Characterization of the Organization of the Genome of Methanogens and Development of Genetic Exchange Systems for Methanococcus Vannielii: Final Report for the Period July 1, 1981-June 30, 1987

摘要

The goals of this contract were to characterize gene organization and expression in methane microorganisms and to develop gene transfer systems for these unusual anaerobes. Antibiotic resistant mutants were isolated, potential shuttle vectors constructed and a number of methanogen genes cloned which when functionally expressed in Escherichia coli complemented auxotrophic mutations of eubacterial species. DNA sequencing of cloned genes was used to establish evolutionary relationships between methanogens and showed the conservation of DNA sequences in archaebacterial, eubacterial and eucaryotic species. Sequencing demonstrated that methanogens use the standard genetic code, have polypeptide-encoding genes organized into operons, initiate translation using ribosome binding sites (RBS) and terminate transcription by forming double-stranded loops in RNA molecules. All these are molecular biological features typical of eubacteria. In contrast, the presence of conserved sequences and protection of DNA from nuclease digestion by bound methanogen-derived RNA polymerases indicated that methanogen promoters do not resemble eubacterial promoters. The initial cloning and sequencing of amino-acid and purine biosynthetic genes from methanogens provided the necessary background information to undertake cloning and analysis of genes which encode enzymes directly involved in methanogenesis. The most abundant enzyme in all methanogens is methyl coenzyme-M reductase which catalyzes the terminal reaction in methanogenesis. The genes which encode the subunits of component C of this enzyme in Methanococcus vannielii have been cloned and sequenced. Their organization and structure indicates that, in vivo, component C may contain previously unrecognized subunits and its synthesis may be regulated at the level of translation. Genes encoding subunits of the major non-F sub 420 reducing hydrogenase of Methanobacterium thermoautotrophicum have also been cloned and sequenced. 16 refs., 1 fig. (ERA citation 13:003206)