1. Methods Mol Biol. 2018;1737:77-88. doi: 10.1007/978-1-4939-7634-8_5.

Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification
Coupled to RNA Sequencing.

Carrier MC(1), Laliberté G(1), Massé E(2).

Author information: 
(1)Department of Biochemistry, RNA Group, Université de Sherbrooke, Sherbrooke,
QC, Canada.
(2)Department of Biochemistry, RNA Group, Université de Sherbrooke, Sherbrooke,
QC, Canada. eric.masse@usherbrooke.ca.

Small regulatory RNAs (sRNAs) are ubiquitous regulatory molecules expressed in
living cells. In prokaryotes, sRNAs usually bind to target mRNAs to either
promote their degradation or interfere with translation initiation. Because a
single sRNA can regulate a considerable number of target mRNAs, we seek to
identify those targets rapidly and reliably. Here, we present a robust method
based on the co-purification of target mRNAs bound to MS2-tagged sRNAs expressed 
in vivo. After purification of the tagged-sRNA, we use RNAseq to determine the
identity of all RNA interacting partners and their enrichment level. We describe 
how to analyze the RNAseq data through the Galaxy Project Platform bioinformatics
tools to identify new mRNA targets. This technique is applicable to most sRNAs of
E. coli and Salmonella.

DOI: 10.1007/978-1-4939-7634-8_5 
PMID: 29484588 


2. BMC Evol Biol. 2018 Feb 12;18(1):21. doi: 10.1186/s12862-018-1134-0.

A novel short L-arginine responsive protein-coding gene (laoB) antiparallel
overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 
Sakai originated by overprinting.

Hücker SM(1)(2), Vanderhaeghen S(1), Abellan-Schneyder I(1)(3), Wecko R(1), Simon
S(4), Scherer S(1)(5), Neuhaus K(6)(7).

Author information: 
(1)Chair for Microbial Ecology, Wissenschaftszentrum Weihenstephan, Technische
Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
(2)Fraunhofer ITEM-R, Am Biopark 9, 93053, Regensburg, Germany.
(3)Core Facility Microbiome/NGS, ZIEL - Institute for Food & Health, Technische
Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
(4)Department of Computer and Information Science, University of Konstanz, Box
78, 78457, Konstanz, Germany.
(5)ZIEL - Institute for Food & Health, Technische Universität München,
Weihenstephaner Berg 3, 85354, Freising, Germany.
(6)Chair for Microbial Ecology, Wissenschaftszentrum Weihenstephan, Technische
Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
neuhaus@tum.de.
(7)Core Facility Microbiome/NGS, ZIEL - Institute for Food & Health, Technische
Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
neuhaus@tum.de.

BACKGROUND: Due to the DNA triplet code, it is possible that the sequences of two
or more protein-coding genes overlap to a large degree. However, such non-trivial
overlaps are usually excluded by genome annotation pipelines and, thus, only a
few overlapping gene pairs have been described in bacteria. In contrast,
transcriptome and translatome sequencing reveals many signals originated from the
antisense strand of annotated genes, of which we analyzed an example gene pair in
more detail.
RESULTS: A small open reading frame of Escherichia coli O157:H7 strain Sakai
(EHEC), designated laoB (L-arginine responsive overlapping gene), is embedded in 
reading frame -2 in the antisense strand of ECs5115, encoding a CadC-like
transcriptional regulator. This overlapping gene shows evidence of transcription 
and translation in Luria-Bertani (LB) and brain-heart infusion (BHI) medium based
on RNA sequencing (RNAseq) and ribosomal-footprint sequencing (RIBOseq). The
transcriptional start site is 289 base pairs (bp) upstream of the start codon and
transcription termination is 155 bp downstream of the stop codon. Overexpression 
of LaoB fused to an enhanced green fluorescent protein (EGFP) reporter was
possible. The sequence upstream of the transcriptional start site displayed
strong promoter activity under different conditions, whereas promoter activity
was significantly decreased in the presence of L-arginine. A strand-specific
translationally arrested mutant of laoB provided a significant growth advantage
in competitive growth experiments in the presence of L-arginine compared to the
wild type, which returned to wild type level after complementation of laoB in
trans. A phylostratigraphic analysis indicated that the novel gene is restricted 
to the Escherichia/Shigella clade and might have originated recently by
overprinting leading to the expression of part of the antisense strand of
ECs5115.
CONCLUSIONS: Here, we present evidence of a novel small protein-coding gene laoB 
encoded in the antisense frame -2 of the annotated gene ECs5115. Clearly, laoB is
evolutionarily young and it originated in the Escherichia/Shigella clade by
overprinting, a process which may cause the de novo evolution of bacterial genes 
like laoB.

DOI: 10.1186/s12862-018-1134-0 
PMCID: PMC5810103
PMID: 29433444 


3. PLoS One. 2017 Sep 13;12(9):e0184119. doi: 10.1371/journal.pone.0184119.
eCollection 2017.

Discovery of numerous novel small genes in the intergenic regions of the
Escherichia coli O157:H7 Sakai genome.

Hücker SM(1)(2), Ardern Z(1)(2), Goldberg T(3), Schafferhans A(3), Bernhofer
M(3), Vestergaard G(4), Nelson CW(5), Schloter M(4), Rost B(3), Scherer S(1)(2), 
Neuhaus K(1)(6).

