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Journal of Bacteriology, July 2009, p. 4365-4371, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00204-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC {triangledown}

Qiang Ge,{dagger} Yoichi Yamada,{dagger} and Helen Zgurskaya*

Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Room 208, Norman, Oklahoma 73019

Received 26 February 2009/ Accepted 22 April 2009

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Room 208, Norman, OK 73019. Phone: (405)325-1678. Fax: (405) 325-6111. E-mail: elenaz{at}ou.edu

{triangledown} Published ahead of print on 1 May 2009.

{dagger} These authors contributed equally to this work.

Journal of Bacteriology, July 2009, p. 4365-4371, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00204-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Tikhonova, E. B., Dastidar, V., Rybenkov, V. V., Zgurskaya, H. I. (2009). Kinetic control of TolC recruitment by multidrug efflux complexes. Proc. Natl. Acad. Sci. USA 106: 16416-16421 [Abstract] [Full Text]  


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