Metabolic Biochemistry of Reductive Dehalogenation in Dehalococcoides and Relatives

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

It is now well established that certain microorganisms can grow by reductively dehalogenating many halogenated organic compounds, including priority pollutants, a process termed dehalorespiration. The Zinder laboratory isolated an organism, Dehalococcoides ethenogenes strain 195, that can dechlorinate the solvents and pollutants tetrachloroethene (PCE) and trichloroethene (TCE) to ethene. This organism was the first isolate from a subphylum of the Chloroflexi (green nonsulfur bacteria) that includes other organisms implicated in reductive dehalogenation of chlorobenzenes, polychlorinated biphenyls, and dioxins. Two reductive dehalogenases (RDs), a PCE RD dehalogenating PCE to TCE, and a TCE RD dehalogenating TCE to VC and slowly to ethene have been described in strain 195. The nearly completed genome sequence of strain 195 reveals seventeen open reading frames potentially encoding RDs, suggesting that it can utilize numerous other halogenated substrates. The Zinder laboratory has recently shown that strain 195 can dehalogenate hexa- penta- and tetra-chlorobenzenes. They have also recently isolated the Alameda strain, an organism related to D. ethenogenes that conserves energy by dehalogenating the carcinogen vinyl chloride (VC) to ethene, a process that does not support growth of strain 195. In future studies, D. ethenogenes and the Alameda strain will be examined for the abilities to utilize and grow on a broader range of halogenated substrates including further studies on chlorobenzenes, and tests of chlorophenols, bromophenols, chlorobenzoates, fluorochloroethenes, dioxins, and PCBs. The expression of the different RD genes during growth with various chlorinated compounds will be examined. The RD genes will be heterologously expressed in Escherichia coli or another readily grown organism, which if functional enzyme is obtained, will provide large amounts of pure RDs. For the Alameda strain, enough VC and TCE RDs will be isolated from a relatively easily-grown mixed culture to obtain sequence information which then can be used to clone the RD genes. These studies will increase our understanding of the environmentally important process of reductive dehalogenation and will educate a graduate student, a postdoctoral fellow, and several undergraduates in microbiology, biochemistry, and molecular biology. Tetrachloroethylene (PCE) and trichloroethylene (TCE) are solvents used for dry-cleaning clothes and degreasing metal parts, and are among the most pervasive and persistent groundwater pollutants. Dehalococcoides ethenogenes, a novel organism isolated in our laboratory, is the first organism that can completely dechlorinate PCE and TCE to the non-toxic compound ethylene. Recently, the genome of this organism was sequenced, and a preliminary analysis indicates that it possesses seventeen genes coding for reductive dehalogenase enzymes, suggesting that it can dehalogenate many more halogenated compounds. This project will study the ability of D. ethenogenes, and a related organism that rapidly detoxifies the carcinogen vinyl chloride, to utilize other halogenated organic compounds, and will determine which dehalogenase genes are expressed when the organism is growing with different chlorinated compounds. Because D. ethenogenes is so difficult to grow, its reductive dehalogenase genes will be moved into a more tractable organism, such as Escherichia coli, so that sufficient amounts of each dehalogenase will be available for study. These studies will increase our understanding of the environmentally important process of reductive dehalogenation and will educate a graduate student, a postdoctoral fellow, and several undergraduates in microbiology, biochemistry, and molecular biology.