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. Mol Microbiol. 2018 Feb 24. doi: 10.1111/mmi.13941. [Epub ahead of print]

The evolutionary impact of Intragenic FliA Promoters in Proteobacteria.

Fitzgerald DM(1), Smith C(2), Lapierre P(2), Wade JT(1)(2).

Author information: 
(1)Department of Biomedical Sciences, School of Public Health, University at
Albany, Albany, New York, USA.
(2)Wadsworth Center, New York State Department of Health, Albany, New York, USA.

In Escherichia coli, one Sigma factor recognizes the majority of promoters, and
six "alternative" Sigma factors recognize specific subsets of promoters. The
alternative Sigma factor FliA (σ28 ) recognizes promoters upstream of many
flagellar genes. We previously showed that most E. coli FliA binding sites are
located inside genes. However, it was unclear whether these intragenic binding
sites represent active promoters. Here, we construct and assay transcriptional
promoter-lacZ fusions for all 52 putative FliA promoters previously identified by
ChIP-seq. These experiments, coupled with integrative analysis of published
genome-scale transcriptional datasets, strongly suggest that most intragenic FliA
binding sites are active promoters that transcribe highly unstable RNAs.
Additionally, we show that widespread intragenic FliA-dependent transcription may
be a conserved phenomenon, but that specific promoters are not themselves
conserved. We conclude that intragenic FliA-dependent promoters and the resulting
RNAs are unlikely to have important regulatory functions. Nonetheless, one
intragenic FliA promoter is broadly conserved, and constrains evolution of the
overlapping protein-coding gene. Thus, our data indicate that intragenic
regulatory elements can influence bacterial protein evolution, and suggest that
the impact of intragenic regulatory sequences on genome evolution should be
considered more broadly. This article is protected by copyright. All rights
reserved.

© 2018 John Wiley & Sons Ltd.

DOI: 10.1111/mmi.13941 
PMID: 29476659 


3. mSystems. 2018 Feb 13;3(1). pii: e00172-17. doi: 10.1128/mSystems.00172-17.
eCollection 2018 Jan-Feb.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the
Prophages along the Escherichia coli K-12 Genome.

Yamamoto K(1)(2), Yamanaka Y(2), Shimada T(2)(3), Sarkar P(1)(4), Yoshida M(1),
Bhardwaj N(4), Watanabe H(1), Taira Y(1), Chatterji D(4), Ishihama A(2).

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Tokyo, Japan.
(2)Micro-Nano Technology Research Center, Hosei University, Tokyo, Japan.
(3)Meiji University, School of Agriculture, Kawasaki, Kanagawa, Japan.
(4)Indian Institute of Science, Molecular Biophysics Unit, Bangalore, India.

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting
of the core enzyme with the subunit structure α2ββ'ω and one of the σ subunits
with promoter recognition properties. The smallest subunit, omega (the rpoZ gene 
product), participates in subunit assembly by supporting the folding of the
largest subunit, β', but its functional role remains unsolved except for its
involvement in ppGpp binding and stringent response. As an initial approach for
elucidation of its functional role, we performed in this study ChIP-chip
(chromatin immunoprecipitation with microarray technology) analysis of wild-type 
and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective
RNAP was identified mostly within open reading frames, in particular, of the
genes inside prophages. For the genes that exhibited increased or decreased
distribution of RpoZ-defective RNAP, the level of transcripts increased or
decreased, respectively, as detected by reverse transcription-quantitative PCR
(qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of
ligands by exponential enrichment), the distribution of constitutive promoters
that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS
within prophages. Since all 10 prophages in E. coli K-12 carry only a small
number of promoters, the altered occupancy of RpoZ-defective RNAP and of
transcripts might represent transcription initiated from as-yet-unidentified host
promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP
are located in the regions of low-level H-NS binding. By using phenotype
microarray (PM) assay, alterations of some phenotypes were detected for the
rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some
genes. Possible mechanisms of altered distribution of RNAP inside prophages are
discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ
gene product) of Escherichia coli RNA polymerase plays a structural role in the
formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit 
(β', of 1,407 residues in length), but except for binding of the stringent signal
ppGpp, little is known of its role in the control of RNAP function. After
analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the
ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open
reading frames, in particular, of the genes within prophages. For a set of the
genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription 
was found to be altered as observed by qRT-PCR assay. All the observations here
described indicate the involvement of RpoZ in recognition of some of the prophage
genes. This study advances understanding of not only the regulatory role of omega
subunit in the functions of RNAP but also the regulatory interplay between
prophages and the host E. coli for adjustment of cellular physiology to a variety
of environments in nature.

DOI: 10.1128/mSystems.00172-17 
PMCID: PMC5811629
PMID: 29468196 


4. MBio. 2018 Feb 20;9(1). pii: e02096-17. doi: 10.1128/mBio.02096-17.

The Essential Genome of Escherichia coli K-12.

Goodall ECA(#)(1), Robinson A(#)(1), Johnston IG(1), Jabbari S(1), Turner KA(2), 
Cunningham AF(1), Lund PA(1), Cole JA(1), Henderson IR(3).

Author information: 
(1)Institute of Microbiology and Infection, University of Birmingham, Birmingham,
United Kingdom.
(2)Discuva Ltd., Cambridge, United Kingdom.
(3)Institute of Microbiology and Infection, University of Birmingham, Birmingham,
United Kingdom i.r.henderson@bham.ac.uk.
(#)Contributed equally

Transposon-directed insertion site sequencing (TraDIS) is a high-throughput
method coupling transposon mutagenesis with short-fragment DNA sequencing. It is 
commonly used to identify essential genes. Single gene deletion libraries are
considered the gold standard for identifying essential genes. Currently, the
TraDIS method has not been benchmarked against such libraries, and therefore, it 
remains unclear whether the two methodologies are comparable. To address this, a 
high-density transposon library was constructed in Escherichia coli K-12.
Essential genes predicted from sequencing of this library were compared to
existing essential gene databases. To decrease false-positive identification of
essential genes, statistical data analysis included corrections for both gene
length and genome length. Through this analysis, new essential genes and genes
previously incorrectly designated essential were identified. We show that manual 
analysis of TraDIS data reveals novel features that would not have been detected 
by statistical analysis alone. Examples include short essential regions within
genes, orientation-dependent effects, and fine-resolution identification of
genome and protein features. Recognition of these insertion profiles in
transposon mutagenesis data sets will assist genome annotation of less well
characterized genomes and provides new insights into bacterial physiology and
biochemistry.IMPORTANCE Incentives to define lists of genes that are essential
for bacterial survival include the identification of potential targets for
antibacterial drug development, genes required for rapid growth for exploitation 
in biotechnology, and discovery of new biochemical pathways. To identify
essential genes in Escherichia coli, we constructed a transposon mutant library
of unprecedented density. Initial automated analysis of the resulting data
revealed many discrepancies compared to the literature. We now report more
extensive statistical analysis supported by both literature searches and detailed
inspection of high-density TraDIS sequencing data for each putative essential
gene for the E. coli model laboratory organism. This paper is important because
it provides a better understanding of the essential genes of E. coli, reveals the
limitations of relying on automated analysis alone, and provides a new standard
for the analysis of TraDIS data.

Copyright © 2018 Goodall et al.

DOI: 10.1128/mBio.02096-17 
PMCID: PMC5821084
PMID: 29463657 


5. 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 


6. Nucleic Acids Res. 2018 Jan 31. doi: 10.1093/nar/gky069. [Epub ahead of print]

Systems assessment of transcriptional regulation on central carbon metabolism by 
Cra and CRP.

Kim D(1)(2), Seo SW(1)(3), Gao Y(4), Nam H(5), Guzman GI(1), Cho BK(6)(7),
Palsson BO(1)(8)(7).

Author information: 
(1)Department of Bioengineering, University of California, San Diego, La Jolla,
CA 92093, USA.
(2)Department of Genetic Engineering, College of Life Sciences, Kyung Hee
University, Yongin 446-701, Republic of Korea.
(3)School of Chemical and Biological Engineering, Institute of Chemical Prcocess,
Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of
Korea.
(4)Division of Biological Science, University of California, San Diego, La Jolla,
CA 92093, USA.
(5)School of Information and Communication, Gwangju Institute of Science and
Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, Republic of Korea.
(6)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, Daejeon 34141, Republic of Korea.
(7)The Novo Nordisk Foundation Center for Biosustainabiliy, Danish Technical
University, 6 Kogle Alle, Hørsholm, Denmark.
(8)Department of Pediatrics, University of California, San Diego, La Jolla, CA
92093, USA.

Two major transcriptional regulators of carbon metabolism in bacteria are Cra and
CRP. CRP is considered to be the main mediator of catabolite repression. Unlike
for CRP, in vivo DNA binding information of Cra is scarce. Here we generate and
integrate ChIP-exo and RNA-seq data to identify 39 binding sites for Cra and 97
regulon genes that are regulated by Cra in Escherichia coli. An integrated
metabolic-regulatory network was formed by including experimentally-derived
regulatory information and a genome-scale metabolic network reconstruction.
Applying analysis methods of systems biology to this integrated network showed
that Cra enables optimal bacterial growth on poor carbon sources by redirecting
and repressing glycolysis flux, by activating the glyoxylate shunt pathway, and
by activating the respiratory pathway. In these regulatory mechanisms, the
overriding regulatory activity of Cra over CRP is fundamental. Thus, elucidation 
of interacting transcriptional regulation of core carbon metabolism in bacteria
by two key transcription factors was possible by combining genome-wide
experimental measurement and simulation with a genome-scale metabolic model.

© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic
Acids Research.

DOI: 10.1093/nar/gky069 
PMID: 29394395 


7. Cell. 2018 Feb 8;172(4):771-783.e18. doi: 10.1016/j.cell.2017.12.027. Epub 2018
Jan 18.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and
Condensin Proteins.

Lioy VS(1), Cournac A(2), Marbouty M(2), Duigou S(1), Mozziconacci J(3), Espéli
O(4), Boccard F(5), Koszul R(6).

Author information: 
(1)Institut de Biologie Intégrative de la Cellule, CEA, CNRS, Université
Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France.
(2)Institut Pasteur, Département Génomes et Génétique, Groupe Régulation spatiale
des génomes, 75015 Paris, France; CNRS, UMR 3525, 75015 Paris, France.
(3)Sorbonne Universités, Laboratoire de Physique Théorique de la Matière
Condensée, UMR 7600, Université Pierre et Marie Curie, 75005 Paris, France.
(4)Centre Interdisciplinaire de Recherche en Biologie, Collège de France,
UMR-CNRS 7241, INSERM U1050, Paris, France.
(5)Institut de Biologie Intégrative de la Cellule, CEA, CNRS, Université
Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France.
Electronic address: frederic.boccard@i2bc.paris-saclay.fr.
(6)Institut Pasteur, Département Génomes et Génétique, Groupe Régulation spatiale
des génomes, 75015 Paris, France; CNRS, UMR 3525, 75015 Paris, France. Electronic
address: romain.koszul@pasteur.fr.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic
information is generally carried on a circular chromosome with a single origin of
replication from which two replication forks proceed bidirectionally toward the
opposite terminus region. Here, we investigate the higher-order architecture of
the Escherichia coli genome, showing its partition into two structurally distinct
entities by a complex and intertwined network of contacts: the replication
terminus (ter) region and the rest of the chromosome. Outside of ter, the
condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote 
DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF 
activity, and contacts are restricted to ∼280 kb, creating a domain with distinct
structural properties. We also show how other NAPs contribute to nucleoid
organization, such as H-NS, which restricts short-range interactions. Combined,
these results reveal the contributions of major evolutionarily conserved proteins
in a bacterial chromosome organization.

Copyright © 2017 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.cell.2017.12.027 
PMID: 29358050 


8. Methods Mol Biol. 2018;1703:87-94. doi: 10.1007/978-1-4939-7459-7_6.

Mapping E. coli Topoisomerase IV Binding and Activity Sites.

El Sayyed H(1), Espéli O(2).

Author information: 
(1)Center for Interdisciplinary Research in Biology (CIRB), College de France,
CNRS/UMR 7241 - INSERM U1050, PSL Research University, 11, Place Marcelin
Berthelot, Paris Cedex 05, 75231, France.
(2)Center for Interdisciplinary Research in Biology (CIRB), College de France,
CNRS/UMR 7241 - INSERM U1050, PSL Research University, 11, Place Marcelin
Berthelot, Paris Cedex 05, 75231, France. olivier.espeli@college-de-france.fr.

