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Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6.

ABSTRACT: 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.

SUBMITTER: Hosaka M

PROVIDER: S-EPMC3811363 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6.

Hosaka Masaru M Kamimura Naofumi N Toribami Shotaro S Mori Kosuke K Kasai Daisuke D Fukuda Masao M Masai Eiji E

Applied and environmental microbiology 20130802 19

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 tp ...[more]

PMID: 23913423

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Project description:This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of beta-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.

Project description:The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In Sphingobium sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (ddvK) and SLG_07780 (ddvR), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A ddvK mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA O-demethylase oxygenase component gene (ligXa), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled ligXa transcriptional regulatory system. A Sphingobium japonicum UT26S transformant expressing ddvK acquired DDVA uptake capacity, indicating that ddvK encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of ddvK overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (ligI) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a ddvK-expression plasmid into a ligI mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.IMPORTANCE The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in Sphingobium sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.

Dataset Information

Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6.

Publications

Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6.

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