Development of a Gene Cloning System in Methanogens.

Research on the genetics of methane producing archaebacteria is directed at fundamental problems of mutant construction, gene transfer by transformation, and construction of vectors which facilitate gene transfer. From the thermophilic methanogen, Methanobacterium thermoautotrophicum, a set of mutants was obtained which included strains resistant to antibiotics or antimetabolites. Also, by using a new mutagenesis protocol, formic acid auxotrophs were obtained, whose physiology is of great interest. Genetic transfer via DNA-dependent natural transformation was achieved for two markers, 5-fluorouracil-resistance, and 6-mercaptopurine resistance. These markers were partially linked, although the genetic distance has not been determined. Vector construction is underway to link methanogen genes with an Escherichia coli plasmid. The recombinant DNA will be mutagenized by transposon insertion and then transformed back into M. thermoautotrophicum. The insertion can be used to locate the wild-type sequences from the mehtanogen by southern blotting of restriction fragments. The characterization of auxotropic and resistant mutants, and the isolation of new mutants is ongoing, as is optimization of the transformation system. In the ten months since the start of the project, significant progress towards the long term goal of understanding genetic regulation in methanogens has been made.