Environmental release of a large number of synthetic chlorinated compounds in the form of herbicides/pesticides, solvents, refrigerants etc., has created major concerns with regard to their effect on human health because of the persistence of many such compounds. The persistence of these compounds is a reflection of the inability of natural microorganisms to utilize them as a sole source of carbon and energy. Many microorganisms can utilize simple chlorinated compounds such as 3-chlorobenzoate (3Cba) or 2,4-dichlorophenoxyacetate (2,4-D) as their sole carbon source but cannot utilize higher chlorinated forms such as 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and others. Under strong selection in a chemostat with 2,4,5-T as the only major source of carbon (directed evolution), it has been possible to isolate a strain of Pseudomonas cepacia AC1100 that can utilize 2,4,5-T as its sole source of carbon and energy. Molecular cloning of the genes both for 3Cba/2,4-D as well as 2,4,5,-T degradation has shown that while the chlorocatechol (clc) genes for 3Cba and 2,4-D dissimilation are highly homologous, the 2,4,5-T degradative (tft) genes show no homology with any of the genomic DNA of a large number of pseudomonads and other bacteria. The applicant proposes experiments to understand the nature of the tft genes and their products to determine exactly how 2,4,5-T is degraded by AC1100 and its mode of regulation. Sequencing and hyperexpression of a number of tft genes will be attempted to obtain information about the role of enzymes that allow efficient permeation of 2,4,5-T and its subsequent dechlorination and oxidation. Similarly, a regulatory protein involved in the positive regulation of catechol (cat) genes will be purified and its mode of binding with the promoter of the structural genes will be delineated to define the nature of this protein-DNA interaction for functional expression of the cat genes. These data would provide valuable information about the mode of evolution of structural and regulatory genes in nature as well as in chemostats in response to the release and availability of chlorinated compounds.
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