From sequence to activity: the HgaI-homologous restriction modification system RM.MhoVI of Mycoplasma hominis.

Restriction-modification (RM) systems are widespread defense mechanisms in prokaryotes that protect the host from potentially harmful foreign DNA. They typically consist of a DNA methyltransferase (MTase), which methylates the host genome at an adenine (6 mA methylation) or cytosine (4mC or 5mC), and a restriction endonuclease (REase), which cleaves foreign, unmethylated DNA. In addition to the 2023 published family of 5mC-MTases, an HgaI-homolog RM system was detected in Mycoplasma hominis with the more rare constellation of two 5mC MTase genes, called RM.MhoVI. A qPCR screening of 239 randomly selected M. hominis isolates revealed a prevalence of the MhoVI-RM system of 12.97% (n = 31/239). Notably, in all tested MhoVI-positive isolates, the MhoVI-RM cassette localized between MHO_3110 and MHO_3120 and comprised an XRE-family transcriptional regulator gene in addition to the RM genes. Intra-species conservation of the encoded MhoVI-enzymes was high (> 99% identities), and inter-species conservation was the lowest compared to the eponymous species Haemophilus gallinarum (46.6% M1.MhoVI; 48.1% M2.MhoVI; 27.4% R.MhoVI). A polycistronic organization of the MhoVI-genes was strongly suspected due to the discovery of gene-overlapping mRNA regions. The MTases activity was demonstrated in RM.MhoVI positive M. hominis isolates by protection of genomic DNA from cleavage by the methylation-sensitive endonuclease HgaI; and bioinformatics analysis using the Dorado basecaller on the Oxford Nanopore sequenced genomes revealed methylation rates of the respective motifs, 5'-GA^mCGC-3'/5'-G^mCGTC-3', above 95% in MhoVI-positives, with a higher methylation frequency of 5'-GA^mCGC-3' than 5'-G^mCGTC-3 in most isolates. A final proof of MhoVI-RM representing an HgaI-RM-like methylation activity was demonstrated through expression and analysis of recombinant rM2.MhoVI in E. coli.