The coenzyme B precursor 5,6-dimethylbenzimidazole is a flavin antagonist in .

subsp. sv. Typhimurium str. LT2 (hereafter Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB) only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report and evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the (encodes D-Ala/D-Ser transporter) and (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.