How to Unmask an Unknown: The Restriction-Modification System MhoVII of Mycoplasma hominis Expresses Two Complementary Methylation Activities in One Enzyme

Restriction-modification (RM) systems contribute to genome plasticity in <i>Mycoplasma hominis</i>, a facultative pathogen with an extremely small but highly heterogeneous genome. The <i>Mho</i>VII RM system, which contains a fusion of two methyltransferases (MTases), M1 and M2, was recently identified within a family of Type II RM systems, but its specificity and biological function remained unknown. Phylogenetic analysis revealed that M1 and M2 belong to distinct MTase classes clustering within the <i>Yhd</i>J and MTaseD12 branches, respectively. In this study, the dissemination, expression and function of the <i>Mho</i>VII system was analyzed in detail using Oxford Nanopore-based methylation analysis, recombinant expression of the individual RM components in <i>Escherichia coli</i>, and methylation-sensitive restriction assays. It was thus possible to demonstrate that M1 and M2 methylate the complementary non-palindromic motifs GATG and CATC, and that the associated restriction endonuclease cleaves only DNA lacking 6mA methylation at these sites. The transcriptional analysis of mid-to-late logarithmic cultures indicated a polycistronic organization of the <i>Mho</i>VII genes, and GATG/CATC-driven methylation analysis revealed culture-dependent methylation differences, suggesting a post-transcriptional regulation, whereas in the infection of HeLa cells, <i>Mho</i>VII transcription was highest at the beginning and was then gradually downregulated in the later stages of infection. These findings establish <i>Mho</i>VII as a previously uncharacterized Type II RM system.