Escherichia coli K-12 strains contain the orphan cytosine-5 DNA methyltransferase enzyme Dcm (DNA cytosine methyltransferase). Two recent reports indicate that Dcm has an influence on stationary phase gene expression in E. coli. Herein, we demonstrate that dcm knockout cells overexpress the drug resistance transporter SugE, which has been linked to ethidium bromide (ETBR) resistance. SugE expression also increased in the presence of the DNA methylation inhibitor 5-azacytidine, suggesting that Dcm-mediated DNA methylation normally represses sugE expression. The effect of Dcm on sugE expression is primarily restricted to early stationary phase, and RpoS is required for robust sugE expression. Dcm knockout cells are more resistant to ETBR than wild-type cells, and complementation with a plasmid-borne dcm gene restores ETBR sensitivity. SugE knockout cells are more sensitive to ETBR than wild-type cells. These data indicate that Dcm influences the sensitivity to an antimicrobial compound through changes in gene expression.

In Escherichia coli, the second cytosine in the sequence 5′CC(A/T)GG3′ is methylated by the Dcm enzyme. Our laboratory has previously shown that DNA methylation represses expression of ribosomal proteins. The laboratory is currently investigating other targets of Dcm‐mediated DNA methylation. The sugE gene has been shown to confer resistance to quaternary ammonium compounds, and the sugE gene has one dcm recognition site in its 5′ flanking region, three in the open reading frame and one in the 3′ flanking region. We used qPCR to measure sugE RNA levels at both log and stationary phases in wild‐type and dcm knockout cells. Our data demonstrate that the sugE gene is overexpressed at both phases in dcm knockout cells. SugE expression also increased in the presence of 5‐azacytidine, an inhibitor of cytosine DNA methylation. In order to determine if dcm influences sensitivity to different antibacterial compounds, the sensitivity to ethidium bromide (EtBr) in wild‐type, dcm knockout, and sugE knockout strains was compared using Kirby‐Bauer disc diffusions assays, MIC assays and growth curve analysis. Our data indicate that sugE knockout cells are hypersensitive to EtBr, whereas dcm knockout cells have increased resistance to EtBr. We are currently testing a panel of quaternary ammonium compounds in these assays. In summary, DNA methylation can influence the sensitivity to antibacterial compounds.

Loss of DNA cytosine methylation increases ethidium bromide resistance in Escherichia coli Alexandra H Mandarano, Olga Varechtchouk, Robert D Simon, Kevin T Militello. Biology, State University of New York at Geneseo, Geneseo, NY In E. coli, the second cytosine in the sequence 5’CC(A/T)GG3’ is methylated by the Dcm enzyme. The sugE gene, which codes for a membrane transporter, has one dcm recognition site in its 5’ flanking region, three in the open reading frame and one in the 3’ flanking region. We used qPCR to measure sugE RNA levels in wild‐type and dcm knockout cells. Our data demonstrate that the sugE gene is overexpressed at early stationary phase in dcm knockout cells. Expression of sugE also increased in the presence of the DNA methyltransferase inhibitor 5‐azacytidine. Furthermore, stationary phase sigma factor rpoS knockout cells showed significant decreases in sugE expression, which is consistent with a known inverse relationship between Dcm and RpoS expression. The sugE gene has been shown to confer resistance to quaternary ammonium compounds, and has been linked to ethidium bromide (EtBr) resistance. In order to determine if dcm influences the sensitivity to antibacterial compounds through changes in sugE expression, the sensitivity of wild‐type, dcm knockout, and sugE knockout strains was compared using Kirby‐Bauer disc diffusions assays, MIC assays and growth curve analyses. Our data indicate that sugE knockout cells are hypersensitive to EtBr, whereas dcm knockout cells have increased resistance to EtBr. We are continuing to investigate the effects of other drugs on these E. coli strains and aim to elucidate the mechanism of the effect of Dcm on SugE‐mediated drug resistance. Grant Funding Source: Geneseo Foundation

Cytochrome's P450 (CYPs) are a group of enzymes that have a major role in metabolizing drugs, and their variable expression can lead to pharmacokinetic and pharmacodynamic differences within the population. Genetic variants in CYPs have been extensively investigated, but they only explain a part of the interindividual variation in hepatic CYP expression. A recent study has proposed SLC10A1, a bile acid uptake carrier, to be a novel regulator of CYP expression. Hence, we hypothesized that functional SNPs (single nucleotide polymorphisms) in SLC10A1 may have an effect on CYP expression. Towards this end we resequenced all exons, promoter, and 3′‐UTR of SLC10A1 in 98 human livers (43 over‐ and 55 under‐expressing) to look for functional SNPs. We found a total of eight SNPs. An exon 1 synonymous SNP (rs4646285) occurred with a higher frequency in livers overexpressing SLC10A1, whereas a 3′‐UTR SNP (rs45593332) occurred with a higher frequency in the livers underexpressing SLC10A1. The genotypes of rs4646285 correlated with total mRNA of SLC10A1 as well as the downstream target CYP3A4 (p=0.02). An allelic expression imbalance assay demonstrated samples having imbalanced SLC10A1 expression, although none of the SLC10A1 SNPs explained the cause of this imbalance. In conclusion, an exonic SLC10A1 SNP led to increased SLC10A1 mRNA expression and increased CYP3A4 expression and may help explain some of the variation in the pharmacokinetics of CYP3A4 substrate drugs. This work was funded by the Pediatric Oncology Education (POE) summer program at St. Jude Children's Hospital (O.V.).