An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae

Abstract

5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an 'orphan' or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 59 C in the degenerate sequence 59-RCCGGY-39. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

“…Solitary REases and MTases might be components of R-M systems encoded apart in the genome. Several of these have been found (35,36,81). In this case, one would expect a similar number of solitary REases and MTases.…”

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription

“…An alternative argument is that this might be a mechanism to ensure that the VSR system can efficiently detect and correct mutant sites with minimal confusion arising from 'normal' C[C/T]W[G/A]G sites; there might be an interplay of both forces in determining the final oligonucleotide usage in the E. coli genome. However, several other organisms might, in fact, make use of cytosine methylation to generate genotypic diversity during stationary phase; for example, Vibrio cholerae encodes a Dcm (targeting the external cytosine in RCCGGY motif), but not a VSR repair system 50 . Analysis of the genome of V. cholerae shows that a striking 97% of 'methylatable' cytosines are at non-synonymous positions, suggesting a strong preference for having methyl-dependent mutations at nonsynonymous sites (3,964 of 4,074 sites on either strand of both chromosomes).…”

Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles

“…The specificity subunit recognizes a specific DNA motif for methylation (3, 6) and has been shown to function together with a dimer of the MTase (7); there are also solitary DNA MTases that do not have a restriction enzyme counterpart. Examples of the latter are the N 6 -adenine MTase Dam, CcrM (8) and the C-5 cytosine methylase Dcm (9, 10), and VchM (9, 11). While R-M systems are known for their role in the defense against foreign DNA (10), DNA methylation enzymes also seem to play functional roles in the timing of DNA replication, chromosome partitioning, DNA repair, control of transposition, and conjugal transfer of plasmids and gene regulation (12–20, 61, 62).…”