title: Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.
abstract: Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.
mesh_terms: D000421:Alcaligenes / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009154:Mutation; D010105:Oxygenases / Q000235:genetics*; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C044174:2,4-dichlorophenoxyacetic acid monooxygenase; D010105:Oxygenases