Gene cloning and characterization of VcrM, a Na+-coupled multidrug efflux pump, from Vibrio cholerae non-O1.
We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.
Huda
Md Nazmul
MN
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, Okayama 700-8530, Japan.
Chen
Jing
J
Morita
Yuji
Y
Kuroda
Teruo
T
Mizushima
Tohru
T
Tsuchiya
Tomofusa
T
D000167:Acriflavine / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D002414:Cations, Monovalent / Q000494:pharmacology; D003001:Cloning, Molecular; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005798:Genes, Bacterial; D057927:Hydrophobic and Hydrophilic Interactions; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D014734:Vibrio cholerae / Q000187:drug effects / Q000235:genetics*
D000900:Anti-Bacterial Agents; D017920:Antiporters; D002414:Cations, Monovalent; D011993:Recombinant Fusion Proteins; D000167:Acriflavine; D012964:Sodium