Development of a Gene Cloning System in Methanogens

摘要

The methane-producing archaebacteria have a unique biochemistry and molecular biology. In this work, a collection of mutants in the thermophilic autotroph Methanobacterium thermoautotrophicum was obtained. Several of these were resistant to pyrimidine and purine analogs (e.g. 5-fluorouracil and 6-mercaptopurine). A formate auxotroph was isolated, which represents a novel growth requirement. Characterization of this stain will reveal new aspects of one-carbon metabolism in methanogens. A complete genomic library of restriction fragments of the methanogen DNA was prepared in Escherichia coli plasmid pBR322. Transposon sequences will be introduced into these fragments, and linearized, transposon-containing plasmids used to transform the methanogen. This technique (additive transformation) followed by selective enrichment will yield mutants. The mutants and the ability to introduce DNA into recipient strains will provide the basis for genetic mapping and engineering of methanogens. Keywords: Gene cloning. (aw)