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In order to study the Salmonella typhimurium cobalamin biosynthetic pathway, the S. typhimurium cob operon was isolated and cloned into Escherichia coli. This approach has given the new host of the cob operon the ability to make cobalamins de novo, an ability that had probably been lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombinant strains have been shown to produce up to 100 times more corrin than the parent S. typhimurium strain. These measurements have been performed with a quantitative cobalamin microbiological assay which is detailed in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are grown 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 cobl genes. When grown in the absence of endogenous cobalt, the oxidized forms of precorrin-2 and precorrin-3, factor II and factor III, respectively, were found to accumulate in the cytosol of the corrin-producing E. coli. Together with the finding that S. typhimurium cbiL mutants are not complemented with the homologous Pseudomonas denitrificans gene, these results lend further credence to the theory that cobalt is required at an early stage in the biosynthesis of cobalamins in S. typhimurium.

Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli