AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening.
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.
Faculty of Cellular and Molecular Biosciences, School of Life Sciences, Arizona State University, Tempe, AZ, USA.
Jon W
JW
Weeks
Teresa
T
Celaya-Kolb
Sara
S
Pecora
Rajeev
R
Misra
D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000254:growth & development; D003087:Colicins / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics; D017434:Protein Structure, Tertiary; D013489:Suppression, Genetic; D053585:Virus Attachment
C083497:AcrA protein, E coli; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C073126:tolC protein, E coli