Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Fang, Gang; Múnera, Diana Paulina; Friedman, David I.; Mandlik, Anjali; Chao, Michael C.; Banerjee, Onureena; Feng, Zhixing; Losic, Bojan; Mahajan, Milind; Jabado, Omar; Deikus, Gintaras; Clark, Tyson A.; Luong, Khai; Murray, Iain A.; Davis, Brigid M.; Keren-Paz, Alona; Chess, Andrew; Roberts, Richard J.; Korlach, Jonas; Turner, Steve; Kumar, Vipin; Waldor, Matthew K.; Schadt, Eric E.

doi:10.1038/nbt.2432

Cited by 325 publications

(395 citation statements)

References 49 publications

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“…Similar redundancies have been observed in Escherichia coli, which has three separate enzymes that methylate GATC (3,22). In addition, some strains of Salmonella enterica may have up to five copies of dam genes (see Table S3 in the supplemental material).…”

Section: Resultssupporting

confidence: 60%

“…The specific type of DNA methylation can often be determined from the polymerase kinetics, e.g., N6-methyladenine or 4-methylcytosine. With SMRT sequencing, it is now possible to identify the complete set of methylated sequence motifs within a microbial genome and the methylation state for each instance of a motif (22)(23)(24). This represents a powerful tool for characterizing the functional roles of DNA methylation in a wide variety of bacteria.…”

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confidence: 99%

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Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

et al. 2013

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We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.

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Section: Resultssupporting

confidence: 60%

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confidence: 99%

Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

et al. 2013

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“…SMRT sequencing has been used to characterize the methylomes of bacteria (47)(48)(49)(50)(51)(52)(53)(54). During SMRT sequencing, DNA polymerase kinetics are monitored in real time; modified bases in the sequencing template lead to altered polymerase kinetics relative to those obtained with an unmodified template (55,56).…”

Section: Resultsmentioning

confidence: 99%

Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing

et al. 2015

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Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged, reducing treatment options for these infections. MDR E. faecalis strains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDR E. faecalis. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can be distinguished from self DNA. A cognate restriction endonuclease digests improperly modified nonself DNA. Little is known about R-M in E. faecalis. Here, we use genome resequencing to identify DNA modifications occurring in the oral isolate OG1RF. OG1RF has one of the smallest E. faecalis genomes sequenced to date and possesses few MGEs. Single-molecule real-time (SMRT) and bisulfite sequencing revealed that OG1RF has global 5-methylcytosine (m5C) methylation at 5=-GC-WGC-3= motifs. A type II R-M system confers the m5C modification, and disruption of this system impacts OG1RF electrotransformability and conjugative transfer of an antibiotic resistance plasmid. A second DNA MTase was poorly expressed under laboratory conditions but conferred global N 4 -methylcytosine (m4C) methylation at 5=-CCGG-3= motifs when expressed in Escherichia coli. Based on our results, we conclude that R-M can act as a barrier to MGE acquisition and likely influences antibiotic resistance gene dissemination in the E. faecalis species. IMPORTANCEThe horizontal transfer of antibiotic resistance genes among bacteria is a critical public health concern. Enterococcus faecalis is an opportunistic pathogen that causes life-threatening infections in humans. Multidrug resistance acquired by horizontal gene transfer limits treatment options for these infections. In this study, we used innovative DNA sequencing methodologies to investigate how a model strain of E. faecalis discriminates its own DNA from foreign DNA, i.e., self versus nonself discrimination. We also assess the role of an E. faecalis genome modification system in modulating conjugative transfer of an antibiotic resistance plasmid. These results are significant because they demonstrate that differential genome modification impacts horizontal gene transfer frequencies in E. faecalis. Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the gastrointestinal tracts of humans and other animals (1). It is an opportunistic pathogen that causes life-threatening infections, such as bacteremia and endocarditis in compromised individuals (2). E. faecalis is among the leading causes of hospital-acquired infections in the United States, making it a major public health concern (3). Rising antibiotic resistance...

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“…In an effort to identify all three oxi-mC species simultaneously in the C. cinerea DNA, we turned to SMRT sequencing (29)(30)(31). 5mC and 5hmC are only weakly detected by SMRT sequencing (15), whereas 5caC yields a strong signal (16).…”

Section: Resultsmentioning

confidence: 99%

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

Chávez

Huang

Luong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.T he discovery that oxidative modifications of DNA bases are catalyzed by the TET/JBP family of 2-oxoglutarate and irondependent dioxygenases (1-4) opened a major area of research into the epigenetics of various eukaryotic lineages (reviewed in refs. 5-7). In metazoans, the TET/JBP family is represented by TET proteins, which are present in all animals that are known to possess DNA cytosine methylation (3,8,9). The three TET paralogs of vertebrates have been shown to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (2), which is progressively oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (10-12). The discovery of these oxidized methylcytosine (oxi-mC) modifications triggered a flurry of studies on the mammalian TET paralogs; the reports increasingly point toward important roles for these oxidative modifications as intermediates in the enigmatic process of DNA demethylation, and also as epigenetic marks in their own right (13, 14) (reviewed in refs. 5-7). TET proteins have roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell function, and cancer (5), but the mechanisms underlying their biological activities are still poorly defined.Several methods have been developed to profile individual oxi-mC species at base resolution in genomic DNA (reviewed in ref. 5). However, none of these methods can simultaneously map all three oxi-mC species-5hmC, 5fC, and 5caC-at the same time; rather, they rely on chemical or enzymatic conversion of individual oxi-mCs followed by bisulfite sequencing. Two recent sequencing technologies, single-molecule, real-time (SMRT) sequencing and protein nanopore sequencing, are capable of recognizing modified bases in unamplified genomic DNA (15-18). In SMRT sequencing, 5mC and 5hmC are barely detectable in unmodified DNA, whereas 5fC and 5caC yield a robust kinetic signature (16). As 5hmC is an abundant oxi-mC modification in ...

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Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Cited by 325 publications

References 49 publications

Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

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