Isolation and characterization of VceC gain-of-function mutants that can function with the AcrAB multiple-drug-resistant efflux pump of Escherichia coli.
VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.
Vediyappan
Govindsamy
G
Department of Microbiology and Immunology, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Borisova
Tatyana
T
Fralick
Joe A
JA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005838:Genotype; D008074:Lipoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C511952:VceC protein, E coli; C073126:tolC protein, E coli