Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. , an environmental saprophyte frequently used as surrogate for the obligate human pathogen , carries approximately 30 genes predicted to be involved in cobalamin biosynthesis. also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to , possesses a cobalamin-independent methionine synthase, , suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling expression in Here, we apply a targeted mass spectrometry-based approach to confirm cobalamin biosynthesis in during aerobic growth We also demonstrate that can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B) and its precursor, dicyanocobinamide ([CN]Cbi). However, the uptake of CNCbl and (CN)Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of are strongly attenuated by endogenous cobalamin. These results support the inference that essentiality in results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between and provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity. Alterations in cobalamin-dependent metabolism have marked the evolution of into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte , we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive cobalamin biosynthesis in , in contrast with , which appears to lack cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B) and its precursors is restricted in , apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.