This methods article described a protocol aiming at mapping E. coli Topoisomerase
IV (Topo IV) binding and cleavage activity sites on the genome. The approach is
readily applicable to any Type II topoisomerase on a broad variety of
gram-positive and gram-negative bacterial species. Conventional ChIP-seq of flag 
tagged Topo IV subunits and a novel method aimed at trapping only DNA bound to
active Topo IV (called NorfliP) are described. NorfliP relies on the ability of
norfloxacin, a quinolone drug, to cross-link the 5' ends of the DNA breaks with
the catalytic tyrosine of bacterial Type II topoisomerases. These methods give
complementary results and their combination brought important insights on both
the function and regulation of Topo IV.

DOI: 10.1007/978-1-4939-7459-7_6 
PMID: 29177735 


9. Nat Commun. 2017 Nov 17;8(1):1596. doi: 10.1038/s41467-017-01613-1.

Global role of the bacterial post-transcriptional regulator CsrA revealed by
integrated transcriptomics.

Potts AH(1), Vakulskas CA(1)(2), Pannuri A(1), Yakhnin H(3), Babitzke P(3), Romeo
T(4).

Author information: 
(1)Department of Microbiology and Cell Science, University of Florida, Institute 
of Food and Agricultural Sciences, Gainesville, FL, 32611-0700, USA.
(2)Integrated DNA Technologies, Molecular Genetics Department, 1710 Commercial
Park, Coralville, IA, 52241, USA.
(3)Department of Biochemistry and Molecular Biology, Center for RNA Molecular
Biology, Pennsylvania State University, University Park, Pennsylvania, PA, 16802,
USA.
(4)Department of Microbiology and Cell Science, University of Florida, Institute 
of Food and Agricultural Sciences, Gainesville, FL, 32611-0700, USA.
tromeo@ufl.edu.

CsrA is a post-transcriptional regulatory protein that is widely distributed
among bacteria. This protein influences bacterial lifestyle decisions by binding 
to the 5' untranslated and/or early coding regions of mRNA targets, causing
changes in translation initiation, RNA stability, and/or transcription
elongation. Here, we assess the contribution of CsrA to gene expression in
Escherichia coli on a global scale. UV crosslinking immunoprecipitation and
sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo,
while ribosome profiling and RNA-seq uncover the impact of CsrA on translation,
RNA abundance, and RNA stability. This combination of approaches reveals
unprecedented detail about the regulatory role of CsrA, including novel binding
targets and physiological roles, such as in envelope function and iron
homeostasis. Our findings highlight the integration of CsrA throughout the E.
coli regulatory network, where it orchestrates vast effects on gene expression.

DOI: 10.1038/s41467-017-01613-1 
PMCID: PMC5694010
PMID: 29150605 


10. 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]


11. 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]


12. Methods Mol Biol. 2017;1624:85-97. doi: 10.1007/978-1-4939-7098-8_8.

Nucleoid-Associated Proteins: Genome Level Occupancy and Expression Analysis.

Singh P(1)(2), Seshasayee ASN(3).

Author information: 
(1)National Centre for Biological Sciences, Tata Institute of Fundamental
Research, Bangalore, 560065, Karnataka, India. singh.parul686@gmail.com.
(2)SASTRA University, Thanjavur, 613401, Tamil Nadu, India.
singh.parul686@gmail.com.
(3)National Centre for Biological Sciences, Tata Institute of Fundamental
Research, Bangalore, 560065, Karnataka, India. aswin@ncbs.res.in.

The advent of Chromatin Immunoprecipitation sequencing (ChIP-Seq) has allowed the
identification of genomic regions bound by a DNA binding protein in-vivo on a
genome-wide scale. The impact of the DNA binding protein on gene expression can
be addressed using transcriptome experiments in appropriate genetic settings.
Overlaying the above two sources of data enables us to dissect the direct and
indirect effects of a DNA binding protein on gene expression. Application of
these techniques to Nucleoid Associated Proteins (NAPs) and Global Transcription 
Factors (GTFs) has underscored the complex relationship between DNA-protein
interactions and gene expression change, highlighting the role of combinatorial
control. Here, we demonstrate the usage of ChIP-Seq to infer binding properties
and transcriptional effects of NAPs such as Fis and HNS, and the GTF CRP in the
model organism Escherichia coli K12 MG1655 (E. coli).

DOI: 10.1007/978-1-4939-7098-8_8 
PMID: 28842878 


13. PLoS One. 2017 May 10;12(5):e0176290. doi: 10.1371/journal.pone.0176290.
eCollection 2017.

Novel genes associated with enhanced motility of Escherichia coli ST131.

Kakkanat A(1)(2), Phan MD(1)(2), Lo AW(1)(2), Beatson SA(1)(2)(3), Schembri
MA(1)(2).

Author information: 
(1)School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, Queensland, Australia.
(2)Australian Infectious Disease Research Centre, University of Queensland,
Brisbane, Queensland, Australia.
(3)Australian Centre for Ecogenomics, University of Queensland, Brisbane,
Queensland, Australia.

Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract
infections (UTIs) and is increasingly associated with multidrug resistance. This 
includes UPEC strains from the recently emerged and globally disseminated
sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant
UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella.
Here, we applied a combination of saturated Tn5 mutagenesis and transposon
directed insertion site sequencing (TraDIS) as a high throughput genetic screen
and identified 30 genes associated with enhanced motility of the reference ST131 
strain EC958. This included 12 genes that repress motility of E. coli K-12, four 
of which (lrhA, ihfA, ydiV, lrp) were confirmed in EC958. Other genes represented
novel factors that impact motility, and we focused our investigation on
characterisation of the mprA, hemK and yjeA genes. Mutation of each of these
genes in EC958 led to increased transcription of flagellar genes (flhD and fliC),
increased expression of the FliC flagellin, enhanced flagella synthesis and a
hyper-motile phenotype. Complementation restored all of these properties to
wild-type level. We also identified Tn5 insertions in several intergenic regions 
(IGRs) on the EC958 chromosome that were associated with enhanced motility; this 
included flhDC and EC958_1546. In both of these cases, the Tn5 insertions were
associated with increased transcription of the downstream gene(s), which resulted
in enhanced motility. The EC958_1546 gene encodes a phage protein with similarity
to esterase/deacetylase enzymes involved in the hydrolysis of sialic acid
derivatives found in human mucus. We showed that over-expression of EC958_1546
led to enhanced motility of EC958 as well as the UPEC strains CFT073 and UTI89,
demonstrating its activity affects the motility of different UPEC strains.
Overall, this study has identified and characterised a number of novel factors
associated with enhanced UPEC motility.

DOI: 10.1371/journal.pone.0176290 
PMCID: PMC5425062
PMID: 28489862  [Indexed for MEDLINE]


14. 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]


15. 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 


16. 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 


17. BMC Syst Biol. 2017 Jan 6;11(1):1. doi: 10.1186/s12918-016-0376-y.

Coordinated regulation of acid resistance in Escherichia coli.

Aquino P(1)(2), Honda B(1), Jaini S(1), Lyubetskaya A(3), Hosur K(1)(2), Chiu
JG(2), Ekladious I(2), Hu D(2), Jin L(2), Sayeg MK(2), Stettner AI(2), Wang J(2),
Wong BG(2), Wong WS(2), Alexander SL(2), Ba C(2), Bensussen SI(2), Bernstein
DB(2), Braff D(2), Cha S(2), Cheng DI(2), Cho JH(2), Chou K(2), Chuang J(2),
Gastler DE(2), Grasso DJ(2), Greifenberger JS(2), Guo C(2), Hawes AK(2), Israni
DV(2), Jain SR(2), Kim J(2), Lei J(2), Li H(2), Li D(2), Li Q(2), Mancuso CP(2), 
Mao N(2), Masud SF(2), Meisel CL(2), Mi J(2), Nykyforchyn CS(2), Park M(2),
Peterson HM(2), Ramirez AK(2), Reynolds DS(2), Rim NG(2), Saffie JC(2), Su H(2), 
Su WR(2), Su Y(2), Sun M(2), Thommes MM(2), Tu T(2), Varongchayakul N(2), Wagner 
TE(2), Weinberg BH(2), Yang R(2), Yaroslavsky A(2), Yoon C(2), Zhao Y(2),
Zollinger AJ(2), Stringer AM(4), Foster JW(5), Wade J(4)(6), Raman S(5), Broude
N(1), Wong WW(1), Galagan JE(7)(8)(9).

Author information: 
(1)Department of Biomedical Engineering, Boston University, Boston, USA.
(2)BE605 Course, Biomedical Engineering, Boston University, Boston, USA.
(3)Bioinformatics program, Boston University, Boston, USA.
(4)Wadsworth Center, New York State Department of Health, Albany, NY, USA.
(5)Department of Microbiology and Immunology, University of South Alabama College
of Medicine, Mobile, AL, 36688, USA.
(6)Department of Biomedical Sciences, University at Albany, Albany, NY, USA.
(7)Department of Biomedical Engineering, Boston University, Boston, USA.
jgalag@bu.edu.
(8)Bioinformatics program, Boston University, Boston, USA. jgalag@bu.edu.
(9)National Emerging Infectious Diseases Laboratory, Boston University, Boston,
USA. jgalag@bu.edu.

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of
the stomach through multiple acid resistance (AR) mechanisms. The most effective 
system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic
protons and excrete GABA. The first described system, AR1, does not require an
external amino acid. Its mechanism has not been determined. The regulation of the
multiple AR systems and their coordination with broader cellular metabolism has
not been fully explored.
RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to
experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and 
csiR. Our data identified all previously in vivo confirmed direct interactions
and revealed several others previously inferred from gene expression data. Our
data demonstrate that nac and csiR directly modulate AR, and leads to a
regulatory network model in which all four TFs participate in coordinating acid
resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a 
novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with
internally derived glutamate. This hypothesis makes several testable predictions 
that we confirmed experimentally.
CONCLUSIONS: Our data suggest that the regulatory network underlying AR is
complex and deeply interconnected with the regulation of GABA and glutamate
metabolism, nitrogen metabolism. These connections underlie and experimentally
validated model of AR1 in which the decarboxylation enzymes of AR2 are used with 
internally derived glutamate.

DOI: 10.1186/s12918-016-0376-y 
PMCID: PMC5217608
PMID: 28061857  [Indexed for MEDLINE]


18. Front Mol Biosci. 2016 Nov 16;3:74. eCollection 2016.

Genome-Wide Transcriptional Regulation and Chromosome Structural Arrangement by
GalR in E. coli.

Qian Z(1), Trostel A(1), Lewis DE(1), Lee SJ(2), He X(3), Stringer AM(4), Wade
JT(5), Schneider TD(6), Durfee T(7), Adhya S(1).

Author information: 
(1)Laboratory of Molecular Biology, National Institutes of Health, National
Cancer Institute Bethesda, MD, USA.
(2)Microbiomics and Immunity Research Center, Korea Research Institute of
Bioscience and Biotechnology Daejeon, Korea.
(3)Laboratory of Metabolism, National Institutes of Health, National Cancer
Institute Bethesda, MD, USA.
(4)Wadsworth Center, New York State Department of Health Albany, NY, USA.
(5)Wadsworth Center, New York State Department of HealthAlbany, NY, USA;
Department of Biomedical Sciences, School of Public Health, University of
AlbanyAlbany, NY, USA.
(6)Gene Regulation and Chromosome Biology Laboratory, National Institutes of
Health, National Cancer Institute, Center for Cancer Research Frederick, MD, USA.
(7)DNASTAR, Inc. Madison, WI, USA.

The regulatory protein, GalR, is known for controlling transcription of genes
related to D-galactose metabolism in Escherichia coli. Here, using a combination 
of experimental and bioinformatic approaches, we identify novel GalR binding
sites upstream of several genes whose function is not directly related to
D-galactose metabolism. Moreover, we do not observe regulation of these genes by 
GalR under standard growth conditions. Thus, our data indicate a broader
regulatory role for GalR, and suggest that regulation by GalR is modulated by
other factors. Surprisingly, we detect regulation of 158 transcripts by GalR,
with few regulated genes being associated with a nearby GalR binding site. Based 
on our earlier observation of long-range interactions between distally bound GalR
dimers, we propose that GalR indirectly regulates the transcription of many genes
by inducing large-scale restructuring of the chromosome.

DOI: 10.3389/fmolb.2016.00074 
PMCID: PMC5110547
PMID: 27900321 


19. 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]


20. 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]


21. Plasmid. 2016 Sep - Nov;87-88:17-27. doi: 10.1016/j.plasmid.2016.07.004. Epub
2016 Aug 1.

Characterization of Acr2, an H-NS-like protein encoded on A/C2-type plasmids.