Author information: 
(1)Chair for Microbial Ecology, Technische Universität München, Freising,
Germany.
(2)ZIEL - Institute for Food & Health, Technische Universität München, Freising, 
Germany.
(3)Department of Informatics-Bioinformatics & TUM-IAS, Technische Universität
München, Garching, Germany.
(4)Research Unit Environmental Genomics, Helmholtz Zentrum München, Neuherberg,
Germany.
(5)Sackler Institute for Comparative Genomics, American Museum of Natural History
New York, New York, United States of America.
(6)Core Facility Microbiome/NGS, ZIEL - Institute for Food & Health, Technische
Universität München, Freising, Germany.

In the past, short protein-coding genes were often disregarded by genome
annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of
annotated genes have usually been interpreted to indicate either ncRNA or
pervasive transcription. Therefore, in addition to the transcriptome, the
translatome (RIBOseq) of the enteric pathogen Escherichia coli O157:H7 strain
Sakai was determined at two optimal growth conditions and a severe stress
condition combining low temperature and high osmotic pressure. All intergenic
open reading frames potentially encoding a protein of ≥ 30 amino acids were
investigated with regard to coverage by transcription and translation signals and
their translatability expressed by the ribosomal coverage value. This led to
discovery of 465 unique, putative novel genes not yet annotated in this E. coli
strain, which are evenly distributed over both DNA strands of the genome. For 255
of the novel genes, annotated homologs in other bacteria were found, and a
machine-learning algorithm, trained on small protein-coding E. coli genes,
predicted that 89% of these translated open reading frames represent bona fide
genes. The remaining 210 putative novel genes without annotated homologs were
compared to the 255 novel genes with homologs and to 250 short annotated genes of
this E. coli strain. All three groups turned out to be similar with respect to
their translatability distribution, fractions of differentially regulated genes, 
secondary structure composition, and the distribution of evolutionary constraint,
suggesting that both novel groups represent legitimate genes. However, the
machine-learning algorithm only recognized a small fraction of the 210 genes
without annotated homologs. It is possible that these genes represent a novel
group of genes, which have unusual features dissimilar to the genes of the
machine-learning algorithm training set.

DOI: 10.1371/journal.pone.0184119 
PMCID: PMC5597208
PMID: 28902868  [Indexed for MEDLINE]


4. 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 


5. 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]


6. 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]


7. BMC Genomics. 2017 Feb 28;18(1):216. doi: 10.1186/s12864-017-3586-9.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933
(EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and 
a peptide, RyhP.

Neuhaus K(1)(2), Landstorfer R(3), Simon S(4), Schober S(5), Wright PR(6), Smith 
C(6), Backofen R(6), Wecko R(3), Keim DA(4), Scherer S(3).

Author information: 
(1)Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Weihenstephaner Berg 3, D-85354, Freising,
Germany. neuhaus@tum.de.
(2)Core Facility Microbiome/NGS, ZIEL Institute for Food & Health,
Weihenstephaner Berg 3, D-85354, Freising, Germany. neuhaus@tum.de.
(3)Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Weihenstephaner Berg 3, D-85354, Freising,
Germany.
(4)Informatik und Informationswissenschaft, Universität Konstanz, D-78457,
Konstanz, Germany.
(5)Institut für Nachrichtentechnik, Universität Ulm, Albert-Einstein-Allee 43,
D-89081, Ulm, Germany.
(6)Bioinformatics Group, Department of Computer Science and BIOSS Centre for
Biological Signaling Studies, Cluster of Excellence, University of Freiburg,
D-79110, Freiburg, Germany.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains
difficult to distinguish ncRNAs from short mRNAs. To detect potentially
translated RNAs, we developed an improved protocol for bacterial ribosomal
footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A
high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV)
is expected to indicate a translated RNA, while a low RCV should point to a
non-translated RNA.
RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were
identified as putative ncRNAs, representing both antisense and intergenic
transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics
analysis predicted statistically significant target regulons for 15 of the
intergenic transcripts; experimental analysis revealed 4-fold or higher
differential expression of 46 novel ncRNA in different growth media. Out of 329
annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of
those, 16 had RIBOseq patterns matching annotated genes in other
enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting 
that such ncRNAs may encode small proteins instead of being solely non-coding. To
support that the RIBOseq signals are reflecting translation, we tested the
ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded
peptide in iron-limiting condition.
CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step 
differentiation between bacterial ncRNAs and small mRNAs. Further, many known
ncRNAs may encode proteins as well.

DOI: 10.1186/s12864-017-3586-9 
PMCID: PMC5331693
PMID: 28245801  [Indexed for MEDLINE]


8. ACS Appl Mater Interfaces. 2017 Mar 22;9(11):10047-10060. doi:
10.1021/acsami.7b02380. Epub 2017 Mar 10.

Transcriptome Analysis Reveals Silver Nanoparticle-Decorated Quercetin
Antibacterial Molecular Mechanism.

Sun D(1), Zhang W(1), Mou Z(1), Chen Y(1), Guo F(1), Yang E(1), Wang W(1).

Author information: 
(1)School of Life Sciences, Anhui Agricultural University , Hefei 230036, China.

