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

Abstract

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, tfdC I D I E I F I (module I) and tfdD II C II E II F II (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-tfdC I D I E I F I grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing 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. 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.

Formation of Protoanemonin from 2-Chloro-cis,cis-Muconate by the Combined Action of Muconate Cycloisomerase and Muconolactone Isomerase

“…When growing on 3-chlorobenzoate, the wild-type strain excretes significant amounts of 2-chloromuconate (14) since both chloromuconate cycloisomerases of this strain are poorly effective with this substrate (11,13). Thus, 2-chloromuconate is available to be transformed by muconate cycloisomerase, which is simultaneously induced, when JMP134 grows on 3-chlorobenzoate (14).…”

Molecular and Population Analyses of a Recombination Event in the Catabolic Plasmid pJP4

“…The growth substrates were LB, fructose, benzoate, 2-MPA, 3-CB, 2,4-D, or MCPA. LB and fructose are carbon sources that do not require tfd genes for their catabolization, whereas MCPA, 2,4-D, 2-MPA, and 3-CB strictly require tfd functions for initial degradation (25,27,28). The degradation of benzoate is chromosomally encoded, although some tfd functions may use benzoate intermediates (27).…”

Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ 1 H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis -Dienelactone

“…A cell extract of strain JMP222 (pBBR1M-I) was therefore prepared in phosphate buffer, and a total of 5 to 8 mg of protein was loaded onto a MonoQ HR5/5 column and eluted with a linear gradient of 0 to 0.5 M NaCl in 50 mM phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed for dienelactone hydrolase activity as described previously (21). Under these conditions, dienelactone hydrolase eluted at a NaCl concentration of 0.03 mM, whereas the bulk of the maleylacetate reductase activity (approximately 60 to 70% of the applied activity) eluted at a NaCl concentration of 0.35 mM.…”

“…In order to characterize the structure of the dienelactone hydrolysis product under physiological conditions, R. eutropha JMP222(pBBR1M-1) was grown on 3-chlorobenzoate as recently described (21). R. eutropha JMP222 is a derivative of R. eutropha JMP134 (9) cured of plasmid pJP4.…”

“…Only module I was present in JMP222(pBBR1M-I). Enzyme activities were estimated in cell extracts by methods described previously (21). R. eutropha JMP222(pBBR1M-I) expresses high levels of dienelactone hydrolase (1,200 U/g of protein with 80 M cis-dienelactone as the substrate) during growth on 3-chlorobenzoate.…”