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

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American Society for Microbiology

RESUMO

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: tfdDIICIIEIIFII and tfdBII (in short, the tfdII cluster), by analogy to tfdCDEF and tfdB (the tfdI 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, tfdDII, suggesting an operon-like organization for the tfdII genes. By expressing each ORF in Escherichia coli, we confirmed that tfdDII coded for a chloromuconate cycloisomerase, tfdCII coded for a chlorocatechol 1,2-dioxygenase, tfdEII coded for a dienelactone hydrolase, tfdFII coded for a maleylacetate reductase, and tfdBII coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfdI and tfdII genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfdII genes seem to be redundant with respect to those of the tfdI cluster. One reason why the tfdII 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.

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