Genetic Analysis of Methanogenesis by Methanosarcina Species

ABSTRACTWm。甲烷生成作为全球碳循环和重要温室气体生产的关键步骤，是至关重要的。它也是替代燃料战略的核心，在农业和废物处理行业都是必不可少的。甲烷菌被认为是最重要的甲烷产生者之一，它们都是所谓的“第三种生命形式”——古细菌的成员。尽管它们很重要，但人们对这些不寻常的生物或甲烷生产过程（甲烷生成）所知相对较少。本研究的目的是利用遗传分析来研究甲烷生成的代谢过程，这些代谢过程涉及到Methanosarcina属的成员。由于缺乏针对这些独特生物体的可靠遗传方法，此类研究在以前是不可能的；然而，最近开发的方法现在使这成为可能。遗传方法在甲烷生成研究中的应用很可能揭示了迄今为止用于研究该过程的生化方法尚未解决的甲烷生成过程的新方面。通过使用遗传学方法，我们的分析不仅将确定主要的产甲烷途径，还将确定直接和间接影响产甲烷的代谢、调节和结构成分。将要进行的实验将利用甲烷藻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出不能由某些底物产生甲烷的突变体，同时保留利用其他底物的能力。重要的是，Methanosarcina物种是唯一遗传上可处理的产甲烷生物，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古细菌，以及缺乏遗传分析方法的更遥远的生物。该项目的具体目标是：1)使用这些最近开发但已被证实的遗传方法对甲烷藻物种进行遗传分析；2)改进和修改现有的遗传工具，以简化这些实验的执行，并扩大这种生物可能的研究范围。本项目旨在开发和利用遗传工具来研究古菌甲烷生成的代谢基础。这是一个极其重要却鲜为人知的过程。这项工作的潜在成果是了解甲烷生成的遗传基础和分子遗传学工具的发展，以检查系统发育域的其他成员。

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.