1887

Abstract

The regulation of the -hydroxybenzoate hydroxylase gene () of WCS358 involved in the catabolism of -hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the β-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria has been found genetically linked to a regulator called which activates expression in response to PHB. In this study the identification and characterization of the - locus of WCS358 is presented. The -hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated PobC. Using the promoter transcriptionally fused to a promoterless 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-PCA response was genetically demonstrated by making use of ::Tn and ::Tn mutants of strain WCS358 constructed in this study. In mutants both the -hydroxybenzoic and PCA response were not observed, whereas in the mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent activation was still observed. Finally, the activation of by PHB varied according to the concentration and it was observed that in the ::Tn regulatory mutant of strain WCS358 the promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the β-ketoadipate pathway, one of which is . It was postulated that the reduction of activation in ::Tn mutants was because there was no expression of the gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.

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2001-06-01
2019-08-21
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