Genetic Analysis of Methylotrophic Methanogenesis in Methanosarcina Species

本项目的长期目标是通过对实验驯化的产甲烷古菌的研究，了解产甲烷古菌的生理、代谢和生态学。这些实验的主要重点将是鉴定和表征影响单碳（C-1）化合物（如产甲烷底物）使用的基因，包括那些通过调节所需基因的表达间接影响过程的基因。特别是，涉及调节甲醇分解代谢所需基因表达的传感和信号转导机制将被检查；还将调查甲基硫化物的生产和消费的遗传基础，甲基硫化物是在全球气候中起关键作用的化合物。为了实现这些目标，将解决以下具体问题：(1)甲烷菌的环境感知和信号转导的分子机制是什么；(2)哪些Methanosarcina基因作为古细菌特异性Msr调控蛋白家族的成员受到调控；(3)甲烷菌中C-1化合物的使用还需要哪些基因？(4)甲烷藻转录后基因调控的程度和机制是什么？实验将利用最新开发的甲烷菌遗传分析方法；然而，生理、生化和分子方法将补充遗传方法。这一策略预计将产生既新颖又补充以往研究结果的数据。拟议研究的智力价值涉及产甲烷微生物在许多社会问题中的核心作用，包括从生物材料生产替代燃料，产甲烷生物在农业和废物处理中的作用，以及全球变暖（甲烷是一种强效温室气体）。拟议的研究结果有望通过揭示甲基化营养途径的新方面以及通过深入了解将环境条件与适当基因表达联系起来的机制，对产甲烷菌在生物圈中的作用产生广泛的影响。此外，对古细菌转录和翻译控制的分子机制的更清晰理解应该源于对高度调控的甲基化营养基因的研究。更广泛的影响拟议研究的更广泛的影响包括培训未来的科学家和学者。两名研究生将获得申请资金的直接资助；另外几名学生也将参与这个项目。该实验室通常会邀请其他大学的学生来提供实验方法的实践培训。基于过去的结果，这项研究也可能产生新的遗传工具和方法，这些工具和方法将对更广泛的微生物研究界有用并且免费提供。遗传工具和突变株通常会提供给其他研究小组，而不管它们是否已经发表。最后，实验室定期为当地的中小学教师提供设施、资源和建议，让他们的学生接触到令人兴奋的微生物世界。

Intellectual MeritThe long-term goal of this project is to understand the physiology, metabolism and ecology of methanogenic archaea through the study of experimentally tractable Methanosarcina species. The primary emphasis of these experiments will be the identification and characterization of genes that affect the use of one-carbon (C-1) compounds such as methanogenic substrates, including those that indirectly affect the process by regulating expression of the required genes. In particular, the sensing and signal transduction mechanisms involved in regulating the expression of genes needed for methanol catabolism will be examined; the genetic basis for production and consumption of methylsulfides, compounds that play a critical role in the global climate will also be investigated. To achieve these goals the following specific questions will be addressed: (1) What are the molecular mechanisms of environmental sensing and signal transduction in Methanosarcina; (2) Which Methanosarcina genes are regulated as members of the archaeal-specific Msr family of regulatory proteins; (3) What other genes are required for use of C-1 compounds in Methanosarcina; and (4) What is the extent and mechanism of post-transcriptional gene regulation in Methanosarcina? The experiments will utilize recently developed methods for genetic analysis of Methanosarcina; however, physiological, biochemical and molecular approaches will complement the genetic approach. This strategy is expected to generate data that are both novel and complementary to the results of preceding studies. The intellectual merit of the proposed research relates to the central role of methanogenic microorganisms in a number of societal problems including the production of alternative fuels from biological materials, the role of methane-producing organisms in agriculture and waste treatment, and global warming (methane is a potent greenhouse gas). The results of the proposed studies are expected to have broad implications with respect to the role of methanogens in the biosphere by revealing novel aspects of the methylotrophic pathway and by lending insight into the mechanisms for linking environmental conditions to appropriate gene expression. Moreover, a clearer understanding of the molecular mechanisms involved in archaeal transcriptional and translational control should result from examination of the highly regulated methylotrophic genes. Broader ImpactThe broader impacts of the proposed research include the training of future scientists and academics. Two graduate students will be directly supported by the requested funds; several additional students will also work on the project. The lab commonly hosts students from other universities to provide hands-on training in the labs methods. Based on past results, this study is also likely to produce new genetic tools and methods that will be useful, and freely available, to the wider microbial research community. Genetic tools and mutant strains are routinely provided to other research groups, regardless of whether they have been published. Finally, the laboratory routinely provides facilities, resources and advice to local elementary and secondary school teachers who wish to expose their students to the excitement of the microbial world.