Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
Appl Environ Microbiol. 2013 Oct;79(19):6148-55. doi: 10.1128/AEM.01600-13. Epub 2013 Aug 2.
It has been suggested that a novel type of aromatic acid transporter, which is similar to the tripartite tricarboxylate transporter (TTT), is involved in terephthalate (TPA) uptake by Comamonas sp. strain E6. This suggestion was based on the presence of the putative TPA-binding protein gene, tphC, in the TPA catabolic operon. The tphC gene is essential for growth on TPA and is similar to the genes encoding TTT-like substrate-binding proteins. Here we identified two sets of E6 genes, tctBA and tpiBA, which encode TTT-like cytoplasmic transmembrane proteins. Disruption of tctA showed no influence on TPA uptake but resulted in a complete loss of the uptake of citrate. This loss suggests that tctA is involved in citrate uptake. On the other hand, disruption of tpiA or tpiB demonstrated that both genes are essential for TPA uptake. Only when both tphC and tpiBA were introduced with the TPA catabolic genes into cells of a non-TPA-degrading Pseudomonas strain did the resting cells of the transformant acquire the ability to convert TPA. From all these results, it was concluded that the TPA uptake system consists of the TpiA-TpiB membrane components and TPA-binding TphC. Interestingly, not only was the tpiA mutant of E6 unable to grow on TPA or isophthalate, it also showed significant growth delays on o-phthalate and protocatechuate. These results suggested that the TpiA-TpiB membrane components are able to interact with multiple substrate-binding proteins. The tpiBA genes were constitutively transcribed as a single operon in E6 cells, whereas the transcription of tphC was positively regulated by TphR. TPA uptake by E6 cells was completely inhibited by a protonophore, carbonyl cyanide m-chlorophenyl hydrazone, indicating that the TPA uptake system requires a proton motive force.
有人提出，一种新型芳香酸转运蛋白与三羧酸转运蛋白（TTT）相似，可能参与了 Comamonas sp. 菌株 E6 对对苯二甲酸（TPA）的摄取。这一观点是基于 TPA 代谢操纵子中存在假定的 TPA 结合蛋白基因 tphC。tphC 基因对于 TPA 的生长是必需的，并且与编码 TTT 样底物结合蛋白的基因相似。在这里，我们鉴定了两组 E6 基因，tctBA 和 tpiBA，它们分别编码 TTT 样细胞质跨膜蛋白。tctA 的缺失对 TPA 的摄取没有影响，但导致柠檬酸的摄取完全丧失。这一损失表明 tctA 参与了柠檬酸的摄取。另一方面，tpiA 或 tpiB 的缺失表明这两个基因对于 TPA 的摄取都是必需的。只有当 tphC 和 tpiBA 与 TPA 代谢基因一起引入不能降解 TPA 的 Pseudomonas 菌株的细胞中时，转化细胞的静止细胞才获得转化 TPA 的能力。从所有这些结果可以得出结论，TPA 摄取系统由 TpiA-TpiB 膜组件和 TPA 结合蛋白 TphC 组成。有趣的是，E6 的 tpiA 突变体不仅不能在 TPA 或间苯二甲酸上生长，而且在邻苯二甲酸和原儿茶酸上的生长延迟也很明显。这些结果表明，TpiA-TpiB 膜组件能够与多种底物结合蛋白相互作用。tpiBA 基因在 E6 细胞中作为一个单一操纵子组成型转录，而 tphC 的转录则受 TphR 的正调控。E6 细胞的 TPA 摄取完全被质子载体羰基氰化物 m-氯苯腙抑制，表明 TPA 摄取系统需要质子动力。