Lang KS(1), Johnson TJ(2).

Author information: 
(1)University of Minnesota, Department of Veterinary and Biomedical Sciences, St.
Paul, MN 55108, United States.
(2)University of Minnesota, Department of Veterinary and Biomedical Sciences, St.
Paul, MN 55108, United States. Electronic address: joh04207@umn.edu.

Conjugation plays an important role in the horizontal movement of DNA between
bacterial species and even genera. Large conjugative plasmids in Gram-negative
bacteria are associated with multi-drug resistance and have been implicated in
the spread of these phenotypes to pathogenic organisms. A/C plasmids often carry 
genes that confer resistance to multiple classes of antibiotics. Recently,
transcription factors were characterized that regulate A/C conjugation. In this
work, we expanded the regulon of the negative regulator Acr2. We developed an A/C
variant, pARK01, by precise removal of resistance genes carried by the plasmid in
order to make it more genetically tractable. Using pARK01, we conducted RNA-Seq
and ChAP-Seq experiments to characterize the regulon of Acr2, an H-NS-like
protein. We found that Acr2 binds several loci on the plasmid. We showed, in
vitro, that Acr2 can bind specific promoter regions directly and identify key
amino acids which are important for this binding. This study further
characterizes Acr2 and suggests its role in modulating gene expression of
multiple plasmid and chromosomal loci.

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

DOI: 10.1016/j.plasmid.2016.07.004 
PMID: 27492737  [Indexed for MEDLINE]


22. PLoS Genet. 2016 May 12;12(5):e1006025. doi: 10.1371/journal.pgen.1006025.
eCollection 2016 May.

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

El Sayyed H(1)(2), Le Chat L(1), Lebailly E(3), Vickridge E(1)(2), Pages C(3),
Cornet F(3), Cosentino Lagomarsino M(4), Espéli O(1).

Author information: 
(1)Center for Interdisciplinary Research in Biology (CIRB), Collège de France,
UMR-CNRS 7241, Paris, France.
(2)Université Paris-Saclay, Gif-sur-Yvette, France.
(3)Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM),
CNRS-Université Toulouse III, Toulouse, France.
(4)UMR 7238, Computational and quantitative biology, Institut de biologie Paris
Seine, Paris, France.

Catenation links between sister chromatids are formed progressively during DNA
replication and are involved in the establishment of sister chromatid cohesion.
Topo IV is a bacterial type II topoisomerase involved in the removal of
catenation links both behind replication forks and after replication during the
final separation of sister chromosomes. We have investigated the global
DNA-binding and catalytic activity of Topo IV in E. coli using genomic and
molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the
E. coli chromosome is controlled by DNA replication. During replication, Topo IV 
has access to most of the genome but only selects a few hundred specific sites
for its activity. Local chromatin and gene expression context influence site
selection. Moreover strong DNA-binding and catalytic activities are found at the 
chromosome dimer resolution site, dif, located opposite the origin of
replication. We reveal a physical and functional interaction between Topo IV and 
the XerCD recombinases acting at the dif site. This interaction is modulated by
MatP, a protein involved in the organization of the Ter macrodomain. These
results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to 
the final step of chromosome management during the cell cycle.

DOI: 10.1371/journal.pgen.1006025 
PMCID: PMC4865107
PMID: 27171414  [Indexed for MEDLINE]


23. 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]


24. 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]


25. PLoS Genet. 2016 Jan 20;12(1):e1005796. doi: 10.1371/journal.pgen.1005796.
eCollection 2016 Jan.

H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during
Their Integration in Host Chromosomes.

Higashi K(1), Tobe T(2), Kanai A(3), Uyar E(4), Ishikawa S(4), Suzuki Y(3),
Ogasawara N(4), Kurokawa K(1)(5), Oshima T(4).

Author information: 
(1)Department of Biological Information, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan.
(2)Department of Biomedical Informatics, Graduate School of Medicine, Osaka
University, Suita, Osaka, Japan.
(3)Department of Medical Genome Sciences, Graduate School of Frontier Sciences,
The University of Tokyo, Kashiwa-shi, Chiba, Japan.
(4)Graduate School of Biological Sciences, Nara Institute of Science and
Technology, Nara, Japan.
(5)Earth-Life Science Institute, Tokyo Institute of Technology, Meguro-ku, Tokyo,
Japan.

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate
expression of transferred genes, however, can disrupt the physiology of the host 
bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated
protein, H-NS, which preferentially binds to horizontally transferred genes to
control their expression. Once expression is optimized, the horizontally
transferred genes may actually contribute to E. coli survival in new habitats.
Therefore, we investigated whether and how H-NS contributes to this optimization 
process. A comparison of H-NS binding profiles on common chromosomal segments of 
three E. coli strains belonging to different phylogenetic groups indicated that
the positions of H-NS-bound regions have been conserved in E. coli strains. The
sequences of the H-NS-bound regions appear to have diverged more so than
H-NS-unbound regions only when H-NS-bound regions are located upstream or in
coding regions of genes. Because these regions generally contain regulatory
elements for gene expression, sequence divergence in these regions may be
associated with alteration of gene expression. Indeed, nucleotide substitutions
in H-NS-bound regions of the ybdO promoter and coding regions have diversified
the potential for H-NS-independent negative regulation among E. coli strains. The
ybdO expression in these strains was still negatively regulated by H-NS, which
reduced the effect of H-NS-independent regulation under normal growth conditions.
Hence, we propose that, during E. coli evolution, the conservation of H-NS
binding sites resulted in the diversification of the regulation of horizontally
transferred genes, which may have facilitated E. coli adaptation to new
ecological niches.

DOI: 10.1371/journal.pgen.1005796 
PMCID: PMC4720273
PMID: 26789284  [Indexed for MEDLINE]


26. PLoS One. 2015 Dec 16;10(12):e0145035. doi: 10.1371/journal.pone.0145035.
eCollection 2015.

Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems.

Zere TR(1), Vakulskas CA(1), Leng Y(1), Pannuri A(1), Potts AH(1), Dias R(1),
Tang D(1), Kolaczkowski B(1), Georgellis D(2), Ahmer BM(3), Romeo T(1).

Author information: 
(1)Department of Microbiology and Cell Science, Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, United States of
America.
(2)Departamento de Genética Molecular, Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México, México D.F., México.
(3)Department of Microbial Infection and Immunity, The Ohio State University,
Columbus, OH, United States of America.

The two-component signal transduction system BarA-UvrY of Escherichia coli and
its orthologs globally regulate metabolism, motility, biofilm formation, stress
resistance, virulence of pathogens and quorum sensing by activating the
transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These
sRNAs act by sequestering the RNA binding protein CsrA (RsmA) away from lower
affinity mRNA targets. In this study, we used ChIP-exo to identify, at single
nucleotide resolution, genomic sites for UvrY (SirA) binding in E. coli and
Salmonella enterica. The csrB and csrC genes were the strongest targets of
crosslinking, which required UvrY phosphorylation by the BarA sensor kinase.
Crosslinking occurred at two sites, an inverted repeat sequence far upstream of
the promoter and a site near the -35 sequence. DNAse I footprinting revealed
specific binding of UvrY in vitro only to the upstream site, indicative of
additional binding requirements and/or indirect binding to the downstream site.
Additional genes, including cspA, encoding the cold-shock RNA-binding protein
CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We 
conclude that the global effects of UvrY/SirA on gene expression are primarily
mediated by activating csrB and csrC transcription. We also used in vivo
crosslinking and other experimental approaches to reveal new features of
csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA.
Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be 
uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with
fliW, which encodes a protein that binds to CsrA and antagonizes its activity in 
Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA 
antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform 
this function in other species.

DOI: 10.1371/journal.pone.0145035 
PMCID: PMC4682653
PMID: 26673755  [Indexed for MEDLINE]


27. MBio. 2015 Dec 15;6(6):e01947-15. doi: 10.1128/mBio.01947-15.

Impact of Anaerobiosis on Expression of the Iron-Responsive Fur and RyhB
Regulons.

Beauchene NA(1), Myers KS(1), Chung D(2), Park DM(1), Weisnicht AM(1), Keleş
S(2), Kiley PJ(3).

Author information: 
(1)Department of Biomolecular Chemistry, University of Wisconsin-Madison,
Madison, Wisconsin, USA.
(2)Departments of Statistics and Biostatistics and Medical Informatics,
University of Wisconsin-Madison, Madison, Wisconsin, USA.
(3)Department of Biomolecular Chemistry, University of Wisconsin-Madison,
Madison, Wisconsin, USA pjkiley@wisc.edu.

Iron, a major protein cofactor, is essential for most organisms. Despite the
well-known effects of O2 on the oxidation state and solubility of iron, the
impact of O2 on cellular iron homeostasis is not well understood. Here we report 
that in Escherichia coli K-12, the lack of O2 dramatically changes expression of 
genes controlled by the global regulators of iron homeostasis, the transcription 
factor Fur and the small RNA RyhB. Using chromatin immunoprecipitation sequencing
(ChIP-seq), we found anaerobic conditions promote Fur binding to more locations
across the genome. However, by expression profiling, we discovered that the major
effect of anaerobiosis was to increase the magnitude of Fur regulation, leading
to increased expression of iron storage proteins and decreased expression of most
iron uptake pathways and several Mn-binding proteins. This change in the pattern 
of gene expression also correlated with an unanticipated decrease in Mn in
anaerobic cells. Changes in the genes posttranscriptionally regulated by RyhB
under aerobic and anaerobic conditions could be attributed to O2-dependent
changes in transcription of the target genes: aerobic RyhB targets were enriched 
in iron-containing proteins associated with aerobic energy metabolism, whereas
anaerobic RyhB targets were enriched in iron-containing anaerobic respiratory
functions. Overall, these studies showed that anaerobiosis has a larger impact on
iron homeostasis than previously anticipated, both by expanding the number of
direct Fur target genes and the magnitude of their regulation and by altering the
expression of genes predicted to be posttranscriptionally regulated by the small 
RNA RyhB under iron-limiting conditions.IMPORTANCE: Microbes and host cells
engage in an "arms race" for iron, an essential nutrient that is often scarce in 
the environment. Studies of iron homeostasis have been key to understanding the
control of iron acquisition and the downstream pathways that enable microbes to
compete for this valuable resource. Here we report that O2 availability affects
the gene expression programs of two Escherichia coli master regulators that
function in iron homeostasis: the transcription factor Fur and the small RNA
regulator RyhB. Fur appeared to be more active under anaerobic conditions,
suggesting a change in the set point for iron homeostasis. RyhB preferentially
targeted iron-containing proteins of respiration-linked pathways, which are
differentially expressed under aerobic and anaerobic conditions. Such findings
may be relevant to the success of bacteria within their hosts since zones of
reduced O2 may actually reduce bacterial iron demands, making it easier to win
the arms race for iron.

Copyright © 2015 Beauchene et al.

DOI: 10.1128/mBio.01947-15 
PMCID: PMC4676285
PMID: 26670385  [Indexed for MEDLINE]


28. 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]


29. Proc Natl Acad Sci U S A. 2015 Aug 25;112(34):E4735-42. doi:
10.1073/pnas.1424269112. Epub 2015 Aug 10.

Quantitative genomic analysis of RecA protein binding during DNA double-strand
break repair reveals RecBCD action in vivo.

Cockram CA(1), Filatenkova M(2), Danos V(2), El Karoui M(3), Leach DR(4).

Author information: 
(1)Institute of Cell Biology, School of Biological Sciences, University of
Edinburgh, Edinburgh EH9 3BF, United Kingdom;
(2)Life Sciences Institute, School of Informatics, University of Edinburgh,
Edinburgh EH8 9LE, United Kingdom; SynthSys, Centre for Synthetic and Systems
Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.
(3)Institute of Cell Biology, School of Biological Sciences, University of
Edinburgh, Edinburgh EH9 3BF, United Kingdom; SynthSys, Centre for Synthetic and 
Systems Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom
Meriem.Elkaroui@ed.ac.uk D.Leach@ed.ac.uk.
(4)Institute of Cell Biology, School of Biological Sciences, University of
Edinburgh, Edinburgh EH9 3BF, United Kingdom; Meriem.Elkaroui@ed.ac.uk
D.Leach@ed.ac.uk.

