Characterization of the pcaR

The pca branch of the 0-ketoadipate pathway in Pseudomonas putida is responsible for the complete degradation ofp-hydroxybenzoate through ortho cleavage of the initial pathway metabolite, protocatechuate. The pcaR regulatory locus has been found to be required for both induction of all of the genes within the pca regulon (pcaBDC, pcaUj, and peaF) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using TnS and mini-TnS transposons, was used to locate, clone, and sequence this pcaR regulatory gene. The peaR gene product, when overexpressed in Escherichia coli, possessed a specific affinity for the pcaIl promoter region and demonstrated that the entire PcaR protein was required for this function. The deduced amino acid sequence of the PcaR regulatory peptide bears little resemblance to its counterpart in the other branch of the pathway, CatR, but exhibits significant homology to its regulatory antecedent, PobR, which regulates the initial breakdown ofp-hydroxybenzoate into protocatechuate. Comparisons of the pcal and pcaR promoter regions revealed conservation of a 15-bp sequence centered around the -10 region in both sequences. This, together with previously defined deletional studies with thepcal promoter region, suggests that PcaR exerts its regulatory effect through protein-DNA interactions within this region, which would be unusually close to the transcriptional start site ofpcalJ for a positive regulator.