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Antimicrob. Agents Chemother. doi:10.1128/AAC.00475-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli

Molecular, Cellular and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Ames, IA 50011; Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322; Laboratories of Microbial Pathogenesis, VA Medical Center, Decatur, Georgia 30033; Department of Physics and Astronomy, Iowa State University, Ames, IA 50011

	Abstract

Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of Neisseria gonorrhoeae and YdhE of Escherichia coli have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capability, we cloned, expressed, and purified these two efflux proteins, and characterized their functions both in vivo and in vitro. E. coli expressing norM or ydhE showed elevated (two-fold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavin, crystal violet, barberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When expressed in E. coli, both transporters reduced ethidum bromide and norfloxacin accumulation through a mechanism requiring the proton motive force and direct measurements of efflux confirmed that NorM behaves as a Na⁺-dependent transporter. The capacity of NorM and YdhE to recognize structurally divergent compounds was confirmed using steady-state fluorescence polarization assays, and the results revealed that these transporters bind antimicrobials with dissociation constants (K_D) in the micromolar region.