Understanding molecular mechanisms in the context of living cells requires the
development of new methods of in vivo biochemical analysis to complement
established in vitro biochemistry. A critically important molecular mechanism is 
genetic recombination, required for the beneficial reassortment of genetic
information and for DNA double-strand break repair (DSBR). Central to
recombination is the RecA (Rad51) protein that assembles into a spiral filament
on DNA and mediates genetic exchange. Here we have developed a method that
combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq)
and mathematical modeling to quantify RecA protein binding during the active
repair of a single DSB in the chromosome of Escherichia coli. We have used
quantitative genomic analysis to infer the key in vivo molecular parameters
governing RecA loading by the helicase/nuclease RecBCD at recombination
hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the 
lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus
region of the chromosome, over 1 Mb away.

DOI: 10.1073/pnas.1424269112 
PMCID: PMC4553759
PMID: 26261330  [Indexed for MEDLINE]


30. 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]


31. Sci Rep. 2015 May 28;5:10469. doi: 10.1038/srep10469.

Characterization of the Escherichia coli σ(S) core regulon by Chromatin
Immunoprecipitation-sequencing (ChIP-seq) analysis.

Peano C(1), Wolf J(2), Demol J(3), Rossi E(4), Petiti L(1), De Bellis G(1),
Geiselmann J(3), Egli T(2), Lacour S(3), Landini P(4).

Author information: 
(1)Institute of Biomedical Technologies, National Research Council (ITB-CNR),
Segrate (MI), Italy.
(2)EAWAG, Swiss Federal Institute for Environmental Science and Technology,
Dübendorf, Switzerland.
(3)1] Lab. Adaptation et Pathogénie des Micro-organismes (LAPM), Univ. Grenoble
Alpes, F-38000 Grenoble, France [2] UMR 5163, Centre National de Recherche
Scientifique (CNRS), Grenoble, France.
(4)Department of Biosciences, Università degli Studi di Milano, Milan, Italy.

In bacteria, selective promoter recognition by RNA polymerase is achieved by its 
association with σ factors, accessory subunits able to direct RNA polymerase
"core enzyme" (E) to different promoter sequences. Using Chromatin
Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the
σ(S)-associated RNA polymerase form (Eσ(S)) during transition from exponential to
stationary phase. We identified 63 binding sites for Eσ(S) overlapping known or
putative promoters, often located upstream of genes (encoding either ORFs or
non-coding RNAs) showing at least some degree of dependence on the σ(S)-encoding 
rpoS gene. Eσ(S) binding did not always correlate with an increase in
transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S)
might remain poised in a pre-initiation state upon binding. A large fraction of
Eσ(S)-binding sites corresponded to promoters recognized by RNA polymerase
associated with σ(70) or other σ factors, suggesting a considerable overlap in
promoter recognition between different forms of RNA polymerase. In particular,
Eσ(S) appears to contribute significantly to transcription of genes encoding
proteins involved in LPS biosynthesis and in cell surface composition. Finally,
our results highlight a direct role of Eσ(S) in the regulation of non coding
RNAs, such as OmrA/B, RyeA/B and SibC.

DOI: 10.1038/srep10469 
PMCID: PMC4447067
PMID: 26020590  [Indexed for MEDLINE]


32. Genome Biol. 2015 May 15;16:98. doi: 10.1186/s13059-015-0666-5.

Visualizing translocation dynamics and nascent transcript errors in paused RNA
polymerases in vivo.

Imashimizu M(1), Takahashi H(2), Oshima T(3), McIntosh C(4), Bubunenko M(5),
Court DL(6), Kashlev M(7).

Author information: 
(1)Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702,
USA. imashimizum@mail.nih.gov.
(2)Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku,
Chiba, 260-8673, Japan. hiroki.takahashi@chiba-u.jp.
(3)Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Ikoma, Nara, 630-0192, Japan. taku@bs.naist.jp.
(4)Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702,
USA. mcintoshc@mail.nih.gov.
(5)Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702,
USA. bubunenm@mail.nih.gov.
(6)Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702,
USA. courtd@mail.nih.gov.
(7)Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702,
USA. kashlevm@mail.nih.gov.

Erratum in
    Genome Biol. 2015;16:270.

BACKGROUND: Transcription elongation is frequently interrupted by pausing signals
in DNA, with downstream effects on gene expression. Transcription errors also
induce prolonged pausing, which can lead to a destabilized genome by interfering 
with DNA replication. Mechanisms of pausing associated with translocation blocks 
and misincorporation have been characterized in vitro, but not in vivo.
RESULTS: We investigate the pausing pattern of RNA polymerase (RNAP) in
Escherichia coli by a novel approach, combining native elongating transcript
sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We
reveal that the G-dC base pair at the 5' end of the RNA-DNA hybrid interferes
with RNAP translocation. The distance between the 5' G-dC base pair and the 3'
end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can
induce pausing in post-translocated, pre-translocated, and backtracked states of 
RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active
site of RNAP inhibits elongation and induces G-to-A errors, which leads to
backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the
backtracked complexes. We also find that pausing events are enriched in the 5'
untranslated region and antisense transcription of mRNA genes and are reduced in 
rRNA genes.
CONCLUSIONS: In E. coli, robust transcriptional pausing involves RNAP interaction
with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with
translocation. CpG DNA sequences induce transcriptional pausing and G-to-A
errors.

DOI: 10.1186/s13059-015-0666-5 
PMCID: PMC4457086
PMID: 25976475  [Indexed for MEDLINE]


33. Nucleic Acids Res. 2015 Mar 31;43(6):3079-88. doi: 10.1093/nar/gkv150. Epub 2015 
Mar 3.

The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli.

Cho S(1), Cho YB(2), Kang TJ(3), Kim SC(1), Palsson B(4), Cho BK(5).

Author information: 
(1)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, Daejeon 305-701, Republic of Korea KI for the BioCentury, Korea
Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.
(2)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, Daejeon 305-701, Republic of Korea.
(3)Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, 
Seoul 100-715, Republic of Korea.
(4)Department of Bioengineering, University of California, San Diego, La Jolla,
CA, USA Department of Pediatrics, University of California, San Diego, La Jolla, 
CA, USA Center for Biosustainability, Technical University of Denmark, Hørsholm, 
Denmark.
(5)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, Daejeon 305-701, Republic of Korea KI for the BioCentury, Korea
Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 
bcho@kaist.ac.kr.

DNA-binding motifs that are recognized by transcription factors (TFs) have been
well studied; however, challenges remain in determining the in vivo architecture 
of TF-DNA complexes on a genome-scale. Here, we determined the in vivo
architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using
high-throughput sequencing of exonuclease-treated chromatin-immunoprecipitated
DNA (ChIP-exo). The ChIP-exo has a unique peak-pair pattern indicating 5' and 3' 
ends of ArgR-binding region. We identified 62 ArgR-binding loci, which were
classified into three groups, comprising single, double and triple peak-pairs.
Each peak-pair has a unique 93 base pair (bp)-long (±2 bp) ArgR-binding sequence 
containing two ARG boxes (39 bp) and residual sequences. Moreover, the three
ArgR-binding modes defined by the position of the two ARG boxes indicate that DNA
bends centered between the pair of ARG boxes facilitate the non-specific contacts
between ArgR subunits and the residual sequences. Additionally, our approach may 
also reveal other fundamental structural features of TF-DNA interactions that
have implications for studying genome-scale transcriptional regulatory networks.

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

DOI: 10.1093/nar/gkv150 
PMCID: PMC4381063
PMID: 25735747  [Indexed for MEDLINE]


34. Elife. 2014 Dec 17;3:e03318. doi: 10.7554/eLife.03318.

Metagenomic chromosome conformation capture (meta3C) unveils the diversity of
chromosome organization in microorganisms.

Marbouty M(1), Cournac A(1), Flot JF(2), Marie-Nelly H(1), Mozziconacci J(3),
Koszul R(1).

Author information: 
(1)Groupe Régulation Spatiale des Génomes, Département Génomes et Génétique,
Institut Pasteur, Paris, France.
(2)Biological Physics and Evolutionary Dynamics Group, Max Planck Institute for
Dynamics and Self-Organization, Göttingen, Germany.
(3)Department of Physics, Laboratoire de physique théorique de la matière
condensée, Université Pierre et Marie Curie, Paris, France.

Genomic analyses of microbial populations in their natural environment remain
limited by the difficulty to assemble full genomes of individual species.
Consequently, the chromosome organization of microorganisms has been investigated
in a few model species, but the extent to which the features described can be
generalized to other taxa remains unknown. Using controlled mixes of bacterial
and yeast species, we developed meta3C, a metagenomic chromosome conformation
capture approach that allows characterizing individual genomes and their average 
organization within a mix of organisms. Not only can meta3C be applied to species
already sequenced, but a single meta3C library can be used for assembling,
scaffolding and characterizing the tridimensional organization of unknown
genomes. By applying meta3C to a semi-complex environmental sample, we confirmed 
its promising potential. Overall, this first meta3C study highlights the
remarkable diversity of microorganisms chromosome organization, while providing
an elegant and integrated approach to metagenomic analysis.

DOI: 10.7554/eLife.03318 
PMCID: PMC4381813
PMID: 25517076  [Indexed for MEDLINE]


35. PLoS One. 2014 Nov 6;9(11):e111962. doi: 10.1371/journal.pone.0111962.
eCollection 2014.

Characterization of the YdeO regulon in Escherichia coli.

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

Author information: 
(1)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.
(2)Graduate School of Information Sciences, Nara Institute of Science and
Technology, Ikoma, Nara, Japan.
(3)Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan;
Micro-Nano Technology Research Center, Hosei University, Koganei, Tokyo, Japan.

Enterobacteria are able to survive under stressful conditions within animals,
such as acidic conditions in the stomach, bile salts during transfer to the
intestine and anaerobic conditions within the intestine. The glutamate-dependent 
(GAD) system plays a major role in acid resistance in Escherichia coli, and
expression of the GAD system is controlled by the regulatory cascade consisting
of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip
to interrogate the E. coli genome for candidate YdeO binding sites. All of the
seven operons identified by ChIP-chip as being potentially regulated by YdeO were
confirmed as being under the direct control of YdeO using RT-qPCR, EMSA,
DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified 
four stress-response transcription factors, DctR, NhaR, GadE, and GadW and
enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation 
of the GAD system and NhaR is an activator for the sodium/proton antiporter gene.
In conjunction with co-transcribed Slp, DctR is involved in protection against
metabolic endoproducts under acidic conditions. Taken all together, we suggest
that YdeO is a key regulator of E. coli survival in both acidic and anaerobic
conditions.

DOI: 10.1371/journal.pone.0111962 
PMCID: PMC4222967
PMID: 25375160  [Indexed for MEDLINE]


36. PLoS Genet. 2014 Oct 2;10(10):e1004649. doi: 10.1371/journal.pgen.1004649.
eCollection 2014 Oct.

Comprehensive mapping of the Escherichia coli flagellar regulatory network.

Fitzgerald DM(1), Bonocora RP(2), Wade JT(3).

Author information: 
(1)Department of Biomedical Sciences, University at Albany, Albany, New York,
United States of America.
(2)Wadsworth Center, New York State Department of Health, Albany, New York,
United States of America.
(3)Department of Biomedical Sciences, University at Albany, Albany, New York,
United States of America; Wadsworth Center, New York State Department of Health, 
Albany, New York, United States of America.

Erratum in
    PLoS Genet. 2015 Sep;11(9):e1005456.
    PLoS Genet. 2014 Oct;10(10):e1004807.

Flagellar synthesis is a highly regulated process in all motile bacteria. In
Escherichia coli and related species, the transcription factor FlhDC is the
master regulator of a multi-tiered transcription network. FlhDC activates
transcription of a number of genes, including some flagellar genes and the gene
encoding the alternative Sigma factor FliA. Genes whose expression is required
late in flagellar assembly are primarily transcribed by FliA, imparting temporal 
regulation of transcription and coupling expression to flagellar assembly. In
this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC 
and FliA regulons. We define a surprisingly restricted FlhDC regulon, including
two novel regulated targets and two binding sites not associated with detectable 
regulation of surrounding genes. In contrast, we greatly expand the known FliA
regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. 
Two of these intragenic promoters are associated with detectable noncoding RNAs, 
while the others either produce highly unstable RNAs or are inactive under these 
conditions. Together, our data redefine the E. coli flagellar regulatory network,
and provide new insight into the temporal orchestration of gene expression that
coordinates the flagellar assembly process.

