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Antimicrobial Agents and Chemotherapy, March 2007, p. 923-929, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.01048-06

Substrate Competition Studies Using Whole-Cell Accumulation Assays with the Major Tripartite Multidrug Efflux Pumps of Escherichia coli{triangledown}

Christopher A. Elkins* and Lisa B. Mullis

Division of Microbiology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, Arkansas 72079-9502

Received 21 August 2006/ Returned for modification 25 September 2006/ Accepted 26 December 2006

AcrAB-TolC is the major, constitutively expressed tripartite multidrug efflux system in Escherichia coli that recognizes various structurally unrelated molecules, including many antibiotics, dyes, and steroids. The AcrB inner membrane pump portion of the efflux system has been shown in recent structural studies to bind substrates at multiple sites, suggesting that particular substrate "sets" may compete for efflux by interfering with a certain binding site(s). However, our data indicate that the general structural class does not appear to dictate a particular substrate binding site that can be competitively inhibited in whole cells. In our study, substrate competition failed to increase cell-associated levels of steroids or dyes to levels characteristic of AcrB- or AcrB/EmrAB-deficient genomic mutants or achieved with the pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In addition, this general observation was sustained even with (i) a cocktail containing seven-pump substrates supplied slightly below their respective wild-type MIC levels, (ii) competing drug substrates of the same structural class (steroids or macrolides), and (iii) hyper-MIC levels of the exogenously supplied agents. Thus, this pump system (and possibly EmrAB-TolC) may have an extraordinary capacity to simultaneously handle multiple-drug substrates that is not necessarily reflected in MIC analyses. In addition, our study has extended the range of substrates recognized by the AcrAB- and EmrAB-TolC systems.

* Corresponding author. Mailing address: Division of Microbiology, National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, AR 72079-9502. Phone: (870) 543-7547. Fax: (870) 543-7307. E-mail: chris.elkins{at}fda.hhs.gov.

{triangledown} Published ahead of print on 8 January 2007.

Antimicrobial Agents and Chemotherapy, March 2007, p. 923-929, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.01048-06






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