important consequences for accurately reconstructing transcriptional regulatory
networks, for motif discovery, for furthering our understanding of local and
non-local factors in protein-DNA interactions and for extending the usefulness
horizon of the ChIP-chip platform.

DOI: 10.1093/nar/gkq848 
PMCID: PMC3061075
PMID: 21051353  [Indexed for MEDLINE]


54. J Bacteriol. 2010 Nov;192(21):5778-87. doi: 10.1128/JB.00489-10. Epub 2010 Sep 3.

RNA polymerase trafficking in Bacillus subtilis cells.

Ishikawa S(1), Oshima T, Kurokawa K, Kusuya Y, Ogasawara N.

Author information: 
(1)Graduate School of Information Science, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the
Bacillus subtilis genome, we analyzed the distribution of the σ(A) and β'
subunits of RNAP and the NusA elongation factor on the genome in exponentially
growing cells using chromatin affinity precipitation coupled with gene chip
mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often
accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly
distributed from the promoter to the coding sequences. This finding suggests
that, in general, B. subtilis RNAP recruited to the promoter promptly
translocates away from the promoter to form the elongation complex and proceeds
without intragenic transcription attenuation. We detected RNAP accumulation in
the promoter-proximal regions of some genes, most of which can be identified as
transcription attenuation systems in the leader region. Our findings suggest that
the differences in RNAP behavior between E. coli and B. subtilis during
initiation and elongation steps might result in distinct strategies for
postinitiation control of transcription. The E. coli mechanism involves trapping 
at the promoter and promoter-proximal pausing of RNAP in addition to
transcription attenuation, whereas transcription attenuation in leader sequences 
is mainly employed in B. subtilis.

DOI: 10.1128/JB.00489-10 
PMCID: PMC2953687
PMID: 20817769  [Indexed for MEDLINE]


55. J Bacteriol. 2010 Sep;192(18):4720-31. doi: 10.1128/JB.00591-10. Epub 2010 Jul
16.

Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7
plasmid pCAR1, is a key global regulator that alters host function.

Yun CS(1), Suzuki C, Naito K, Takeda T, Takahashi Y, Sai F, Terabayashi T,
Miyakoshi M, Shintani M, Nishida H, Yamane H, Nojiri H.

Author information: 
(1)Biotechnology Research Center and Agricultural Bioinformatics Research Unit,
Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins
(NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is
transmissible among various Pseudomonas strains and carries a gene encoding the
H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which
harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765
(TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse 
transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not
affect the transcription of these five genes and that only pmr, turA, and turB
were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed
that Pmr strongly interacted with itself and with TurA, TurB, and TurE.
Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1)
strains indicated that pmr disruption had greater effects on the host
transcriptome than did pCAR1 carriage. The transcriptional levels of some genes
that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and 
parI, reverted with pmr disruption to levels in pCAR1-free KT2440.
Transcriptional levels of putative horizontally acquired host genes were not
altered by pCAR1 carriage but were altered by pmr disruption. Identification of
genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification
coupled with high-density tiling chip) analysis demonstrated that Pmr
preferentially binds to horizontally acquired DNA regions. The Pmr binding sites 
overlapped well with the location of the genes differentially transcribed
following pmr disruption on both the plasmid and the chromosome. Our findings
indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and
the host chromosome.

DOI: 10.1128/JB.00591-10 
PMCID: PMC2937398
PMID: 20639326  [Indexed for MEDLINE]


56. Nucleic Acids Res. 2010 Sep;38(17):5735-45. doi: 10.1093/nar/gkq363. Epub 2010
May 11.

Identification of {beta}-catenin binding regions in colon cancer cells using
ChIP-Seq.

Bottomly D(1), Kyler SL, McWeeney SK, Yochum GS.

Author information: 
(1)Oregon Clinical and Translational Research Institute, Oregon Health and
Science University, Portland, OR, USA.

Deregulation of the Wnt/β-catenin signaling pathway is a hallmark of colon
cancer. Mutations in the adenomatous polyposis coli (APC) gene occur in the vast 
majority of colorectal cancers and are an initiating event in cellular
transformation. Cells harboring mutant APC contain elevated levels of the
β-catenin transcription coactivator in the nucleus which leads to abnormal
expression of genes controlled by β-catenin/T-cell factor 4 (TCF4) complexes.
Here, we use chromatin immunoprecipitation coupled with massively parallel
sequencing (ChIP-Seq) to identify β-catenin binding regions in HCT116 human colon
cancer cells. We localized 2168 β-catenin enriched regions using a concordance
approach for integrating the output from multiple peak alignment algorithms.
Motif discovery algorithms found a core TCF4 motif (T/A-T/A-C-A-A-A-G), an
extended TCF4 motif (A/T/G-C/G-T/A-T/A-C-A-A-A-G) and an AP-1 motif
(T-G-A-C/T-T-C-A) to be significantly represented in β-catenin enriched regions. 
Furthermore, 417 regions contained both TCF4 and AP-1 motifs. Genes associated
with TCF4 and AP-1 motifs bound β-catenin, TCF4 and c-Jun in vivo and were
activated by Wnt signaling and serum growth factors. Our work provides evidence
that Wnt/β-catenin and mitogen signaling pathways intersect directly to regulate 
a defined set of target genes.

