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

Abstract

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...

Biogeographic conservation of the cytosine epigenome in the globally important marine, nitrogen‐fixing cyanobacterium Trichodesmium

“…Alternatively, we used bisulfite sequencing which uses sodium bisulfite to chemically deaminate unmethylated m5C cytosine residues to uracil while leaving mostly all methylated cytosines as cytosines. This method thus enables reliable detection of m5C methylation when compared with a reference genome (Clark et al ., ; Huo et al ., ). Hence, although Blow and colleagues () were unable to comprehensively address m5C methylation, two m5C dcm genes were predicted to associate with two consistently methylated motifs also identified in this study (see below).…”

“…Blow and colleagues (2016) used single molecular real time (SMRT) sequencing, which measures the rate of base incorporation on a single DNA molecule by DNA polymerase via fluorescence pulses. The interpulse duration (IDP) (i.e., speed of incorporation) yields certain kinetic signatures that can distinguish between m6A, m4C and m5C modifications, but due to the small size of the m5C methyl group and its positioning in the major groove, only subtle changes in polymerase kinetics are produced thereby yielding inconsistent m5C detection (Clark et al, 2013;Huo et al, 2015). Alternatively, we used bisulfite sequencing which uses sodium bisulfite to chemically deaminate unmethylated m5C cytosine residues to uracil while leaving mostly all methylated cytosines as cytosines.…”

Enterococcus faecalisCRISPR-Cas is a robust barrier to conjugative antibiotic resistance dissemination in the murine intestine

“…At the desired time point, a 5 mL culture aliquot was collected from planktonic cultures conditions, or biofilms were collected from agar plates using 2 mL of PBS supplemented with 2 mM EDTA. Total RNA was isolated using RNA-Bee and chloroform precipitation as previously described (36). 200 ng RNA was used as template for cDNA synthesis using qScript cDNA Supermix (Quanta Biosiences).…”

A Type I Restriction-Modification System Associated withEnterococcus faeciumSubspecies Separation

“…If an E. faecium query protein length was less than half of its subject’s length, the match was removed from the prediction list. Due to the sequence diversity of REases which complicates their bioinformatic identification (15), guilt-by-association was used to identify full R-M systems as we have previously described (16). The proteins encoded near candidate DNA MTases were analyzed using BLAST and Pfam for conserved domains consistent with REase activities and/or sequence identity to confirmed REases.…”

“…Genes encoding the specificity and methylation subunits of Efa733I (EFSG_05028-EFSG_05027) were PCR-amplified in their entirety, including the upstream region to retain the native promoter, using primers 733_T1A_SM_F and 733_T1A_SM_R (see Table S1 for primer sequences). The PCR product was digested with Not I and ligated into Not I-digested pWH03 (16) using T4 DNA Ligase (NEB), generating pHA102. pWH03 is a pLT06 derivative for expression of genes from a previously validated neutral genomic insertion site (EF2238-EF2239) for expression (GISE) (16, 17).…”