Intercellular mosaic methylation in fast-growing <i>Mycobacterium tuberculosis</i> clinical isolates

Orphan DNA adenine methyltransferases (MTases) of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) exhibit diversity across clinical isolates, but the forces driving this variation are not entirely clear. Recently, we observed several isolates exhibiting anomalous hypomethylation by Type I MTase Mycobacterial Adenine Methyltransferase C (MamC) despite a wild-type <i>mamC</i> genotype ('MamC-anomalous' isolates). Investigating this hypomethylation through multiple analyses revealed three key findings. First, heterogeneity analysis revealed intercellular mosaic methylation (IMM) in MamC-anomalous isolates. While they often exhibit phase-variable heterogeneity, this is the first report of IMM by a prokaryotic Type I MTase. Second, MamC-anomalous isolates exhibited a large, stable difference in chromosome copy number along the replication axis (a proxy for bacterial growth rate), suggesting a distinct growth phase accompanied by MamC hypomethylation. Third, MamC methylation efficiency decreased progressively with distance from the origin of replication on both strands, with a marked exaggeration in MamC-anomalous isolates. In contrast, other <i>Mtb</i> MTases (MamA and MamB) exhibited lower methylation levels away from the origin only on the lagging strand, and without exaggeration in MamC-anomalous isolates. We conclude that, among <i>Mtb</i> MTases, MamC DNA methylation is uniquely linked to growth dynamics.