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Journal of Bacteriology, March 2002, p. 1370-1377, Vol. 184, No. 5
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.5.1370-1377.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

*** Jian-Lin Yu,1 Leo Grinius,2 and David C. Hooper1*

Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114,1 Health Care Research Center, Procter and Gamble Pharmaceuticals, Mason, Ohio 450402

Received 20 June 2001/ Accepted 6 December 2001

Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K+-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.

* Corresponding author. Mailing address: Division of Infectious Diseases, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696. Phone: (617) 726-3812. Fax: (617) 726-7416. E-mail: dhooper{at}partners.org.

Journal of Bacteriology, March 2002, p. 1370-1377, Vol. 184, No. 5
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.5.1370-1377.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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