Facile and simple method is developed to synthesize silver-nanoparticle-decorated
quercetin nanoparticles (QA NPs). Modification suggests that synergistic
quercetin (Qe) improves the antibacterial effect of silver nanoparticles (Ag
NPs). Characterization experiment indicates that QA NPs have a diameter of
approximately 10 nm. QA NPs show highly effective antibacterial activities
against drug-resistant Escherichia coli (E. coli) and Staphylococcus aureus (S.
aureus). We explore antibacterial mechanisms using S. aureus and E. coli treated 
with QA NPs. Through morphological changes in E. coli and S. aureus, mechanisms
are examined for bacterial damage caused by particulate matter from local
dissociation of silver ion and Qe from QA NPs trapped inside membranes. Moreover,
we note that gene expression profiling methods, such as RNA sequencing, can be
used to predict discover mechanisms of toxicity of QA NPs. Gene ontology (GO)
assay analyses demonstrate the molecular mechanism of the antibacterial effect of
QA NPs. Regarding cellular component ontology, "cell wall organization or
biogenesis" (GO: 0071554) and "cell wall macromolecule metabolic process" (GO:
0044036) are the most represented categories. The present study reports that
transcriptome analysis of the mechanism offers novel insights into the molecular 
mechanism of antibacterial assays.

DOI: 10.1021/acsami.7b02380 
PMID: 28240544 


9. Biotechnol Biofuels. 2017 Feb 3;10:32. doi: 10.1186/s13068-017-0720-5.
eCollection 2017.

Development of a genetically programed vanillin-sensing bacterium for
high-throughput screening of lignin-degrading enzyme libraries.

Sana B(1), Chia KHB(2), Raghavan SS(1), Ramalingam B(3), Nagarajan N(2), Seayad
J(3), Ghadessy FJ(1).

Author information: 
(1)p53 Laboratory, Agency for Science Technology And Research (ASTAR), 8A
Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore, 138648 Singapore.
(2)Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore, 
138672 Singapore.
(3)Institute of Chemical and Engineering Sciences, 8 Biomedical Grove, Neuros,
#07-01, Singapore, 138665 Singapore.

BACKGROUND: Lignin is a potential biorefinery feedstock for the production of
value-added chemicals including vanillin. A huge amount of lignin is produced as 
a by-product of the paper industry, while cellulosic components of plant biomass 
are utilized for the production of paper pulp. In spite of vast potential, lignin
remains the least exploited component of plant biomass due to its extremely
complex and heterogenous structure. Several enzymes have been reported to have
lignin-degrading properties and could be potentially used in lignin biorefining
if their catalytic properties could be improved by enzyme engineering. The much
needed improvement of lignin-degrading enzymes by high-throughput selection
techniques such as directed evolution is currently limited, as robust methods for
detecting the conversion of lignin to desired small molecules are not available.
RESULTS: We identified a vanillin-inducible promoter by RNAseq analysis of
Escherichia coli cells treated with a sublethal dose of vanillin and developed a 
genetically programmed vanillin-sensing cell by placing the 'very green
fluorescent protein' gene under the control of this promoter. Fluorescence of the
biosensing cell is enhanced significantly when grown in the presence of vanillin 
and is readily visualized by fluorescence microscopy. The use of
fluorescence-activated cell sorting analysis further enhances the sensitivity,
enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is 
highly specific to vanillin and no major response is elicited by the presence of 
lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic
chemicals.
CONCLUSIONS: We developed an engineered E. coli cell that can detect vanillin at 
a concentration as low as 200 µM. The vanillin-sensing cell did not show
cross-reactivity towards lignin or major lignin degradation products including
vanillin analogues. This engineered E. coli cell could potentially be used as a
host cell for screening lignin-degrading enzymes that can convert lignin to
vanillin.

DOI: 10.1186/s13068-017-0720-5 
PMCID: PMC5291986
PMID: 28174601 


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. Methods. 2017 Mar 15;117:28-34. doi: 10.1016/j.ymeth.2016.11.011. Epub 2016 Nov
19.

Identification of unknown RNA partners using MAPS.

Lalaouna D(1), Prévost K(1), Eyraud A(1), Massé E(2).

Author information: 
(1)Department of Biochemistry, RNA Group, Université de Sherbrooke, Sherbrooke,
Québec J1E 4K8, Canada.
(2)Department of Biochemistry, RNA Group, Université de Sherbrooke, Sherbrooke,
Québec J1E 4K8, Canada. Electronic address: eric.masse@usherbrooke.ca.

Recent advances in high-throughput sequencing have led to an explosion in the
rate of small regulatory RNAs (sRNAs) discovery among bacteria. However, only a
handful of them are functionally characterized. Most of the time, little to no
targets are known. In Lalaouna et al. (2015), we proposed a new technology to
uncover sRNAs targetome, which is based on the MS2-affinity purification (MAPS). 
We were able to prove its efficiency by applying it on well-characterized sRNAs
of Escherichia coli. Thereafter, we adapted the procedure to other kind of RNA
(mRNAs and tRNA-derived RNA fragments) and bacteria (pathogenic or Gram-positive 
strains). Here, we clearly report all improvements and adjustments made to MAPS
technology since it was originally reported.

