Abstract
In prokaryotes, DNA methylation plays roles in DNA repair, gene expression, cell cycle progression, and immune recognition of foreign DNA. Genome-wide methylation patterns can vary between strains, influencing phenotype, and gene transfer. However, broader evolutionary studies on bacterial epigenomic variation remain limited. In this study, we conducted an epigenomic analysis using single-molecule real-time sequencing on 21 strains of Sinorhizobium meliloti, a facultative plant nitrogen-fixing alphaproteobacterium. This species is notable for its multipartite genome structure, consisting of a chromosome, chromid, and megaplasmid, leading to significant genomic and phenotypic diversity. We identified 16 palindromic and nonpalindromic methylated DNA motifs, including N4-methylcytosine and N6-methyladenine modifications, and analyzed their associated methyltransferases. Some motifs were methylated across all strains, forming a core set of epigenomic signatures, while others exhibited variable methylation frequencies, indicating a dispensable (shell) epigenome. Additionally, we observed differences in methylation frequency between replicons and within coding sequences versus regulatory regions, suggesting that methylation patterns may reflect multipartite genome evolution and influence gene regulation. Overall, our findings reveal extensive epigenomic diversity in S. meliloti, with complex epigenomic signatures varying across replicons and genomic regions. These results enhance our understanding of multipartite genome evolution and highlight the potential role of epigenomic diversity in phenotypic variation.