PcaR-mediated activation and repression of pca genes from Pseudomonas putida are propagated by its binding to both the -35 and the -10 promoter elements

Abstract

Degradation of protocatechuate in Pseudomonas putida is accomplished by the products of the pca genes (pcaH, G, pcaBDC, pcal, J and pcaF). In P. putida, all these genes (with the exception of pcaH, G) are activated by the regulatory protein PcaR, in association with the pathway intermediate β-ketoadipate. Having previously cloned and characterized the pcaR locus, we have overexpressed and purified the PcaR protein to homogeneity. The purified PcaR protein was shown to form a homodimer in solution and to bind specifically to its own promoter, as well as to the promoter regions of pcal, J and pcaF. Subsequent footprint analyses demonstrated that the binding of PcaR to its own promoter occurs within a footprint that extends from the -20 to the +4 position. In contrast, PcaR appeared to interact with the inducible pcal, J promoter as a dimer of dimers; binding in tandem within a dual footprint encompassing both the '-35' and the '-10' regions of the promoter sequence. The similarities and differences between the two binding patterns correlate well with the very different effects that PcaR has upon transcription at each of the promoter sequences. The interactions at the pcal, J promoter are reminiscent of those exhibited by the MerR family of regulatory proteins. However, as PcaR bears very little primary sequence homology to any of the regulatory proteins within this family, the results presented here reveal the possible existence of a new series of functionally related transcriptional inducers.