Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, ^6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(G_PS^6mA) dinucleotides in the G_PS^6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(G_PSA) motifs per genome, with ^6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and ^6mA in another consensus sequence, G_PS^6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in G_PSATC) and Dam methyltransferase from E. coli DH10B (^6mA in G^6mATC). Based on this model, in vitro studies revealed reduced Dam activity in G_PSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed ^6mA in all 2,058 G_PSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that ^6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.