Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium

An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.