Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

Lluch‐Senar, María; Luong, Khai; Lloréns‐Rico, Verónica; Delgado, Javier; Fang, Gang; Spittle, Kristi; Clark, Tyson A.; Schadt, Eric E.; Turner, Stephen W.; Korlach, Jonas; Serrano, Luís

doi:10.1371/journal.pgen.1003191

Cited by 96 publications

(91 citation statements)

References 62 publications

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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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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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“…Five of the nonmethylated sites were in type I R-M motifs, two were located in GATC motifs, and one was found in an ATCGAT motif (Table 1; see also Table S2). Nonmethylated and hemimethylated sites have been detected in other bacteria (8,20,22,23,51,52), and these sites are often protected from methyltransferases by DNA binding proteins.…”

Section: Resultsmentioning

confidence: 99%

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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“…In contrast to other m C and h C detection techniques that rely on m C-specific chemical reactions and/or enzymatic kinetics, our system detects the methylation directly. Unlike single-molecule detection via SMRT (22,34), the methylation signal in MspA-based nanopore sequencing is carried in the primary signal: the ion current. Polymerase kinetics (22) may be used as an additional indicator of modified bases in our method.…”

Section: Discussionmentioning

confidence: 99%

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA

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et al. 2013

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

274

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Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.nanopore DNA sequencing | DNA methylation | DNA hydroxymethylation | nanotechnology | next generation sequencing

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Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

Cited by 96 publications

References 62 publications

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

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

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

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA

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