title: VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA.
abstract: VceR, a member of the TetR family of transcriptional regulators, is a repressor of the vceCAB operon, which encodes a multidrug efflux pump in Vibrio cholerae. VceR binds to a 28 bp inverted-repeat within the vceR-vceC intergenic region and is dissociated from this site with CCCP, a pump substrate. The rate of the CCCP-induced conformational change in VceR was determined by stopped-flow fluorescence spectroscopy, revealing a highly co-operative process that occurs with a Hill coefficient of approximately 4. The apparent affinity for CCCP decreased in a linear manner with increasing concentrations of DNA, indicative of competition between the CCCP and DNA for binding to VceR. These data are consistent with an equilibrium between mutually exclusive conformations that are supported by the binding of DNA and CCCP to the N and C termini of VceR, respectively. Size-exclusion chromatography and dynamic light-scattering studies indicate that VceR exists predominantly as a dimer; however, a pair of dimers binds to the DNA. In order to account for the fact that VceR is a dimer in the absence of DNA but binds CCCP with a Hill co-efficient of 4, implying that it has at least four binding-sites, we propose that the VceR monomer possesses a pair of binding sites that can be simultaneously occupied by CCCP. Using a gene-reporter system and stopped-flow spectroscopy, we established that the equilibrium between free VceR and VceR-CCCP plays a critical role in controlling expression of the pump. The co-operative transition between these states allows the repressor to respond to relatively small changes in drug concentration. Thus, repression and induction can be readily switched about a critical drug concentration which will prove toxic to the cell.
authors.affiliation: Centre for Infectious Diseases, Department of Biological and Biomedical Sciences, University of Durham, Wolfson Research Institute, Queen's Campus, Stockton-on-Tees TS17 6BH, UK.
authors.forename: M Ines
authors.initials: MI
authors.lastname: Borges-Walmsley
authors.forename: Dijun
authors.initials: D
authors.lastname: Du
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: McKeegan
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Sharples
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Walmsley
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D021901:DNA, Intergenic / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024881:Drug Resistance, Bacterial; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D021901:DNA, Intergenic; D012097:Repressor Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004247:DNA