Long-read methylome analysis of Oleidesulfovibrio alaskensis G20 biofilm under copper stress

This study represents the first investigation of 5-methyl cytosine (5mC) DNA methylation patterns in sulfate-reducing bacterial (SRB) biofilms under copper (Cu) stress, utilizing Oxford Nanopore Technologies (ONT) sequencing. DNA methylation is a crucial epigenetic modification that is dynamic and regulates the signals to modulate molecular mechanisms across biological systems. The regulatory roles of DNA methylation in prokaryotic systems remain comparatively understudied than in eukaryotes. Bacteria are highly sensitive to environmental changes and therefore may utilize additional mechanisms like DNA methylation to combat the stresses. Our previous studies, utilizing microscopy and growth analyses, revealed that Oleidesulfovibrio alaskensis G20 (OA G20) biofilms responded to Cu stress. However, the DNA methylation patterns associated with this response remain unexplored, leaving a critical gap in our understanding of the epigenetic mechanisms regulating OA G20 biofilms under Cu stress. This study aims to address this knowledge gap by identifying 5mC DNA methylation in biofilms of OA G20 under Cu stress. To achieve our goal OA G20 biofilms cultivated under 30 µM-Cu ion stress along with control and sequenced through ONT sequencing. DNA methylation analysis was performed using the MicrobeMod pipeline identifying three methylated motifs: TCCG, CCCGCCCG, and CGGGAT in control (0 µM-Cu). TCCG was identified as the predominant methylated motif, with analysis revealing 78,022 genomic positions in the control condition. Of these, 61.7% exhibited 5mC modifications, 33.9% remained unmodified, and 4.4% showed uncharacterized modifications. In contrast, the 30 µM-Cu biofilm showed methylation in only two motifs, TCCG and GCANCTGCGS. Analysis of TCCG revealed 63,315 genomic positions, with 62.7% (39,706 sites) showing methylation and 33.2% (20,990 sites) remaining unmethylated. A total of 1418 common methylated positions were identified for both conditions and there were 341 and 424 genomic positions identified for motif TCCG above 75% methylation in the 0 µM and 30 µM-Cu biofilm samples, respectively. Differential methylation analysis revealed significant variations in methylation patterns across several key genes of crucial molecular pathways, important for biofilm formation, including ATP-Binding Cassettes (ABC) transporters, phosphohydrolase, flagellar biosynthesis, chemotaxis, cobalamin synthase, histidine kinase, and uncharacterized proteins.