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

智力优势：这项研究将阐明呼吸酶大家族成员的功能和底物特异性。 这些酶是还原性脱卤酶（RDases），并且它们在最近发现的严格厌氧细菌群Dehalococcoides（Dhc）中被发现。 RDases从有毒的氯化有机化合物（如多氯联苯（PCB））中去除氯，并且该脱氯过程为Dhc生长提供能量，同时使PCB解毒。Dhc的各个菌株各自具有许多不同的RDases，这可能使它们能够使广泛的底物脱氯，但这些酶的了解很少，只有三种酶的功能和底物特异性是已知的。该实验室已经确定了两种Dhc菌株，每种菌株都可以对一系列具有4至9个氯的多氯联苯（PCB）同系物进行脱氯和解毒。该项目提供了一个独特的机会，以获得大量的功能和底物特异性的许多成员，这个大的和新的呼吸道蛋白家族的见解。 将确定多氯联苯脱氯的多种呼吸RDases的分子序列和功能，并随后进行比较和研究。 由于各种各样的PCB分子与不同的氯取代模式是已知的还原脱卤酶在这些DHC菌株的底物，洞察力将获得这些酶的区域特异性。 还将获得关于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的工具来解决四个主要目标。更广泛的影响：水生沉积物中的微生物还原性多氯联苯脱氯提供了一种自然和高效的解毒手段。 这项研究将首次确定负责脱氯和解毒的特定多氯联苯是最令人关注的RDases，并将使编码关键脱氯酶的特定RDase基因的深入研究成为可能。 这项研究也是朝着开发成本效益高的方法促进多氯联苯就地补救的目标迈出的重要一步，也是朝着设计分子生物学工具的目标迈出的重要一步，这些工具将检测、量化和监测受污染环境中的多氯联苯脱氯生物。 该项目将支持一名访问科学家，一名博士后研究员，并将使6名本科生有机会获得生化分析，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性研究。 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。 为了推广，PI将参加一个针对中学女生的青年妇女科学计划。 这包括在学年和夏季通过演示和实验室会议向女孩介绍她的研究。 她还将指导高中女生的科学项目，她将开发和演示“比小说更奇怪：微生物的极端生活方式”在当地高中的多个团体。

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.