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Unraveling the Specific Regulation of the Central Pathway for Anaerobic Degradation of 3-Methylbenzoate*

  • Javier F. Juárez
    Footnotes
    Affiliations
    Department of Environmental Biology, Centro de Investigaciones Biológicas-Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
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  • Huixiang Liu
    Footnotes
    Affiliations
    Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom
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  • María T. Zamarro
    Affiliations
    Department of Environmental Biology, Centro de Investigaciones Biológicas-Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
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  • Stephen McMahon
    Affiliations
    Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom
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  • Huanting Liu
    Affiliations
    Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom
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  • James H. Naismith
    Footnotes
    Affiliations
    Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom
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  • Christian Eberlein
    Affiliations
    Institute for Biology II, University of Freiburg, 79104 Freiburg, Germany
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  • Matthias Boll
    Affiliations
    Institute for Biology II, University of Freiburg, 79104 Freiburg, Germany
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  • Manuel Carmona
    Affiliations
    Department of Environmental Biology, Centro de Investigaciones Biológicas-Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
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  • Eduardo Díaz
    Correspondence
    To whom correspondence should be addressed. Tel.: 34-918373112; Fax: 34-915360432
    Affiliations
    Department of Environmental Biology, Centro de Investigaciones Biológicas-Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
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  • Author Footnotes
    * This work was supported in part by Ministry of Economy and Competitiveness of Spain Grants BIO2009-10438, BIO2012-39501, and CSD2007-00005 and European Union FP7 Grant 311815. Crystallography was supported by a Biotechnology and Biological Sciences Research Council grant and a Wellcome Trust award.
    1 Supported by a predoctoral fellowship from the Comunidad Autónoma de Madrid. Present address: Dept. of Genetics, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02155.
    2 Present address: College of Plant Protection, Shandong Agricultural University, 61 Daizong Rd., Taian, Shandong 271018, China.
    3 A Royal Society Wolfson Merit Award holder.
Open AccessPublished:March 20, 2015DOI:https://doi.org/10.1074/jbc.M115.637074
      The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the β-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB1, and P3R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
      Background: The specific transcriptional regulation of the mbd pathway for anaerobic 3-methylbenzoate degradation is unknown.
      Results: The MbdR/3-methylbenzoyl-CoA couple controls the induction of the mbd genes.
      Conclusion: MbdR is the regulator of the mbd pathway in Azoarcus sp. CIB.
      Significance: This work highlights the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.

      Introduction

      Aromatic compounds are included among the most widespread organic compounds in nature, and some of them are man-made environmental pollutants (
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      Towardhabitat-oriented systems biology of “Aromatoleum aromaticum” EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.
      ). Microorganisms play a fundamental role in the degradation of these aromatic compounds in diverse ecological niches (
      • Fuchs G.
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      Microbial degradation of aromatic compounds–from one strategy to four.
      ,
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      Cleaning up with genomics: applying molecular biology to bioremediation.
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      Anaerobic metabolism of aromatic compounds.
      ,
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      Metabolic networks, microbial ecology and 'omics' technologies: toward understanding in situ biodegradation processes.
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      Aerobic degradation of aromatic compounds.
      ). Many habitats containing large amounts of aromatic compounds are often anoxic. In the last decades, biochemical studies concerning the anaerobic degradation of aromatic compounds have been steadily accumulating, with benzoyl-CoA representing the intermediate to which most monocyclic aromatic compounds are converted (
      • Fuchs G.
      • Boll M.
      • Heider J.
      Microbial degradation of aromatic compounds–from one strategy to four.
      ,
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      Towardhabitat-oriented systems biology of “Aromatoleum aromaticum” EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.
      ,
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      Cleaning up with genomics: applying molecular biology to bioremediation.
      ,
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      Anaerobic catabolism of aromatic compounds: a genetic and genomic view.
      • Meckenstock R.U.
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      Anaerobic degradation of non-substituted aromatic hydrocarbons.
      ,
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      Different strategies in anaerobic biodegradation of aromatic compounds: nitrate reducers versus strict anaerobes.
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      Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: organisms, strategies and key enzymes.
      ). On the contrary, the study on the specific regulatory systems controlling the expression of the gene clusters involved in the anaerobic degradation of aromatic compounds has been mainly restricted to the characterization of a few transcriptional regulators.
      Anaerobic benzoate degradation via benzoyl-CoA was shown to be controlled by the two-component BamVW regulatory system (
      • Juárez J.F.
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      • Barragán M.J.
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      Identification of the Geobacter metallireducens BamVW two-component system, involved in transcriptional regulation of aromatic degradation.
      ) or the BgeR regulator (
      • Ueki T.
      Identification of a transcriptional repressor involved in benzoate metabolism in Geobacter bemidjiensis.
      ) in the obligate anaerobes Geobacter strains, and by the BadR/BadM (
      • Egland P.G.
      • Harwood C.S.
      BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member.
      ,
      • Peres C.M.
      • Harwood C.S.
      BadM is a transcriptional repressor and one of three regulators that control benzoyl coenzyme A reductase gene expression in Rhodopseudomonas palustris.
      ) and BzdR/BoxR (
      • Barragán M.J.
      • Blázquez B.
      • Zamarro M.T.
      • Mancheño J.M.
      • García J.L.
      • Díaz E.
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      BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators.
      • Durante-Rodríguez G.
      • Valderrama J.A.
      • Mancheño J.M.
      • Rivas G.
      • Alfonso C.
      • Arias-Palomo E.
      • Llorca O.
      • García J.L.
      • Díaz E.
      • Carmona M.
      Biochemical characterization of the transcriptional regulator BzdR from Azoarcus sp. CIB.
      ,
      • Durante-Rodríguez G.
      • Mancheño J.M.
      • Rivas G.
      • Alfonso C.
      • García J.L.
      • Díaz E.
      • Carmona M.
      Identification of a missing link in the evolution of an enzyme into a transcriptional regulator.
      • Valderrama J.A.
      • Durante-Rodríguez G.
      • Blázquez B.
      • García J.L.
      • Carmona M.
      • Díaz E.
      Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways.
      ) regulators in the facultative anaerobes Rhodopseudomonas palustris and Azoarcus strains, respectively. Moreover, a few global regulators, e.g. AadR, AcpR, and AccR, that influence the anaerobic expression of the benzoyl-CoA central pathway have been reported (
      • Egland P.G.
      • Harwood C.S.
      BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member.
      ,
      • Durante-Rodríguez G.
      • Zamarro M.T.
      • García J.L.
      • Díaz E.
      • Carmona M.
      Oxygen-dependent regulation of the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus sp. strain CIB.
      ,
      • Valderrama J.A.
      • Shingler V.
      • Carmona M.
      • Díaz E.
      AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB.
      ). A TdiSR (TutC1B1) two-component regulatory system was described for the regulation of the bss/bbs genes encoding the peripheral pathway that converts toluene into benzoyl-CoA in denitrifying bacteria (
      • Rabus R.
      • Trautwein K.
      • Wöhlbrand L.
      Towardhabitat-oriented systems biology of “Aromatoleum aromaticum” EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.
      ,
      • Coschigano P.W.
      • Young L.Y.
      Identification and sequence analysis of two regulatory genes involved in anaerobic toluene metabolism by strain T1.
      ,
      • Leuthner B.
      • Heider J.
      A two-component system involved in regulation of anaerobic toluene metabolism in Thauera aromatica.
      ,
      • Coschigano P.W.
      • Bishop B.J.
      Role of benzylsuccinate in the induction of the tutE tutFDGH gene complex of T. aromatica strain T1.
      ). It was also reported that the regulation of the peripheral routes that funnel 4-hydroxybenzoate and p-coumarate into the benzoyl-CoA central pathway in the phototrophic R. palustris strain is accomplished by the HbaR and CouR proteins, respectively (
      • Egland P.G.
      • Harwood C.S.
      HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris.
      ,
      • Hirakawa H.
      • Schaefer A.L.
      • Greenberg E.P.
      • Harwood C.S.
