Genetics of Particulate Methane Monooxygenase

甲烷氧化细菌，或甲烷氧化菌，含有一种不寻常的膜结合铜单加氧酶，pMMO，它将甲烷氧化成甲醇。编码该酶的结构基因（pmo基因）在甲烷氧化菌中存在双拷贝，这两个基因都已被证明在甲烷氧化菌荚膜甲基球菌浴中起作用。该项目的具体目的是确定这两组pmo基因在不同条件下的表达方式，以及这对甲烷氧化能力、生长速率和生长产量等生理参数的影响。接下来的方法是利用基因融合来定义每组基因在野生型中不同条件下的表达，并对每组pmo基因进行缺失菌株的转录本分析，确定基因的转录组织。然后，在为另一组基因删除的菌株中评估每组pmo基因的表达，以确定基因剂量的变化如何影响pMMO活性、生长速率和生长产量。最后，将分离和表征调控突变体，开始分析铜调控的分子机制。该项目的结果将为pMMO系统提供重要的新视角。一旦获得这些信息，就有可能确定其他参与产生活性pMMO的基因，并确定其表达的细节。甲烷氧化细菌或甲烷氧化菌含有一种不寻常的膜结合酶pMMO，它能将甲烷氧化成甲醇。由于以下原因，这种酶引起了极大的兴趣。首先，它具有氧化多种有毒化合物的能力，这些化合物是常见的环境污染物。其中许多反应具有潜在的应用，可用于清理受污染的水和土壤，以及在工业过程中进行环境友好的化学合成。此外，甲烷氧化菌在土壤温室气体循环中发挥着重要作用，对未来全球变暖具有重要意义。由于这些原因，了解pMMO及其在这些细菌生长中的核心作用是很重要的。这个项目包括通过分析pMMO的基因以及它们如何产生这种酶来研究pMMO。此外，该项目将处理影响pMMO生产的因素，以及我们如何为环境和工业应用操纵这些因素。* * *

9630645 Lidstrom Methane-oxidizing bacteria, or methanotrophs, contain an unusual membrane-bound copper monooxygenase, the pMMO, that oxidizes methane to methanol. The structural genes encoding this enzyme (pmo genes) are present in dual copies in methanotrophs, both of which have been shown to be functional in the methanotroph Methylococcus capsulatus Bath. The specific objective of this project is to determine how both sets of pmo genes are expressed and how this affects physiological parameters such as methane oxidation capacity, growth rate and growth yield under different conditions. The approach that will be followed is to use gene fusions to define the expression of each set of genes under different conditions in the wild type, and to carry out transcript analysis in strains deleted for each set of pmo genes, to determine the transcriptional organization of the genes. Then, expression of each set of pmo genes will be assessed in the strain deleted for the other set, to determine how this change in gene dosage affects pMMO activity, growth rate and growth yield. Finally, regulatory mutants will be isolated and characterized, to begin to analyze the molecular mechanism involved in copper regulation. The results of this project should provide major new in sights into the pMMO system. Once this information is available, it should be possible to define other genes involved in generating active pMMO, and determine the details of their expression. %%% Methane-oxidizing bacteria, or methanotrophs, contain an unusual membrane-bound enzyme, the pMMO, that oxidizes methane to methanol. This enzyme has attracted a great deal of interest for the following reasons. First, it has the ability to oxidize a variety of toxic compounds that are common environmental pollutants. Many of these reactions have potential applications for cleaning up contaminated water and soil, as well as carrying out environmentally-friendly chemical syntheses in industrial processes. In addition, methanotrophs are important in the cycling of greenhouse gases in the soil, and they play a significant role in the future of global warming. For these reasons, it is important to understand the pMMO and its central role in the growth of these bacteria. This project involves a study of the pMMO by analyzing its genes and how they produce the enzyme. In addition, the project will address the factors that influence production of pMMO and how we can manipulate those factors for both environmental and industrial applications. ***