Nichols N N, Harwood C S
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
J Bacteriol. 1995 Dec;177(24):7033-40. doi: 10.1128/jb.177.24.7033-7040.1995.
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 tricarboxylic acid cycle intermediates. Structural genes (pca genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the beta-ketoadipate pathway have been characterized, and a specific transport system for 4-hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processes of 4-hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-hydroxybenzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently. PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes. In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate. This previously unrecognized layer of regulatory control in the beta-ketoadipate pathway appears to extend to the first two steps of 4-hydroxybenzoate degradation, since levels of 4-hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-hydroxybenzoate and benzoate. The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate. These findings indicate that 4-hydroxybenzoate 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.
恶臭假单胞菌PRS2000通过两条平行的反应序列降解芳香酸苯甲酸酯和4-羟基苯甲酸酯，这两条序列在β-酮己二酸处汇聚，β-酮己二酸的一种衍生物被裂解形成三羧酸循环中间体。通过β-酮己二酸途径的原儿茶酸分支完全降解4-羟基苯甲酸所需的结构基因（pca基因）已得到表征，并且最近描述了一种针对4-羟基苯甲酸的特定转运系统。为了更好地理解恶臭假单胞菌如何协调4-羟基苯甲酸的转运和代谢过程以实现完全降解，对4-羟基苯甲酸转运基因pcaK以及苯甲酸酯和4-羟基苯甲酸降解所需的基因pcaF的调控进行了比较。引物延伸分析和lacZ融合表明，位于染色体上相邻位置的pcaK和pcaF是独立转录的。PcaR是β-酮己二酸途径几个基因的转录激活因子，pcaF和pcaK的表达都需要它，并且途径中间体β-酮己二酸可诱导这两个基因。除了这些预期的调控元件外，苯甲酸酯会抑制pcaK的表达，但不会抑制pcaF的表达。β-酮己二酸途径中这一先前未被认识的调控层面似乎延伸到了4-羟基苯甲酸降解的前两个步骤，因为当细胞在4-羟基苯甲酸和苯甲酸酯的混合物上生长时，4-羟基苯甲酸羟化酶和原儿茶酸3,4-双加氧酶的活性水平也会降低。苯甲酸酯抑制的明显结果是细胞优先降解苯甲酸酯而非4-羟基苯甲酸。这些发现表明，4-羟基苯甲酸的转运是恶臭假单胞菌β-酮己二酸途径的一个组成特征，并且转运在确立苯甲酸酯比4-羟基苯甲酸优先降解方面发挥作用。这些结果还证明了β-酮己二酸途径的两个分支之间存在信息交流。
