Cytosine methylation by ScmA contributes to the fitness of Caulobacter crescentus cells naturally expressing a Vsr-like protein

While methylated cytosines are known to play important roles in eukaryotes, their significance in bacteria remains poorly understood especially when they are added on genomes by DNA methyltransferases that are not parts of restriction-modification systems. The newly named ScmA protein of Caulobacter crescentus is one of these solitary cytosine methyltransferases. Here, we show that it methylates YGCCGGCR motifs introducing thousands of m5C bases on the C. crescentus genome. We also find that wild-type C. crescentus cells easily outcompete{Delta} scmA cells during competition experiments. Transcriptome comparisons and using single-cell fluorescent reporters reveal that a DNA damage response is turned on in a significant proportion of{Delta} scmA cells. We then show that this particular response is strictly dependent on the presence of a Vsr-like protein named VsrA. Eventhough the vsrA gene is surprisingly neither genetically linked with the scmA gene nor with another cytosine methyltransferase gene, VsrA is predicted to encode an endonuclease preventing m5C-to-T mutations. Fluorescence microscopy experiments then show that GFP-VsrA forms foci relatively frequently in stationary phase cells, which may correspond to active repair sites. We propose that VsrA may accidentally create double strand breaks when detecting mismatched bases on the genome of{Delta} scmA cells, leading to their observed loss of fitness. Thus, the presence of vsr-like genes can potentially stabilize genes encoding cytosine methyltransferases in bacterial genomes, providing a potential explanation for the relatively high prevalence of solitary cytosine methyltransferases in bacteria even if some of these do not appear to play an easily detectable regulatory role.