DOI: 10.1371/journal.pgen.1004649 
PMCID: PMC4183435
PMID: 25275371  [Indexed for MEDLINE]


37. 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 


38. J Bacteriol. 2014 Oct;196(20):3534-45. doi: 10.1128/JB.01589-14. Epub 2014 Jul
21.

Escherichia coli genes and pathways involved in surviving extreme exposure to
ionizing radiation.

Byrne RT(1), Chen SH(1), Wood EA(1), Cabot EL(2), Cox MM(3).

Author information: 
(1)Department of Biochemistry, University of Wisconsin-Madison, Madison,
Wisconsin, USA.
(2)Genome Center, University of Wisconsin, Madison, Wisconsin, USA.
(3)Department of Biochemistry, University of Wisconsin-Madison, Madison,
Wisconsin, USA cox@biochem.wisc.edu.

To further an improved understanding of the mechanisms used by bacterial cells to
survive extreme exposure to ionizing radiation (IR), we broadly screened
nonessential Escherichia coli genes for those involved in IR resistance by using 
transposon-directed insertion sequencing (TraDIS). Forty-six genes were
identified, most of which become essential upon heavy IR exposure. Most of these 
were subjected to direct validation. The results reinforced the notion that
survival after high doses of ionizing radiation does not depend on a single
mechanism or process, but instead is multifaceted. Many identified genes affect
either DNA repair or the cellular response to oxidative damage. However,
contributions by genes involved in cell wall structure/function, cell division,
and intermediary metabolism were also evident. About half of the identified genes
have not previously been associated with IR resistance or recovery from IR
exposure, including eight genes of unknown function.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

DOI: 10.1128/JB.01589-14 
PMCID: PMC4187691
PMID: 25049088  [Indexed for MEDLINE]


39. 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]


40. Science. 2014 Jun 13;344(6189):1285-9. doi: 10.1126/science.1253458.

Interactions between RNA polymerase and the "core recognition element" counteract
pausing.

Vvedenskaya IO(1), Vahedian-Movahed H(2), Bird JG(3), Knoblauch JG(1), Goldman
SR(1), Zhang Y(2), Ebright RH(4), Nickels BE(5).

Author information: 
(1)Department of Genetics and Waksman Institute, Rutgers University, Piscataway, 
NJ 08854, USA.
(2)Department of Chemistry and Waksman Institute, Rutgers University, Piscataway,
NJ 08854, USA.
(3)Department of Genetics and Waksman Institute, Rutgers University, Piscataway, 
NJ 08854, USA. Department of Chemistry and Waksman Institute, Rutgers University,
Piscataway, NJ 08854, USA.
(4)Department of Chemistry and Waksman Institute, Rutgers University, Piscataway,
NJ 08854, USA. bnickels@waksman.rutgers.edu ebright@waksman.rutgers.edu.
(5)Department of Genetics and Waksman Institute, Rutgers University, Piscataway, 
NJ 08854, USA. bnickels@waksman.rutgers.edu ebright@waksman.rutgers.edu.

Comment in
    Science. 2014 Jun 13;344(6189):1226-7.

Transcription elongation is interrupted by sequences that inhibit nucleotide
addition and cause RNA polymerase (RNAP) to pause. Here, by use of native
elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables 
analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze
transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a
consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to 
the position of the RNA 3' end). We demonstrate that sequence-specific
interactions between RNAP core enzyme and a core recognition element (CRE) that
stabilize transcription initiation complexes also occur in transcription
elongation complexes and facilitate pause read-through by stabilizing RNAP in a
posttranslocated register. Our findings identify key sequence determinants of
transcriptional pausing and establish that RNAP-CRE interactions modulate
pausing.

Copyright © 2014, American Association for the Advancement of Science.

DOI: 10.1126/science.1253458 
PMCID: PMC4277259
PMID: 24926020  [Indexed for MEDLINE]


41. PLoS Genet. 2014 Apr 3;10(4):e1004264. doi: 10.1371/journal.pgen.1004264.
eCollection 2014 Apr.

Determining the control circuitry of redox metabolism at the genome-scale.

Federowicz S(1), Kim D(2), Ebrahim A(2), Lerman J(1), Nagarajan H(2), Cho BK(2), 
Zengler K(3), Palsson B(3).

Author information: 
(1)Department of Bioengineering, University of California San Diego, La Jolla,
California, United States of America; Bioinformatics and Systems Biology Program,
University of California San Diego, La Jolla, California, United States of
America.
(2)Department of Bioengineering, University of California San Diego, La Jolla,
California, United States of America.
(3)Department of Bioengineering, University of California San Diego, La Jolla,
California, United States of America; Novo Nordisk Foundation Center for
Biosustainability, Technical University of Denmark, Lyngby, Denmark.

Determining how facultative anaerobic organisms sense and direct cellular
responses to electron acceptor availability has been a subject of intense study. 
However, even in the model organism Escherichia coli, established mechanisms only
explain a small fraction of the hundreds of genes that are regulated during
electron acceptor shifts. Here we propose a qualitative model that accounts for
the full breadth of regulated genes by detailing how two global transcription
factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act 
on a genome-wide basis to regulate anabolic, catabolic, and energy generation
pathways. We first fill gaps in our knowledge of this transcriptional regulatory 
network by carrying out ChIP-chip and gene expression experiments to identify 463
regulatory events. We then interfaced this reconstructed regulatory network with 
a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate 
>80% of total metabolic flux and 96% of differential gene expression across
fermentative and nitrate respiratory conditions. Based on the data, we propose a 
feedforward with feedback trim regulatory scheme, given the extensive repression 
of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr.
We further corroborated this regulatory scheme by showing a 0.71 r(2) (p<1e-6)
correlation between changes in metabolic flux and changes in regulatory activity 
across fermentative and nitrate respiratory conditions. Finally, we are able to
relate the proposed model to a wealth of previously generated data by
contextualizing the existing transcriptional regulatory network.

DOI: 10.1371/journal.pgen.1004264 
PMCID: PMC3974632
PMID: 24699140  [Indexed for MEDLINE]


42. J Biosci. 2014 Mar;39(1):53-61.

Inhibition of factor-dependent transcription termination in Escherichia coli
might relieve xenogene silencing by abrogating H-NS-DNA interactions in vivo.

Chandraprakash D(1), Seshasayee AS.

Author information: 
(1)National Centre for Biological Sciences, Tata Institute of Fundamental
Research, GKVK, Bellary Road, Bangalore 560 065, India.

Many horizontally acquired genes (xenogenes) in the bacterium Escherichia coli
are maintained in a silent transcriptional state by the nucleoid-associated
transcription regulatory protein H-NS. Recent evidence has shown that
antibiotic-mediated inhibition of the transcription terminator protein Rho leads 
to de-repression of horizontally acquired genes, akin to a deletion of hns. The
mechanism behind this similarity in outcomes between the perturbations of two
distinct processes remains unclear. Using ChIP-seq of H-NS in wild-type cells, in
addition to that in cells treated with bicyclomycin--a specific inhibitor of Rho,
we show that bicyclomycin treatment leads to a decrease in binding signal for
H-NS to the E. coli chromosome. Rho inhibition leads to RNA polymerase
readthrough, which in principle could displace H-NS from the DNA, thus leading to
transcriptional derepression of H-NS-silenced genes. Other possible mediators of 
the effect of Rho on H-NS are discussed. A possible positive feedback between Rho
and H-NS might help reinforce xenogene silencing.


PMID: 24499790  [Indexed for MEDLINE]


43. J Bacteriol. 2014 Feb;196(3):660-71. doi: 10.1128/JB.01007-13. Epub 2013 Nov 22.

Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal
noncanonical targets and an expanded core regulon.

Stringer AM(1), Currenti S, Bonocora RP, Baranowski C, Petrone BL, Palumbo MJ,
Reilly AA, Zhang Z, Erill I, Wade JT.

Author information: 
(1)Wadsworth Center, New York State Department of Health, Albany, New York, USA.

Escherichia coli AraC is a well-described transcription activator of genes
involved in arabinose metabolism. Using complementary genomic approaches,
chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we
identify direct regulatory targets of AraC, including five novel target genes:
ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established
connection to arabinose metabolism, suggesting that AraC has a broader function
than previously described. We demonstrate arabinose-dependent repression of ydeNM
by AraC, in contrast to the well-described arabinose-dependent activation of
other target genes. We also demonstrate unexpected read-through of transcription 
at the Rho-independent terminators downstream of araD and araE, leading to
significant increases in the expression of polB and ygeA, respectively. AraC is
highly conserved in the related species Salmonella enterica. We use ChIP
sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. 
enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with
a bioinformatic analysis of other related species, reveals a conserved regulatory
network across the family Enterobacteriaceae comprised of 10 genes associated
with arabinose transport and metabolism.

DOI: 10.1128/JB.01007-13 
PMCID: PMC3911152
PMID: 24272778  [Indexed for MEDLINE]


44. PLoS Genet. 2013 Nov;9(11):e1003902. doi: 10.1371/journal.pgen.1003902. Epub 2013
Nov 7.

Transposable prophage Mu is organized as a stable chromosomal domain of E. coli.

Saha RP(1), Lou Z, Meng L, Harshey RM.

Author information: 
(1)Department of Molecular Biosciences & Institute of Cellular and Molecular
Biology, University of Texas at Austin, Austin, Texas, United States of America.

The E. coli chromosome is compacted by segregation into 400-500 supercoiled
domains by both active and passive mechanisms, for example, transcription and
DNA-protein association. We find that prophage Mu is organized as a stable domain
bounded by the proximal location of Mu termini L and R, which are 37 kbp apart on
the Mu genome. Formation/maintenance of the Mu 'domain' configuration, reported
by Cre-loxP recombination and 3C (chromosome conformation capture), is dependent 
on a strong gyrase site (SGS) at the center of Mu, the Mu L end and MuB protein, 
and the E. coli nucleoid proteins IHF, Fis and HU. The Mu domain was observed at 
two different chromosomal locations tested. By contrast, prophage λ does not form
an independent domain. The establishment/maintenance of the Mu domain was
promoted by low-level transcription from two phage promoters, one of which was
domain dependent. We propose that the domain confers transposition readiness to
Mu by fostering topological requirements of the reaction and the proximity of Mu 
ends. The potential benefits to the host cell from a subset of proteins expressed
by the prophage may in turn help its long-term stability.

DOI: 10.1371/journal.pgen.1003902 
PMCID: PMC3820752
PMID: 24244182  [Indexed for MEDLINE]

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


45. PLoS Genet. 2013;9(10):e1003839. doi: 10.1371/journal.pgen.1003839. Epub 2013 Oct
17.

The bacterial response regulator ArcA uses a diverse binding site architecture to
regulate carbon oxidation globally.

Park DM(1), Akhtar MS, Ansari AZ, Landick R, Kiley PJ.

Author information: 
(1)Department of Biomolecular Chemistry, University of Wisconsin-Madison,
Madison, Wisconsin, United States of America.

Despite the importance of maintaining redox homeostasis for cellular viability,
how cells control redox balance globally is poorly understood. Here we provide
new mechanistic insight into how the balance between reduced and oxidized
electron carriers is regulated at the level of gene expression by mapping the
regulon of the response regulator ArcA from Escherichia coli, which responds to
the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our
genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic
conditions such that carbon oxidation pathways that recycle redox carriers via
respiration are transcriptionally repressed by ArcA. We propose that this
strategy favors use of catabolic pathways that recycle redox carriers via
fermentation akin to lactate production in mammalian cells. Unexpectedly,
bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that
most ArcA binding sites contain additional direct repeat elements beyond the two 
required for binding an ArcA dimer. DNase I footprinting assays suggest that
non-canonical arrangements of cis-regulatory modules dictate both the length and 
concentration-sensitive occupancy of DNA sites. We propose that this plasticity
in ArcA binding site architecture provides both an efficient means of encoding
binding sites for ArcA, σ(70)-RNAP and perhaps other transcription factors within
the same narrow sequence space and an effective mechanism for global control of
carbon metabolism to maintain redox homeostasis.

DOI: 10.1371/journal.pgen.1003839 
PMCID: PMC3798270
PMID: 24146625  [Indexed for MEDLINE]

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


46. PLoS Comput Biol. 2013;9(10):e1003246. doi: 10.1371/journal.pcbi.1003246. Epub
2013 Oct 17.

dPeak: high resolution identification of transcription factor binding sites from 
PET and SET ChIP-Seq data.

Chung D(1), Park D, Myers K, Grass J, Kiley P, Landick R, Keleş S.

Author information: 
(1)Department of Statistics, University of Wisconsin, Madison, Wisconsin, United 
States of America.

Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq)
has been successfully used for genome-wide profiling of transcription factor
binding sites, histone modifications, and nucleosome occupancy in many model
organisms and humans. Because the compact genomes of prokaryotes harbor many
binding sites separated by only few base pairs, applications of ChIP-Seq in this 
domain have not reached their full potential. Applications in prokaryotic genomes
are further hampered by the fact that well studied data analysis methods for
ChIP-Seq do not result in a resolution required for deciphering the locations of 
nearby binding events. We generated single-end tag (SET) and paired-end tag (PET)
ChIP-Seq data for σ⁷⁰ factor in Escherichia coli (E. coli). Direct comparison of 
these datasets revealed that although PET assay enables higher resolution
identification of binding events, standard ChIP-Seq analysis methods are not
equipped to utilize PET-specific features of the data. To address this problem,
we developed dPeak as a high resolution binding site identification
(deconvolution) algorithm. dPeak implements a probabilistic model that accurately
describes ChIP-Seq data generation process for both the SET and PET assays. For
SET data, dPeak outperforms or performs comparably to the state-of-the-art
high-resolution ChIP-Seq peak deconvolution algorithms such as PICS, GPS, and
GEM. When coupled with PET data, dPeak significantly outperforms SET-based
analysis with any of the current state-of-the-art methods. Experimental
validations of a subset of dPeak predictions from σ⁷⁰ PET ChIP-Seq data indicate 
that dPeak can estimate locations of binding events with as high as 2 to 21 bp
resolution. Applications of dPeak to σ⁷⁰ ChIP-Seq data in E. coli under aerobic
and anaerobic conditions reveal closely located promoters that are differentially
occupied and further illustrate the importance of high resolution analysis of
ChIP-Seq data.

DOI: 10.1371/journal.pcbi.1003246 
PMCID: PMC3798280
PMID: 24146601  [Indexed for MEDLINE]


47. PLoS Genet. 2013 Jun;9(6):e1003565. doi: 10.1371/journal.pgen.1003565. Epub 2013 
Jun 20.

Genome-scale analysis of escherichia coli FNR reveals complex features of
transcription factor binding.

Myers KS(1), Yan H, Ong IM, Chung D, Liang K, Tran F, Keleş S, Landick R, Kiley
PJ.

Author information: 
(1)Microbiology Doctoral Training Program, University of Wisconsin-Madison,
Madison, Wisconsin, USA.

FNR is a well-studied global regulator of anaerobiosis, which is widely conserved
across bacteria. Despite the importance of FNR and anaerobiosis in microbial
lifestyles, the factors that influence its function on a genome-wide scale are
poorly understood. Here, we report a functional genomic analysis of FNR action.
We find that FNR occupancy at many target sites is strongly influenced by
nucleoid-associated proteins (NAPs) that restrict access to many FNR binding
sites. At a genome-wide level, only a subset of predicted FNR binding sites were 
bound under anaerobic fermentative conditions and many appeared to be masked by
the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog 
StpA showed increased FNR occupancy at sites bound by H-NS in WT strains,
indicating that large regions of the genome are not readily accessible for FNR
binding. Genome accessibility may also explain our finding that genome-wide FNR
occupancy did not correlate with the match to consensus at binding sites,
suggesting that significant variation in ChIP signal was attributable to
cross-linking or immunoprecipitation efficiency rather than differences in
binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with
transcriptomic data showed that less than half of the FNR-regulated operons could
be attributed to direct FNR binding. Conversely, FNR bound some promoters without
regulating expression presumably requiring changes in activity of
condition-specific transcription factors. Such combinatorial regulation may allow
Escherichia coli to respond rapidly to environmental changes and confer an
ecological advantage in the anaerobic but nutrient-fluctuating environment of the
mammalian gut.

DOI: 10.1371/journal.pgen.1003565 
PMCID: PMC3688515
PMID: 23818864  [Indexed for MEDLINE]

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


48. Mol Microbiol. 2013 Jun;88(5):936-50. doi: 10.1111/mmi.12234. Epub 2013 May 5.

Integrated stress response of Escherichia coli to methylglyoxal: transcriptional 
readthrough from the nemRA operon enhances protection through increased
expression of glyoxalase I.

Ozyamak E(1), de Almeida C, de Moura AP, Miller S, Booth IR.

Author information: 
(1)School of Medical Sciences, Institute of Medical Sciences, University of
Aberdeen, Aberdeen, AB25 2ZD, UK. ozyamak@gmail.com

Methylglyoxal (MG) elicits activation of K(+) efflux systems to protect cells
against the toxicity of the electrophile. ChIP-chip targeting RNA polymerase,
supported by a range of other biochemical measurements and mutant creation, was
used to identify genes transcribed in response to MG and which complement this
rapid response. The SOS DNA repair regulon is induced at cytotoxic levels of MG, 
even when exposure to MG is transient. Glyoxalase I alone among the core MG
protective systems is induced in response to MG exposure. Increased expression is
an indirect consequence of induction of the upstream nemRA operon, encoding an
enzyme system that itself does not contribute to MG detoxification. Moreover,
this induction, via nemRA only occurs when cells are exposed to growth inhibitory
concentrations of MG. We show that the kdpFABCDE genes are induced and that this 
expression occurs as a result of depletion of cytoplasmic K(+) consequent upon
activation of the KefGB K(+) efflux system. Finally, our analysis suggests that
the transcriptional changes in response to MG are a culmination of the damage to 
DNA and proteins, but that some integrate specific functions, such as DNA repair,
to augment the allosteric activation of the main protective system, KefGB.

© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

DOI: 10.1111/mmi.12234 
PMCID: PMC3739934
PMID: 23646895  [Indexed for MEDLINE]


49. Nucleic Acids Res. 2013 Jul;41(12):6058-71. doi: 10.1093/nar/gkt325. Epub 2013
Apr 30.

Genome conformation capture reveals that the Escherichia coli chromosome is
organized by replication and transcription.

Cagliero C(1), Grand RS, Jones MB, Jin DJ, O'Sullivan JM.

Author information: 
(1)Gene Regulation and Chromosome Biology Laboratory, Frederick National
Laboratory for Cancer Research, National Cancer Institute, National Institutes of
Health, Frederick, MD 21702, USA.

To fit within the confines of the cell, bacterial chromosomes are highly
condensed into a structure called the nucleoid. Despite the high degree of
compaction in the nucleoid, the genome remains accessible to essential biological
processes, such as replication and transcription. Here, we present the first
high-resolution chromosome conformation capture-based molecular analysis of the
spatial organization of the Escherichia coli nucleoid during rapid growth in rich
medium and following an induced amino acid starvation that promotes the stringent
response. Our analyses identify the presence of origin and terminus domains in
exponentially growing cells. Moreover, we observe an increased number of
interactions within the origin domain and significant clustering of SeqA-binding 
sequences, suggesting a role for SeqA in clustering of newly replicated
chromosomes. By contrast, 'histone-like' protein (i.e. Fis, IHF and H-NS)
-binding sites did not cluster, and their role in global nucleoid organization
does not manifest through the mediation of chromosomal contacts. Finally, genes
that were downregulated after induction of the stringent response were spatially 
clustered, indicating that transcription in E. coli occurs at transcription foci.

DOI: 10.1093/nar/gkt325 
PMCID: PMC3695519
PMID: 23632166  [Indexed for MEDLINE]


50. BMC Genomics. 2013 Apr 15;14:254. doi: 10.1186/1471-2164-14-254.

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

Bonocora RP, Fitzgerald DM, Stringer AM, Wade JT.

BACKGROUND: ChIP-chip and ChIP-seq are widely used methods to map protein-DNA
interactions on a genomic scale in vivo. Waldminghaus and Skarstad recently
reported, in this journal, a modified method for ChIP-chip. Based on a comparison
of our previously-published ChIP-chip data for Escherichia coli σ32 with their
own data, Waldminghaus and Skarstad concluded that many of the σ32 targets
identified in our earlier work are false positives. In particular, we identified 
many non-canonical σ32 targets that are located inside genes or are associated
with genes that show no detectable regulation by σ32. Waldminghaus and Skarstad
propose that such non-canonical sites are artifacts, identified due to flaws in
the standard ChIP methodology. Waldminghaus and Skarstad suggest specific changes
to the standard ChIP procedure that reportedly eliminate the claimed artifacts.
RESULTS: We reanalyzed our published ChIP-chip datasets for σ32 and the datasets 
generated by Waldminghaus and Skarstad to assess data quality and
reproducibility. We also performed targeted ChIP/qPCR for σ32 and an unrelated
transcription factor, AraC, using the standard ChIP method and the modified ChIP 
method proposed by Waldminghaus and Skarstad. Furthermore, we determined the
association of core RNA polymerase with disputed σ32 promoters, with and without 
overexpression of σ32. We show that (i) our published σ32 ChIP-chip datasets have
a consistently higher dynamic range than those of Waldminghaus and Skarstad, (ii)
our published σ32 ChIP-chip datasets are highly reproducible, whereas those of
Waldminghaus and Skarstad are not, (iii) non-canonical σ32 target regions are
enriched in a σ32 ChIP in a heat shock-dependent manner, regardless of the ChIP
method used, (iv) association of core RNA polymerase with some disputed σ32
target genes is induced by overexpression of σ32, (v) σ32 targets disputed by
Waldminghaus and Skarstad are predominantly those that are most weakly bound, and
(vi) the modifications to the ChIP method proposed by Waldminghaus and Skarstad
reduce enrichment of all protein-bound genomic regions.
CONCLUSIONS: The modifications to the ChIP-chip method suggested by Waldminghaus 
and Skarstad reduce rather than increase the quality of ChIP data. Hence, the
non-canonical σ32 targets identified in our previous study are likely to be
genuine. We propose that the failure of Waldminghaus and Skarstad to identify
many of these σ32 targets is due predominantly to the lower data quality in their
study. We conclude that surprising ChIP-chip results are not artifacts to be
ignored, but rather indications that our understanding of DNA-binding proteins is
incomplete.

DOI: 10.1186/1471-2164-14-254 
PMCID: PMC3738151
PMID: 23586855  [Indexed for MEDLINE]


51. 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]


52. PLoS One. 2012;7(10):e47314. doi: 10.1371/journal.pone.0047314. Epub 2012 Oct 5.

Genome-wide PhoB binding and gene expression profiles reveal the hierarchical
gene regulatory network of phosphate starvation in Escherichia coli.

Yang C(1), Huang TW, Wen SY, Chang CY, Tsai SF, Wu WF, Chang CH.

Author information: 
(1)Institute of Biomedical Informatics, Center for Systems and Synthetic Biology,
National Yang Ming University, Taipei, Taiwan.

The phosphate starvation response in bacteria has been studied extensively for
the past few decades and the phosphate-limiting signal is known to be mediated
via the PhoBR two-component system. However, the global DNA binding profile of
the response regulator PhoB and the PhoB downstream responses are currently
unclear. In this study, chromatin immunoprecipitation for PhoB was combined with 
high-density tiling array (ChIP-chip) as well as gene expression microarray to
reveal the first global down-stream responses of the responding regulator, PhoB
in E. coli. Based on our ChIP-chip experimental data, forty-three binding sites
were identified throughout the genome and the known PhoB binding pattern was
updated by identifying the conserved pattern from these sites. From the gene
expression microarray data analysis, 287 differentially expressed genes were
identified in the presence of PhoB activity. By comparing the results obtained
from our ChIP-chip and microarray experiments, we were also able to identify
genes that were directly or indirectly affected through PhoB regulation. Nineteen
out of these 287 differentially expressed genes were identified as the genes
directly regulated by PhoB. Seven of the 19 directly regulated genes (including
phoB) are transcriptional regulators. These transcriptional regulators then
further pass the signal of phosphate starvation down to the remaining
differentially expressed genes. Our results unveiled the genome-wide binding
profile of PhoB and the downstream responses under phosphate starvation. We also 
present the hierarchical structure of the phosphate sensing regulatory network.
The data suggest that PhoB plays protective roles in membrane integrity and
oxidative stress reduction during phosphate starvation.

DOI: 10.1371/journal.pone.0047314 
PMCID: PMC3465305
PMID: 23071782  [Indexed for MEDLINE]


53. 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]


54. PLoS One. 2012;7(7):e40207. doi: 10.1371/journal.pone.0040207. Epub 2012 Jul 2.

T7 RNA polymerase functions in vitro without clustering.

Finan K(1), Torella JP, Kapanidis AN, Cook PR.

Author information: 
(1)Sir William Dunn School of Pathology, University of Oxford, Oxford, United
Kingdom.

