Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance

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. Typhimu-rium), 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 (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the10 conserved region, the tran-scriptional 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 PramA 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 (KD [equilibrium dissociation constant] 191 nM) for the 2-bp-deleted PramA of anMDR S. Typhimurium clinical isolate than for the wild-type PramA (KD 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcrip-tion and precisely define how an alteration of its binding site can give rise to anMDR phenotype. Multidrug resistance (MDR) achieved by intrinsic efflux sys-tems is a major resistance mechanism used by bacteria to resist antimicrobial treatments, and it therefore represents a seri-ous health problemworldwide.Most bacteria intrinsically possess severalmembrane transport systemswhich can decrease the intra