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Journal of Bacteriology, August 2009, p. 4905-4915, Vol. 191, No. 15
0021-9193/09/$08.00+0 doi:10.1128/JB.00331-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1
Beatriz Cámara,1,
Patricia Nikodem,1,
Piotr Bielecki,2
Roberto Bobadilla,3
Howard Junca,1,
and
Dietmar H. Pieper1*
Department of Microbial Pathogenesis,1 Division of Molecular Biotechnology, HZI Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany,2 Departamento de Prevención de Riesgos y Medio Ambiente Universidad Tecnológica Metropolitana, Dieciocho No. 390, Santiago, Chile3
Received 10 March 2009/ Accepted 17 May 2009
Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12OccaA) and ccaB (MCIccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12OccaA and MCIccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaB and the previously identified C12OsalD, rather than C12OccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.
* Corresponding author. Mailing address: Department of Microbial Pathogenesis, HZI Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. Phone: (49) 531 6181 4200. Fax: (49) 531 6181 4499. E-mail: dpi{at}helmholtz-hzi.de
Published ahead of print on 22 May 2009.
Present address: Department of Microbiology and Centre for Molecular Microbiology and Infection, Division of Investigative Sciences, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom.
Present address: Novo Nordisk A/S, Hallas Allée, 4400 Kalundborg, Denmark.
Present address: Centro Colombiano de Genómica y Bioinformática de Ambientes Extremos (GeBiX), Grupo de Genética Molecular, Corporación CorpoGen, Carrera 5 No. 66A-35, Bogotá, Colombia.
Journal of Bacteriology, August 2009, p. 4905-4915, Vol. 191, No. 15
0021-9193/09/$08.00+0 doi:10.1128/JB.00331-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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