Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Appl Environ Microbiol. 2010 Dec;76(24):8093-101. doi: 10.1128/AEM.01863-10. Epub 2010 Oct 15.
The protocatechuate (PCA) 4,5-cleavage (PCA45) pathway is the essential catabolic route for the degradation of various aromatic acids in the genus Comamonas. All of the PCA45 pathway genes, orf1-pmdKEFDABC, as well as another PCA 4,5-dioxygenase gene, pmdA(II)B(II), were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the β subunit of PCA 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate (PDC) hydrolase, respectively, resulted in a growth defect on PCA, indicating that these genes are essential for the growth of E6 on PCA. On the other hand, inactivation of pmdB(II) did not affect the growth of E6 on PCA. Disruption of pmdK, which is related to a 4-hydroxybenzoate/PCA transporter of Pseudomonas putida, resulted in growth retardation on PCA. The insertional inactivation of orf1 in E6, whose deduced amino acid sequence has no similarity with proteins of known function, led to the complete loss of growth on PCA and the accumulation of PDC and 4-oxalomesaconate (OMA) from PCA. These results indicated the involvement of orf1 in the PCA45 pathway, and this gene, designated pmdU, was suggested to code for OMA tautomerase. Reverse transcription-PCR analysis suggested that the pmdUKEFDABC genes constitute an operon. The transcription start site of the pmd operon was mapped at 167 nucleotides upstream of the initiation codon of pmdU. The pmd promoter activity was enhanced 20-fold when the cells were grown in the presence of PCA. Inducers of the pmd operon were found to be PCA and PDC, but PDC was the more effective inducer.
原儿茶酸（PCA）4,5-裂合（PCA45）途径是降解丛毛单胞菌属中各种芳香酸的必需分解途径。所有 PCA45 途径基因，orf1-pmdKEFDABC，以及另一个 PCA 4,5-双加氧酶基因 pmdA(II)B(II)，均从邻苯二甲酸降解菌丛毛单胞菌属 E6 中分离得到。E6 中 pmdB 和 pmdD 的缺失，分别编码 PCA 4,5-双加氧酶的β亚基和 2-吡喃酮-4,6-二羧酸（PDC）水解酶，导致在 PCA 上生长缺陷，表明这些基因对 E6 在 PCA 上的生长是必需的。另一方面，pmdB(II)的失活并不影响 E6 在 PCA 上的生长。pmdK 的失活，与恶臭假单胞菌的 4-羟基苯甲酸/PCA 转运体有关，导致在 PCA 上生长迟缓。E6 中 orf1 的插入失活，其推导的氨基酸序列与已知功能的蛋白质没有相似性，导致在 PCA 上完全丧失生长能力，并积累 PDC 和 4-氧代戊二酸（OMA）从 PCA 中。这些结果表明 orf1 参与了 PCA45 途径，该基因被命名为 pmdU，被建议编码 OMA 互变异构酶。反转录-PCR 分析表明，pmdUKEFDABC 基因构成一个操纵子。pmd 操纵子的转录起始位点位于 pmdU 起始密码子上游 167 个核苷酸处。当细胞在 PCA 存在下生长时，pmd 启动子活性增强了 20 倍。发现 pmd 操纵子的诱导物是 PCA 和 PDC，但 PDC 是更有效的诱导物。
