title: Analysis of the topology of Vibrio cholerae NorM and identification of amino acid residues involved in norfloxacin resistance.
abstract: NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. We demonstrate that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple sequence alignment of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using beta-lactamase and chloramphenicol acetyltransferase fusions. The amino acid residues G(184), K(185), G(187), P(189), E(190), G(192), and G(195) in the periplasmic loops and L(381), R(382), G(383), Y(384), K(385), and D(386) in the cytoplasmic loops, as well as all the acidic and cysteine residues of NorM, were mutated. Mutants G184V, G184W, K185I, P189S, E190K, and E190A lost the norfloxacin resistance-imparting phenotype characteristic of NorM. Mutants E124V, D155V, G187V, G187R, C196S, Y384H, Y384S, and Y384F exhibited partial resistance to norfloxacin. Mutants with replacements of G(184) or G(187) by A, K(185) by R, and E(190) by D retained the norfloxacin resistance phenotype of NorM. Analysis of the accumulation of norfloxacin in intact cells of Escherichia coli expressing NorM or its mutants in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone supported the results obtained through susceptibility testing and argued in favor of NorM-mediated efflux as the determining factor in norfloxacin susceptibility in the genetically manipulated strains. Taken together, these results suggested that E(124), D(155), G(184), K(185), G(187), P(189), E(190), C(196), and Y(384) are likely involved in NorM-dependent norfloxacin efflux. Except for D(155), C(196), and Y(384), all of these residues are located in periplasmic loops.
authors.affiliation: Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Rajen
authors.initials: R
authors.lastname: Haldar
authors.forename: Debabrata
authors.initials: D
authors.lastname: Mandal
authors.forename: Manikuntala
authors.initials: M
authors.lastname: Kundu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D015153:Blotting, Western; D003545:Cysteine / Q000502:physiology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium; D005456:Fluorescent Dyes; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009643:Norfloxacin / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014734:Vibrio cholerae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D001618:beta-Lactamases; D004996:Ethidium; D003545:Cysteine; D009643:Norfloxacin