Intercellular mosaic methylation in fast-growing Mycobacterium tuberculosis clinical isolates

Orphan DNA adenine methyltransferases (MTases) of Mycobacterium tuberculosis (Mtb) 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 mamC 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 Mtb 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 Mtb MTases, MamC DNA methylation is uniquely linked to growth dynamics.