      Anaerobic p-coumarate degradation by Rhodopseudomonas palustris and identification of CouR, a MarR repressor protein that binds p-coumaroyl coenzyme A.
      ). However, no specific-transcriptional regulators that control anaerobic degradation pathways, other than that of benzoyl-CoA and some peripheral routes that converge to the latter, have been described so far.
      Azoarcus sp. CIB is a denitrifying β-proteobacterium able to anaerobically degrade different aromatic compounds, including some hydrocarbons such as toluene, via benzoyl-CoA, and m-xylene, via 3-methylbenzoyl-CoA (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). The Azoarcus sp. CIB bzd genes responsible for the anaerobic degradation of benzoate are clustered and consist of the PN promoter-driven bzdNOPQMSTUVWXYZA catabolic operon and the bzdR regulatory gene (
      • López Barragán M.J.
      • Carmona M.
      • Zamarro M.T.
      • Thiele B.
      • Boll M.
      • Fuchs G.
      • García J.L.
      • Díaz E.
      The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. strain CIB.
      ). BzdR-mediated repression of PN is alleviated by the inducer molecule benzoyl-CoA, the first intermediate of the catabolic pathway (
      • Barragán M.J.
      • Blázquez B.
      • Zamarro M.T.
      • Mancheño J.M.
      • García J.L.
      • Díaz E.
      • Carmona M.
      BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators.
      ,
      • Durante-Rodríguez G.
      • Valderrama J.A.
      • Mancheño J.M.
      • Rivas G.
      • Alfonso C.
      • Arias-Palomo E.
      • Llorca O.
      • García J.L.
      • Díaz E.
      • Carmona M.
      Biochemical characterization of the transcriptional regulator BzdR from Azoarcus sp. CIB.
      ). In addition, the PN promoter is also subject to control by the benzoyl-CoA-dependent BoxR repressor, a BzdR paralog that regulates the expression of the box genes responsible for the aerobic degradation of benzoate in Azoarcus sp. CIB (
      • Valderrama J.A.
      • Durante-Rodríguez G.
      • Blázquez B.
      • García J.L.
      • Carmona M.
      • Díaz E.
      Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways.
      ). The mbd cluster of Azoarcus sp. CIB encodes the central pathway responsible for the degradation of the 3-methylbenzoyl-CoA formed during the anaerobic degradation of m-xylene and 3-methylbenzoate (Fig. 1) (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). The mbd cluster is organized in at least three operons, i.e. the mbdO-orf9, mbdB1-mbdA, and mbdR operons (Fig. 1A). The mbdB1-mbdA operon is driven by the PB1 promoter and encodes a putative 3-methylbenzoate ABC transporter (MbdB1B2B3B4B5) and the 3-methylbenzoate-CoA ligase (MbdA) that activates 3-methylbenzoate to 3-methylbenzoyl-CoA (peripheral pathway) (Fig. 1B). The mbdO-orf9 operon is regulated by the PO promoter and encodes the enzymes for the anaerobic conversion of 3-methylbenzoyl-CoA to a hydroxymethylpimelyl-CoA (MbdMNOPQWXYZ) (upper central pathway) and the further degradation of the latter to the central metabolism (Orf1–9) (lower central pathway) (Fig. 1) (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). The mbdR gene was proposed to encode a transcriptional regulator of the TetR family that might regulate the inducible expression of the catabolic mbd genes (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). The efficient expression of the bzd and mbd genes required the oxygen-dependent AcpR activator, and it was under the control of AccR-mediated carbon catabolite repression by some organic acids and amino acids (
      • Valderrama J.A.
      • Shingler V.
      • Carmona M.
      • Díaz E.
      AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB.
      ,
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ).