Many nucleic acid polymerases function in clusters known as factories. We
investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when
active. Using 'pulldowns' and fluorescence correlation spectroscopy we find that 
elongation complexes do not interact in vitro with a K(d)<1 µM. Chromosome
conformation capture also reveals that genes located 100 kb apart on the E. coli 
chromosome do not associate more frequently when transcribed by T7 RNAP. We
conclude that if clustering does occur in vivo, it must be driven by weak
interactions, or mediated by a phage-encoded protein.

DOI: 10.1371/journal.pone.0040207 
PMCID: PMC3388079
PMID: 22768341  [Indexed for MEDLINE]


55. Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11336-41. doi:
10.1073/pnas.1208595109. Epub 2012 Jun 25.

Galactose repressor mediated intersegmental chromosomal connections in
Escherichia coli.

Qian Z(1), Dimitriadis EK, Edgar R, Eswaramoorthy P, Adhya S.

Author information: 
(1)Laboratory of Molecular Biology, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892, USA.

By microscopic analysis of fluorescent-labeled GalR, a regulon-specific
transcription factor in Escherichia coli, we observed that GalR is present in the
cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. 
To investigate whether these foci represent GalR-mediated association of some of 
the GalR specific DNA binding sites (gal operators), we used the chromosome
conformation capture (3C) method in vivo. Our 3C data demonstrate that, in
stationary phase cells, many of the operators distributed around the chromosome
are interacted. By the use of atomic force microscopy, we showed that the
observed remote chromosomal interconnections occur by direct interactions between
DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with
three or five operators positioned at defined loci showed GalR-dependent loops of
expected sizes of the intervening DNA segments. Our findings provide unique
evidence that a transcription factor participates in organizing the chromosome in
a three-dimensional structure. We believe that these chromosomal connections
increase local concentration of GalR for coordinating the regulation of widely
separated target genes, and organize the chromosome structure in space, thereby
likely contributing to chromosome compaction.

DOI: 10.1073/pnas.1208595109 
PMCID: PMC3396475
PMID: 22733746  [Indexed for MEDLINE]


56. 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]


57. Nucleic Acids Res. 2012 Apr;40(8):3524-37. doi: 10.1093/nar/gkr1236. Epub 2011
Dec 17.

Genomic analysis of DNA binding and gene regulation by homologous
nucleoid-associated proteins IHF and HU in Escherichia coli K12.

Prieto AI(1), Kahramanoglou C, Ali RM, Fraser GM, Seshasayee AS, Luscombe NM.

Author information: 
(1)Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QP, UK.

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong
to the same protein family but interact differently with the DNA. IHF is a
sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the 
most conserved NAP, which binds non-specifically to duplex DNA with a particular 
preference for targeting nicked and bent DNA. Despite their importance, the in
vivo interactions of the two proteins to the DNA remain to be described at a high
resolution and on a genome-wide scale. Further, the effects of these proteins on 
gene expression on a global scale remain contentious. Finally, the contrast
between the functions of the homo- and heterodimeric forms of proteins deserves
the attention of further study. Here we present a genome-scale study of HU- and
IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also
perform microarray analysis of gene expression in single- and double-deletion
mutants of each protein to identify their regulons. The sequence-specific binding
profile of IHF encompasses ∼30% of all operons, though the expression of <10% of 
these is affected by its deletion suggesting combinatorial control or a molecular
backup. The binding profile for HU is reflective of relatively non-specific
binding to the chromosome, however, with a preference for A/T-rich DNA. The HU
regulon comprises highly conserved genes including those that are essential and
possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments,
where possible, of each subunit of IHF and HU in the absence of the other
subunit, we define genome-wide maps of DNA binding of the proteins in their
hetero- and homodimeric forms.

DOI: 10.1093/nar/gkr1236 
PMCID: PMC3333857
PMID: 22180530  [Indexed for MEDLINE]


58. Nat Chem Biol. 2011 Nov 13;8(1):65-71. doi: 10.1038/nchembio.710.

Deciphering the transcriptional regulatory logic of amino acid metabolism.

Cho BK(1), Federowicz S, Park YS, Zengler K, Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California at San Diego, La Jolla,
California, USA. bkcho01@gmail.com

Comment in
    Nat Chem Biol. 2012 Jan;8(1):23-4.

Although metabolic networks have been reconstructed on a genome scale, the
corresponding reconstruction and integration of governing transcriptional
regulatory networks has not been fully achieved. Here we reconstruct such an
integrated network for amino acid metabolism in Escherichia coli. Analysis of
ChIP-chip and gene expression data for the transcription factors ArgR, Lrp and
TrpR showed that 19 out of 20 amino acid biosynthetic pathways are either
directly or indirectly controlled by these regulators. Classifying the regulated 
genes into three functional categories of transport, biosynthesis and metabolism 
leads to the elucidation of regulatory motifs that constitute the integrated
network's basic building blocks. The regulatory logic of these motifs was
determined on the basis of relationships between transcription factor binding and
changes in the amount of transcript in response to exogenous amino acids.
Remarkably, the resulting logic shows how amino acids are differentiated as
signaling and nutrient molecules, revealing the overarching regulatory principles
of the amino acid stimulon.

DOI: 10.1038/nchembio.710 
PMCID: PMC3777760
PMID: 22082910  [Indexed for MEDLINE]


59. Science. 2011 Sep 9;333(6048):1445-9. doi: 10.1126/science.1204697.

Chromosome organization by a nucleoid-associated protein in live bacteria.

Wang W(1), Li GW, Chen C, Xie XS, Zhuang X.

Author information: 
(1)Department of Physics, Harvard University, Cambridge, MA 02138, USA.

Bacterial chromosomes are confined in submicrometer-sized nucleoids. Chromosome
organization is facilitated by nucleoid-associated proteins (NAPs), but the
mechanisms of action remain elusive. In this work, we used super-resolution
fluorescence microscopy, in combination with a chromosome-conformation capture
assay, to study the distributions of major NAPs in live Escherichia coli cells.
Four NAPs--HU, Fis, IHF, and StpA--were largely scattered throughout the
nucleoid. In contrast, H-NS, a global transcriptional silencer, formed two
compact clusters per chromosome, driven by oligomerization of DNA-bound H-NS
through interactions mediated by the amino-terminal domain of the protein. H-NS
sequestered the regulated operons into these clusters and juxtaposed numerous DNA
segments broadly distributed throughout the chromosome. Deleting H-NS led to
substantial chromosome reorganization. These observations demonstrate that H-NS
plays a key role in global chromosome organization in bacteria.

DOI: 10.1126/science.1204697 
PMCID: PMC3329943
PMID: 21903814  [Indexed for MEDLINE]


60. Nucleic Acids Res. 2011 Aug;39(15):6456-64. doi: 10.1093/nar/gkr307. Epub 2011
May 13.

The PurR regulon in Escherichia coli K-12 MG1655.

Cho BK(1), Federowicz SA, Embree M, Park YS, Kim D, Palsson BØ.

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

The PurR transcription factor plays a critical role in transcriptional regulation
of purine metabolism in enterobacteria. Here, we elucidate the role of PurR under
exogenous adenine stimulation at the genome-scale using high-resolution chromatin
immunoprecipitation (ChIP)-chip and gene expression data obtained under in vivo
conditions. Analysis of microarray data revealed that adenine stimulation led to 
changes in transcript level of about 10% of Escherichia coli genes, including the
purine biosynthesis pathway. The E. coli strain lacking the purR gene showed that
a total of 56 genes are affected by the deletion. From the ChIP-chip analysis, we
determined that over 73% of genes directly regulated by PurR were enriched in the
biosynthesis, utilization and transport of purine and pyrimidine nucleotides, and
20% of them were functionally unknown. Compared to the functional diversity of
the regulon of the other general transcription factors in E. coli, the functions 
and size of the PurR regulon are limited.

DOI: 10.1093/nar/gkr307 
PMCID: PMC3159470
PMID: 21572102  [Indexed for MEDLINE]


61. Nucleic Acids Res. 2011 Mar;39(6):2073-91. doi: 10.1093/nar/gkq934. Epub 2010 Nov
21.

Direct and indirect effects of H-NS and Fis on global gene expression control in 
Escherichia coli.

Kahramanoglou C(1), Seshasayee AS, Prieto AI, Ibberson D, Schmidt S, Zimmermann
J, Benes V, Fraser GM, Luscombe NM.

Author information: 
(1)Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QP, UK.

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in
Escherichia coli, which affect DNA conformation by bending, wrapping and bridging
the DNA. Two of these--H-NS and Fis--bind to specific DNA sequences and
structures. Because of their importance to global gene expression, the binding of
these NAPs to the DNA was previously investigated on a genome-wide scale using
ChIP-chip. However, variation in their binding profiles across the growth phase
and the genome-scale nature of their impact on gene expression remain poorly
understood. Here, we present a genome-scale investigation of H-NS and Fis binding
to the E. coli chromosome using chromatin immunoprecipitation combined with
high-throughput sequencing (ChIP-seq). By performing our experiments under
multiple time-points during growth in rich media, we show that the binding
regions of the two proteins are mutually exclusive under our experimental
conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent
with the linear spread of H-NS binding from high- to surrounding lower-affinity
sites; the length of binding regions is associated with the degree of
transcriptional repression imposed by H-NS. For Fis, a majority of binding events
do not lead to differential expression of the proximal gene; however, it has a
significant indirect effect on gene expression partly through its effects on the 
expression of other transcription factors. We propose that direct transcriptional
regulation by Fis is associated with the interaction of tandem arrays of Fis
molecules to the DNA and possible DNA bending, particularly at operon-upstream
regions. Our study serves as a proof-of-principle for the use of ChIP-seq for
global DNA-binding proteins in bacteria, which should become significantly more
economical and feasible with the development of multiplexing techniques.

DOI: 10.1093/nar/gkq934 
PMCID: PMC3064808
PMID: 21097887  [Indexed for MEDLINE]


62. BMC Genomics. 2010 Jul 5;11:414. doi: 10.1186/1471-2164-11-414.

ChIP on Chip: surprising results are often artifacts.

Waldminghaus T(1), Skarstad K.

Author information: 
(1)Department of Cell Biology, Institute for Cancer Research, The Norwegian
Radium Hospital, Oslo University Hospital and University of Oslo, 0310 Oslo,
Norway.

BACKGROUND: The method of chromatin immunoprecipitation combined with microarrays
(ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding.
However, a high background signal is a common phenomenon.
RESULTS: Reinvestigation of the chromatin immunoprecipitation procedure led us to
discover four causes of high background: i) non-unique sequences, ii) incomplete 
reversion of crosslinks, iii) retention of protein in spin-columns and iv)
insufficient RNase treatment. The chromatin immunoprecipitation method was
modified and applied to analyze genome-wide binding of SeqA and sigma(32) in
Escherichia coli.
CONCLUSIONS: False positive findings originating from these shortcomings of the
method could explain surprising and contradictory findings in published ChIP-Chip
studies. We present a modified chromatin immunoprecipitation method greatly
reducing the background signal.

DOI: 10.1186/1471-2164-11-414 
PMCID: PMC2996942
PMID: 20602746  [Indexed for MEDLINE]


63. Proc Natl Acad Sci U S A. 2009 Sep 8;106(36):15406-11. doi:
10.1073/pnas.0903846106. Epub 2009 Aug 20.

Rho directs widespread termination of intragenic and stable RNA transcription.

Peters JM(1), Mooney RA, Kuan PF, Rowland JL, Keles S, Landick R.

Author information: 
(1)Department of Biochemistry, University of Wisconsin, Madison, WI 5370, USA.

The transcription termination factor Rho is a global regulator of RNA polymerase 
(RNAP). Although individual Rho-dependent terminators have been studied
extensively, less is known about the sites of RNAP regulation by Rho on a
genome-wide scale. Using chromatin immunoprecipitation and microarrays
(ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in 
response to the Rho-specific inhibitor bicyclomycin (BCM). We found approximately
200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at
the ends of genes) and intragenic (within genes). The intergenic class contained 
noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing
a previously unappreciated role of Rho in termination of stable RNA synthesis.
The intragenic class of terminators included a previously uncharacterized set of 
short antisense transcripts, as judged by a shift in the distribution of RNAP in 
BCM-treated cells that was opposite to the direction of the corresponding gene.
These Rho-terminated antisense transcripts point to a role of noncoding
transcription in E. coli gene regulation that may resemble the ubiquitous
noncoding transcription recently found to play myriad roles in eukaryotic gene
regulation.

