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Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate.

ABSTRACT: The pca branch of the beta-ketoadipate pathway in Pseudomonas putida is responsible for the complete degradation of p-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, pcaIJ, and pcaF) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using Tn5 and mini-Tn5 transposons, was used to locate, clone, and sequence this pcaR regulatory gene. The pcaR gene product, when overexpressed in Escherichia coli, possessed a specific affinity for the pcaIJ 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 of p-hydroxybenzoate into protocatechuate. Comparisons of the pcaIJ 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 the pcaIJ 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 of pcaIJ for a positive regulator.

SUBMITTER: Romero-Steiner S

PROVIDER: S-EPMC196781 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate.

Romero-Steiner S S Parales R E RE Harwood C S CS Houghton J E JE

Journal of bacteriology 19940901 18

The pca branch of the beta-ketoadipate pathway in Pseudomonas putida is responsible for the complete degradation of p-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, pcaIJ, and pcaF) and the chemotactic response of the bacteria to aromatic compounds. Insertional inactivation mutagenesis, using Tn5 and mini-Tn5 transposons, was u ...[more]

PMID: 8083169

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Project description:Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, and regulate expression of the nicotine-degrading genes in the middle of nic2 gene cluster, which are not controlled by the previously reported Phsp promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the -35/-10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters (Pspm and Phsp), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase.IMPORTANCE We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, which controls expression of nicotine-degrading genes that are not controlled by the Phsp promoter. We identified specific nucleotides of the Pspm promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the Pspm and Phsp promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.

Project description:Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4-hydroxybenzoate via two parallel sequences of reactions that converge at beta-ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates. Structural genes (pca genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the beta-ketoadipate pathway have been characterized, and a specific transport system for 4-hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processes of 4-hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-hydroxybenzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently. PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes. In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate. This previously unrecognized layer of regulatory control in the beta-ketoadipate pathway appears to extend to the first two steps of 4-hydroxybenzoate degradation, since levels of 4-hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-hydroxybenzoate and benzoate. The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate. These findings indicate that 4-hydroxybenzoate transport is an integral feature of the beta-ketoadipate pathway in P. putida and that transport plays a role in establishing the preferential degradation of benzoate over 4-hydroxybenzoate. These results also demonstrate that there is communication between the two branches of the beta-ketoadipate pathway.

Dataset Information

Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate.

Publications

Characterization of the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate.

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