ABSTRACT Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis , an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis , carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition to metH , M. smegmatis possesses a cobalamin-independent methionine synthase, metE , suggesting that enzyme selection – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis . Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro . We also demonstrate that M. smegmatis transports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B 12 ) and its precursor, dicyanocobinamide ((CN) 2 Cbi). Interestingly, the uptake of CNCbl and (CN) 2 Cbi appears restricted in M. smegmatis and 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 metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity. IMPORTANCE Accumulating evidence suggests that alterations in cobalamin-dependent metabolism marked the evolution of Mycobacterium tuberculosis from an environmental ancestor to an obligate human pathogen. However, the roles of cobalamin in mycobacterial physiology and pathogenicity remain poorly understood. We used the non-pathogenic saprophyte, M. smegmatis , to investigate the production of cobalamin, transport and assimilation of cobalamin precursors, and the potential role of cobalamin in regulating methionine biosynthesis. We provide biochemical and genetic evidence confirming constitutive de novo cobalamin biosynthesis in M. smegmatis under standard laboratory conditions, in contrast with M. tuberculosis , which appears to lack de novo cobalamin biosynthetic capacity. We also demonstrate that the uptake of cyanocobalamin (vitamin B 12 ) and its precursors is restricted in M. smegmatis , apparently depending on the need to service the co-factor requirements of the cobalamin-dependent methionine synthase. These observations support the utility of M. smegmatis as a model to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

<i>De novo</i>cobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis in<i>Mycobacterium smegmatis</i>

De novocobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis inMycobacterium smegmatis