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

The role of methylated cytosines introduced into bacterial genomes by solitary DNA methyltransferases often remains elusive. Here, we show that the solitary cytosine methyltransferase ScmA of <i>Caulobacter crescentus</i> methylates thousands of YGCCGGCR motifs in its genome and that wild-type cells outcompete <i>ΔscmA</i> mutant cells. Transcriptomic and single-cell analyses reveal that a DNA damage response is turned on in a significant proportion of <i>ΔscmA</i> cells and that this response is strictly dependent on the Vsr-like protein VsrA. Although the <i>vsrA</i> gene is not genetically linked to <i>scmA</i> or to any other cytosine methyltransferase gene, VsrA is predicted to be a mismatch repair (MMR) endonuclease preventing 5mC-to-T mutations. In <i>ΔscmA</i> cells, VsrA may accidentally cause double-strand breaks (DSBs) during MMR, leading to reduced fitness. These findings suggest that <i>vsr</i>-like genes can stabilize solitary cytosine methyltransferase genes in bacterial genomes, potentially explaining their prevalence despite the frequent absence of detectable regulatory roles.