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AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening
Author(s) -
Weeks Jon W.,
CelayaKolb Teresa,
Pecora Sara,
Misra Rajeev
Publication year - 2010
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2010.07068.x
Subject(s) - periplasmic space , biology , efflux , mutant , escherichia coli , colicin , bacterial outer membrane , microbiology and biotechnology , biochemistry , gene
Summary In Escherichia coli , the TolC–AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four‐residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC‐mediated phage or colicin infection. However, despite the null‐like drug sensitivity phenotype, chemical cross‐linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA‐mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB‐dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state.