Repression of 4-hydroxybenzoate transport and degradation by benzoate: A new layer of regulatory control in the Pseudomonas putida β-ketoadipate pathway

Abstract

Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4- hydroxybenzoate via two parallel sequences of reactions that converge at β- ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates. Structural genes (pea genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the β- 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 β-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate β-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 β-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 β-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 β-ketoadipate pathway.