Metabolic Biochemistry of Reductive Dehalogenation in Dehalococcoides and Relatives

现在已经确定，某些微生物可以通过还原脱卤化许多卤化有机化合物来生长，包括优先污染物，这一过程被称为脱卤素呼吸。Zinder实验室分离出一种能够将溶剂和污染物四氯乙烯(PCE)和三氯乙烯(TCE)脱氯为乙烯的微生物--产乙烯脱卤球菌属195号菌株。该细菌是氯杆菌亚门(绿色非硫细菌)的第一个分离物，包括与氯苯、多氯联苯和二恶英的还原脱卤化有关的其他生物。在菌株195中描述了两种还原脱卤酶(RDS)，一种是将PCE脱卤化为TCE的PCE脱卤酶，另一种是将TCE脱卤化为VC并缓慢转化为乙烯的TCE脱卤酶。菌株195几乎完成的基因组序列显示了17个可能编码RDS的开放阅读框，这表明它可以利用许多其他卤化底物。Zinder实验室最近表明，菌株195可以对六氯、五氯和四氯苯进行脱卤化。他们最近还分离出了Alameda菌株，这是一种与产烯D菌有亲缘关系的有机体，通过将致癌物质氯乙烯(VC)脱卤化成乙烯来节省能量，这一过程不支持菌株195的生长。在未来的研究中，将测试产乙烯双胞菌和Alameda菌株在更广泛的卤化底物上的利用和生长能力，包括对氯苯的进一步研究，以及对氯苯酚、溴苯酚、氯苯甲酸酯、氟氯乙烯、二恶英和多氯联苯的测试。将检测不同的RD基因在不同含氯化合物的生长过程中的表达。RD基因将在大肠杆菌或其他容易生长的生物体中异源表达，如果获得功能酶，将提供大量纯RDS。对于Alameda菌株，将从相对容易生长的混合培养物中分离出足够的VC和TCE RDS，以获得序列信息，然后可以用于克隆RD基因。这些研究将增加我们对环境重要的还原脱卤化过程的理解，并将培养一名研究生、一名博士后研究员和几名本科生在微生物学、生物化学和分子生物学方面的知识。四氯乙烯(PCE)和三氯乙烯(TCE)是用于干洗衣服和金属部件脱脂的溶剂，是最普遍和最持久的地下水污染物之一。本实验室分离到的一种新的微生物--产乙烯脱卤球菌属，是第一个能将PCE和TCE完全脱氯为无毒化合物乙烯的微生物。最近，该菌的基因组被测序，初步分析表明，它具有17个编码还原脱卤酶的基因，这表明它可以脱卤化更多的化合物。该项目将研究产乙烯双胞菌和一种能够快速解毒致癌物质氯乙烯的相关生物体利用其他卤化有机化合物的能力，并将确定当有机体与不同的氯化化合物生长时，哪些脱卤酶基因表达。由于产乙烯双胞菌很难生长，其还原脱卤酶基因将被转移到更易驯化的生物体中，如大肠杆菌，以便有足够数量的每种脱卤酶可供研究。这些研究将增加我们对环境重要的还原脱卤化过程的理解，并将培养一名研究生、一名博士后研究员和几名本科生在微生物学、生物化学和分子生物学方面的知识。

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.