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DNA Cytosine Methyltransferase Increases Stationary Phase Fitness under Temperature Stress in Escherichia coli
Author(s) -
Huss Rebecca A,
Davidson Samantha,
Militello Kevin T
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.581.2
Subject(s) - plasmid , cytosine , escherichia coli , microbiology and biotechnology , dna , gene , biology , dna methyltransferase , methyltransferase , restriction enzyme , genetics , methylation
Escherichia coli contains two orphan DNA methyltransferases. DNA adenine methyltransferase (Dam) methylates 5′GATC3′ sequences, and DNA cytosine methyltransferase (Dcm) methylates the interior cytosine in 5′CCWGG3′ sequences. Numerous functions of the Dam enzyme have been elucidated, however the role of Dcm is less defined. Growth curve assays in our laboratory have shown no significant difference in growth rate between strains containing dcm and those without it. However, our laboratory's DNA microarray analyses have revealed numerous gene expression changes in dcm knockout cells in early stationary phase, suggesting dcm influences stationary phase biology. Thus, we tested the role of Dcm in stationary phase fitness. A dcm containing plasmid was constructed by amplifying the dcm gene as well as the putative promoter upstream of the gene by PCR, and inserting it into the pET‐28a+ plasmid. The pET‐28a+ plasmid with and without dcm were introduced into T7 express bacteria, which has a large chromosomal deletion that includes the dcm gene. We call the new strains T7E/pDcm and T7E/pET‐28a+. In order to demonstrate that the cells containing dcm methylated their DNA, a restriction enzyme isoschizomer assay was conducted with PspGI and BstnI. The results indicated that the cells containing T7E/pDcm methylated their DNA at 5′CCWGG3′ sites. The T7E/pET‐28a+ and T7E/pDcm strains were competed over long‐term stationary phase at 37°C. Colony PCR assays were used to determine which strain was outcompeting the other at the conclusion of one and three month intervals. At 37°C, T7E/pET‐28a+ outcompeted T7E/pDcm. As our laboratory has revealed in recent microarray studies, the cold‐shock protein B gene ( cspb ) was upregulated in bacteria treated with the DNA methyltransferase inhibitor, 5‐azacytidine. This suggests a possible biological role for Dcm at colder temperatures. We then competed T7E/pET‐28a+ and T7E/pDcm at 20°C over long‐term stationary phase. When competing at 20°C, T7E/pDcm outcompeted T7E/pET‐28a+. In order to verify the surviving T7E/pDcm colonies still methylated their DNA, restriction enzyme isoschizomer assays were performed, with the results indicating methylation occurs in only the T7E/pDcm colonies. Our work suggests a new and unexpected phenotype for Dcm . Dcm provides a fitness advantage during stationary phase at low temperatures. We aim to determine the biological mechanism for this phenotype. Support or Funding Information This work was supported by the Geneseo Foundation.