COBALAMIN (VITAMIN-B-12) BIOSYNTHESIS - FUNCTIONAL-CHARACTERIZATION OF THE BACILLUS-MEGATERIUM CBI GENES REQUIRED TO CONVERT UROPORPHYRINOGEN-III INTO COBYRINIC ACID A,C-DIAMIDE

The function of individual genes of the Bacillus megaterium cobI opero n genes in cobalamin (vitamin B-12) biosynthesis was investigated by t heir ability to complement defined Salmonella typhimurium cab mutants. This strategy confirmed the role of cbiA, -D, -F, -J, -L and cysG(A). Furthermore the operon as a whole was used to restore corrin biosynth esis in Escherichia coli, which, although closely related to S. typhim urium, does not possess the CobI pathway. When the B. megaterium cab o peron was cloned into a plasmid and transformed into an E. coli strain containing the S. typhimurium cbiP, it conferred upon the host strain the ability to make the cobyric acid de novo. However, cobyric acid s ynthesis was observed only when the strain was grown anaerobically. De rivatives of the corrin-producing E. coli strain were constructed in w hich genes of the B. megaterium cob operon had been inactivated. These strains were used to demonstrate that, whereas B. megaterium cbiD, -G and -X are essential for cobyric acid synthesis, the cbiW and -Y gene s could be deleted without detriment to cobyric acid production in E. coli.