TY - JOUR
T1 - AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening
AU - Weeks, Jon W.
AU - Celaya-Kolb, Teresa
AU - Pecora, Sara
AU - Misra, Rajeev
PY - 2010/3
Y1 - 2010/3
N2 - 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.
AB - 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.
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U2 - 10.1111/j.1365-2958.2010.07068.x
DO - 10.1111/j.1365-2958.2010.07068.x
M3 - Article
C2 - 20132445
AN - SCOPUS:77949360141
SN - 0950-382X
VL - 75
SP - 1468
EP - 1483
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 6
ER -