REPRESSION OF 4-HYDROXYBENZOATE TRANSPORT AND DEGRADATION BY BENZOATE- A NEW LAYER OF REGULATORY CONTROL IN THE PSEUDOMONAS-PUTIDA BETA-KETOADIPATE PATHWAY

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 tricarbo xylic acid cycle intermediates. Structural genes (pea genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechu ate branch of the beta-ketoadipate pathway have been characterized, an d a specific transport system for 4-hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processe s of 4-hydroxybenzoate transport and metabolism to achieve complete de gradation, the regulation of pcaK, the 4-hydroxybenzoate transport gen e, and that of pcaF, a gene required for both benzoate and 4-hydroxybe nzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosom e, are transcribed independently. PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expres sion of both pcaF and pcaK, and the pathway intermediate beta-ketoadip ate induces both genes. In addition to these expected regulatory eleme nts, expression of pcaK, but not pcaF, is repressed by benzoate, This previously unrecognized layer of regulatory control in the beta-ketoad ipate pathway appears to extend to the first two steps of 4-hydroxyben zoate degradation, since levels of 4-hydroxybenzoate hydroxylase and p rotocatechuate 3,4-dioxygenase activities were also depressed when cel ls were grown on a mixture of 4-hydroxybenzoate and benzoate, The appa rent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate. These findings indicate that 4-hy droxybenzoate 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.