DOI: 10.1073/pnas.0903846106 
PMCID: PMC2741264
PMID: 19706412  [Indexed for MEDLINE]


64. Mol Cell. 2009 Aug 14;35(3):255-6. doi: 10.1016/j.molcel.2009.07.019.

Can the protein occupancy landscape show the topologically isolated chromosomal
domains in the E. coli genome?: An exciting prospect.

Cho BK(1), Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California, San Diego, La Jolla,
92093, USA.

Comment on
    Mol Cell. 2009 Jul 31;35(2):247-53.

In a recent issue of Molecular Cell, Vora et al. (2009) introduce an in vivo
protein occupancy display (IPOD) technology and propose that the domains
extensively occupied by proteins with inactive transcription in bacteria are
mainly topologically isolated chromosomal domains.

DOI: 10.1016/j.molcel.2009.07.019 
PMID: 19683488  [Indexed for MEDLINE]


65. Mol Microbiol. 2009 Aug;73(4):680-94. doi: 10.1111/j.1365-2958.2009.06799.x. Epub
2009 Jul 27.

NsrR targets in the Escherichia coli genome: new insights into DNA sequence
requirements for binding and a role for NsrR in the regulation of motility.

Partridge JD(1), Bodenmiller DM, Humphrys MS, Spiro S.

Author information: 
(1)Department of Molecular and Cell Biology, The University of Texas at Dallas,
Richardson, TX 75080, USA.

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of
transcription. The NsrR-binding site is predicted to comprise two copies of an 11
bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we
confirmed that both 11 bp motifs are required for maximal NsrR repression of the 
ytfE promoter. We used chromatin immunoprecipitation and microarray analysis
(ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes.
Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the
consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo.
NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and
mqsR-ygiT transcription units, which encode proteins involved in motility and
biofilm development. Reporter fusion assays confirmed that NsrR negatively
regulates the fliA and fliL promoters. A mutation in the predicted 11 bp
NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented 
detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative
regulator of motility in E. coli K12 and in a uropathogenic strain; surface
attachment assays revealed decreased levels of attached growth in the absence of 
NsrR.

DOI: 10.1111/j.1365-2958.2009.06799.x 
PMID: 19656291  [Indexed for MEDLINE]


66. Mol Cell. 2009 Jul 31;35(2):247-53. doi: 10.1016/j.molcel.2009.06.035.

Protein occupancy landscape of a bacterial genome.

Vora T(1), Hottes AK, Tavazoie S.

Author information: 
(1)Department of Molecular Biology, Princeton University, Princeton, NJ 08544,
USA.

Comment in
    Mol Cell. 2009 Aug 14;35(3):255-6.

Protein-DNA interactions are fundamental to core biological processes, including 
transcription, DNA replication, and chromosomal organization. We have developed
in vivo protein occupancy display (IPOD), a technology that reveals protein
occupancy across an entire bacterial chromosome at the resolution of individual
binding sites. Application to Escherichia coli reveals thousands of protein
occupancy peaks, highly enriched within and in close proximity to noncoding
regulatory regions. In addition, we discovered extensive (>1 kilobase) protein
occupancy domains (EPODs), some of which are localized to highly expressed genes,
enriched in RNA-polymerase occupancy. However, the majority are localized to
transcriptionally silent loci dominated by conserved hypothetical ORFs. These
regions are highly enriched in both predicted and experimentally determined
binding sites of nucleoid proteins and exhibit extreme biophysical
characteristics such as high intrinsic curvature. Our observations implicate
these transcriptionally silent EPODs as the elusive organizing centers, long
proposed to topologically isolate chromosomal domains.

DOI: 10.1016/j.molcel.2009.06.035 
PMCID: PMC2763621
PMID: 19647521  [Indexed for MEDLINE]


67. Mol Cell. 2009 Jan 16;33(1):97-108. doi: 10.1016/j.molcel.2008.12.021.

Regulator trafficking on bacterial transcription units in vivo.

Mooney RA(1), Davis SE, Peters JM, Rowland JL, Ansari AZ, Landick R.

Author information: 
(1)Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

The trafficking patterns of the bacterial regulators of transcript elongation
sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for
promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E.
coli ChIP-chip revealed distinct association patterns of regulators as RNAP
transcribes away from promoters (rho first, then NusA, then NusG). However, the
interactions of elongating complexes with these regulators did not differ
significantly among most transcription units. A modest variation of NusG signal
among genes reflected increased NusG interaction as transcription progresses,
rather than functional specialization of elongating complexes. Promoter-proximal 
RNAP peaks were offset from sigma(70) peaks in the direction of transcription and
co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected
elongating, rather than initiating, complexes. However, inhibition of rho did not
increase RNAP levels within genes downstream from the RNAP peaks, suggesting the 
peaks are caused by a mechanism other than rho-dependent attenuation.

DOI: 10.1016/j.molcel.2008.12.021 
PMCID: PMC2747249
PMID: 19150431  [Indexed for MEDLINE]


68. Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19462-7. doi:
10.1073/pnas.0807227105. Epub 2008 Dec 3.

Genome-scale reconstruction of the Lrp regulatory network in Escherichia coli.

Cho BK(1), Barrett CL, Knight EM, Park YS, Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California at San Diego, 9500
Gilman Dr., La Jolla, CA 92093-0412, USA.

Broad-acting transcription factors (TFs) in bacteria form regulons. Here, we
present a 4-step method to fully reconstruct the leucine-responsive protein (Lrp)
regulon in Escherichia coli K-12 MG 1655 that regulates nitrogen metabolism. Step
1 is composed of obtaining high-resolution ChIP-chip data for Lrp, the RNA
polymerase and expression profiles under multiple environmental conditions. We
identified 138 unique and reproducible Lrp-binding regions and classified their
binding state under different conditions. In the second step, the analysis of
these data revealed 6 distinct regulatory modes for individual ORFs. In the third
step, we used the functional assignment of the regulated ORFs to reconstruct 4
types of regulatory network motifs around the metabolites that are affected by
the corresponding gene products. In the fourth step, we determined how leucine,
as a signaling molecule, shifts the regulatory motifs for particular metabolites.
The physiological structure that emerges shows the regulatory motifs for
different amino acid fall into the traditional classification of amino acid
families, thus elucidating the structure and physiological functions of the
Lrp-regulon. The same procedure can be applied to other broad-acting TFs, opening
the way to full bottom-up reconstruction of the transcriptional regulatory
network in bacterial cells.

DOI: 10.1073/pnas.0807227105 
PMCID: PMC2614783
PMID: 19052235  [Indexed for MEDLINE]


69. J Bacteriol. 2008 Sep;190(18):6170-7. doi: 10.1128/JB.00508-08. Epub 2008 Jul 25.

Escherichia coli NsrR regulates a pathway for the oxidation of 3-nitrotyramine to
4-hydroxy-3-nitrophenylacetate.

Rankin LD(1), Bodenmiller DM, Partridge JD, Nishino SF, Spain JC, Spiro S.

Author information: 
(1)Department of Molecular and Cell Biology, the University of Texas at Dallas,
800 W Campbell Road, Richardson, TX 75080, USA.

Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the
nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to
the promoters of the tynA and feaB genes. These genes encode the first two
enzymes of a pathway that is required for the catabolism of phenylethylamine
(PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR
caused small increases in the activities of the tynA and feaB promoters in
cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and
caused a marked repression of the tynA and feaB promoters. Both the growth defect
and the promoter repression were reversed in the presence of a source of NO.
These results are consistent with NsrR mediating repression of the tynA and feaB 
genes by binding (in an NO-sensitive fashion) to the sites identified by
ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for
growth, conditions which partially induce the tynA and feaB promoters. Mutation
of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant
phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine
is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the
expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed 
that 3-nitrotyramine and its oxidation product
(4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by
tynA and feaB, respectively. The results suggest that an additional physiological
role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that
may accumulate in cells exposed to NO.

DOI: 10.1128/JB.00508-08 
PMCID: PMC2546798
PMID: 18658270  [Indexed for MEDLINE]


70. Methods Mol Biol. 2008;439:131-45. doi: 10.1007/978-1-59745-188-8_9.

Genomewide identification of protein binding locations using chromatin
immunoprecipitation coupled with microarray.

Cho BK(1), Knight EM, Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California-San Diego, La Jolla,
CA, USA.

Interactions between cis-acting elements and proteins play a key role in
transcriptional regulation of all known organisms. To better understand these
interactions, researchers developed a method that couples chromatin
immunoprecipitation with microarrays (also known as ChIP-chip), which is capable 
of providing a whole-genome map of protein-DNA interactions. This versatile and
high-throughput strategy is initiated by formaldehyde-mediated cross-linking of
DNA and proteins, followed by cell lysis, DNA fragmentation, and
immunopurification. The immunoprecipitated DNA fragments are then purified from
the proteins by reverse-cross-linking followed by amplification, labeling, and
hybridization to a whole-genome tiling microarray against a reference sample. The
enriched signals obtained from the microarray then are normalized by the
reference sample and used to generate the whole-genome map of protein-DNA
interactions. The protocol described here has been used for discovering the
genomewide distribution of RNA polymerase and several transcription factors of
Escherichia coli.

DOI: 10.1007/978-1-59745-188-8_9 
PMID: 18370100  [Indexed for MEDLINE]


71. Genome Res. 2008 Jun;18(6):900-10. doi: 10.1101/gr.070276.107. Epub 2008 Mar 13.

Genome-wide analysis of Fis binding in Escherichia coli indicates a causative
role for A-/AT-tracts.

Cho BK(1), Knight EM, Barrett CL, Palsson BØ.

Author information: 
(1)Department of Bioengineering, University of California-San Diego, La Jolla,
California 92093-0412, USA.

We determined the genome-wide distribution of the nucleoid-associated protein Fis
in Escherichia coli using chromatin immunoprecipitation coupled with
high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated
regions across the E. coli genome. A significant number of these binding sites
were found within open reading frames (33%) and between divergently transcribed
transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important
signal for preferred Fis-binding sites, and that A(6)-tracts in particular
constitute a high-affinity signal that dictates Fis phasing in stretches of DNA
containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we
find evidence for an average of two Fis-binding regions per supercoiling domain
in the chromosome of exponentially growing cells. Transcriptome analysis shows
that approximately 21% of genes are affected by the deletion of fis; however, the
changes in magnitude are small. To address the differential Fis bindings under
growth environment perturbation, ChIP-chip analysis was performed using cells
grown under aerobic and anaerobic growth conditions. Interestingly, the
Fis-binding regions are almost identical in aerobic and anaerobic growth
conditions-indicating that the E. coli genome topology mediated by Fis is
superficially identical in the two conditions. These novel results provide new
insight into how Fis modulates DNA topology at a genome scale and thus advance
our understanding of the architectural bases of the E. coli nucleoid.

DOI: 10.1101/gr.070276.107 
PMCID: PMC2413157
PMID: 18340041  [Indexed for MEDLINE]


72. J Bacteriol. 2005 Sep;187(17):6166-74.

Immobilization of Escherichia coli RNA polymerase and location of binding sites
by use of chromatin immunoprecipitation and microarrays.

Herring CD(1), Raffaelle M, Allen TE, Kanin EI, Landick R, Ansari AZ, Palsson BØ.

Author information: 
(1)Department of Engineering, UC San Diego Bioengineering, 9500 Gilman Drive,
Dept 0412, La Jolla, CA 92093-0412, USA.

The genome-wide location of RNA polymerase binding sites was determined in
Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip).
Cross-linked chromatin was isolated in triplicate from rifampin-treated cells,
and DNA bound to RNA polymerase was precipitated with an antibody specific for
the beta' subunit. The DNA was amplified and hybridized to "tiled"
oligonucleotide microarrays representing the whole genome at 25-bp resolution. A 
total of 1,139 binding sites were detected and evaluated by comparison to gene
expression data from identical conditions and to 961 promoters previously
identified by established methods. Of the detected binding sites, 418 were
located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA
polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of 
the known sigma70-specific promoters occurring upstream of an expressed open
reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters
were not detected by chIP-chip, leading to an estimated 26% negative-detection
rate. Most of the detected binding sites could be associated with expressed
transcription units, but 299 binding sites occurred near inactive transcription
units. This map of RNA polymerase binding sites represents a foundation for
studies of transcription factors in E. coli and an important evaluation of the
chIP-chip technique.

DOI: 10.1128/JB.187.17.6166-6174.2005 
PMCID: PMC1196165
PMID: 16109958  [Indexed for MEDLINE]