Importance of DifferenttfdGenes for Degradation of Chloroaromatics byRalstonia eutrophaJMP134

Abstract

The tfdC I D I E I F I, and tfdD II C II E II F II gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd I -encoded enzymes was usually higher than that of tfd II -encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD II -encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro-and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.

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

“…Growth in liquid culture was performed as previously described (18), with the mineral salts medium of Dorn et al (6) modified such that the concentration of the buffer was twice that described and with benzoate concentrations of up to 15 mM or 2,4-dichlorophenoxyacetate concentrations of 5 mM. Cell extracts were prepared as described previously (18).…”

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

“…Although the maleylacetate reductase gene, tfdF, of Ralstonia eutropha JMP134(pJP4) was initially misinterpreted as encoding a chlorodienelactone isomerase (Don et al, 1985), the chlorocatechol gene clusters of pJP4, pAC27, Pseudomonas sp. B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002). Sequence similarities suggest that this is true also for the chlorocatechol gene clusters of various other plasmids or strains.…”

“…B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002 During growth with 4-fluorobenzoate, R. eutropha 335 T and R. eutropha JMP222, a pJP4-free derivative of the 2,4-dichlorophenoxyacetate-utilizing strain JMP134, induce a maleylacetate reductase, but not the other enzymes typical for chlorocatechol catabolism (Schlömann et al, 1990a). Thus, these strains contain a presumably chromosomal maleylacetate reductase gene of still unknown metabolic affiliation.…”

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).…”