Characterization of the Global DNA Methylome in Escherichia coli Under Silver Stress

Bacteria are continually exposed to stressors, making their ability to sense, acclimate and adapt to changing conditions essential for fitness. Two-component response systems (TCRS) play a primary role in mediating these responses. However, the role of epigenetic modifications in acclimation and adaptation is not well understood. We characterized N6-methyladenine (6mA) and N4-methylcytosine (4mC) DNA methylation patterns in wild-type Escherichia coli K-12 MG1655, four cusS mutants and six silver-adapted mutants with and without exposure to 50 ng/mL AgNO₃ for 1 h using Single-Molecule Real-Time sequencing. The cusS mutants were selected to study the impact of the mutations on DNA methylation. Global mean 6mA fractions were high across all strains with and without silver and were largely driven by the dominant GATC motif in all strains and the AACN₆GTGC/GCACN₆GTT motif in wild-type and silver-adapted mutants, while global mean putative 4mC fractions were lower. Across conditions, variation was observed in extended degenerate motifs with moderate-to-low methylation. Our findings suggest that motif-specific methylation patterns vary across silver exposure and genetic backgrounds, while global methylation levels in E. coli remain remarkably resilient. This study improves understanding of microbial stress adaptation and stress response, raising the possibility that methylation contributes to TCRS-mediated stress tolerance.