Characterization of the Isophthalate Degradation Genes of Comamonas sp Strain E6
Characterization of the Isophthalate Degradation Genes of Comamonas sp Strain E6
复制标题
DOI：
10.1128/aem.01270-09
复制
发表时间：
2010-01-15
影响因子：
4.4
通讯作者：
Masai, Eiji
中科院分区：
文献类型：
--
作者：
Fukuhara, Yuki;Inakazu, Keisuke;Masai, Eiji
从丛毛单胞菌（Comamonas sp.）E6菌株中分离到负责IPA转化为原儿茶酸（PCA）的IPA降解基因簇（iphACBDR），该菌株通过PCA 4，5-裂解途径利用邻苯二甲酸酯异构体作为唯一碳源和能源。基于氨基酸序列相似性，iphA、iphC、iphB、iphD和iphR基因分别编码IPA双加氧酶（IPADO）的加氧酶组分、周质IPA结合受体、1，2-二羟基-3，5-环己二烯-1，5-二羧酸（1，5-DCD）脱氢酶、IPADO的还原酶组分和IclR型转录调节因子。iphACBDR基因构成单个转录单位，并且在E6在IPA上生长期间诱导了iphACBDR分解代谢操纵子的转录。iphA、iphD和iphB基因在大肠杆菌中表达。IphA和IphD粗品在NADPH存在下将IPA转化为产物，该产物通过IphB转化为PCA。这些结果表明，IPADO是一种双组分双加氧酶，由末端加氧酶组分（IphA）和还原酶组分（IphD）组成，iphB编码1，5-DCD脱氢酶。iphA和iphB的破坏导致E6在IPA上的生长完全丧失。灭活的iphD显着影响IPA上的生长，和iphC突变体不生长在IPA在中性pH值。这些结果表明，iphACBD基因是必不可少的IPA在E6中的catenase。iphR的破坏导致IPA上E6的更快生长，表明iphR编码iph分解代谢操纵子的阻遏物。操纵子的启动子分析支持了这一观点。
The isophthalate (IPA) degradation gene cluster (iphACBDR) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from Comamonas sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the iphA, iphC, iphB, iphD, and iphR genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The iphACBDR genes constitute a single transcriptional unit, and transcription of the iph catabolic operon was induced during growth of E6 on IPA. The iphA, iphD, and iphB genes were expressed in Escherichia coli. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that iphB encodes the 1,5-DCD dehydrogenase. Disruption of iphA and iphB resulted in complete loss of growth of E6 on IPA. Inactivation of iphD significantly affected growth on IPA, and the iphC mutant did not grow on IPA at neutral pH. These results indicated that the iphACBD genes are essential for the catabolism of IPA in E6. Disruption of iphR resulted in faster growth of E6 on IPA, suggesting that iphR encodes a repressor for the iph catabolic operon. Promoter analysis of the operon supported this notion.