Dataset Information

Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (?-Resorcylate) in Thauera aromatica Strain AR-1.

ABSTRACT: Thauera aromatica strain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) by Azoarcus anaerobius. Southern hybridization of a T. aromatica strain AR-1 gene library identified a 25-kb chromosome region based on its homology with A. anaerobius main pathway genes. Sequence analysis defined 20 open reading frames. Knockout mutations of the most relevant genes in the pathway were generated by reverse genetics. Physiological and biochemical analyses identified the genes for the three main steps in the pathway which were homologous to those described in A. anaerobius and suggested the function of several auxiliary genes possibly involved in enzyme maturation and intermediate stabilization. However, T. aromatica strain AR-1 had an additional enzyme to metabolize hydroxyhydroquinone, a putative cytoplasmic quinone oxidoreductase. In addition, a specific tripartite ATP-independent periplasmic (TRAP) transport system was required for efficient growth on 3,5-DHB. Reverse transcription-PCR (RT-PCR) analysis showed that the pathway genes were organized in five 3,5-DHB-inducible operons, three of which have been shown to be under the control of a single LysR-type transcriptional regulator, DbdR. Despite sequence homology, the genetic organizations of the clusters in T. aromatica strain AR-1 and A. anaerobius differed substantially.

SUBMITTER: Molina-Fuentes A

PROVIDER: S-EPMC4579451 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1.

Molina-Fuentes Águeda Á Pacheco Daniel D Marín Patricia P Philipp Bodo B Schink Bernhard B Marqués Silvia S

Applied and environmental microbiology 20150807 20

Thauera aromatica strain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) by Azoarcus anaerobius. Southern hybridization of a T. ...[more]

PMID: 26253674

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Project description:The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCE Thauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

Project description:The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.

Project description:Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate ("phenylphosphate carboxylase"). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcus strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.

Project description:The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen.

Dataset Information

Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (?-Resorcylate) in Thauera aromatica Strain AR-1.

Publications

Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1.

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