SALMONELLA-TYPHIMURIUM COBALAMIN (VITAMIN-B-12) BIOSYNTHETIC GENES - FUNCTIONAL-STUDIES IN SALMONELLA-TYPHIMURIUM AND ESCHERICHIA-COLI

In order to study the Salmonella typhimurium cobalamin biosynthetic pa thway, the S. typhimurium cob operon was isolated and cloned into Esch erichia coli. This approach has given the new host of the cob operon t he ability to make cobalamins de novo, an ability that had probably be en lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombin ant strains have been shown to produce up to 100 times more corrin tha n the parent S. typhimurium strain. These measurements have been perfo rmed with a quantitative cobalamin microbiological assay which is deta iled in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are gro wn under anaerobic conditions. Derivatives of the cobalamin-producing E. coli strains were constructed in which genes of the cob operon were inactivated. These strains, together with S. typhimurium cob mutants, have permitted the determination of the genes necessary for cobalamin production and classification of cbiD and cbiG as cobI genes. When gr own in the absence of endogenous cobalt, the oxidized forms of precorr in-2 and precorrin-3, factor II and factor III, respectively, were fou nd to accumulate in the cytosol of the corrin-producing E. coli. Toget her with the finding that S. typhimurium cbiL, mutants are not complem ented with the homologous Pseudomonas denitrificans gene, these result s lend further credence to the theory that cobalt is required at an ea rly stage in the biosynthesis of cobalamins in S. typhimurium.