Methanococcus maripaludis is emerging as a premier model for the methanogens and the Archaea. It grows unusually well, has an extensive set of facile genetic tools, and has a known complete annotated genome sequence. Systems have been established for reliable growth in chemostats under defined nutrient conditions. Expression arrays have been implemented, as well as wide-coverage quantitative proteomics. Novel protein-protein interactions have been demonstrated in pull-down experiments under anaerobic conditions. No other species of methanogens or of the Archaea boasts such a combination of effective technologies. Many basic questions that arise in the study of other methanogens, Archaea, and even eukaryotes can be best addressed in M. maripaludis. Although the community of researchers currently focusing on M. maripaludis is small, the number is rapidly growing. Leigh's and Whitman's labs have long-standing projects that focus on M. maripaludis. Many additional labs have recently expressed interest in using the M. maripaludis system and some have begun doing so (letters provided). In this proposal, further genetic tools will be developed by improving on an existing transposon insertion method and constructing a bank of insertion mutants available to the community. A set of markerless in-frame deletion mutations will also be produced, focusing on a selected set of genes of wide interest. Vectors will be designed for expression of cloned genes using known regulated promoters. These vectors will facilitate protein production, protein purification, studies of protein-protein interactions, a rigorous test of gene essentiality, and other genetic manipulations. Continued studies of global gene expression will be facilitated by improving capabilities for chemostat culture, setting up a complete Affymetrix oligonucleotide array, and continuing to develop proteomics by optimizing data collection on a recently-purchased LTQ linear ion trap mass spectrometer. Arrays and proteomics will be used to generate datasets that define the global regulatory response to conditions of wide biological interest. In addition, database systems will be improved for analyzing, correlating, and displaying the results of chemostat growth, array analysis, and proteomic analysis. Information processing systems in the Archaea resemble those of eukaryotes. Hence, the Archaea provide simple prokaryotic models for eukaryotic systems with relevance to human biology. In addition, although pathogenic Archaea have not been found, a number of enzymes and biochemical pathways discovered in Archaea have been subsequently discovered in some pathogenic bacteria. ? ? ?
