Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358

The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomo nas putida WC5358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic ac id (PCA) is then degraded via the beta -ketoadipate pathway to form tricarb oxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA ex pression in response to PHB, In this study the identification and character ization of the pobC-pobA locus of P, putida WCS358 is presented. The p-hydr oxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high id entity to other PobR proteins studied and belonged to the AraC family of re gulatory proteins, hence it has been designated PobC, Using the pobA promot er transcriptionally fused to a promoterless lad gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA, This PobC-P CA response was genetically demonstrated by making use of pobC::Tn5 and pca H::Tn5 mutants of strain WC5358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in th e pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, th e protocatechuic-acid-dependent pobA activation was still observed. Finally , the activation of pobA by PHB varied according to the concentration and i t was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced. PcaR is a regulator involved in the re gulation of several loci of the beta -ketoadipate pathway, one of which is pcaK, It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the P HB transport protein resulting in lower levels of PHB present inside the ce ll.