Horizontal acquisition of the Type I restriction-modification system enhances bacterial pathogenicity by mediating methylation of transcription factor-encoding genes.

The Type I restriction-modification (RM) system, encoded by the hsdR, hsdM, and hsdS genes, plays a crucial role in shaping the prokaryotic DNA methylation landscape. Although known for defending against foreign DNA, key aspects of its evolutionary trajectory and functional implications after stable inheritance remain poorly understood. In this study, we identified four primary types of Type I RM systems across 4273 prokaryotic genomes based on gene arrangement. Among these, the 5'-hsdR, hsdM, hsdS-3' (RMS) configuration emerged as the most evolutionarily advanced form. Phylogenetic reconstruction revealed that RMS was formed through gene duplication, horizontal gene transfer, and gene loss, and it now stably exists in bacteria. Functional characterization demonstrated that RMS deletion in bacteria led to the absence of flagella and a significant reduction in their ability to colonize and infect mice. Integrated multi-omics analysis uncovered a potential regulatory cascade where RMS modulates the expression of transcription factors via DNA methylation, which in turn regulate downstream flagellar and chemotaxis genes, thereby influencing bacterial pathogenicity. These findings establish a complete evolutionary-functional paradigm, elucidating how (evolutionary trajectory) and why (functional constraints) RMS has been stably inherited in bacterial genomes, and revealing the molecular mechanism through which RMS orchestrates bacterial pathogenicity.