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.
Weeks
Jon W
JW
Faculty of Cellular and Molecular Biosciences, School of Life Sciences, Arizona State University, Tempe, AZ, USA.
Celaya-Kolb
Teresa
T
Pecora
Sara
S
Misra
Rajeev
R
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