MICROBIAL DEGRADATION OF AGENT ORANGE

描述：橙子剂是两种除草剂的混合物，丁酯 2，4-二氯苯氧乙酸（2，4-D）和2，4，5- 三氯苯氧乙酸（2，4，5-T）。2，4-D可生物降解， 生物降解，类似于氯苯甲酸盐或三氯苯， 通过氯儿茶酚和氯粘康酸盐的形成进行， 中间体，最终通过氯-β- 酮己二酸和通过三羧酸（TCA）循环。与此相反， 2，4，5-T通常不受微生物的侵袭。在强 在2，4，5-T作为主要来源的存在下的恒化选择 碳，洋葱假单胞菌AC 1100菌株出现 可以利用2，4，5-T作为碳和能源的唯一来源， 中间体形成2，4，5-三氯苯酚（2，4，5-TCP）、2，5- 二氯对苯二酚（DCHQ）、5-氯羟基对苯二酚（CHQ）和氯-β- 酮己二酸。查克拉巴蒂博士描述了这个组织 氯儿茶酚降解（clc）基因的调控进化为 putida菌株中的质粒，并比较其组织结构 和调节相似的染色体儿茶酚 降解（猫）基因。他同样对两个基因簇进行了测序 参与2，4，5-T降解的两个酶，即tftAB和tftEFGH。tftAB基因 簇参与2，4，5-T到2，4，5-TCP的转换，而 tftEFGH基因簇参与了CHQ的降解，这是一个重要的 2，4，5-T降解的中间体以及其他高浓度的 氯化化合物如五氯苯酚或γ-氯苯酚 六氯环己烷。这项建议的主要目标是克隆 指定2，4，5-TCP转化为CHQ的基因，以及研究 参与CHQ降解的酶的性质。调控 这些基因的位置将被研究。相对于 为了研究clc基因的进化，他计划研究调控基因是如何被激活的。 蛋白ClcR，其正调节clcABD的表达（F） 操纵子，结合操纵子的启动子区，以及这种结合是否 导致DNA发生弯曲。诱导剂氯-顺， 粘康酸在结合和推定的DNA弯曲的ClcR将是 评估。clcABD启动子区的关键核苷酸和 与启动子区域接触的ClcR的关键氨基酸将 接受检查。cat操纵子和clc操纵子都具有内部 下游中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IBS 在启动子激活过程中很重要。有两个IBS 在clcABD操纵子的下游clcA基因中。实验将 进行以确定IBS在clcABD激活中的作用 启动子

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.