Lawrence Berkeley National Laboratory

We performed whole genome analyses of DNA methylation in Shewanella17 oneidensis MR-1 to examine its possible role in regulating gene expression and18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing19 revealed extensive methylation of adenine (N6mA) throughout the20 genome. These methylated bases were located in five sequence motifs,21 including three novel targets for Type I restriction/modification enzymes. The22 sequence motifs targeted by putative methyltranferases were determined via23 SMRT sequencing of gene knockout mutants. In addition, we found S.24 oneidensis MR-1 cultures grown under various culture conditions displayed25 different DNA methylation patterns. However, the small number of differentially26 methylated sites could not be directly linked to the much larger number of27 differentially expressed genes in these conditions, suggesting DNA methylation is28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment29 of methylated GATC motifs in the origin of replication indicate DNA methylation30 may regulate genome replication in a manner similar to that seen in Escherichia31 coli. Furthermore, comparative analyses suggest that many32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.