Identification of Dehalococcoides Genes Encoding Reductive Dehalogenases That Dechlorinate Specific PCB Congeners

智力价值：这项研究将阐明呼吸酶大家族成员的功能和底物专一性。这些酶是还原脱卤酶(RDase)，它们是在最近发现的一组严格厌氧菌-脱卤球菌属(DHC)中发现的。RDase从有毒的氯化有机化合物(如多氯联苯)中去除氯，这种脱氯过程为DHC的生长提供能量，同时使多氯联苯解毒。DHC的每个菌株都有许多不同的RDase，这些RDase可能允许它们对广泛的底物进行脱氯，但人们对这些酶了解很少，只知道其中三种酶的功能和底物特异性。该实验室已经确定了两种DHC菌株，每种菌株都可以用4到9个氯对一系列多氯联苯(PCBs)同系物进行脱氯和解毒。这个项目提供了一个独特的机会，可以深入了解这个庞大而新颖的呼吸蛋白家族中许多成员的功能和底物专一性。多个呼吸RDase脱氯多氯联苯的分子序列和功能将被鉴定，并随后进行比较和研究。由于已知各种具有不同氯取代模式的多氯联苯分子是这些DHC菌株中还原脱卤酶的底物，因此将深入了解这些酶的区域特异性。还将获得有关这些酶如何适应仅存在80年的多氯联苯的信息。这个项目将使用生物化学、微生物学、分子生物学和基因组学中的工具来解决四个主要目标。更广泛的影响：水中沉积物中的微生物还原多氯联苯脱氯提供了一种自然和高效的解毒方法。这项研究将首次确定负责对最受关注的特定多氯联苯进行脱氯和解毒的RDase，并将能够深入研究编码关键脱氯酶的特定RDase基因。这项研究也是朝着开发成本效益高的方法促进就地修复多氯联苯的目标迈出的重要一步，并朝着设计分子生物学工具的方向迈出了一大步，这些工具将检测、量化和监测污染环境中多氯联苯的脱氯生物。该项目将支持一名访问科学家和一名博士后研究员，并将为6名本科生提供机会，获得在生化分析、微生物学、分子生物学和生物信息学方面的宝贵研究技能，并参与最先进的原创研究。他们还将有机会在科学会议上介绍他们的成果，并共同撰写出版物。在外展方面，少年科学协会将参加一项针对中学女生的青年妇女科学方案。这包括在学年和暑期通过演讲和实验室会议向女孩们介绍她的研究。她还将在科学项目上指导高中女孩，她将在当地一所高中为多个小组开发和演讲《比小说更奇怪：微生物的极端生活方式》。

Intellectual Merit: This research will elucidate the function and substrate specificity of members of a large family of respiratory enzymes. These enzymes are reductive dehalogenases (RDases) and they are found in a recently discovered group of strictly anaerobic bacteria, Dehalococcoides (Dhc). The RDases remove chlorines from toxic chlorinated organic compounds such as polychlorinated biphenyls (PCBs) and this dechlorination process provides energy for Dhc growth while detoxifying the PCBs. Individual strains of Dhc each possess many different RDases which presumably allow them to dechlorinate a broad range of substrates, but these enzymes are poorly understood and the function and substrate specificity of only three are known. This laboratory has identified two strains of Dhc that can each dechlorinate and detoxify a broad suite of polychlorinated biphenyl (PCB) congeners with 4 to 9 chlorines. This project provides a unique opportunity to gain substantial insights into the function and substrate specificity of many members of this large and novel respiratory protein family. The molecular sequence and function of multiple respiratory RDases that dechlorinate PCBs will be identified and subsequently compared and studied . Because a wide variety of PCB molecules with different chlorine substitution patterns are known to be substrates for the reductive dehalogenases in these Dhc strains, insights will be gained into the regiospecificity of these enzymes. Information will also be gained on how these enzymes have adapted to PCBs which have existed for only 80 years. This project will use tools in biochemistry, microbiology, molecular biology, and genomics to address four main objectives. Broader Impacts: Microbial reductive PCB dechlorination in aquatic sediments provides a natural and highly effective means of detoxification. This research will for the first time identify the RDases responsible for dechlorinating and detoxifying the particular PCBs that are of most concern and will enable the in-depth study of the specific RDase genes that code for the key dechlorination enzymes. The research is also a major step toward the goal of developing cost-effective methods for promoting in situ PCB remediation and toward the design of molecular biological tools that will detect, quantify and monitor PCB-dechlorinating organisms in contaminated environments. This project will support a visiting scientist, a post-doctoral fellow, and will give 6 undergraduates the opportunity to acquire valuable research skills in biochemical analysis, microbiology, molecular biology, and bioinformatics and to participate in state-of-the-art original research. They will also have opportunities to present their results at scientific meetings and to co-author publications. For outreach the PI will participate in a Young Women in Science program aimed at middle school girls. This involves introducing the girls to her research through presentations and lab sessions during the academic year and summer. She will also mentor high school girls on science projects, and she will develop and give presentations on "Stranger than Fiction: Extreme Lifestyles of Microorganisms" to multiple groups at a local high school.