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.
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