Novel Tripartite Aromatic Acid Transporter Essential for Terephthalate Uptake in Comamonas sp Strain E6

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.