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Journal of Bacteriology, March 2005, p. 1923-1929, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.1923-1929.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aminoglycosides Are Captured from both Periplasm and Cytoplasm by the AcrD Multidrug Efflux Transporter of Escherichia coli

Julio Ramos Aires and Hiroshi Nikaido*

Department of Molecular and Cell Biology, University of California, Berkeley, California

Received 13 July 2004/ Accepted 10 September 2004

To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the Escherichia coli AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the proton flux accompanying substrate transport. When the interior of the proteoliposomes was acidified, the addition of aminoglycosides to the external medium stimulated proton efflux and the intravesicular accumulation of radiolabeled gentamicin, suggesting that aminoglycosides can be captured and transported from the external medium in this system (corresponding to cytosol). This activity required the presence of AcrA within the proteoliposomes. Interestingly, the increase in proton efflux also occurred when aminoglycosides were present only in the intravesicular space. This result suggested that AcrD can also capture aminoglycosides from the periplasm to extrude them into the medium in intact cells, acting as a "periplasmic vacuum cleaner."

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 426 Barker Hall, University of California, Berkeley, CA 94720-3202. Phone: (510) 642-2027. Fax: (510) 643-9290. E-mail: nhiroshi{at}uclink4.berkeley.edu.

Journal of Bacteriology, March 2005, p. 1923-1929, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.1923-1929.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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