Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli

ABSTRACT Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd. Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

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Tombusvirus p19 captures RNase III-cleaved double-stranded RNAs formed by overlapping sense and antisense transcripts in E. coli

10.1101/510842 ◽

2019 ◽

Author(s):

Linfeng Huang ◽

Padraig Deighan ◽

Jingmin Jin ◽

Yingxue Li ◽

Elaine Lee ◽

...

Keyword(s):

Stationary Phase ◽

Ectopic Expression ◽

Antisense Transcription ◽

Bacterial Cells ◽

Rnase Iii ◽

Antisense Transcripts ◽

E Coli ◽

Physiological Importance ◽

Protein Levels ◽

Bacterial Genes

AbstractAntisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form long double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. Ectopic expression of plant tombusvirus p19 in E. coli stabilizes ~21 bp dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA and total RNA. dsRNA formed at most bacterial genes in the bacterial chromosome and in a plasmid. The most abundant dsRNA clusters were mostly formed by divergent transcription of sense and antisense transcripts overlapping at their 5’-ends. The most abundant clusters included small RNAs, such as ryeA/ryeB, 4 toxin-antitoxin genes, and 3 tRNAs, and some longer coding genes, including rsd and cspD. The sense and antisense transcripts in abundant dsRNA clusters were more plentiful and had longer half-lives in RNase III mutant strains, suggesting that formation of dsRNAs promoted RNA decay at these loci. However, widespread changes in protein levels did not occur in RNase III mutant bacteria. Nonetheless, some proteins involved in antioxidant responses and glycolysis changed reproducibly. dsRNAs accumulated in bacterial cells lacking RNase III, increasing in stationary phase, and correlated with increased cell death in RNase III mutant bacteria in late stationary phase. The physiological importance of widespread antisense transcription in bacteria remains unclear but it may become important during environmental stress. Ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in bacteria.

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Widespread Antisense Transcription in Escherichia coli

mBio ◽

10.1128/mbio.00024-10 ◽

2010 ◽

Vol 1 (1) ◽

Cited By ~ 121

Author(s):

James E. Dornenburg ◽

Anne M. DeVita ◽

Michael J. Palumbo ◽

Joseph T. Wade

Keyword(s):

Escherichia Coli ◽

Transcription Initiation ◽

Antisense Transcription ◽

Antisense Transcripts ◽

Protein Coding ◽

Content Type ◽

Antisense Rnas ◽

Regulatory Molecule ◽

Overlapping Gene ◽

Transcription Complexes

ABSTRACT The vast majority of annotated transcripts in bacteria are mRNAs. Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli. We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene. IMPORTANCE The vast majority of known genes in bacteria are protein coding, and there are very few known antisense transcripts within these genes, i.e., RNAs that are encoded opposite the gene. Here we demonstrate the existence of ~1,000 antisense RNAs in the model bacterium Escherichia coli. Given the high potential for these RNAs to base pair with mRNA of the overlapping gene and the likelihood of clashes between transcription complexes of antisense and sense transcripts, we propose that antisense RNAs represent an important but overlooked class of regulatory molecule.

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Influence of Type I Fimbriae and Fluid Shear Stress on Bacterial Behavior and Multicellular Architecture of Early Escherichia coli Biofilms at Single-Cell Resolution

Applied and Environmental Microbiology ◽

10.1128/aem.02343-17 ◽

2018 ◽

Vol 84 (6) ◽

Cited By ~ 8

Author(s):

Liyun Wang ◽

Robert Keatch ◽

Qi Zhao ◽

John A. Wright ◽

Clare E. Bryant ◽

...

Keyword(s):

Escherichia Coli ◽

Shear Stress ◽

Single Cell ◽

Biofilm Formation ◽

Fluid Shear Stress ◽

Cell Membrane Permeability ◽

Type I ◽

Fluid Shear ◽

Content Type ◽

Type I Fimbriae

ABSTRACT Biofilm formation on abiotic surfaces in the food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine the cellular architecture of early biofilms and the bacterial behavior of the constituent cells remains largely unknown. In this study, we examined the specific role of type I fimbriae in nascent stages of biofilm formation and the response of microcolonies to environmental flow shear at the single-cell resolution. The results show that type I fimbriae are not required for reversible adhesion from plankton, but they are critical for the irreversible adhesion of Escherichia coli strain MG1655 cells that form biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing firm cell surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E. coli on the surface. After the application of shear stress, bacterial retention is dominated by the three-dimensional architecture of colonies, independent of the population size, and the multilayered structure could protect the embedded cells from being insulted by fluid shear, while the cell membrane permeability mainly depends on the biofilm population size and the duration of the shear stress. IMPORTANCE Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level; thus, little is known about how individual bacterium behavior within biofilms and the multicellular architecture are influenced by bacterial appendages (e.g., pili/fimbriae) and environmental factors during early biofilm formation. We applied confocal laser scanning microscopy (CLSM) to visualize Escherichia coli microcolonies at a single-cell resolution. Our findings suggest that type I fimbriae are vital to the initiation of bacterial proliferation on surfaces. We also found that the fluid shear stress affects the biofilm architecture and cell membrane permeability of the constituent bacteria in a different way: the onset of the biofilm is linked with the three-dimensional morphology, while membranes are regulated by the overall population of microcolonies.

