Bertani Iris, Kojic Milan, Venturi Vittorio
Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy1.
Microbiology (Reading). 2001 Jun;147(Pt 6):1611-1620. doi: 10.1099/00221287-147-6-1611.
The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the beta-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB. In this study the identification and characterization of the pobC-pobA locus of P. putida WCS358 is presented. The p-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 pobA promoter transcriptionally fused to a promoterless lacZ 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 pobC::Tn5 and pcaH::Tn5 mutants of strain WCS358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed. Finally, the activation of pobA by PHB varied according to the concentration and it 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 regulation 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 PHB transport protein resulting in lower levels of PHB present inside the cell.
研究了恶臭假单胞菌WCS358中对羟基苯甲酸羟化酶基因（pobA）的调控，该基因参与对羟基苯甲酸（PHB）分解代谢生成中心中间体原儿茶酸的过程。原儿茶酸（PCA）随后通过β-酮己二酸途径降解，形成三羧酸中间体。在几种革兰氏阴性细菌中，已发现pobA在基因上与一种名为pobR的调节因子相连，pobR可响应PHB激活pobA的表达。在本研究中，介绍了恶臭假单胞菌WCS358的pobC - pobA基因座的鉴定和特征。对羟基苯甲酸羟化酶（PobA）与其他已鉴定的PobA蛋白高度同源，而调节蛋白PobC与其他研究的PobR蛋白的同源性并不高，属于AraC家族调节蛋白，因此将其命名为POBC：使用与无启动子lacZ基因转录融合的pobA启动子，观察到当存在PHB时，通过PobC的诱导非常有效，在存在PCA时诱导水平较低但仍很显著。利用本研究构建的WCS358菌株的pobC::Tn5和pcaH::Tn5突变体，从遗传学角度证明了这种PobC - PCA反应。在pobC突变体中，未观察到对羟基苯甲酸和PCA反应，而在缺乏功能性原儿茶酸3,4 - 双加氧酶的pcaH突变体中，仍观察到原儿茶酸依赖性的pobA激活。最后，PHB对pobA的激活因浓度而异，并且观察到在WCS358菌株的pcaR::Tn5调节突变体中，pobA启动子活性降低。PcaR是一种参与β-酮己二酸途径多个基因座调控的调节因子，其中之一是pcaK。据推测，pcaR::Tn5突变体中pobA激活的降低是因为编码PHB转运蛋白的pcaK基因没有表达，导致细胞内PHB水平较低。
