title: Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-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.
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
authors.forename: Águeda
authors.initials: Á
authors.lastname: Molina-Fuentes
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pacheco
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Marín
authors.affiliation: Westfälische Wilhelms-Universität Münster, Institut für Molekulare Mikrobiologie und Biotechnologie, Münster, Germany University of Konstanz, Department of Biology, Constance, Germany.
authors.forename: Bodo
authors.initials: B
authors.lastname: Philipp
authors.affiliation: University of Konstanz, Department of Biology, Constance, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schink
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain silvia@eez.csic.es.
authors.forename: Silvia
authors.initials: S
authors.lastname: Marqués
mesh_terms: D000693:Anaerobiosis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism*; D005810:Multigene Family / Q000235:genetics; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020628:Thauera / Q000235:genetics / Q000378:metabolism*
chemical_list: D062385:Hydroxybenzoates