title: Methods for Isolation of Auxotrophic Mutants of Methanobacterium ivanovii and Initial Characterization of Acetate Auxotrophs.
abstract: To develop a biochemical genetic approach to understanding cell carbon synthesis or metabolic pathways in methanogens, Methanobacterium ivanovii was selected as a model organism for genetic manipulation studies. The organism displayed a colony size of 3 to 6 mm in less than 2 weeks and had a plating efficiency of about 90%, which made it suitable for replica plating. Mutagenesis and selection techniques were developed for selection of acetate auxotrophs. Chemical mutagenesis with ethyl methanesulfonate, followed by enrichment with bacitracin as a selective agent, resulted in stable acetate auxotrophs. M. ivanovii was very sensitive to UV, but UV-induced acetate auxotrophs were unstable and reverted within two to four transfers. The acetate auxotrophs were analyzed in relation to wild type for carbon monoxide dehydrogenase enzyme activity.
authors.affiliation: Michigan Biotechnology Institute and the Departments of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: M K
authors.initials: MK
authors.lastname: Jain
authors.forename: J G
authors.initials: JG
authors.lastname: Zeikus