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Antimicrob. Agents Chemother. doi:10.1128/AAC.00460-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Analysis of the topology of NorM of Vibrio cholerae and identification of amino acid residues involved in norfloxacin resistance

Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700009, India

^* To whom correspondence should be addressed. Email: manikuntala{at}vsnl.net.

	Abstract

NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion (MATE) family of transporters. We demonstrate that NorM confers resistance to norfloxacin (NOR), ciprofloxacin (CIP), and ethidium bromide (ETBR). Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple-sequence alignments of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using -lactamase- and chloramphenicol acetyl transferase (CAT) fusions. The amino acid residues G¹⁸⁴, K¹⁸⁵, G¹⁸⁷, P¹⁸⁹, E¹⁹⁰, G¹⁹² and G¹⁹⁵ in the periplasmic loops, L³⁸¹, R³⁸², G³⁸³, Y³⁸⁴, K³⁸⁵ and D³⁸⁶ 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 G¹⁸⁴ or G¹⁸⁷ by A, K¹⁸⁵ by R and E¹⁹⁰ by D retained the norfloxacin resistance phenotype of NorM. Analysis of accumulation of norfloxacin in intact cells of Escherichia coli expressing NorM or its mutants, in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) 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 E¹²⁴, D¹⁵⁵, G¹⁸⁴, K¹⁸⁵, G¹⁸⁷, P¹⁸⁹, E¹⁹⁰, C¹⁹⁶ and Y³⁸⁴ are likely involved in NorM-dependent norfloxacin efflux. Except D¹⁵⁵, C¹⁹⁶ and Y³⁸⁴, all these residues were located in periplasmic loops.

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