title: Identification, cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from Vibrio cholerae O395.
abstract: A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 microg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 microg/ml), 62.5-fold for rifampin (MIC = 50 microg/ml), >30-fold for erythromycin (MIC = 50 microg/ml) and minocycline (MIC = 2 microg/ml), 20-fold for trimethoprim (MIC = 0.12 microg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 microg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 microg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 microg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.
authors.affiliation: Department of Biology, Eastern New Mexico University, Roosevelt Hall, Room 101, Station 33, Portales, NM 88130, USA.
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Smith
authors.forename: Sanath
authors.initials: S
authors.lastname: Kumar
authors.forename: Manuel F
authors.initials: MF
authors.lastname: Varela
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D014734:Vibrio cholerae / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D004996:Ethidium