Type I restriction-modification systems regulate gene expression through DNA topology.

This study examines how DNA methylation by both phase-variable and canonical restriction-modification (RM) systems contributes to bacterial gene regulation, using the SpnD39III system of Streptococcus pneumoniae and the prototypical EcoKI system of Escherichia coli. RNA-seq analysis of pneumococcal phase-locked strains expressing individual SpnD39III specificities revealed widespread but promoter-context-dependent differential gene expression associated with methylation. Targeted luciferase reporter assays confirmed that methylation of specific intergenic sites can either activate or repress transcription, validating the transcriptomic observations. Genetic dissection demonstrated that these regulatory effects depend on methyltransferase activity mediated by HsdM and are independent of restriction endonuclease function. These methylation-dependent responses were conserved across pneumococcal strain backgrounds. Mechanistic analyses revealed that methylation alters DNA topology, with methylated plasmids exhibiting reduced negative supercoiling. Consistent with a topology-driven mechanism, modulation of the nucleoid-associated protein HlpA (HU) reshaped promoter responses in a manner dependent on methylation state and promoter topological responsiveness. Extension of these analyses to E. coli showed analogous methylation-dependent regulation by EcoKI, supported by reporter assays, quantitative polymerase chain reaction, RNA-seq, and methylome profiling. Together, these findings demonstrate that RM system-mediated DNA methylation functions as an epigenetic regulator across bacterial species by altering local DNA topology and promoter accessibility, thereby dynamically shaping bacterial gene regulatory networks.