Isolation and characterization of a mutant defective in the production of methanol dehydrogenase from a new restricted facultative methanol-oxidizing bacterium.

A new restricted facultative methanol-oxidizing bacterium Methylobacillus sp. strain SK1 (DSM 8269) was subjected to insertion mutagenesis studies with the transposon Tn5 to generate mutants deficient in the production of methanol dehydrogenase (MDH). The transposon was conjugally transferred into the methylotroph by using the triparental mating procedure. The mutants induced by Tn5 were selected directly from a plate containing succinate by using the allyl alcohol selection procedure. The transposition of Tn5 to the bacterium showed a moderate transposition frequency (10(-5) - 10(-6)). Southern hybridization analysis confirmed that the transposon Tn5 was inserted into the chromosomal DNA of the mutants. In addition, the mutants had no RNA transcripts produced from the mdh gene, as judged by Northern blot analysis. The mutants could neither grow on methanol nor produce MDH protein, as determined by Western blot analysis with anti-MDH antibody. These results suggest that Tn5 mutagenesis maybe a useful tool for the molecular analysis of a facultative methanol-oxidizing bacterium.