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<p>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&#xa0;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 <i>Hga</i>I-homolog RM system was detected in <i>Mycoplasma hominis</i> with the more rare constellation of two 5mC MTase genes, called RM.<i>Mho</i>VI. A qPCR screening of 239 randomly selected <i>M. hominis</i> isolates revealed a prevalence of the <i>Mho</i>VI-RM system of 12.97% (<i>n</i> = 31/239). Notably, in all tested <i>Mho</i>VI-positive isolates, the <i>Mho</i>VI-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 <i>Mho</i>VI-enzymes was high (&gt; 99% identities), and inter-species conservation was the lowest compared to the eponymous species <i>Haemophilus gallinarum</i> (46.6% M1.<i>Mho</i>VI; 48.1% M2.<i>Mho</i>VI; 27.4% R.<i>Mho</i>VI). A polycistronic organization of the <i>Mho</i>VI-genes was strongly suspected due to the discovery of gene-overlapping mRNA regions. The MTases activity was demonstrated in RM.<i>Mho</i>VI positive <i>M. hominis</i> isolates by protection of genomic DNA from cleavage by the methylation-sensitive endonuclease <i>Hga</i>I; and bioinformatics analysis using the Dorado basecaller on the Oxford Nanopore sequenced genomes revealed methylation rates of the respective motifs, 5’-GA<sup>m</sup>CGC-3’/5’-G<sup>m</sup>CGTC-3’, above 95% in <i>Mho</i>VI-positives, with a higher methylation frequency of 5’-GA<sup>m</sup>CGC-3’ than 5’-G<sup>m</sup>CGTC-3 in most isolates. A final proof of <i>Mho</i>VI-RM representing an <i>Hga</i>I-RM-like methylation activity was demonstrated through expression and analysis of recombinant rM2.<i>Mho</i>VI in <i>E. coli</i>.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月25日

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  • Lars Vogelgsang,
  • Manuel Dolgopolow-Schmidt,
  • Azlan Nisar,
  • Dana Bäcker,
  • Alexander T. Dilthey,
  • Birgit Henrich

摘要

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’-GAmCGC-3’/5’-GmCGTC-3’, above 95% in MhoVI-positives, with a higher methylation frequency of 5’-GAmCGC-3’ than 5’-GmCGTC-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.