C1-Oxidation Pathways in Proteobacteria: Function and Regulation

该奖项由2009年《美国复苏和再投资法案》(公法111-5)资助。环境中常见的单碳化合物包括甲烷、甲醇、甲基胺、卤代甲烷和甲磺酸盐。这些化合物特别引起环境关注，因为它们中的一些(甲烷、甲基磺酸盐)直接导致温室效应，而另一些(甲醇和甲基胺)则是产甲烷的前体。微生物氧化甲醇和甲基胺是碳和氮环境循环的重要组成部分，也是防止甲烷和N2O等温室气体形成的重要步骤。虽然这些C1化合物的一些初级氧化途径已经很好地建立起来了，但其他的仍然缺乏特征。氨基酸介导的甲胺利用途径曾被认为是植物和微生物代谢的重要组成部分，然而这些途径对于基于基因组的预测仍然是不可见的。这项研究旨在获得对微生物利用甲胺和甲醇的代谢机制的新的遗传学见解。揭示这些机制将允许开发探测环境中这些系统的探测器，从而在全球意义上提供对环境中C1循环的更完整的理解。该项目将对单碳化合物代谢的基本理解以及作为代谢能力的甲基营养的演变提供有价值的见解。纯化和鉴定的酶将扩大生产N-甲基氨基酸(如N-甲基苯丙氨酸衍生物和茶氨酸)的酶的数据库，并可进一步探索工业应用。简而言之，具体目标包括以下几个方面：目的1.获得一种新的甲基营养菌基因组的草稿序列并对其进行注释。目的2.通过基因表达比较研究和转座子诱变，筛选甲胺和甲醇氧化相关基因。目的3.通过蛋白质纯化和蛋白质组学研究确定参与甲胺初级氧化的蛋白质，补充目标2的数据。目的4.使用遗传和生化方法对感兴趣的基因进行功能分析。这些分析将补充来自具体目标2和3的数据，并最终确认作为目标2和3的一部分提出的活动和功能。更广泛的影响：该项目将支持广泛的本科生研究，并将与现有的外展计划相结合。该项目将支持让高中生参与研究项目的实验室传统，并为他们提供参与生物发现的机会。参与该项目的研究生、本科生和高中生将被鼓励参加当地一所K-6小学的两个外展项目，其中包括描述细菌对人类、植物和环境的重要性的讲座，以及一次“动手”的科学体验。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Single carbon (C1) compounds commonly found in the environment include methane, methanol, methylated amines, halogenated methanes, and methylsulfonates. These compounds are of particular environmental concern as some of them (methane, methylsulfonates) directly contribute to the greenhouse effect, while others (methanol, and methylated amines) serve as precursors for methanogenesis. Microbial oxidation of methanol and methylated amines are important parts of environmental cycling of C and N as well as an essential step in preventing formation of greenhouse gases such as methane and N2O. While some of the pathways for primary oxidation of these C1 compounds are well established, others remain poorly characterized. The amino acid-mediated pathways for methylamine utilization were once discussed as an important part of both plant and microbial metabolism, however these pathways are still invisible for genome-based predictions. This research aims to obtain new genetic insights into the metabolic mechanisms of microbial utilization of methylamine and methanol. Uncovering these mechanisms will allow for development of probes for detecting these systems in the environment, and thus in turn will provide a more complete understanding of C1 cycling in the environment, in a global sense. The project will provide valuable insights into the fundamental understanding of metabolism of single carbon compounds, as well as the evolution of methylotrophy as a metabolic capacity. The purified and characterized enzyme will expand the database of enzymes for production of N-methyl amino acids (such as N-methyl-phenylalanine derivative and theanine) and could be further explored for industrial applications. Briefly, the specific aims include the following: Aim 1. Obtain draft sequence and annotate the genome of a novel methylotroph. Aim 2. Identify genes involved in methylamine and methanol oxidation via comparative gene expression studies and transposon mutagenesis. Aim 3. Identify proteins involved in primary oxidation of methylamine via protein purification and proteomic studies, complementing the data from Aim 2. Aim 4. Perform functional analysis of the genes of interest, using genetic and biochemical approaches. These analyses will complement the data from specific Aims 2 and 3 and ultimately confirm the activities and functions proposed as parts of Aims 2 and 3. Broader Impacts: This project will support extensive undergraduate research and will interface with existing outreach programs. This project will support the laboratory tradition of involving high school students in research projects, and provide them with opportunities to participate in biological discoveries. Graduate, undergraduate and high school students involved in the project will be encouraged to participate in two outreach programs at a local K-6 elementary school that include lectures describing the importance of bacteria for humans, plants and the environment as well as a "hands-on" science experience.