Lars Barquist (@lbarquist.bsky.social)

Methylome and transcriptome mapping reveal miniscule DNA methyltransferase regulons in Salmonella enterica serovar Typhimurium DNA methylation is a regulator of bacterial gene expression and adaptation, influencing traits such as virulence and antimicrobial resistance. The dynamic nature of DNA methylation enables rapid responses to changing environments and is a source of heterogeneity in bacterial populations. However, condition-dependent DNA methylation and consequences for transcriptional output remain poorly understood. We applied Oxford Nanopore sequencing to profile DNA methylation during exponential growth and late stationary phase of Salmonella enterica serovar Typhimurium and integrated these data with transcriptomic analyses. We found that each DNA methyltransferase (MTases) exhibits a distinct activity pattern across growth stages, which could not be explained by transcriptional levels of the corresponding enzymes. As predicted, DNA methylation patterns determined by regulatory MTases were dynamic across growth conditions whereas methylation patterns of MTases belonging to R-M systems were comparatively stable. We identified growth stage–specific methylation patterns for all studied MTases and correlations between methylation states and gene expression patterns. Together, these findings chart DNA methylation networks in the epigenetic regulation of bacterial physiology. Author summary DNA methylation in bacteria is best known for its role protecting DNA from endonucleases, such as restriction–modification, and coordinating chromosome replication and mutation repair, yet DNA methylation also regulates gene expression and cell physiology. Previous studies primarily examined bacterial DNA methylation at single time points or in limited genomic regions, providing only a partial view of its biological significance. In this study, we used Oxford Nanopore sequencing to compare DNA methylation patterns in Salmonella enterica during exponential growth and late stationary phase then integrated these data with corresponding gene expression profiles. We identified numerous methylation target motifs, all of which demonstrated constitutively methylated or unmethylated regions. This systems-level analysis clarifies the role of DNA methylation in bacterial adaptation across growth stages and demonstrates the utility of Oxford Nanopore sequencing for genome-wide methylation profiling. ### Competing Interest Statement The authors have declared no competing interest. European Union, https://ror.org/019w4f821, Marie Skłodowska-Curie grant agreement No. 896441 University of Regina Fir cluster of the Digital Research Alliance of Canada https://www.biorxiv.org/content/10.64898/2026.01.27.702048v1