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Applied and Environmental Microbiology, April 2006, p. 2783-2792, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2783-2792.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

T. Ledger,¹ D. H. Pieper,² and B. González^1*

Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Millennium Nucleus on Microbial Ecology and Environmental Microbiology and Biotechnology, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile,¹ Biodegradation Laboratory, Division of Microbiology, GBF, Braunschweig, Germany²

Received 15 December 2005/ Accepted 3 February 2006

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB_I and tfdB_II genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB_I and tfdB_II genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB_I contributes to a significantly higher extent than TfdB_II. Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

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* Corresponding author. Mailing address: Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. Phone: 56-2-6862845. Fax: 56-2-2225515. E-mail: bgonzalez{at}bio.puc.cl.

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Applied and Environmental Microbiology, April 2006, p. 2783-2792, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2783-2792.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Larrain-Linton, J., De la Iglesia, R., Melo, F., Gonzalez, B. (2006). Molecular and Population Analyses of a Recombination Event in the Catabolic Plasmid pJP4.. J. Bacteriol. 188: 6793-6801 [Abstract] [Full Text]