Binding of the RamR Repressor to Wild-Type and Mutated Promoters of theramAGene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

Abstract

ABSTRACTThe transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenicEnterobacteriaceae. InSalmonella entericaserovar Typhimurium (S.Typhimurium),ramAexpression 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 theramApromoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site oframA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramAas 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 (KD[equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramAof an MDRS.Typhimurium clinical isolate than for the wild-type PramA(KD= 66 nM). These results confirm the direct regulatory role of RamR in the repression oframAtranscription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.