MICROBIAL DEGRADATION OF AGENT ORANGE

描述：橙剂是两种除草剂丁酯的混合物。 2，4-二氯苯氧基乙酸(2，4-D)和2，4，5-二氯苯氧基乙酸 三氯苯氧基乙酸(2，4，5-T)。2，4-D是可生物降解的，其 生物降解，类似于氯苯甲酸酯或三氯苯， 通过形成氯代邻苯二酚和氯磺酸盐作为 中间体，最终通过氯-β-代谢- 并通过三羧酸(TCA)循环。相比之下， 2，4，5-T通常对微生物的侵袭具有顽固性。下强下 以2，4，5-T为主要来源的化学药物选择 出现了洋葱假单胞菌AC1100菌株Carbon 可以利用2，4，5-T作为唯一的碳和能源来源，通过 2，4，5-三氯苯酚(2，4，5-TCP)，2，5-三氯苯酚的中间体 二氯对苯二酚(DCHQ)、5-氯羟基苯酚(CHQ)和氯-β-苯二酚- 己二酸酮酯。查克拉巴蒂博士描述了该组织和 氯代儿茶酚降解性基因(CLC)的部分进化调控 恶臭假单胞菌中的一个质粒组分，并比较其组织结构 与染色体儿茶酚的调控相似之处 降解性(CAT)基因。他同样对两个基因簇进行了测序 参与了2，4，5-T的降解，即TftAB和tftEFGH。TftAB基因 CLUSTER参与了2，4，5-T向2，4，5-TCP的转化，而 TftEFGH基因簇参与了CHQ的降解，是一种重要的 2，4，5-T降解的中间体以及其他高度降解的中间体 氯化化合物，如五氯苯酚或伽马 六氯环己烷。这项提议的主要目标是克隆 2，4，5-磷酸三钙转化为CHQ的基因研究 与CHQ降解有关的酶的性质。这项规定 并将研究这些基因的位置。关于 CLC基因的进化，他计划研究如何调控 蛋白CLcr，正向调节clcABD(F)的表达 操纵子，结合操纵子的启动子区域，以及这种结合 会导致DNA的弯曲。诱导剂氯-顺式，顺式- CLcr结合和可能的DNA弯曲中的变锥将是 已评估。ClcABD启动子区域和 CLcr与启动子区域接触的关键氨基酸将 接受检查。猫操纵子和《中图法》操纵子都有内部 下游CatR和CLcr结合的结合位点(IBS) 结构基因。在CatBC操纵子的情况下，已经显示了IBS 在启动子激活过程中起重要作用。有两个IBS 在clcABD操纵子下游的clca基因中。实验将是 进行以确定IBS在激活clcABD中的作用 推动者。

DESCRIPTION: Agent Orange is a mixture of two herbicides, butyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetic acid (2,4,5-T). 2,4-D is biodegradable and its biodegradation, similar to that of chlorobenzoates or trichlorobenzenes, proceeds through formation of chlorocatechol and chloromuconate as intermediates, which are finally metabolized through chloro-beta- ketoadipate and through the tricarboxylic acid (TCA) cycle. In contrast, 2,4,5-T is normally recalcitrant to microbial attack. Under strong chemostatic selection in presence of 2,4,5-T as the major source of carbon, a strain of Pseudomonas (Burkholderia) cepacia AC1100 emerged that could utilize 2,4,5-T as a sole source of carbon and energy through intermediate formation of 2,4,5-trichlorophenol (2,4,5-TCP), 2,5- dichlorohydroquinone (DCHQ), 5-chlorohydroxyquinol (CHQ) and chloro-beta- ketoadipate. Dr. Chakrabarty has delineated the organization and regulation of the chlorocatechol degradative (clc) genes evolved as part of a plasmid in a strain of P. putida and compared its organizational and regulatory similarities to that of the chromosomal catechol degradative (cat) genes. He has similarly sequenced two gene clusters involved in 2,4,5-T degradation, viz. tftAB and tftEFGH. The tftAB gene cluster is involved in the conversion of 2,4,5-T to 2,4,5-TCP while the tftEFGH gene cluster is involved in the degradation of CHQ, an important intermediate of 2,4,5-T degradation as well as that of other highly chlorinated compounds such as pentachlorophenol or gamma hexachlorocyclohexane. The major goal of this proposal is to clone the genes specifying conversion of 2,4,5-TCP to CHQ as well as study the nature of the enzymes involved in the degradation of CHQ. The regulation and location of these genes will be studied. With regard to the evolution of the clc genes, he plans to examine how the regulatory protein ClcR, which regulates positively the expression of the clcABD(F) operon, binds the promoter region of the operon and whether such binding causes a bend in the DNA. The role of the inducer chloro-cis,cis- muconate in the binding and putative DNA bending by ClcR will be evaluated. The critical nucleotides of the clcABD promoter region and the critical amino acids of ClcR that contact the promoter region will be examined. Both the cat operon and the clc operon possess internal binding sites (IBS) for CatR and ClcR binding in the downstream structural genes. In case of the catBC operon, the IBS has been shown to be important in the promoter activation process. There are two IBSs in the downstream clcA gene of the clcABD operon. Experiments will be conducted to define the role of the IBSs in the activation of the clcABD promoter.