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Journal of Bacteriology, January 2008, p. 691-698, Vol. 190, No. 2
0021-9193/08/$08.00+0     doi:10.1128/JB.01276-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Fitting Periplasmic Membrane Fusion Proteins to Inner Membrane Transporters: Mutations That Enable Escherichia coli AcrA To Function with Pseudomonas aeruginosa MexB

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

Received 7 August 2007/ Accepted 5 November 2007

AcrAB-TolC from Escherichia coli is a multidrug efflux complex capable of transenvelope transport. In this complex, AcrA is a periplasmic membrane fusion protein that establishes a functional connection between the inner membrane transporter AcrB of the RND superfamily and the outer membrane channel TolC. To gain insight into the mechanism of the functional association between components of this complex, we replaced AcrB with its close homolog MexB from Pseudomonas aeruginosa. Surprisingly, we found that AcrA is promiscuous and can form a partially functional complex with MexB and TolC. The chimeric AcrA-MexB-TolC complex protected cells from sodium dodecyl sulfate, novobiocin, and ethidium bromide but failed with other known substrates of MexB. We next identified single and double mutations in AcrA and MexB that enabled the complete functional fit between AcrA, MexB, and TolC. Mutations in either the -helical hairpin of AcrA making contact with TolC or the β-barrel domain lying on MexB improved the functional alignment between components of the complex. Our results suggest that three components of multidrug efflux pumps do not associate in an "all-or-nothing" fashion but accommodate a certain degree of flexibility. This flexibility in the association between components affects the transport efficiency of RND pumps.

Published ahead of print on 16 November 2007.