Copyright © 2016 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ymeth.2016.11.011 
PMID: 27876680  [Indexed for MEDLINE]


12. FEMS Microbiol Lett. 2017 Jan;364(2). pii: fnw262. doi: 10.1093/femsle/fnw262.
Epub 2016 Nov 16.

Transcriptional and translational regulation by RNA thermometers, riboswitches
and the sRNA DsrA in Escherichia coli O157:H7 Sakai under combined cold and
osmotic stress adaptation.

Hücker SM(1), Simon S(2), Scherer S(1), Neuhaus K(3).

Author information: 
(1)Chair for Microbial Ecology, Technische Universität München, Weihenstephaner
Berg 3, 85354 Freising, Germany.
(2)Chair for Data Analysis and Visualization, Department of Computer and
Information Science, University of Konstanz, Box 78, 78457 Konstanz, Germany.
(3)Chair for Microbial Ecology, Technische Universität München, Weihenstephaner
Berg 3, 85354 Freising, Germany neuhaus@wzw.tum.de.

The enteric pathogen Escherichia coli O157:H7 Sakai (EHEC) is able to grow at
lower temperatures compared to commensal E. coli Growth at environmental
conditions displays complex challenges different to those in a host. EHEC was
grown at 37°C and at 14°C with 4% NaCl, a combination of cold and osmotic stress 
as present in the food chain. Comparison of RNAseq and RIBOseq data provided a
snap shot of ongoing transcription and translation, differentiating
transcriptional and post-transcriptional gene regulation, respectively. Indeed,
cold and osmotic stress related genes are simultaneously regulated at both
levels, but translational regulation clearly dominates. Special emphasis was
given to genes regulated by RNA secondary structures in their 5'UTRs, such as RNA
thermometers and riboswitches, or genes controlled by small RNAs encoded in trans
The results reveal large differences in gene expression between short-time shock 
compared to adaptation in combined cold and osmotic stress. Whereas the majority 
of cold shock proteins, such as CspA, are translationally downregulated after
adaptation, many osmotic stress genes are still significantly upregulated mainly 
translationally, but several also transcriptionally.

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

DOI: 10.1093/femsle/fnw262 
PMID: 27856567  [Indexed for MEDLINE]


13. 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]


14. 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 


15. 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]


16. BMC Genomics. 2016 Feb 24;17:133. doi: 10.1186/s12864-016-2456-1.

Translatomics combined with transcriptomics and proteomics reveals novel
functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).

Neuhaus K(1), Landstorfer R(2), Fellner L(3), Simon S(4), Schafferhans A(5),
Goldberg T(6), Marx H(7), Ozoline ON(8), Rost B(9), Kuster B(10)(11), Keim
DA(12), Scherer S(13).

Author information: 
(1)Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und
Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität
München, Weihenstephaner Berg 3, 85354, Freising, Germany. neuhaus@wzw.tum.de.
(2)Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und
Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität
München, Weihenstephaner Berg 3, 85354, Freising, Germany. r.landstorfer@gmx.de.
(3)Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und
Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität
München, Weihenstephaner Berg 3, 85354, Freising, Germany.
fellnerlea@hotmail.com.
(4)Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und
Informationswissenschaft, Universität Konstanz, Box 78, 78457, Konstanz, Germany.
simon@dbvis.inf.uni-konstanz.de.
(5)Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität
München, Boltzmannstraße 3, 85748, Garching, Germany.
andrea.schafferhans@rostlab.org.
(6)Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität
München, Boltzmannstraße 3, 85748, Garching, Germany. goldberg@rostlab.org.
(7)Chair of Proteomics and Bioanalytics, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising,
Germany. h4r4ld.marx@googlemail.com.
(8)Institute of Cell Biophysics, Russian Academy of Sciences, Moscow Region,
142290, Pushchino, Russia. ozoline@rambler.ru.
(9)Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität
München, Boltzmannstraße 3, 85748, Garching, Germany. rost@rostlab.org.
(10)Chair of Proteomics and Bioanalytics, Wissenschaftszentrum Weihenstephan,
Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising,
Germany. kuster@wzw.tum.de.
(11)Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technische
Universität München, Gregor-Mendel-Str. 4, 85354, Freising, Germany.
kuster@wzw.tum.de.
(12)Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und
Informationswissenschaft, Universität Konstanz, Box 78, 78457, Konstanz, Germany.
keim@informatik.uni-konstanz.de.
(13)Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und
Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität
München, Weihenstephaner Berg 3, 85354, Freising, Germany.
siegfried.scherer@wzw.tum.de.

BACKGROUND: Genomes of E. coli, including that of the human pathogen Escherichia 
coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which,
apparently, have escaped annotation due to their small size and non-essential
function. To find such genes, global gene expression of EHEC EDL933 was examined,
using strand-specific RNAseq (transcriptome), ribosomal footprinting
(translatome) and mass spectrometry (proteome).
RESULTS: Using the above methods, 72 short, non-annotated protein-coding genes
were detected. All of these showed signals in the ribosomal footprinting assay
indicating mRNA translation. Seven were verified by mass spectrometry.
Fifty-seven genes are annotated in other enterobacteriaceae, mainly as
hypothetical genes; the remaining 15 genes constitute novel discoveries. In
addition, protein structure and function were predicted computationally and
compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. 
Based on this comparison, 61 of the 72 novel proteins exhibit predicted
structural and functional features similar to those of annotated proteins. Many
of the novel genes show differential transcription when grown under eleven
diverse growth conditions suggesting environmental regulation. Three genes were
found to confer a phenotype in previous studies, e.g., decreased cattle
colonization.
CONCLUSIONS: These findings demonstrate that ribosomal footprinting can be used
to detect novel protein coding genes, contributing to the growing body of
evidence that hypothetical genes are not annotation artifacts and opening an
additional way to study their functionality. All 72 genes are taxonomically
restricted and, therefore, appear to have evolved relatively recently de novo.

