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Applied and Environmental Microbiology, April 2000, p. 1602-1608, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Role of tfdCIDIEIFI and tfdDIICIIEIIFII Gene Modules in Catabolism of 3-Chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

D. Pérez-Pantoja,1 L. Guzmán,1 M. Manzano,1 D. H. Pieper,2 and B. González1,*

Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile,1 and Division of Microbiology, National Research Centre for Biotechnology---GBF, Braunschweig, Germany2

Received 10 November 1999/Accepted 2 February 2000

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdCIDIEIFI (module I) and tfdDIICIIEIIFII (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdCIDIEIFI grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdDIICIIEIIFII. In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

* 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: bgonzale{at}genes.bio.puc.cl.

Applied and Environmental Microbiology, April 2000, p. 1602-1608, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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