CHARACTERIZATION OF THE PCAR REGULATORY GENE FROM PSEUDOMONAS-PUTIDA,WHICH IS REQUIRED FOR THE COMPLETE DEGRADATION OF P-HYDROXYBENZOATE

The pea branch of the beta-ketoadipate pathway in Pseudomonas putida i s responsible for the complete degradation of p-hydroxybenzoate throug h ortho cleavage of the initial pathway metabolite, protocatechuate. T he pcaR regulatory locus has been found to be required for both induct ion of all of the genes within the pea regulon (pcaBDC, pcaIJ, and pca F) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using Tn5 and mini-Tn5 transpos ons, was used to locate, clone, and sequence this pcaR regulatory gene . The pcaR gene product, when overexpressed in Escherichia coli, posse ssed a specific affinity for the pcaIJ promoter region and demonstrate d that the entire PcaR protein was required for this function. The ded uced amino acid sequence of the PcaR regulatory peptide bears;little r esemblance 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 of p-hydroxybenzoate into prot ocatechuate. Comparisons of the pcaIJ and pcaR promoter regions reveal ed conservation of a 15-bp sequence centered around the -10 region in both sequences. This, together with previously defined deletional stud ies with the pcaIJ promoter region, suggests that PcaR exerts its regu latory effect through protein-DNA interactions within this region, whi ch would be unusually close to the transcriptional start site of pcaIJ for a positive regulator.