DOI: 10.1186/s12864-016-2456-1 
PMCID: PMC4765031
PMID: 26911138  [Indexed for MEDLINE]


17. Sci Rep. 2016 Jan 28;6:19899. doi: 10.1038/srep19899.

Global transcriptomic responses of Escherichia coli K-12 to volatile organic
compounds.

Yung PY(1), Grasso LL(1), Mohidin AF(1), Acerbi E(1), Hinks J(1), Seviour T(1),
Marsili E(1)(2)(3), Lauro FM(1)(4).

Author information: 
(1)Singapore Centre for Environmental Life Sciences Engineering (SCELSE). 60
Nanyang Drive, SBS-01N-27, Singapore 637551.
(2)School of Chemical and Biomedical Engineering, Nanyang Technological
University, 62 Nanyang Drive, Singapore 637459.
(3)School of Biotechnology, Dublin City University, Collins Avenue, Dublin 9,
Ireland.
(4)Asian School of the Environment, Nanyang Technological University, 50 Nanyang 
Avenue, N2-01C-45, Singapore 639798.

Volatile organic compounds (VOCs) are commonly used as solvents in various
industrial settings. Many of them present a challenge to receiving environments, 
due to their toxicity and low bioavailability for degradation. Microorganisms are
capable of sensing and responding to their surroundings and this makes them ideal
detectors for toxic compounds. This study investigates the global transcriptomic 
responses of Escherichia coli K-12 to selected VOCs at sub-toxic levels. Cells
grown in the presence of VOCs were harvested during exponential growth, followed 
by whole transcriptome shotgun sequencing (RNAseq). The analysis of the data
revealed both shared and unique genetic responses compared to cells without
exposure to VOCs. Results suggest that various functional gene categories, for
example, those relating to Fe/S cluster biogenesis, oxidative stress responses
and transport proteins, are responsive to selected VOCs in E. coli. The
differential expression (DE) of genes was validated using GFP-promoter fusion
assays. A variety of genes were differentially expressed even at non-inhibitory
concentrations and when the cells are at their balanced-growth. Some of these
genes belong to generic stress response and others could be specific to VOCs.
Such candidate genes and their regulatory elements could be used as the basis for
designing biosensors for selected VOCs.

DOI: 10.1038/srep19899 
PMCID: PMC4730218
PMID: 26818886  [Indexed for MEDLINE]


18. MBio. 2015 Aug 25;6(4). pii: e00998-15. doi: 10.1128/mBio.00998-15.

A New Noncoding RNA Arranges Bacterial Chromosome Organization.

Qian Z(1), Macvanin M(1), Dimitriadis EK(2), He X(3), Zhurkin V(4), Adhya S(5).

Author information: 
(1)Laboratory of Molecular Biology, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland, USA.
(2)Biomedical Engineering and Physical Science, National Institute of Biomedical 
Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland,
USA.
(3)Laboratory of Metabolism, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland, USA.
(4)Laboratory of Cell Biology, National Cancer Institute, National Institutes of 
Health, Bethesda, Maryland, USA.
(5)Laboratory of Molecular Biology, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland, USA sadhya@helix.nih.gov.

Repeated extragenic palindromes (REPs) in the enterobacterial genomes are usually
composed of individual palindromic units separated by linker sequences. A total
of 355 annotated REPs are distributed along the Escherichia coli genome. RNA
sequence (RNAseq) analysis showed that almost 80% of the REPs in E. coli are
transcribed. The DNA sequence of REP325 showed that it is a cluster of six
repeats, each with two palindromic units capable of forming cruciform structures 
in supercoiled DNA. Here, we report that components of the REP325 element and at 
least one of its RNA products play a role in bacterial nucleoid DNA condensation.
These RNA not only are present in the purified nucleoid but bind to the bacterial
nucleoid-associated HU protein as revealed by RNA IP followed by microarray
analysis (RIP-Chip) assays. Deletion of REP325 resulted in a dramatic increase of
the nucleoid size as observed using transmission electron microscopy (TEM), and
expression of one of the REP325 RNAs, nucleoid-associated noncoding RNA 4
(naRNA4), from a plasmid restored the wild-type condensed structure.
Independently, chromosome conformation capture (3C) analysis demonstrated
physical connections among various REP elements around the chromosome. These
connections are dependent in some way upon the presence of HU and the REP325
element; deletion of HU genes and/or the REP325 element removed the connections. 
Finally, naRNA4 together with HU condensed DNA in vitro by connecting REP325 or
other DNA sequences that contain cruciform structures in a pairwise manner as
observed by atomic force microscopy (AFM). On the basis of our results, we
propose molecular models to explain connections of remote cruciform structures
mediated by HU and naRNA4.IMPORTANCE: Nucleoid organization in bacteria is being 
studied extensively, and several models have been proposed. However, the
molecular nature of the structural organization is not well understood. Here we
characterized the role of a novel nucleoid-associated noncoding RNA, naRNA4, in
nucleoid structures both in vivo and in vitro. We propose models to explain how
naRNA4 together with nucleoid-associated protein HU connects remote DNA elements 
for nucleoid condensation. We present the first evidence of a noncoding RNA
together with a nucleoid-associated protein directly condensing nucleoid DNA.

