bioRxiv2026
Bacteria require rapid adaptation under fluctuating environmental conditions. Commonly recognized global regulators enable bacteria to respond promptly to external changes, though they are either restricted to specific bacterial taxonomies or physiological statuses, suggesting that additional regulators are required for adaptation. DNA methylation is a reversible modification affecting bacterial gene regulation. However, conventional methods can only detect one DNA methylation form each round, leaving the understanding of DNA methylation in bacterial adaptation mostly unknown. This study aimed to identify genome-wide DNA methylation variation (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) in Escherichia coli under different culture conditions using Oxford Nanopore sequencing. DNA samples from six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure) during the exponential and stationary phases were extracted. When culture conditions were compared to the normal condition, E. coli exhibited more differentially methylated sites during the exponential phase than in the stationary phase. During the exponential phase, the genes differentially methylated in all conditions were involved in cellular activities, such as cellular and metabolic processes. During the stationary phase, universally differentially methylated genes were associated with oxidation responses. Subsequent analysis found that although DNA methylation analysis was affected by batch effects, some genes (e.g. rpoS) showed consistently differential methylation across datasets. Our findings suggest that the E. coli DNA methylation profile was affected by growth phases and conditions, and DNA methylation profiling by Oxford Nanopore sequencing could be a potential approach for gene activity estimation in environmental samples. ImportanceBacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. Three major forms in bacteria are N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we characterized genome-wide variation in these methylation types in Escherichia coli under six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure). DNA methylation signatures in E. coli varied with growth conditions. Using the normal condition as a baseline, E. coli during the exponential phase exhibited more differentially methylated genomic loci under stress conditions compared to the stationary phase. Under stress conditions, genes with differential methylation were associated with cellular processes or oxidative responses, depending on the growth phase. Our findings reveal that the DNA methylation signature in E. coli was affected by growth phases and conditions, and Oxford Nanopore-based DNA methylation profiling could be a potential approach for gene activity estimation in environmental samples.