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. ***
