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 (TPPCI) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na⁺ or Li⁺. Moreover, Na⁺ efflux was observed with VcrM when TPPCI was added to Na⁺-loaded cells. Therefore, we conclude that VcrM is a Na⁺/drug antiporter-type multidrug efflux pump.