Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, a nd maleylacetate reductase allow Ralstonia eutrop ha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dich lorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules l ocated in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(I I)F(II) (module II), putatively encoding these enzymes, To assess the role of both ya modules in the degradation of chloroaromatics, each module was c loned into the medium-copy-number plasmid vector pBBR1MCS-2 under the contr ol of the tfdR regulatory gene, These constructs were introduced into R. eu rtropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT 2442, two strains able to transform 3-CB into chlorocatechols. Specific act ivities in cell extracts of chlorocatechol-1,3-dioxygenase (tfdC), chloromu conate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 5 0 times higher for microorganisms containing module I compared to those con taining module II. In contrast, a significantly (50-fold) higher activity o f maleylacetate reductase (tfdF) was observed in cell extracts of microorga nisms containing module II compared to module I, The R, eutropha JMP222 der ivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liqui d cultures with 3-CB as a sole carbon and energy source than in cultures co ntaining tfdR-tfdD(II)C(II)E(II)F(II) 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, eutro pha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely su pplied primarily by module I, while TfdF is likely supplied by module II.