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. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Antimicrobial Agents and Chemotherapy Année : 2012

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.

Sylvie Baucheron
Franck Coste
Sylvie Canepa
  • Fonction : Auteur
Marie-Christine Maurel
  • Fonction : Auteur
Etienne Giraud
Bertrand Castaing
Alain Roussel
Axel Cloeckaert

Résumé

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.

Dates et versions

hal-00721770 , version 1 (30-07-2012)

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Citer

Sylvie Baucheron, Franck Coste, Sylvie Canepa, Marie-Christine Maurel, Etienne Giraud, et al.. 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.. Antimicrobial Agents and Chemotherapy, 2012, 56 (2), pp.942-8. ⟨10.1128/AAC.05444-11⟩. ⟨hal-00721770⟩
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