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Loss of DNA cytosine methylation increases ethidium bromide resistance in Escherichia coli (800.4)
Author(s) -
Mandarano Alexandra,
Varechtchouk Olga,
Simon Robert,
Militello Kevin
Publication year - 2014
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.28.1_supplement.800.4
Subject(s) - ethidium bromide , microbiology and biotechnology , rpos , dna methylation , gene knockout , escherichia coli , methylation , knockout mouse , biology , chemistry , cytosine , gene , gene expression , dna , promoter , genetics
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