title: Inactivation of the acrA gene is partially responsible for chloramphenicol sensitivity of Escherichia coli CM2555 strain expressing the chloramphenicol acetyltransferase gene.
abstract: An Escherichia coli CM2555 strain, sensitive to chloramphenicol when expressing the cat gene and producing active chloramphenicol acetyltransferase (CAT), was described recently. It was proposed that this sensitivity is due to decreased levels of acetyl coenzyme A (Acetyl CoA) in cat-expressing CM2555 cells in the presence of chloramphenicol. CAT catalyzes transfer of the acetyl moiety from Acetyl CoA to a chloramphenicol molecule. Thus, a very efficient acetylation of chloramphenicol may cause deprivation of Acetyl CoA and cell death. A specific mutation causing the chloramphenicol sensitivity phenotype of CM2555 was not reported to date. Therefore, we aimed to identify a genetic defect causing this phenotype. Here, we found that overexpression of the acrEF genes, encoding a transmembrane pump, or the acrE gene alone, results in restoration of chloramphenicol-resistance of cat-expressing CM2555 strain. Although no mutation exists in the CM2555 acrE locus, a nonsense mutation in the 67th codon of the acrA gene, which encodes a component of another transmembrane pump, has been found. Although introduction of the deltaacrAB allele into CM732, a parental strain of CM2555, and into some other commonly used E. coli strains led to their chloramphenicol sensitivity in the presence of CAT, the same genetic manipulation did not result in such a phenotype in other genetic backgrounds, including "wild-type" E. coli MG1655. These results suggest that the acrA dysfunction is one of more mutations responsible for chloramphenicol sensitivity of cat-expressing CM2555 strain.
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002701:Chloramphenicol / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D018389:Codon, Nonsense; D017931:DNA Primers; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008074:Lipoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests
chemical_list: C083497:AcrA protein, E coli; D001426:Bacterial Proteins; D018389:Codon, Nonsense; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase