Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)

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Abstract
Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism ( tfdCDEF and tfdB ) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows: tfdD II C II E II F II and tfdB II (in short, the tfd II cluster), by analogy to tfdCDEF and tfdB (the tfd I cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdD II , suggesting an operon-like organization for the tfd II genes. By expressing each ORF in Escherichia coli , we confirmed that tfdD II coded for a chloromuconate cycloisomerase, tfdC II coded for a chlorocatechol 1,2-dioxygenase, tfdE II coded for a dienelactone hydrolase, tfdF II coded for a maleylacetate reductase, and tfdB II coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfd I and tfd II genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd II genes seem to be redundant with respect to those of the tfd I cluster. One reason why the tfd II genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS .