Vitamin B12 is a critical component of the human diet. B12 deficiency, which plagues over 20% of elderly individuals, can cause hematological disorders, jaundice, loss of coordination, and dementia. Understanding how this vitamin is synthesized may contribute to the overall knowledge and treatment of B12-related disease. Unlike all other vitamins, B12 is synthesized exclusively by microorganisms. In some pathogenic bacteria, B12 is utilized specifically during infection of the host. Therefore, studying the physiology of B12 in bacteria could lead to insights about pathogenesis. Until recently, the enzymes responsible for the production of the "lower ligand" of B12, 5,6-dimethylbenzimidazole (DMB), were unknown. We recently identified BluB, an enzyme that catalyzes the production of DMB in the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. The bluB mutant of S. meliloti is severely impaired in establishing a nitrogen-fixing symbiosis with its legume host, alfalfa. Our structural and biochemical analyses demonstrated that BluB catalyzes the oxygen-dependent transformation of flavin mononucleotide (FMN) to DMB. The proposed work will seek to identify the detailed reaction mechanism of this unique transformation. Putative homologs of bluB exist in numerous bacterial genomes, suggesting that BluB has a ubiquitous role in DMB biosynthesis. However, a separate DMB biosynthetic pathway that does not use FMN as a precursor exists in anaerobic bacteria, and the enzymes involved in this pathway have not been identified. The anaerobic DMB biosynthetic pathway will be investigated by genetic, bioinformatic, and biochemical approaches using the techniques developed in studying BluB. Additionally, the physiological response to B12 deprivation in S. meliloti will be analyzed. In S. meliloti, an altered exopolysaccharide is produced in response to B12 deprivation, and this response is caused by DNA damage. The structure, regulatory mechanism, and physiological relevance of the DNA damage-induced altered exopolysaccharide will be explored. Together, these studies will contribute to the overall understanding of B12 biosynthesis and the function of B12 in bacteria-host interactions.