Genetic Analysis of Methanogenesis by Methanosarcina Species

中文摘要MetcalfMCB-9874459甲烷生成作为全球碳循环和产生重要温室气体的关键步骤至关重要。它也是替代燃料战略的核心，在农业和废物处理行业中至关重要。甲烷八叠球菌属被认为是最重要的甲烷生产者之一，所有这些物种都是所谓的“第三种生命形式”的成员。尽管它们很重要，但对这些不寻常的生物或甲烷产生过程（甲烷生成）的了解相对较少。该研究的目的是利用遗传分析来研究甲烷八叠球菌属成员参与甲烷生成的代谢过程。由于缺乏针对这些独特生物体的强大遗传方法，这种研究以前是不可能的;然而，最近开发的方法现在使这成为可能。遗传方法学的研究甲烷的应用可能会揭示新的方面的产甲烷过程中，还没有解决的生化方法来研究这个过程的日期。通过使用遗传方法，我们的分析将确定不仅是主要的产甲烷途径，而且直接和间接影响产甲烷的代谢，调节和结构组成部分。将要进行的实验将利用甲烷八叠球菌属物种相对于其他产甲烷生物的代谢多样性。这种代谢多样性应该允许分离不能由某些底物提供燃料的产甲烷的突变体，同时保留利用其他底物的能力。重要的是，甲烷八叠球菌属物种是唯一遗传上易于处理的产甲烷生物，用于检查这种代谢多样性。 此外，我们开发的工具应该广泛应用于一般的生物，以及缺乏遗传分析方法的更远的相关生物。该项目的具体目标是：1）使用这些最近开发的，但经过验证的遗传方法对甲烷八叠球菌属物种进行遗传分析，2）改进和修改现有的遗传工具，以简化这些实验的执行，并扩大这种生物体可能的研究范围。该项目旨在开发和利用遗传工具，以研究古菌甲烷八叠球菌中甲烷生成的代谢基础。 这是一个极其重要的过程，但人们对此知之甚少。 这项工作的潜在成果是甲烷生成的遗传基础的理解和分子遗传工具的发展，以检查其他成员的系统发育域。

ABSTRACTWm. MetcalfMCB-9874459Methanogenesis is critically important as a key step in the global carbon cycle and in the production of a significant greenhouse gas. It is also at the center of alternative fuel strategies and is essential in both agriculture and the waste treatment industry. Methanosarcina species are believed to be among the most significant methane producers, all of which are members of the so-called "third form of life" known as the Archaea. Despite their importance, relatively little is known about these unusual organisms, or about the process of methane production (methanogenesis). The aim of the proposed research is to use genetic analysis to investigate the metabolic processes involved in methanogenesis by members of the genus Methanosarcina. Such studies have not previously been possible due to a lack of robust genetic methods for these unique organisms; however, recently developed methods now make this possible. The application of genetic methodologies to the study of methanogenesis is likely to reveal novel aspects of the methanogenic process that have not been addressed by the biochemical methods used to study this process to date. By using a genetic approach, our analysis will identify not only the main methanogenic pathways, but also the metabolic, regulatory, and structural components that directly and indirectly effect methanogenesis. The experiments to be conducted will take advantage of the metabolic diversity of Methanosarcina species relative to other methanogenic organisms. This metabolic diversity should allow isolation of mutants that are incapable of methanogenesis fueled by certain substrates while retaining the ability to utilize others. Importantly, Methanosarcina species are the only genetically tractable methanogenic organisms for examining this metabolic diversity. Moreover, the tools we have developed should have broad application to the Archaea in general, and to more distantly related organisms for which methods of genetic analysis are lacking. The specific goals of the project are; 1) genetic analysis of Methanosarcina species using these recently developed, but proven, genetic methods, and 2) the refinement and modification of currently available genetic tools to simplify the execution of these experiments and to expand the range of the studies possible in this organism. This project is aimed at developing and utilizing genetic tools for examining the metabolic basis of methanogenesis in the archaeon Methanosarcina. This is an extremely important process about which little is known. The potential outcomes of this work are an understanding of the genetic basis of methanogenesis and the development of molecular genetic tools to examine other members of the phylogenetic domain.