Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR

The PtxS and PtxR regulators control the expression of the glucose dehydrogenase genes from the P(gad) promoter in Pseudomonas aeruginosa. These regulators bind to their cognate operators, that are separated by ∼50 nt, within the promoter region and interact with each other creating a DNA-loop that prevents RNA polymerase promoter access. Binding of the 2-ketogluconate effector to PtxS caused PtxS/PtxR complex dissociation and led to the dissolution of the repression loop facilitating the entry of the RNA polymerase and enabling the transcription of the gad gene. We have identified a hydrophobic surface patch on the PtxR putative surface that was hypothesized to correspond to the binding site for PtxS. Two surface-exposed residues in this patch, V173 and W269, were replaced by alanine. Isothermal titration calorimetry assays showed that PtxS does not interact with the mutant variants of PtxR. Electrophoretic mobility shift assay and DNAase I footprinting assays proved that both regulators bind to their target operators and that failure to interact with each other prevented the formation of the DNA-loop. In vitro transcription showed that PtxS per se is sufficient to inhibit transcription from the P(gad) promoter, but that affinity of PtxS for its effector is modulated by PtxR.