Cobalamin (B-12) is an essential nutrient for humans that is only synthesized by microbes and some protists. The biosynthesis of B-12 is rather poorly understood. The complexity and instability of intermediates has made biochemical approaches difficult and most study has been focused on bacteria with very poor genetic systems. We have found that the genetic organism, Salmonella typhimurium, is able to synthesize B-12 de novo when grown under anaerobic conditions. We have initiated a primarily genetic approach to the synthesis of B-12 and how that synthesis is regulated. We hope to identify the structural genes for all of the synthetic enzymes and elucidate the mechanisms whereby these genes are regulated in response to the end product (probably adenosylcobalamin), cAMP, and redox state of the cell. Synthesis, transport, and recycling of B-12 appears to require about 1% of the Salmonella genome. Despite the size of this genetic investment in B- 12, only four enzymes are known in Salmonella that require B-12 as a cofactor; none of these functions appears to be fundamentally important. The B-12 dependent functions include on (of two) routes to methionine, synthesis of a nonessential tRNA modification, and ability to use ethanolamine and propanediol as carbon sources. Which of these, or what undiscovered function, accounts for the importance of B-12 to Salmonella? Why does Salmonella synthesize B-12 only under anaerobic conditions? (Many obligate aerobes are known to be able to synthesize B-12.) What physiological factors account for the distribution of B-12 in nature? (It is absent from plants, required by animals and some protists, and synthesized primarily by microbes.) To approach questions of biological significance of B-12, we will investigate the anaerobic metabolism of Salmonella. Despite the great interest that has been focused on genetics and metabolism of Salmonella and its relative E. coli, anaerobic behavior has received rather little attention. We have to contribute to a better understanding of anaerobic metabolism and its regulation, with special attention to the role of B-12.