title: Binding of the RamR repressor to wild-type and mutated promoters of the RamA gene involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium.
abstract: The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(D) [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P(ramA) of an MDR S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(D) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
authors: INRA, UR1282 Infectiologie Animale et Santé Publique, Nouzilly, France. sylvie.baucheron@tours.inra.fr
authors: Sylvie
authors: S
authors: Baucheron
authors: Franck
authors: F
authors: Coste
authors: Sylvie
authors: S
authors: Canepa
authors: Marie-Christine
authors: MC
authors: Maurel
authors: Etienne
authors: E
authors: Giraud
authors: Françoise
authors: F
authors: Culard
authors: Bertrand
authors: B
authors: Castaing
authors: Alain
authors: A
authors: Roussel
authors: Axel
authors: A
authors: Cloeckaert
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C531999:RamA protein, Salmonella typhimurium