title: The Azoarcus anaerobius 1,3-Dihydroxybenzene (Resorcinol) Anaerobic Degradation Pathway Is Controlled by the Coordinated Activity of Two Enhancer-Binding Proteins.
abstract: The anaerobic resorcinol degradation pathway inAzoarcus anaerobiusis unique in that it uses an oxidative rather than a reductive strategy to overcome the aromatic ring stability in degradation of this compound, in a process that is dependent on nitrate respiration. We show that the pathway is organized in five transcriptional units, three of which are inducible by the presence of the substrate. Three σ54-dependent promoters located upstream from the three operons coding for the main pathway enzymes were identified, which shared a similar structure with conserved upstream activating sequences (UASs) located at 103 to 111 bp from the transcription start site. Expression of the pathway is controlled by the bacterial enhancer-binding proteins (bEBPs) RedR1 and RedR2, two homologous regulators that, despite their high sequence identity (97%), have nonredundant functions: RedR2, the master regulator which also controls RedR1 expression, is itself able to promote transcription from two of the promoters, while RedR1 activity is strictly dependent on the presence of RedR2. The two regulators were shown to interact with each other, suggesting that the natural mode of activation is by forming heterodimers, which become active in the presence of the substrate after its metabolization to hydroxybenzoquinone through the pathway enzymes. The model structure of the N-terminal domain of the proteins is composed of tandem GAF and PAS motifs; the possible mechanisms controlling the activity of the regulators are discussed.IMPORTANCEAzoarcus anaerobiusis a strict anaerobe that is able to use 1,3-dihydroxybenzene as the sole carbon source in a process that is dependent on nitrate respiration. We have shown that expression of the pathway is controlled by two regulators of almost identical sequences: the bEBPs RedR1 and RedR2, which share 97% identity. These regulators control three promoters with similar structure. Despite their sequence identity, the two bEBPs are not redundant and are both required for maximum pathway expression. In fact, the two proteins function as heterodimers and require activation by the pathway intermediate hydroxyhydroquinone. The structure of the domain sensing the activation signal resembles that of regulators that are known to interact with other proteins.
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-Sánchez
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: Patricia
authors.initials: P
authors.lastname: Marín
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
authors.forename: Javier-I
authors.initials: JI
authors.lastname: Medina-Bellver
authors.affiliation: Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
authors.forename: Óscar
authors.initials: Ó
authors.lastname: González-López
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; D020627:Azoarcus / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009876:Operon; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D012118:Resorcinols / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D012118:Resorcinols; C031389:resorcinol
keywords: GAF domain; PAS domain; aromatic anaerobic biodegradation; hydroxybenzoquinone; hydroxyhydroquinone; nitrate-reducing bacteria; transcription activation; σ54 RNA polymerase