      Figure thumbnail gr1
      FIGURE 13-Methylbenzoate anaerobic degradation pathway in Azoarcus sp. CIB. A, scheme of the mbd gene cluster of Azoarcus sp. CIB. Genes are represented by thick arrows, and their predicted function is annotated as follows: gray, regulatory gene; horizontal stripes, genes encoding a 3-methylbenzoate ABC-type transport system; stippling, gene encoding the 3-methylbenzoate-CoA ligase; black, genes encoding the 3-methylbenzoyl-CoA upper central pathway; white, genes involved in the 3-methylbenzoyl-CoA lower pathway (and some genes of unknown function). Bent arrows represent the promoters driving the expression of the mbd genes. The mbdO-orf9 operon and the mbdB1-mbdA operon are indicated by broken arrows. B, scheme of 3-methylbenzoate activation and 3-methylbenzoyl-CoA anaerobic degradation pathway. The enzymes involved are indicated following the same code of A. The Bss-Bbs peripheral pathway that converts m-xylene into 3-methylbenzoyl-CoA is indicated by a dashed arrow. The compounds are as follows: 1, 3-methylbenzoate; 2, 3-methylbenzoyl-CoA; 3a, 3-hydroxy-6-methyl-pimelyl-CoA; 3b, 3-hydroxy-4-methyl-pimelyl-CoA; and 4; m-xylene.
      In this work we have characterized the promoters of the mbd cluster and demonstrated the 3-methylbenzoyl-CoA/MbdR-dependent transcriptional control of the mbd genes in Azoarcus sp. CIB. The studies on the structural-functional relationships of the MbdR protein expand our current view on the transcriptional regulation of anaerobic pathways, and highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.

      DISCUSSION

      Bacterial metabolism of some compounds that usually are nonpreferred carbon sources, e.g. aromatic compounds, is generally strictly regulated at the transcriptional level (
      • Díaz E.
      • Jiménez J.I.
      • Nogales J.
      Aerobic degradation of aromatic compounds.
      ). In this work, we have characterized the specific regulation of the mbd central cluster, which is responsible for anaerobic 3-methylbenzoate degradation in Azoarcus sp. CIB, by the MbdR transcriptional repressor. MbdR is an efficient repressor of the mbd genes whose expression can only be switched on when the Azoarcus sp. CIB cells grow anaerobically on 3-methylbenzoate (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ) but not on benzoate (Fig. 2, A and B). This finding provides an explanation to the fact that Azoarcus sp. CIBdbzdN, a strain lacking a functional benzoate degradation (bzd) pathway, cannot use benzoate anaerobically despite the Mbd enzymes that can activate benzoate to benzoyl-CoA and further metabolize this CoA-derived compound (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). On the other hand, it is worth noting that the bzd genes are not induced when Azoarcus sp. CIB grows anaerobically in 3-methylbenzoate (data not shown). Therefore, these results reveal that there is no cross-induction between the bzd and mbd pathways, supporting the existence of devoted BzdR- and MbdR-dependent regulatory systems that control, respectively, each of these two central catabolic pathways in Azoarcus sp. CIB.
      Analytical ultracentrifugation and crystallographic data indicate that MbdR is a homodimer in solution, a common feature shared by most TetR-like regulators (Fig. 4D) (
      • Ramos J.L.
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      • Yu Z.
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      • Savchenko A.
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      A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.
      ). Like other members of the TetR family, e.g. TetR (
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      • Hillen W.
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      Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system.
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      • Grkovic S.
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      • Schumacher M.A.
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      • Willems A.R.
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      • Lee Z.Z.
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      ), FadR (
      • Agari Y.
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      ,
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      • Agari Y.
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      Transcriptional repression mediated by a TetR family protein, PfmR, from Thermus thermophilus HB8.
      ), and the MbdR monomeric structure includes two domains with nine helices (α1 to α9) linked by loops (Fig. 4A). The N-terminal DNA binding domain (helices α1 to α3) contains the helix-turn-helix motif whose amino acid sequence is rather conserved in other TetR-like transcriptional regulators (Fig. 3). An electrostatic surface representation of the MbdR dimer structure shows a positively charged patch at the N-terminal domain of both monomers (Fig. 4C), which might contact the phosphate backbone of the target operator region as in the cases of other TetR family proteins (
      • Yu Z.
      • Reichheld S.E.
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      • Parkinson J.
      • Davidson A.R.
      A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.
