Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4)

Abstract

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons TnS and Tnl 771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways

“…This work clearly demonstrated that no additional tfd genes or pseudogenes are encoded in pJP4. The sequence of the tfd genes in pJP4 perfectly matched that reported previously (Don et al, 1985;Laemmli et al, 2000). However, an additional G in the position 1020 of the tfdD II gene (ORF 29) produces a protein of 372 aa, which is 23 residues larger than that described previously (Laemmli et al, 2000), but of a size very similar to other reported chloromuconate cycloisomerases.…”

Microbial breakdown of halogenated aromatic pesticides and related compounds

“…The plasmids pJP4 and pAC25 have been extensively characterized and show significant ho-mology, suggesting that they have a common ancestral origin [371,372]. The genes for chlorocatechol degradation (pyrocatechase, cycloisomerase, hydrolase, lactone isomerase) are clustered as one operon [373,374]. The complete nucleotide sequences of chlorocatechol oxidative operons have been determined [374,375].…”

Horizontal transfer of dehalogenase genes on IncP1β plasmids during bacterial adaptation to degrade α-halocarboxylic acids

“…d PCR results show that these strains contain two dehalogenases, both a group I deh (D,L-2MCPA-speci¢c) and a group II deh (L-2MCPA-speci¢c). e Exogenously isolated plasmid pQKH90 carrying strain of R. eutropha JMP222 [24]. UWC11 is isogenic to UWC10.…”