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Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00731-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Dongchang Sun ◽

Xudan Mao ◽

Mingyue Fei ◽

Ziyan Chen ◽

Tingheng Zhu ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Natural Transformation ◽

Inducible Promoter ◽

Regulation Mechanism ◽

Type I ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

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Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σEin Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00079-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2316-2324 ◽

Cited By ~ 17

Author(s):

Yasushi Daimon ◽

Shin-ichiro Narita ◽

Yoshinori Akiyama

Keyword(s):

Escherichia Coli ◽

Stress Responses ◽

Ectopic Expression ◽

Growth Media ◽

Content Type ◽

E Coli ◽

Envelope Stress ◽

Genes Encoding ◽

Σ Factor ◽

Mutant Forms

ABSTRACTσE, an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth ofEscherichia colinot only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of thehicBgene has been reported to suppress the lethality caused by the loss of σE.hicBencodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σEand, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption ofhicAabolished suppression of the σEessentiality in the absence ofhicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σEessentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins,E. colicells with no suppressor mutations grew independently of σE. Taken together, our results indicate that the activation of TA system toxins can suppress the σEessentiality and affect the extracytoplasmic stress responses.IMPORTANCEσEis an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σEis indispensable for the survival ofE. colieven under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σEessentiality, suggesting a connection between TA systems and σEfunction. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

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The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

Journal of Bacteriology ◽

10.1128/jb.02418-14 ◽

2014 ◽

Vol 197 (2) ◽

pp. 337-342 ◽

Cited By ~ 27

Author(s):

Louise Roer ◽

Frank M. Aarestrup ◽

Henrik Hasman

Keyword(s):

Escherichia Coli ◽

Rapid Evolution ◽

Type I ◽

Modification System ◽

Major Barrier ◽

Exogenous Dna ◽

Content Type ◽

Restriction Modification System ◽

K 12 ◽

Restriction Modification

The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strainEscherichia coliK-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intacthsdRgene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disruptedhsdRgene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer.

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Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.00369-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3220-3231 ◽

Cited By ~ 9

Author(s):

Kumiko Kurabayashi ◽

Tomohiro Agata ◽

Hirofumi Asano ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Antimicrobial Agents ◽

Host Cells ◽

Type I ◽

Wild Type ◽

Content Type ◽

Type I Fimbriae ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

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Enteropathogenic Escherichia coli and Vaccinia Virus Do Not Require the Family of WASP-Interacting Proteins for Pathogen-Induced Actin Assembly

Infection and Immunity ◽

10.1128/iai.06148-11 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4071-4077 ◽

Cited By ~ 7

Author(s):

John J. Garber ◽

Fuminao Takeshima ◽

Inés M. Antón ◽

Michiko K. Oyoshi ◽

Anna Lyubimova ◽

...

Keyword(s):

Escherichia Coli ◽

Vaccinia Virus ◽

Family Members ◽

Ectopic Expression ◽

Host Cells ◽

Actin Assembly ◽

Human Pathogens ◽

Interacting Protein ◽

Content Type ◽

Syndrome Protein

ABSTRACTThe human pathogens enteropathogenicEscherichia coli(EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.

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Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01444-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5650-5659 ◽

Cited By ~ 4

Author(s):

Shan Goh ◽

Angela Hohmeier ◽

Timothy C. Stone ◽

Victoria Offord ◽

Francisco Sarabia ◽

...

Keyword(s):

Escherichia Coli ◽

Target Gene ◽

Essential Gene ◽

Transcript Level ◽

Essential Genes ◽

Content Type ◽

Reading Frame ◽

Knockout Mutants ◽

Bacterial Genes ◽

Relationship Of

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

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Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone

mBio ◽

10.1128/mbio.01602-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 18

Author(s):

Brian M. Forde ◽

Minh-Duy Phan ◽

Jayde A. Gawthorne ◽

Melinda M. Ashcroft ◽

Mitchell Stanton-Cook ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Methylation ◽

Real Time ◽

Single Molecule ◽

Mobile Genetic Elements ◽

Sequence Type ◽

Type I ◽

Smrt Sequencing ◽

Content Type ◽

E Coli

ABSTRACTEscherichia colisequence type 131 (ST131) is a clone of uropathogenicE. colithat has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome ofE. coliEC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered threem6A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible form6A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.IMPORTANCEDNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistantE. colisequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-definedE. coliclone.

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