      ). An 18-bp conserved palindromic sequence (ATACN10GTAT) was suggested to be the operator region recognized by MbdR in PO and PB1 promoters (Fig. 5). The MbdR binding regions in PO and PB1 promoters span the transcription initiation sites as well as the −10 and −35 (only in PB1) sequences for recognition of the σ70-dependent RNA polymerase (Fig. 5, C and D), which is in agreement with the observed repressor role of MbdR at both promoters (Fig. 2, A and B), and it supports MbdR as a transcriptional repressor of the mbd cluster. Although the length of the MbdR operator region is similar to that of other TetR operators, their different consensus sequences agree with the fact that the DNA-binding mechanisms differ among the TetR family proteins (
      • Yu Z.
      • Reichheld S.E.
      • Savchenko A.
      • Parkinson J.
      • Davidson A.R.
      A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.
      ).
      In vivo (Fig. 6A) and in vitro (Fig. 6, B and D) experiments revealed that 3-methylbenzoyl-CoA, the first intermediate of the mbd catabolic pathway, is the cognate inducer molecule that interacts with the MbdR repressor allowing transcription from the PO and PB1 promoters. There is an increasing number of regulators, i.e. PaaR (
      • Sakamoto K.
      • Agari Y.
      • Kuramitsu S.
      • Shinkai A.
      Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophilus HB8.
      ) (TetR family), CouR, FerC, HcaR, FerR, and GenR (MarR family) (
      • Hirakawa H.
      • Schaefer A.L.
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      Anaerobic p-coumarate degradation by Rhodopseudomonas palustris and identification of CouR, a MarR repressor protein that binds p-coumaroyl coenzyme A.
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      • Parke D.
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      • Mancheño J.M.
      • García J.L.
      • Díaz E.
      • Carmona M.
      BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators.
      ,
      • Valderrama J.A.
      • Durante-Rodríguez G.
      • Blázquez B.
      • García J.L.
      • Carmona M.
      • Díaz E.
      Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways.
      ), that control aromatic degradation pathways and recognize aromatic CoA thioesters as inducers. Thus, FerR/FerC recognize feruloyl-CoA; CouR/HcaR recognize p-coumaroyl-CoA; BzdR/BoxR/GenR recognize benzoyl-CoA; and PaaX/PaaR recognize phenylacetyl-CoA. In this work, we show that MbdR constitutes the first member of this group of regulators that belongs to the TetR family and controls the anaerobic catabolism of aromatic compounds.
      The C-terminal domain of TetR-like regulators is highly variable, with its specific surfaces required for the dimerization of the protein and for the interaction with the inducer (
      • Ramos J.L.
      • Martínez-Bueno M.
      • Molina-Henares A.J.
      • Terán W.
      • Watanabe K.
      • Zhang X.
      • Gallegos M.T.
      • Brennan R.
      • Tobes R.
      The TetR family of transcriptional repressors.
      ,
      • Yu Z.
      • Reichheld S.E.
      • Savchenko A.
      • Parkinson J.
      • Davidson A.R.
      A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.
      ,
      • Orth P.
      • Schnappinger D.
      • Hillen W.
      • Saenger W.
      • Hinrichs W.
      Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system.
      ). Based on the previously published studies of other TetR-like regulators, ligand binding usually induces a conformational change in the protein that leads to changes in DNA recognition and interaction, causing the dissociation of the repressor from the cognate promoter (
      • Yu Z.
      • Reichheld S.E.
      • Savchenko A.
      • Parkinson J.
      • Davidson A.R.
      A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.
      ). To date, all ligands bind in the same general location at or near the dimer interface. However, it has been shown that in some members of the TetR family, for example AcrR (
      • Li M.
      • Gu R.
      • Su C.C.
      • Routh M.D.
      • Harris K.C.
      • Jewell E.S.
      • McDermott G.
      • Yu E.W.
      Crystal structure of the transcriptional regulator AcrR from Escherichia coli.
      ), the ligand binds in a large internal cavity in the C-terminal region, surrounded by helices α4 through α8 of each monomer. In contrast, MbdR and other members of TetR family, such as QacR (
      • Grkovic S.
      • Brown M.H.
