Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B ₁₂

Abstract

An insight into a previously unknown step in B12 biosynthesis was unexpectedly obtained through our analysis of a mutant of the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. This mutant was identified based on its unusually bright fluorescence on plates containing the succinoglycan binding dye calcofluor. The mutant contains a Tn5 insertion in a gene that has not been characterized previously in S. meliloti. The closest known homolog is the bluB gene of Rhodobacter capsulatus, which is implicated in the biosynthesis of B 12 (cobalamin). The S. meliloti bluB mutant is unable to grow in minimal media and fails to establish a symbiosis with alfalfa, and these defects can be rescued by the addition of vitamin B 12 (cyanocobalamin) or the lower ligand of cobalamin, 5,6-dimethylbenzimidazole (DMB). Biochemical analysis demonstrated that the bluB mutant does not produce cobalamin unless DMB is supplied. Sequence comparison suggests that BluB is a member of the NADH͞flavin mononucleotide (FMN)-dependent nitroreductase family, and we propose that it is involved in the conversion of FMN to DMB.cobalamin ͉ symbiosis ͉ bacteria ͉ vitamin B12 ͉ biosynthesis

Molecular Determinants of a Symbiotic Chronic Infection

“…As a Class II RNR that is both oxygen-independent and oxygen-insensitive (80), this enzyme likely provides a key adaptation for rhizobial persistence within the microaerobic environment of the host cell cytoplasm. The B 12 biosynthetic enzyme BluB, which catalyzes formation of the lower ligand 5,6-dimethylbenzimidazole, was fortuitously found to be required for symbiosis between S. meliloti and M. sativa based on its involvement in succinoglycan biosynthesis (22,162). A bluB mutant appears able to infect the host via normal IT growth, suggesting that any alteration to the succinoglycan does not affect invasion; however, bacteroids are not observed within the host cell cytoplasm and the nodules that develop are unable to fix nitrogen (22).…”

“…The B 12 biosynthetic enzyme BluB, which catalyzes formation of the lower ligand 5,6-dimethylbenzimidazole, was fortuitously found to be required for symbiosis between S. meliloti and M. sativa based on its involvement in succinoglycan biosynthesis (22,162). A bluB mutant appears able to infect the host via normal IT growth, suggesting that any alteration to the succinoglycan does not affect invasion; however, bacteroids are not observed within the host cell cytoplasm and the nodules that develop are unable to fix nitrogen (22). Thus, BluB function in B 12 biosynthesis is necessary for symbiosis due to the requirement for a B 12 -dependent enzyme(s), for which NrdJ is one possible candidate.…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…FMN (but not FAD) also possesses an important biological function in being the biosynthetic precursor of the dimethylbenzimidazol part of coenzyme B 12 (63,366,367). Conversion of FMN to dimethylbenzimidazol involves a unique transformation reaction with no precedent in chemistry, which includes retro-aldol condensation sandwiched between two 2-electron oxidations (154,519).…”

Gut and Root Microbiota Commonalities

“…Vitamin B 12 is an exclusive product of prokaryotes (98), and it is produced by plant root and gut bacteria (99)(100)(101)(102). Essential amino acids and vitamins B and K are produced by gut bacteria (reviewed in reference 58).…”