Drucker, S. G. | Search bioRxiv

bioRxiv2026

Epigenomics identifies three sources of DNA methylation in Streptococcus mutans UA159

DNA methylation is a widespread but incompletely characterized regulatory feature of bacterial genomes. While restriction-modification systems represent well-studied sources of DNA methylation, the full complement of methyltransferases shaping bacterial epigenomes and their physiological consequences remain poorly understood. Here, we used Oxford Nanopore sequencing to comprehensively map DNA methylation in the model oral pathogen Streptococcus mutans UA159. Genome-wide analysis identified extensive N6-methyladenosine (6mA) modification and revealed three predominant methylation motifs. Using targeted deletion mutants, we demonstrate that methylation at GATC sites is mediated by the conserved DpnII restriction-modification system, while a novel bipartite CGANNNNNNNTCY/RGANNNNNNNTCA motif is methylated by the HsdM component of the type I Hsd restriction-modification system. The remaining 6mA sites corresponded to a CTGNAG/CTNCAG motif, defining the activity of a third methyltransferase. Genetic and epigenomic analyses identified SMU.43 as the enzyme responsible for this modification, which we designate DnmA, a novel orphan adenine methyltransferase with homology to regulatory methyltransferases rather than defense-associated systems. Functional characterization of single and double mutants revealed that distinct methylation systems differentially influence biofilm formation and antagonistic interactions with the commensal, Streptococcus sanguinis. Notably, loss of dnmA reversed biofilm and aggregation defects associated with deletion of dpnII, indicating epistatic interactions between methylation pathways. Together, this study resolves the major sources of DNA methylation in S. mutans UA159, identifies a novel regulatory methyltransferase, and highlights the utility of nanopore sequencing for bacterial epigenome discovery. These findings expand our understanding of bacterial DNA methylation and suggest that epigenomic enzymes may represent targets for modulation of microbial physiology and virulence.

microbiology↗

bioRxiv2026

Monounsaturated fatty acid biosynthesis is critical for streptococcal envelope homeostasis and stress tolerance

AbstractThe genus Streptococcus contains some of the most important commensals and pathogens of the human microbiome. To obtain the fatty acids required for cell membranes, Streptococcus either produce fatty acids de novo through the fatty acid biosynthesis (fab) pathway or uptake host fatty acids through the fatty acid kinase (fak) pathway. Although both the fab and fak pathways represent potential therapeutic targets to prevent or treat infection, progress is limited because of an incomplete understanding of taxon-to-taxon variability in streptococcal lipid metabolism. Here, we examined the role of de novo monounsaturated fatty acid (MUFA) synthesis in physiology and virulence-associated traits in Streptococcus mutans, Streptococcus pyogenes, and Streptococcus pneumoniae, three major pathogens that cause disease at distinct body sites. In all three species, deletion of fabM abolished MUFA production and caused severe growth defects, decreased stress tolerance, increased antibiotic susceptibility, and defects in cell viability, morphology, and division. In S. mutans, loss of fabM also markedly reduced competence signaling and production of the mutacin IV bacteriocin. Deletion of fabM increased susceptibility to killing by human neutrophils in S. mutans and S. pneumoniae, but not S. pyogenes. Together, these findings illustrate that MUFA synthesis is broadly important for streptococcal physiology and cell membrane homeostasis, while its contribution to pathogenesis is strongly species- and context-dependent, providing leads to guide development of novel therapeutic and/or preventative strategies.

microbiology↗