      • Schumacher M.A.
      • Brennan R.G.
      • Skurray R.A.
      The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers.
      ), do not have such a cavity (Fig. 4, A and C). By superimposing the apo-MbdR structure with the structure of the QacR·diamidine hexamidine complex (
      • Peters K.M.
      • Schuman J.T.
      • Skurray R.A.
      • Brown M.H.
      • Brennan R.G.
      • Schumacher M.A.
      QacR-cation recognition is mediated by a redundancy of residues capable of charge neutralization.
      ), we could suggest the binding site of 3-methylbenzoyl-CoA in MbdR and a model of the MbdR-3-methylbenzoyl-CoA interaction (Fig. 4E). Binding of 3-methylbenzoyl-CoA would require the movements of helices α5, α6, α8, and α9 in MbdR, similar to that described as the “induced fit” mechanism of QacR bound to its ligand (
      • Peters K.M.
      • Schuman J.T.
      • Skurray R.A.
      • Brown M.H.
      • Brennan R.G.
      • Schumacher M.A.
      QacR-cation recognition is mediated by a redundancy of residues capable of charge neutralization.
      ,
      • Schumacher M.A.
      • Miller M.C.
      • Grkovic S.
      • Brown M.H.
      • Skurray R.A.
      • Brennan R.G.
      Structural mechanisms of QacR induction and multidrug recognition.
      ). Similar to what has been observed in the QacR·ligand complex structure, the movement of α6 after 3-methylbenzoyl-CoA binding to MbdR would induce a rotation of the helix-turn-helix domain (Fig. 4E), and as a consequence, this DNA binding domain would lose its DNA binding ability. Sequence comparison of MbdR and PaaR (Fig. 3), another member of the TetR family which uses phenylacetyl-CoA as inducer (
      • Sakamoto K.
      • Agari Y.
      • Kuramitsu S.
      • Shinkai A.
      Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophilus HB8.
      ), shows two MbdR-specific hydrophobic clusters, Gln-107 to Gly-123 within α6 and the α6/α7 linkage loop, and Ser-165 to Ile-176 within α8. Some residues within these two clusters could be involved in discriminating between the 3-methylbenzoyl group of 3-methylbenzoyl-CoA and the phenylacetyl group of phenylacetyl-CoA (Fig. 4F). Nevertheless, further experiments are needed to determine the structure of the MbdR·3-methylbenzoyl-CoA complex for understanding the inducer specificity determinants and the molecular mechanism of transcriptional de-repression at the target promoters.
      PA and P3R are two additional promoters within the mbd cluster whose activity levels are lower than those of PO and PB1 but that share with the latter the 3-methylbenzoyl-CoA/MbdR-dependent control (Fig. 8). The P3R promoter drives the expression of the regulatory mbdR gene (Fig. 1). Interestingly, the amount of MbdR needed for the retardation of 50% of the P3R probe was at least 1 order of magnitude higher than that needed for the retardation of the PA (Fig. 8A), PO (Fig. 2C), and PB1 (Fig. 2D) promoters. The fact that the activity from the P3R promoter is under auto-repression by MbdR at high protein concentrations underlines the importance of a negative feedback loop that would restrict the intracellular concentration of the transcriptional repressor when it reaches a given concentration. The PA promoter is located within the PB1-driven operon (Fig. 1). The predicted MbdR operator region (ATACN10GTAT) (Fig. 8E) spans the −35 sequence for recognition of the σ70-dependent RNA polymerase in the PA promoter (Fig. 7B), thus supporting the observed repressor role of MbdR on this promoter. Whereas the role of P3R driving the expression of the mbdR regulatory gene is obvious, the role of the PA promoter was puzzling, and therefore, it was further investigated.
