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Journal of Bacteriology, March 2009, p. 1729-1737, Vol. 191, No. 6
0021-9193/09/$08.00+0     doi:10.1128/JB.01441-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism

Yumiko Takatsuka and Hiroshi Nikaido^*

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

Received 14 October 2008/ Accepted 26 November 2008

Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The "linked-trimer" AcrB was expressed well in the inner membrane fraction of acrB recA strains, as a large protein of 300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

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* 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) 643-6334. E-mail: nhiroshi{at}berkeley.edu

Published ahead of print on 5 December 2008.

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Journal of Bacteriology, March 2009, p. 1729-1737, Vol. 191, No. 6
0021-9193/09/$08.00+0     doi:10.1128/JB.01441-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Related articles in JB:

Covalently Linked AcrB Giant Offers a New Powerful Tool for Mechanistic Analysis of Multidrug Efflux in Bacteria

Helen I. Zgurskaya
JB 2009 191: 1727-1728. [Full Text]  

This article has been cited by other articles:

• Zgurskaya, H. I. (2009). Covalently Linked AcrB Giant Offers a New Powerful Tool for Mechanistic Analysis of Multidrug Efflux in Bacteria. J. Bacteriol. 191: 1727-1728 [Full Text]