Epigenetic Regulation in Prokaryotes: Transcriptional and Phenotypic Outcomes of DNA Methyltransferase activity

DNA methyltransferases (DNA MTases) are central epigenetic regulators in bacteria and archaea, with functions extending far beyond classical restriction-modification defence. Diverse MTase classes exist, including canonical and phase-variable restriction-modification systems, orphan MTases, and enzymes with currently undefined roles. MTase activity is associated with regulatory outcomes through both direct and indirect mechanisms. Methylation of promoters or regulatory regions can influence transcription, while broader methylome remodelling may affect genome-wide gene expression. These processes generate distinct epigenetic states associated with phenotypic variation. MTase-mediated regulation has been implicated in virulence, colonisation, immune evasion, biofilm formation, motility, stress tolerance, metabolism, and antibiotic susceptibility. In archaea, MTase systems contribute to genome integrity and ecological specialisation, highlighting shared epigenetic principles across domains of life. A major challenge is to move beyond descriptive methylome surveys and correlative analyses toward experimentally validated links between methylation and phenotype. This review synthesises current understanding of prokaryotic DNA methylation, with primary emphasis on experimentally validated phenotypic outcomes. We also incorporate insights from omics-based studies where these provide context or generate testable hypotheses, while noting when evidence is based on inference rather than direct experimental validation, and include underrepresented archaeal systems.