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Antimicrobial Agents and Chemotherapy, November 2006, p. 3717-3723, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00460-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Analysis of the Topology of Vibrio cholerae NorM and Identification of Amino Acid Residues Involved in Norfloxacin Resistance ^,

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

Received 13 April 2006/ Returned for modification 6 June 2006/ Accepted 24 August 2006

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 ß-lactamase and chloramphenicol acetyltransferase fusions. The amino acid residues G¹⁸⁴, K¹⁸⁵, G¹⁸⁷, P¹⁸⁹, E¹⁹⁰, G¹⁹², and G¹⁹⁵ in the periplasmic loops and 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 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 E¹²⁴, D¹⁵⁵, G¹⁸⁴, K¹⁸⁵, G¹⁸⁷, P¹⁸⁹, E¹⁹⁰, C¹⁹⁶, and Y³⁸⁴ are likely involved in NorM-dependent norfloxacin efflux. Except for D¹⁵⁵, C¹⁹⁶, and Y³⁸⁴, all of these residues are located in periplasmic loops.

Published ahead of print on 5 September 2006.

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