      Inactivation of either the strong (PB1) or the weak (PA) promoters in Azoarcus sp. CIBdmbdB1 and Azoarcus sp. CIBΔPA mutant strains, respectively, revealed that both promoters are essential for the anaerobic growth of strain CIB in 3-methylbenzoate (Fig. 9A). However, whereas PB1 accounts for most of the mbdA expression when the cells are actively growing in 3-methylbenzoate, the PA promoter allows the initial induction of the mbdA expression when the cells start to grow in this aromatic compound (Fig. 9B). Therefore, these results suggest that the fast and modest induction of the PA promoter in the presence of 3-methylbenzoate leads to an increase of mbdA expression that, in turn, would enhance the amount of the inducer molecule 3-methylbenzoyl-CoA triggering the induction of the PB1 promoter. The expression of the mbdA gene driven by the induced PB1 promoter will provide the required amount of MbdA for the efficient degradation of 3-methylbenzoate and thus will allow growth on this aromatic compound. In summary, these studies highlight the main role of some minor regulatory loops that control the expression of CoA ligases for triggering the efficient expression of aromatic catabolic pathways that use aryl-CoA compounds as central intermediates.
      Mbd enzymes are able to activate benzoate and further convert benzoyl-CoA in vitro (
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). We have shown here that MbdR is an efficient repressor of the mbd genes, and it recognizes 3-methylbenzoyl-CoA, but not benzoyl-CoA, as inducer. These results suggest that the broad substrate range mbd catabolic genes have recruited a regulatory system based on the MbdR regulator and its target promoters to evolve to a distinct central aromatic catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as central metabolite. Thus, the existence in Azoarcus sp. CIB of two different central pathways, i.e. the bzd pathway, for the anaerobic degradation of aromatic compounds that generate benzoyl-CoA as central intermediate, and the mbd pathway, for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as central intermediate, could be mainly driven by the high specificity of the corresponding repressors, i.e. BzdR and MbdR, for their cognate inducers, i.e. benzoyl-CoA and 3-methylbenzoyl-CoA, respectively. If correct, this highlights the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.
      The studies presented in this work expand our knowledge on the specific regulation of anaerobic pathways for the catabolism of aromatic compounds (
      • Rabus R.
      • Trautwein K.
      • Wöhlbrand L.
      Towardhabitat-oriented systems biology of “Aromatoleum aromaticum” EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.
      ,
      • Carmona M.
      • Zamarro M.T.
      • Blázquez B.
      • Durante-Rodríguez G.
      • Juárez J.F.
      • Valderrama J.A.
      • Barragán M.J.
      • García J.L.
      • Díaz E.
      Anaerobic catabolism of aromatic compounds: a genetic and genomic view.
      ,
      • Ueki T.
      Identification of a transcriptional repressor involved in benzoate metabolism in Geobacter bemidjiensis.
      ,
      • Barragán M.J.
      • Blázquez B.
      • Zamarro M.T.
      • Mancheño J.M.
      • García J.L.
      • Díaz E.
      • Carmona M.
      BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators.
      ,
      • Valderrama J.A.
      • Durante-Rodríguez G.
      • Blázquez B.
      • García J.L.
      • Carmona M.
      • Díaz E.
      Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways.
      ,
      • Hirakawa H.
      • Schaefer A.L.
      • Greenberg E.P.
      • Harwood C.S.
      Anaerobic p-coumarate degradation by Rhodopseudomonas palustris and identification of CouR, a MarR repressor protein that binds p-coumaroyl coenzyme A.
      ,
      • Juárez J.F.
      • Zamarro M.T.
      • Eberlein C.
      • Boll M.
      • Carmona M.
      • Díaz E.
      Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.
      ). Moreover, it worth noting that 3-methylbenzoyl-CoA is an uncommon metabolite in living cells, and MbdR-responsive promoters are likely to be also very infrequent in nature. Therefore, the PB1 promoter, mbdR regulator, and mbdA genes become potential BioBricks for creating new conditional expression systems that respond to 3-methylbenzoate in a fashion minimally influenced by the host and that has no impact on the host physiology (biological orthogonality), two major desirable traits in current synthetic biology approaches (
      • de Lorenzo V.
      Beware of metaphors: chasses and orthogonality in synthetic biology.
      ).

      Acknowledgments

      We thank A. Valencia for technical assistance, Secugen S.L. for DNA sequencing, and C. A. Botello for ultracentrifugation experiments.

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