Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)
ABSTRACT Within the 5.9-kb DNA region between the tfdR andtfdK 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 IIand tfdB II (in short, thetfd II cluster), by analogy totfdCDEF and tfdB (thetfd 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 bothtfd 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 andtfdS.