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Antimicrobial Agents and Chemotherapy, September 2008, p. 3052-3060, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00475-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli{triangledown} ,{dagger}

Feng Long,1 Corinne Rouquette-Loughlin,2 William M. Shafer,2,3 and Edward W. Yu1,4*

Molecular, Cellular, and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Ames, Iowa 50011,1 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322,2 Laboratories of Microbial Pathogenesis, VA Medical Center, Decatur, Georgia 30033,3 Department of Physics and Astronomy, Iowa State University, Ames, Iowa 500114

Received 9 April 2008/ Accepted 22 June 2008

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 the YdhE protein 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 capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro. E. coli strains expressing norM or ydhE showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in E. coli, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na+-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.

* Corresponding author. Mailing address: Department of Physics and Astronomy, A115 Physics, Iowa State University, Ames, IA 50011. Phone: (515) 294-4955. Fax: (515) 294-6027. E-mail: ewyu{at}iastate.edu

{triangledown} Published ahead of print on 30 June 2008.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, September 2008, p. 3052-3060, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00475-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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