Development of gene transfer systems in Methylobacillus flagellatum KT: Isolation of auxotrophic mutants

A collection of polyauxotrophic mutants of the obligate methylotroph Methylobacillus flagellatum KT was obtained. On the first step two stable auxotrophic mutants with a high requirement for amino acids supplements were isolated by treatment with nitrosoguanidine and selection on complete medium. Spontaneous variants of these mutants with a low requirement for nutrient supplements were the base for obtaining polyauxotrophic strains. It was shown, that the growth of mutants of M. flagellatum KT is inhibited by complete medium. Some amino acids and nucleotides are the inhibitor components of complete media. An approach for selection of auxotrophic mutants of individual genes was worked out on minimal medium. The optimal conditions for nitrosoguanidine mutagenesis of M. flagellatum KT were developed. The possible mechanisms of action of some of the nutrient supplements on the growth of M. flagellatum KT are discussed.