The major goal of this proposal is to obtain an understanding of the evolution, organization and regulation of genes involved in the total degradation of 2,4,-Di-chlorophenoxyacetic acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) - the two components of the herbicide Agent Orange. The occurrence of Agent Orange, particularly one of its components, 2,4,5-T, in large concentrations in various parts of the world has created public health problems, that can only be minimized if such pollutants are removed from the contaminated sites through microbial degradation. Plasmids such as pAC27 are known to encode the degradative pathway for chlorocatechols, while plasmids such as pJP2 and pJP4 encode a complete 2,4-D degradative pathway including chlorocatechols. Since these three plasmids differ in sizes and incompatibility characteristics, yet demonstrate extensive clustering and homology for the chlorocatechol genes, attempts would be made to clone the individual chlorocatechol and the phenoxyacetate degradative genes from plasmids pJP2 and pJP4, determine their complete nucleotide sequences, and compare the open reading frames as well as the upstream sequences to those of pAC27 to see if the chlorocatechol genes are identical. The products for each individual genes will be overproduced under the transcriptional control of the tac promoter and their enzymatic activities studied. Using the 2,4,5-T degrading strain of Pseudomonas cepacia AC1100, where Tn5 mutagenesis has allowed isolation of a large number of 2,4,5-T negative mutants, attempts would be made to localize the presence of the Tn5 either on the plasmid or on the chromosomal DNA, thereby demonstrating the location of the 2,4,5-T genes. In one instance, transposon mutagenesis followed by hybridization with Tn5 DNA have demonstrated the chromosomal location of a 2,4,5-T gene which appears to harbor a repeated sequence on or near this gene. This repeated sequence is absent in the laboratory strains of P. cepacia, P. putida, P. aeruginosa, P. mendocina, etc. Attempts would be made to characterize and sequence this repeated sequence, to determine its occurrence in natural microorganisms other than Pseudomonas, to determine if other types of repeated sequences are involved with other 2,4,5-T degradative genes and to determine the translocatibility of such repeated sequences. Such studies will provide important clues regarding the mode of evolution and organization of the phenoxyacetate herbicide degradative genes in Pseudomonas.
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