Copyright © 2015 Qian et al.

DOI: 10.1128/mBio.00998-15 
PMCID: PMC4550694
PMID: 26307168  [Indexed for MEDLINE]


19. PLoS One. 2015 Jun 30;10(6):e0130902. doi: 10.1371/journal.pone.0130902.
eCollection 2015.

Identification of Candidate Adherent-Invasive E. coli Signature Transcripts by
Genomic/Transcriptomic Analysis.

Zhang Y(1), Rowehl L(2), Krumsiek JM(3), Orner EP(2), Shaikh N(4), Tarr PI(5),
Sodergren E(6), Weinstock GM(6), Boedeker EC(7), Xiong X(8), Parkinson J(9),
Frank DN(10), Li E(2), Gathungu G(3).

Author information: 
(1)Department of Applied Mathematics and Statistics, Stony Brook University,
Stony Brook, New York, United States of America.
(2)Department of Medicine, Stony Brook University, Stony Brook, New York, United 
States of America.
(3)Department of Pediatrics, Stony Brook University, Stony Brook, New York,
United States of America.
(4)Department of Pediatrics, Washington University St. Louis, St. Louis,
Missouri, United States of America.
(5)Department of Pediatrics, Washington University St. Louis, St. Louis,
Missouri, United States of America; Department of Molecular Microbiology,
Washington University St. Louis, St. Louis, Missouri, United States of America.
(6)The Genome Institute, Washington University St. Louis, St. Louis, Missouri,
United States of America.
(7)Department of Medicine, University of New Mexico, Albuquerque, New Mexico,
United States of America.
(8)Program in Molecular Structure and Function, The Hospital for Sick Children,
Toronto, Canada.
(9)Department of Biochemistry & Molecular and Medical Genetics, University of
Toronto, Toronto, Canada.
(10)Department of Medicine, University of Colorado, Denver, Colorado, United
States of America.

Erratum in
    PLoS One. 2015;10(7):e0134759.

Adherent-invasive Escherichia coli (AIEC) strains are detected more frequently
within mucosal lesions of patients with Crohn's disease (CD). The AIEC phenotype 
consists of adherence and invasion of intestinal epithelial cells and survival
within macrophages of these bacteria in vitro. Our aim was to identify candidate 
transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and
might be useful for rapid and accurate identification of AIEC by
culture-independent technology. We performed comparative RNA-Sequence (RNASeq)
analysis using AIEC strain LF82 and NIEC strain HS during exponential and
stationary growth. Differential expression analysis of coding sequences (CDS)
homologous to both strains demonstrated 224 and 241 genes with increased and
decreased expression, respectively, in LF82 relative to HS. Transition metal
transport and siderophore metabolism related pathway genes were up-regulated,
while glycogen metabolic and oxidation-reduction related pathway genes were
down-regulated, in LF82. Chemotaxis related transcripts were up-regulated in LF82
during the exponential phase, but flagellum-dependent motility pathway genes were
down-regulated in LF82 during the stationary phase. CDS that mapped only to the
LF82 genome accounted for 747 genes. We applied an in silico subtractive genomics
approach to identify CDS specific to AIEC by incorporating the genomes of 10
other previously phenotyped NIEC. From this analysis, 166 CDS mapped to the LF82 
genome and lacked homology to any of the 11 human NIEC strains. We compared these
CDS across 13 AIEC, but none were homologous in each. Four LF82 gene loci
belonging to clustered regularly interspaced short palindromic repeats region
(CRISPR)--CRISPR-associated (Cas) genes were identified in 4 to 6 AIEC and absent
from all non-pathogenic bacteria. As previously reported, AIEC strains were
enriched for pdu operon genes. One CDS, encoding an excisionase, was shared by 9 
AIEC strains. Reverse transcription quantitative polymerase chain reaction assays
for 6 genes were conducted on fecal and ileal RNA samples from 22 inflammatory
bowel disease (IBD), and 32 patients without IBD (non-IBD). The expression of Cas
loci was detected in a higher proportion of CD than non-IBD fecal and ileal RNA
samples (p <0.05). These results support a comparative genomic/transcriptomic
approach towards identifying candidate AIEC signature transcripts.

DOI: 10.1371/journal.pone.0130902 
PMCID: PMC4509574
PMID: 26125937  [Indexed for MEDLINE]


20. Front Microbiol. 2014 Aug 13;5:402. doi: 10.3389/fmicb.2014.00402. eCollection
2014.

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder
bacterial ethanologenesis by activating regulatory circuits controlling inhibitor
efflux and detoxification.

