Growth Phase-Related DNA Methylation and Localized DNA Fragment Density Variation in Escherichia coli and Lactobacillus acidophilus Genomes

Background/Objectives: Bacterial DNA methylation is a reversible genetic modification affecting gene regulation without significant DNA sequence alteration, thereby enabling rapid adaptation to various environments. However, traditional motif-based approaches detect DNA methylation at low resolution, limiting the identification of DNA methylation variation and often restricting analysis of multiple methylation forms. Methods: Here, we used Oxford Nanopore Technologies sequencing to examine genome-wide variation in three DNA methylation forms (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) and DNA fragment density between the exponential and stationary phases of Escherichia coli and Lactobacillus acidophilus. Results: During the exponential phase, regions surrounding the replication terminus displayed lower DNA fragment density in both species and lower 5-methylcytosine levels in Escherichia coli. Methylation calling on each genomic locus revealed almost 21% more differentially methylated sites than motif-based detection. Through the locus-based detection, the majority (72%) of differentially methylated genes in Escherichia coli were differentially expressed. The functions of these genes were associated with the physiological response to growth phase transition, such as cell membrane synthesis and translation. Incorporating multiple DNA methylation forms and including all methylated loci, not just those within common motifs, resulted in higher concordance between differentially expressed genes and differentially methylated genes compared to single-form or motif-based approaches. Conclusions: Our findings suggest that the bacterial DNA methylation pattern is affected by growth phase transition, and that a locus-based differential methylation analysis using Nanopore sequencing can estimate microbial activity in some cases, especially in microbiome studies where the transcriptome-genome link is difficult to establish. Impact Statement: Bacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. There are three major DNA methylation forms in bacteria, namely N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we found that DNA methylation levels (N6-methyladenine and 5-methylcytosine) are globally and regionally lower during the exponential phase compared to the stationary phase. Our results showed that differentially methylated genes can robustly estimate the overall transcriptomic profile in Escherichia coli. The inclusion of multiple DNA methylation forms and all methylated loci can improve concordance with differentially expressed genes. Our findings reveal that the bacterial DNA methylation pattern shifts during growth phase transition, and indicate that methylation could be an accessible method for estimating microbial activity.