Co-regulation of the dependent Pu promoter of TOL plasmid: interplay between the LytTR response regulator PprA and the enhanced binding protein XylR

Bacteria colonize a wide range of environments in which carbon and energy sources are often limited. Therefore the presence of a certain catabolic arrangement are key functions in the bacterial fight for survival. One example of this is the degradation of toluene by the Pseudomonas putida encoded by upper and meta operons. Expression from the upper operon is driven by the ?54-dependent promoter Pu that is activated in the presence of aromatic effector by the activator XylR. The physical interaction between XylR and ?54-RNA polymerase, assisted by IHF-induced bent, triggers transcription initiation from Pu. Pu activity is regulated under different physiological and/or environmental conditions through mechanisms which have not been extensively characterized. In this work we aimed to identify novel proteins involved in the physiological regulation of Pu. We screened the P. putida proteome for proteins able to interact with Pu DNA by EMSA assay. This approach indicated that the Pu enhancer region (UAS), which contains the target sites of XylR, could form a specific complex with an unknown protein factor. Following DNA affinity purification and mass spectrometry analysis, this factor was identified as the two-component response regulator PprA. Knock-out inactivation of the pprA gene resulted in the up-regulation of Pu, while PprA expression from a plasmid strongly repressed Pu activity. This negative regulatory role was also verified in vitro with purified components and in an E. coli reporter system. Interestingly, the overlap of the PprA binding site with UAS suggested a direct interplay with XylR as repression mechanism. We speculated that PprA could hinder the XylR cooperative occupation of UAS and eventually impede full Pu activation.