The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of approximately 28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element within cobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3' of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to approximately 30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediate N1-(5-phospho-alpha-D-ribosyl)-5, 6-dimethylbenzimidazole (also known as alpha-ribazole-5'-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 184.108.40.206) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997) J. Biol. Chem. 272, 17662-17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR and YTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996) J. Bacteriol. 178, 7016-7019).