Keating DH(1), Zhang Y(1), Ong IM(1), McIlwain S(1), Morales EH(2), Grass JA(3), 
Tremaine M(1), Bothfeld W(1), Higbee A(1), Ulbrich A(4), Balloon AJ(4), Westphall
MS(5), Aldrich J(6), Lipton MS(6), Kim J(7), Moskvin OV(1), Bukhman YV(1), Coon
JJ(8), Kiley PJ(2), Bates DM(1), Landick R(9).

Author information: 
(1)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA.
(2)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA ; Department of Biomolecular Chemistry, University of
Wisconsin-Madison Madison, WI, USA.
(3)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA ; Department of Biochemistry, University of Wisconsin-Madison
Madison, WI, USA.
(4)Department of Chemistry, University of Wisconsin-Madison Madison, WI, USA.
(5)Department of Biomolecular Chemistry, University of Wisconsin-Madison Madison,
WI, USA ; Department of Chemistry, University of Wisconsin-Madison Madison, WI,
USA.
(6)Pacific Northwest National Laboratory Richland, WA, USA.
(7)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA ; Department of Chemical and Biological Engineering, University 
of Wisconsin-Madison Madison, WI, USA.
(8)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA ; Department of Biomolecular Chemistry, University of
Wisconsin-Madison Madison, WI, USA ; Department of Chemistry, University of
Wisconsin-Madison Madison, WI, USA.
(9)Great Lakes Bioenergy Research Center, University of Wisconsin-Madison
Madison, WI, USA ; Department of Biochemistry, University of Wisconsin-Madison
Madison, WI, USA ; Department of Bacteriology, University of Wisconsin-Madison
Madison, WI, USA.

Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a
key barrier to the economically viable deployment of lignocellulosic biofuels. A 
chief contributor to this barrier is the impact on microbial processes and energy
metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates,
phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and
furfurals. To understand the bacterial pathways induced by inhibitors present in 
ammonia-pretreated biomass hydrolysates, which are less well studied than
acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics
of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory
responses to the inhibitors normally present in ACSH, we measured transcript and 
protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative 
proteomics during fermentation to ethanol of synthetic hydrolysates containing or
lacking the inhibitors. Our study identified four major regulators mediating
these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of
these regulons was correlated with a reduced rate of ethanol production, buildup 
of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose
conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to
be reduced to its alcohol form by the ethanologen during fermentation, whereas
phenolic acid and amide inhibitors were not metabolized. Together, our findings
establish that the major regulatory responses to lignocellulose-derived
inhibitors are mediated by transcriptional rather than translational regulators, 
suggest that energy consumed for inhibitor efflux and detoxification may limit
biofuel production, and identify a network of regulators for future synthetic
biology efforts.

DOI: 10.3389/fmicb.2014.00402 
PMCID: PMC4132294
PMID: 25177315 


21. 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]


22. Proc Natl Acad Sci U S A. 2014 Jun 24;111(25):E2576-85. doi:
10.1073/pnas.1401853111. Epub 2014 Jun 9.

Correcting direct effects of ethanol on translation and transcription machinery
confers ethanol tolerance in bacteria.

Haft RJ(1), Keating DH(1), Schwaegler T(1), Schwalbach MS(1), Vinokur J(1),
Tremaine M(1), Peters JM(2), Kotlajich MV(3), Pohlmann EL(1), Ong IM(1), Grass
JA(1), Kiley PJ(4), Landick R(5).

Author information: 
(1)Great Lakes Bioenergy Research Center and.
(2)Departments of Biochemistry,Genetics.
(3)Departments of Biochemistry.
(4)Great Lakes Bioenergy Research Center andBiomolecular Chemistry, and.
(5)Great Lakes Bioenergy Research Center andDepartments of
Biochemistry,Bacteriology, University of Wisconsin-Madison, Madison, WI 53706
landick@biochem.wisc.edu.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although 
important for biotechnology and bioenergy applications, remain incompletely
understood. Genetic studies have identified potential cellular targets for
ethanol and have revealed multiple mechanisms of tolerance, but it remains
difficult to separate the direct and indirect effects of ethanol. We used
adaptive evolution to generate spontaneous ethanol-tolerant strains of
Escherichia coli, and then characterized mechanisms of toxicity and resistance
using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific
assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and
rpsQ recapitulated most of the observed ethanol tolerance, implicating
translation and transcription as key processes affected by ethanol. Ethanol
induced miscoding errors during protein synthesis, from which the evolved rpsQ
allele protected cells by increasing ribosome accuracy. Ribosome profiling and
RNAseq analyses established that ethanol negatively affects transcriptional and
translational processivity. Ethanol-stressed cells exhibited ribosomal stalling
at internal AUG codons, which may be ameliorated by the adaptive inactivation of 
the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant
intragenic transcription termination for mRNAs with low ribosome density, which
was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol
inhibited transcript elongation by RNA polymerase in vitro. We propose that
ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through
direct effects on ribosomes and RNA polymerase conformations are major
contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ,
rho, and rpsQ help protect these central dogma processes in the presence of
ethanol.

DOI: 10.1073/pnas.1401853111 
PMCID: PMC4078849
PMID: 24927582  [Indexed for MEDLINE]


23. Nucleic Acids Res. 2013 Jan;41(Database issue):D1-7. doi: 10.1093/nar/gks1297.
Epub 2012 Nov 30.

The 2013 Nucleic Acids Research Database Issue and the online molecular biology
database collection.

