How to Unmask an Unknown: The Restriction-Modification System MhoVII of Mycoplasma hominis Expresses Two...

Restriction-modification (RM) systems contribute to genome plasticity in Mycoplasma hominis, a facultative pathogen with an extremely small but highly heterogeneous genome. The MhoVII 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 YhdJ and MTaseD12 branches, respectively. In this study, the dissemination, expression and function of the MhoVII system was analyzed in detail using Oxford Nanopore-based methylation analysis, recombinant expression of the individual RM components in Escherichia coli, 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 MhoVII genes, and GATG/CATC-driven methylation analysis revealed culture-dependent methylation differences, suggesting a post-transcriptional regulation, whereas in the infection of HeLa cells, MhoVII transcription was highest at the beginning and was then gradually downregulated in the later stages of infection. These findings establish MhoVII as a previously uncharacterized Type II RM system.