Characterization of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism on plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)

Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dic hlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutro pha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCD EF and tfdB) already present elsewhere on pJP4. The five ORFs were organize d 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 JMP 134 identified a single transcription start site in front of the first gene of the cluster, tfdD(II) suggesting an operon-like organization for the tf d(II) genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD(II) coded for a chloromuconate cycloisomerase, tfdC(II) coded for a ch lorocatechol 1,2-dioxygenase, tfdE(II) coded for a dienelactone hydrolase, tfdF(II) coded for a maleylacetate reductase, and tfdB(II) coded for a chlo rophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutr opha 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 wh y the tfd(II) genes do not disappear from plasmid pJP4 might be the necessi ty for keeping the regulatory genes for the 2,4-D pathway expression tfdR a nd tfdS.