DOI: 10.1093/nar/gkq363 
PMCID: PMC2943592
PMID: 20460455  [Indexed for MEDLINE]


57. Mol Microbiol. 2009 Dec;74(5):1169-86. doi: 10.1111/j.1365-2958.2009.06929.x.
Epub 2009 Oct 19.

The H-NS-like protein StpA represses the RpoS (sigma 38) regulon during
exponential growth of Salmonella Typhimurium.

Lucchini S(1), McDermott P, Thompson A, Hinton JC.

Author information: 
(1)Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK.
sacha.lucchini@bbsrc.ac.uk

StpA is a paralogue of the nucleoid-associated protein H-NS that is conserved in 
a range of enteric bacteria and had no known function in Salmonella Typhimurium. 
We show that 5% of the Salmonella genome is regulated by StpA, which contrasts
with the situation in Escherichia coli where deletion of stpA only had minor
effects on gene expression. The StpA-dependent genes of S. Typhimurium are a
specific subset of the H-NS regulon that are predominantly under the positive
control of sigma(38) (RpoS), CRP-cAMP and PhoP. Regulation by StpA varied with
growth phase; StpA controlled sigma(38) levels at mid-exponential phase by
preventing inappropriate activation of sigma(38) during rapid bacterial growth.
In contrast, StpA only activated the CRP-cAMP regulon during late exponential
phase. ChIP-chip analysis revealed that StpA binds to PhoP-dependent genes but
not to most genes of the CRP-cAMP and sigma(38) regulons. In fact, StpA
indirectly regulates sigma(38)-dependent genes by enhancing sigma(38) turnover by
repressing the anti-adaptor protein rssC. We discovered that StpA is essential
for the dynamic regulation of sigma(38) in response to increased glucose levels. 
Our findings identify StpA as a novel growth phase-specific regulator that plays 
an important physiological role by linking sigma(38) levels to nutrient
availability.

DOI: 10.1111/j.1365-2958.2009.06929.x 
PMID: 19843227  [Indexed for MEDLINE]


58. Nucleic Acids Res. 2009 Jan;37(Database issue):D464-70. doi: 10.1093/nar/gkn751. 
Epub 2008 Oct 30.

EcoCyc: a comprehensive view of Escherichia coli biology.

Keseler IM(1), Bonavides-Martínez C, Collado-Vides J, Gama-Castro S, Gunsalus RP,
Johnson DA, Krummenacker M, Nolan LM, Paley S, Paulsen IT, Peralta-Gil M,
Santos-Zavaleta A, Shearer AG, Karp PD.

Author information: 
(1)SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025, USA.

EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia
coli biology. EcoCyc integrates information about the genome, genes and gene
products; the metabolic network; and the regulatory network of E. coli. Recent
EcoCyc developments include a new initiative to represent and curate all types of
E. coli regulatory processes such as attenuation and regulation by small RNAs.
EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a 
dataset of E. coli GO terms available through the GO Web site. The curation and
visualization of electron transfer processes has been significantly improved.
Other software and Web site enhancements include the addition of tracks to the
EcoCyc genome browser, in particular a type of track designed for the display of 
ChIP-chip datasets, and the development of a comparative genome browser. A new
Genome Omics Viewer enables users to paint omics datasets onto the full E. coli
genome for analysis. A new advanced query page guides users in interactively
constructing complex database queries against EcoCyc. A Macintosh version of
EcoCyc is now available. A series of Webinars is available to instruct users in
the use of EcoCyc.

DOI: 10.1093/nar/gkn751 
PMCID: PMC2686493
PMID: 18974181  [Indexed for MEDLINE]


59. Bioinformatics. 2008 Oct 15;24(20):2288-95. doi: 10.1093/bioinformatics/btn420.
Epub 2008 Aug 12.

MotifVoter: a novel ensemble method for fine-grained integration of generic motif
finders.

Wijaya E(1), Yiu SM, Son NT, Kanagasabai R, Sung WK.

Author information: 
(1)School of Computing, National University of Singapore, Singapore.

MOTIVATION: Locating transcription factor binding sites (motifs) is a key step in
understanding gene regulation. Based on Tompa's benchmark study, the performance 
of current de novo motif finders is far from satisfactory (with sensitivity
<or=0.222 and precision <or=0.307). The same study also shows that no motif
finder performs consistently well over all datasets. Hence, it is not clear which
finder one should use for a given dataset. To address this issue, a class of
algorithms called ensemble methods have been proposed. Though the existing
ensemble methods overall perform better than stand-alone motif finders, the
improvement gained is not substantial. Our study reveals that these methods do
not fully exploit the information obtained from the results of individual
finders, resulting in minor improvement in sensitivity and poor precision.
RESULTS: In this article, we identify several key observations on how to utilize 
the results from individual finders and design a novel ensemble method,
MotifVoter, to predict the motifs and binding sites. Evaluations on 186 datasets 
show that MotifVoter can locate more than 95% of the binding sites found by its
component motif finders. In terms of sensitivity and precision, MotifVoter
outperforms stand-alone motif finders and ensemble methods significantly on
Tompa's benchmark, Escherichia coli, and ChIP-Chip datasets. MotifVoter is
available online via a web server with several biologist-friendly features.

DOI: 10.1093/bioinformatics/btn420 
PMID: 18697768  [Indexed for MEDLINE]


60. BMC Bioinformatics. 2008 May 6;9:228. doi: 10.1186/1471-2105-9-228.

Inferring the role of transcription factors in regulatory networks.

Veber P(1), Guziolowski C, Le Borgne M, Radulescu O, Siegel A.

Author information: 
(1)Centre INRIA Rennes Bretagne Atlantique, IRISA, Rennes, France.
philippe.veber@googlemail.com

BACKGROUND: Expression profiles obtained from multiple perturbation experiments
are increasingly used to reconstruct transcriptional regulatory networks, from
well studied, simple organisms up to higher eukaryotes. Admittedly, a key
ingredient in developing a reconstruction method is its ability to integrate
heterogeneous sources of information, as well as to comply with practical
observability issues: measurements can be scarce or noisy. In this work, we show 
how to combine a network of genetic regulations with a set of expression
profiles, in order to infer the functional effect of the regulations, as inducer 
or repressor. Our approach is based on a consistency rule between a network and
the signs of variation given by expression arrays.
RESULTS: We evaluate our approach in several settings of increasing complexity.
First, we generate artificial expression data on a transcriptional network of E. 
coli extracted from the literature (1529 nodes and 3802 edges), and we estimate
that 30% of the regulations can be annotated with about 30 profiles. We
additionally prove that at most 40.8% of the network can be inferred using our
approach. Second, we use this network in order to validate the predictions
obtained with a compendium of real expression profiles. We describe a filtering
algorithm that generates particularly reliable predictions. Finally, we apply our
inference approach to S. cerevisiae transcriptional network (2419 nodes and 4344 
interactions), by combining ChIP-chip data and 15 expression profiles. We are
able to detect and isolate inconsistencies between the expression profiles and a 
significant portion of the model (15% of all the interactions). In addition, we
report predictions for 14.5% of all interactions.
CONCLUSION: Our approach does not require accurate expression levels nor times
series. Nevertheless, we show on both data, real and artificial, that a
relatively small number of perturbation experiments are enough to determine a
significant portion of regulatory effects. This is a key practical asset compared
to statistical methods for network reconstruction. We demonstrate that our
approach is able to provide accurate predictions, even when the network is
incomplete and the data is noisy.

DOI: 10.1186/1471-2105-9-228 
PMCID: PMC2422845
PMID: 18460200  [Indexed for MEDLINE]


61. J Bioinform Comput Biol. 2007 Apr;5(2B):549-60.

Intragenic promotor-like sites in the genome of Escherichia coli discovery and
functional implication.

Tutukina MN(1), Shavkunov KS, Masulis IS, Ozoline ON.

Author information: 
(1)Institute of Cell Biophysics Russian Academy of Sciences, Pushchino, Moscow
region, 142290, Russia. maria@icb.psn.ru

Mapping of putative promoters within the entire genome of Escherichia coli (E.
coli) by means of pattern-recognition software PlatProm revealed several thousand
of sites having high probability to perform promoter function. Along with the
expected promoters located upstream of coding sequences, PlatProm identified more
than a thousand potential promoters for antisense transcription and several
hundred very similar signals within coding sequences having the same direction
with the genes. Since recently developed ChIP-chip technology also testified the 
presence of intragenic RNA polymerase binding sites, such distribution of
putative promoters is likely to be a general biological phenomenon reflecting yet
undiscovered regulatory events. Here, we provide experimental evidences that two 
internal promoters are recognized by bacterial RNA polymerase. One of them is
located within the hns coding sequence and may initiate synthesis of RNA from the
antisense strand. Another one is found within the overlapping genes htgA/yaaW and
may control the production of a shortened mRNA or an RNA-product complementary to
mRNA of yaaW. Both RNA-products can form secondary structures with free energies 
of folding close to those of small regulatory RNAs (sRNAs) of the same length.
Folding propensity of known sRNAs was further compared with that of antisense
RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S.
typhimurium). Slightly lower stability observed for aRNAs assumes that their
structural compactness may be less significant for biological function.


PMID: 17636861  [Indexed for MEDLINE]