Fernández-Suárez XM(1), Galperin MY.

Author information: 
(1)nardatabase@gmail.com

The 20th annual Database Issue of Nucleic Acids Research includes 176 articles,
half of which describe new online molecular biology databases and the other half 
provide updates on the databases previously featured in NAR and other journals.
This year's highlights include two databases of DNA repeat elements; several
databases of transcriptional factors and transcriptional factor-binding sites;
databases on various aspects of protein structure and protein-protein
interactions; databases for metagenomic and rRNA sequence analysis; and four
databases specifically dedicated to Escherichia coli. The increased emphasis on
using the genome data to improve human health is reflected in the development of 
the databases of genomic structural variation (NCBI's dbVar and EBI's DGVa), the 
NIH Genetic Testing Registry and several other databases centered on the genetic 
basis of human disease, potential drugs, their targets and the mechanisms of
protein-ligand binding. Two new databases present genomic and RNAseq data for
monkeys, providing wealth of data on our closest relatives for comparative
genomics purposes. The NAR online Molecular Biology Database Collection,
available at http://www.oxfordjournals.org/nar/database/a/, has been updated and 
currently lists 1512 online databases. The full content of the Database Issue is 
freely available online on the Nucleic Acids Research website
(http://nar.oxfordjournals.org/).

DOI: 10.1093/nar/gks1297 
PMCID: PMC3531151
PMID: 23203983  [Indexed for MEDLINE]


24. Nucleic Acids Res. 2012 Nov 1;40(20):e156. doi: 10.1093/nar/gks680. Epub 2012 Jul
19.

RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA
from bacteria.

Stead MB(1), Agrawal A, Bowden KE, Nasir R, Mohanty BK, Meagher RB, Kushner SR.

Author information: 
(1)Department of Genetics, University of Georgia, Athens, GA 30602, USA.

RNAsnap™ is a simple and novel method that recovers all intracellular RNA
quantitatively (>99%), faster (<15 min) and less expensively (∼3 cents/sample)
than any of the currently available RNA isolation methods. In fact, none of the
bacterial RNA isolation methods, including the commercial kits, are effective in 
recovering all species of intracellular RNAs (76-5700 nt) with equal efficiency, 
which can lead to biased results in genome-wide studies involving microarray or
RNAseq analysis. The RNAsnap™ procedure yields ∼60 µg of RNA from 10(8)
Escherichia coli cells that can be used directly for northern analysis without
any further purification. Based on a comparative analysis of specific transcripts
ranging in size from 76 to 5700 nt, the RNAsnap™ method provided the most
accurate measure of the relative amounts of the various intracellular RNAs.
Furthermore, the RNAsnap™ RNA was successfully used in enzymatic reactions such
as RNA ligation, reverse transcription, primer extension and reverse
transcriptase-polymerase chain reaction, following sodium acetate/ethanol
precipitation. The RNAsnap™ method can be used to isolate RNA from a wide range
of Gram-negative and Gram-positive bacteria as well as yeast.

DOI: 10.1093/nar/gks680 
PMCID: PMC3488207
PMID: 22821568  [Indexed for MEDLINE]


25. Nucleic Acids Res. 2012 Sep;40(16):7870-84. doi: 10.1093/nar/gks503. Epub 2012
Jun 11.

Altered tRNA characteristics and 3' maturation in bacterial symbionts with
reduced genomes.

Hansen AK(1), Moran NA.

Author information: 
(1)Department of Ecology and Evolutionary Biology, West Campus, Yale University, 
PO Box 27388 West Haven, CT 06516-7388, USA. allison.hansen@yale.edu

Translational efficiency is controlled by tRNAs and other genome-encoded
mechanisms. In organelles, translational processes are dramatically altered
because of genome shrinkage and horizontal acquisition of gene products. The
influence of genome reduction on translation in endosymbionts is largely unknown.
Here, we investigate whether divergent lineages of Buchnera aphidicola, the
reduced-genome bacterial endosymbiont of aphids, possess altered translational
features compared with their free-living relative, Escherichia coli. Our RNAseq
data support the hypothesis that translation is less optimal in Buchnera than in 
E. coli. We observed a specific, convergent, pattern of tRNA loss in Buchnera and
other endosymbionts that have undergone genome shrinkage. Furthermore, many
modified nucleoside pathways that are important for E. coli translation are lost 
in Buchnera. Additionally, Buchnera's A + T compositional bias has resulted in
reduced tRNA thermostability, and may have altered aminoacyl-tRNA synthetase
recognition sites. Buchnera tRNA genes are shorter than those of E. coli, as the 
majority no longer has a genome-encoded 3' CCA; however, all the expressed,
shortened tRNAs undergo 3' CCA maturation. Moreover, expression of tRNA
isoacceptors was not correlated with the usage of corresponding codons. Overall, 
our data suggest that endosymbiont genome evolution alters tRNA characteristics
that are known to influence translational efficiency in their free-living
relative.

DOI: 10.1093/nar/gks503 
PMCID: PMC3439896
PMID: 22689638  [Indexed for MEDLINE]