De novocobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis inMycobacterium smegmatis
Authors: Kipkorir Terry , Mashabela Gabriel T. , Wet Timothy J. De , Koch Anastasia , Wiesner Lubbe , Mizrahi Valerie , Warner Digby F. 
Published: November 2020

ABSTRACTCobalamin 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 inde novocobalamin biosynthesis.M. smegmatisalso encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition tometH,M. smegmatispossesses 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 controllingmetEexpression inM. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirmde novocobalamin biosynthesis inM. smegmatisduring aerobic growthin vitro. We also demonstrate thatM. smegmatistransports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). Interestingly, the uptake of CNCbl and (CN)2Cbi appears restricted inM. smegmatisand 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 ofmetEare strongly attenuated by endogenous cobalamin. These results support the inference thatmetHessentiality inM. smegmatisresults from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism betweenM. smegmatisandM. tuberculosisprovide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCEAccumulating evidence suggests that alterations in cobalamin-dependent metabolism marked the evolution ofMycobacterium tuberculosisfrom 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 constitutivede novocobalamin biosynthesis inM. smegmatisunder standard laboratory conditions, in contrast withM. tuberculosis, which appears to lackde novocobalamin biosynthetic capacity. We also demonstrate that the uptake of cyanocobalamin (vitamin B12) and its precursors is restricted inM. smegmatis, apparently depending on the need to service the co-factor requirements of the cobalamin-dependent methionine synthase. These observations support the utility ofM. smegmatisas a model to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.