Furthermore, new E. coli environmental samples were isolated as described in the materials and methods from a relatively small geographical region (Western New York). These strains included representatives of the four main phylogenetic groups of A, B1, B2, and D (Clermont et al., 2000). All 162 DNAs tested generated an appropriate size PCR product, indicating the presence of the dcm gene or a highly related dcm homolog. The

presence of the dcm gene was independent of the source, pathogenicity, or phylogenetic group of the strain (Table S1). While all strains tested contained a full-length dcm gene, the PCR assay alone does not prove that each strain contains a functional cytosine Roxadustat concentration DNA methylation and 5mC. Our PCR assay could not rule out dcm mutations that inactivate the enzyme, mutations in regulatory regions that inhibit transcription and translation, or the absence of other molecules required for cytosine DNA methylation.

Therefore, a restriction enzyme isoschizomer assay was used to test for methylation of 5′CCWGG3′ sequences. Genomic DNAs were separately digested with the restriction enzymes BstNI and PspGI. Both enzymes cleave the sequence 5′CCWGG3′, but PspGI is blocked by Dcm-mediated cytosine methylation of the second cytosine. Etoposide nmr The assay was originally optimized with JM109 DNA (dcm+) and ER2925 DNA (dcm−). JM109 DNA was resistant to digestion with PspGI, which is consistent with DNA methylation of 5′CCWGG3′ sequences (Fig. 1b). When ER2925 DNA was cut with PspGI, fragments that were

heterogeneous in size were observed via gel electrophoresis, indicating ER2925 DNA is sensitive to this enzyme and lacks methylation at 5′CCWGG3′ sites. Titration of mixtures of methylated and unmethylated DNA indicated that the isoschizomer assay could detect partial cytosine check DNA methylation down to 10%, but the assay is largely qualitative. DNA samples from all 162 ECOR and environmental strains were resistant to digestion by PspGI. This demonstrates that every strain of E. coli examined in this study has a dcm gene and 5mC in the sequence 5′CCWGG3′. Our data are in contrast to data on the solitary cytosine DNA methyltransferase M.Vch from Vibro cholera, as it was absent in two of 25 strains tested (Banerjee & Chowdhury, 2006). Our experiments cannot determine whether all 5′CCWGG3′ sites are methylated; however, there are reports suggesting the presence of rare, unmethylated 5′CCWGG3′ sites (Ringquist & Smith, 1992; Bormann Chung et al., 2010). Nonetheless, each strain analyzed in our study has a functional cytosine DNA methylation pathway. We were interested in determining the actual levels of 5mC in different strains and used LC MS/MS to detect 5-methyl-2′-deoxycytidine (5mdC) levels in complete DNA digests. The dcm+ laboratory K-12 strains have ~1% 5mdC; JM109 has 0.92% (± 0.02) 5mdC; and BW25113 has 1.02% (± 0.09) 5mdC.