IDENTIFICATION OF THE PCARKF GENE-CLUSTER FROM PSEUDOMONAS-PUTIDA - INVOLVEMENT IN CHEMOTAXIS, BIODEGRADATION, AND TRANSPORT OF 4-HYDROXYBENZOATE

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and oth er aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded vi a the beta-ketoadipate pathway. Isolation of a transposon mutant defec tive in 4-hydroxybenzoate chemotaxis allowed identification of a new g ene cluster designated pcaRKF. DNA sequencing, mutational analysis, an d complementation studies revealed that pcaR encodes a regulatory prot ein required for induction of at least four of the enzymes of the beta -ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encode s a transporter for 4-hydroxybenzoate, and this protein is also requir ed for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membersh ip in the major facilitator superfamily of transport proteins. The pro tein, expressed in Escherichia coil, catalyzed 4-hydroxybenzoate trans port. In addition, whole cells of P. putida pcaK mutants accumulated 4 -hydroxybenzoate at reduced rates compared with that in wild-type cell s. The pcaK mutation did not impair growth at the expense of 4-hydroxy benzoate under most conditions; however, mutant cells grew somewhat mo re slowly than the wild type on 4-hydroxybenzoate at a high pH. The fi nding that 4-hydroxybenzoate chemotaxis can be disrupted without an ac companying effect on metabolism indicates that this chemotactic respon se is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it pl ays an indirect role in chemotaxis. These findings indicate that aroma tic acid detection and transport are integral features of aromatic deg radation pathways.