A Pan‐Methylome Framework for Population‐Scale Bacterial Epigenomics

ABSTRACT Epigenetic modifications furnish a hidden regulatory layer that shapes cellular fate and evolutionary potential without rewriting the genetic code. In eukaryotes, methylome maps have profoundly transformed our understanding of development, disease, and lineage diversification. In bacteria, which represent the most abundant and ecologically versatile forms of life, the epigenome remains a largely uncharted dimension. To enable population‐scale comparison of bacterial methylomes, we develop a principled strategy to prioritize methylation systems that are enzymatically encoded, broadly conserved and phylogenetically informative, thereby defining a stable substrate for quantitative comparative analysis. We reconstruct the first population‐scale bacterial methylation‐informed phylogenies that broadly recapitulate sequence‐based relationships while resolving epi‐phylogroups associated with GC content and environmental stress resilience. We introduce a three‐metric quantitative framework (MPK, MR, and MFR) that converts site‐level methylation calls into standardized, cross‐sample comparable quantitative traits, enabling robust identification of highly methylated core genes from as few as 30 Escherichia coli strains. Finally, co‐methylation network analysis identifies virulence‐enriched modules and coordinated methylation of horizontally acquired virulence loci, providing the first evidence that horizontally acquired virulence loci are epigenetically assimilated into host regulatory circuitry. Together, this framework enables locus‐resolved, population‐scale integration of bacterial methylation data to interrogate epigenetic contributions to evolution, fitness and pathogenicity.