MICROBIAL DEGRADATION OF AGENT ORANGE

这项建议的主要目标是了解 基因的进化、组织和调控 2，4-二氯苯氧基乙酸(2，4-D)的降解 2，4，5-三氯苯氧基乙酸(2，4，5-T) 除草剂橙色。橙剂的出现，特别是一种 其成分2，4，5-T在不同部位大量聚集 世界制造了公共卫生问题，只有在以下情况下才能最大限度地减少 这些污染物通过微生物从受污染的地点移走。 退化。已知的是，pAC27等质粒编码降解物 氯代儿茶酚的途径，而质粒如pJP2和pJP4编码一个 包括氯代儿茶酚在内的完整的2，4-D降解途径。因为这些 三种质粒在大小和不相容特性上存在差异 证明氯代儿茶酚基因具有广泛的聚集性和同源性， 将尝试克隆单个氯代儿茶酚和 来自质粒pJP2和pJP4的苯氧乙酸酯降解基因，确定 它们的完整核苷酸序列，并将开放阅读框比较为 以及pAC27的上游序列，以查看是否 氯代儿茶酚的基因是相同的。每个单独基因的产物 将在tac启动子的转录控制下过度生产 并对其酶活性进行了研究。利用2，4，5-T降解菌 洋葱假单胞菌AC1100，其中Tn5突变允许分离 对于大量2，4，5-T阴性突变体，将尝试 定位Tn5存在于质粒上或在 染色体DNA，从而证明了2，4，5-T基因的位置。 在一个例子中，转座子突变后与Tn5杂交 DNA已经证明了2，4，5-T基因的染色体位置 似乎在这个基因上或附近有重复的序列。这重复了一遍 洋葱假单胞菌、恶臭假单胞菌、恶臭假单胞菌、洋葱假单胞菌、恶臭假单胞菌、洋葱假单胞菌、恶臭假单胞菌、假单胞菌和假单胞菌都没有测序。 铜绿假单胞菌、门多克氏假单胞菌等。 对这个重复序列进行测序，以确定其在自然界中的出现情况 假单胞菌以外的微生物，以确定是否有其他类型的 重复序列与其他2，4，5-T降解基因有关 确定这种重复序列的可移位性。这类研究 将提供关于进化模式的重要线索 苯氧乙酸酯类除草剂降解基因的组织 假单胞菌。

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.