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

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 Dcrn 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 enclonuclease 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 5 ' C in the degenerate sequence 5 '-RCCGGY-3 '. 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.