P025

1:San Diego State University

DNA methylation plays diverse regulatory roles in bacterial pathogens, yet its dynamics remain poorly understood in Mycobacterium tuberculosis (Mtb). Here, we report intercellular mosaic methylation (IMM) by the orphan Type I methyltransferase MamC in a subset of fast-growing clinical isolates despite having wild-type mamC. Through PacBio long-read sequencing, we identified four Mtb complex isolates with anomalous hypomethylation at MamC-targeted sequence motifs. These “MamC-anomalous” isolates exhibited unique methylation heterogeneity, distinct from phase variation, indicating IMM, a phenomenon rarely documented for Type I methyltransferases.
 
We found that hypomethylation patterns in these isolates were linked to chromosomal replication dynamics. MamC methylation efficiency decreased progressively with distance from the origin of replication, with this positional bias markedly intensified in MamC-anomalous strains. By contrast, other Mtb methyltransferases (MamA, MamB) showed strand-specific but less pronounced positional methylation effects.
 
Using a refined peak-to-trough ratio (PTR) metric adapted for long-read sequencing, we show that MamC-anomalous isolates exhibit elevated and highly stable replication-linked coverage bias, consistent with unusually rapid and synchronized growth. These growth dynamics were not reflected in methylation patterns of MamA or MamB, suggesting a unique sensitivity of MamC activity to bacterial growth phase.
 
Together, our results uncover a growth phase-associated modulation of methylation efficiency, manifesting as intercellular mosaic methylation, a previously unknown relationship in Mtb epigenetics. This advances our understanding of Mtb DNA methylation systems and suggests functional links between replication state and epigenetic heterogeneity in clinical Mtb isolates with potential implications for adaptive diversification during rapid growth.