The Role of the Escherichia coli dcm gene in Stationary Phase Fitness and Catalase Activity

While the role of DNA methylation in eukaryotes is well known, little is known about the role of DNA methylation in prokaryotes. Dcm is a DNA cytosine methyltransferase in E. coli that methylates the second cytosine in the sequence 5′CCWGG3′. Although the methylation recognition sites are known, the function of methylation catalyzed by Dcm and the mechanism by which methylation impacts bacterial physiology is not yet known. Our laboratory's microarray data indicates numerous gene expression changes at stationary phase upon addition of a methylation inhibitor, 5‐azacytidine, suggesting a role for Dcm in stationary phase. Stationary phase competition experiments were conducted with a wild‐type strain and a kanamycin resistant dcm knockout strain and demonstrated that the dcm knockout strain was less fit during stationary phase. In subsequent experiments, a kanamycin resistant manA knockout strain was utilized in place of the wild‐type strain to control for possible effects of the addition of a kanR gene on stationary phase fitness. Competition experiments between the manA knockout strain and dcm knockout strain indicated that the lack of a dcm gene conferred a decrease in stationary phase fitness. As previous data have indicated that the dcm knockout strain is less fit than strains with a functional dcm gene at stationary phase, the laboratory is working to determine the mechanism by which cells lacking Dcm are disadvantaged. Dcm‐mediated methylation may regulate genes important to cell survival or play a role in growth advantage in stationary phase (GASP) mutation generation. Cells in stationary phase are characterized by an increase in rpoS expression, a change that can be determined indirectly by measuring the activity of the RpoS‐dependent catalase enzyme. Catalase activity was measured in a wild‐type strain, a dcm knockout strain, and a rpoS knockout strain; the experiments demonstrated that there was significantly more catalase activity in the dcm knockout strain compared to the wild‐type strain and that the rpoS knockout strain had minimal catalase activity. These data suggest that RpoS activity increases significantly in cells lacking the dcm gene. In order to test that the increase in catalase activity was due to the lack of the dcm gene, catalase activity was measured in a dcm knockout strain complemented with an empty plasmid and a knockout strain complemented with a dcm containing plasmid. The dcm knockout strain with the empty plasmid showed increased catalase activity compared to wild‐type strains, but when complemented with a dcm containing plasmid, levels of catalase activity were reduced to that seen in wild‐type strains. Additionally, colonies from the last day of each stationary phase competition experiment are currently being assayed for catalase activity to determine if the colonies that won the competition experiments contain GASP mutations. Published data indicate that rpoS is mutated during GASP, but retains a small amount of activity. In summary, we have demonstrated that lacking functional Dcm results in reduced fitness at stationary phase. Experiments that measure catalase activity as a proxy for RpoS function may implicate rpoS in the process through which loss of the dcm gene confers its effects. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .