@article{Kipkorir2021,
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 catalyzes the final reaction in methionine biosynthesis. In addition to metH , M. smegmatis possesses a cobalamin-independent methionine synthase, metE , suggesting that enzyme use – 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 can transport and assimilate exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B 12 ) and its precursor, dicyanocobinamide ((CN) 2 Cbi). However, the uptake of CNCbl and (CN) 2 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 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 Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis as human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remain poorly understood. Using the non-pathogenic saprophyte, M. smegmatis , we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis , in contrast with M. tuberculosis , which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B 12 ) and its precursors is restricted in M. smegmatis , apparently depending on the co-factor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.},
author = {Kipkorir, Terry and Mashabela, Gabriel T and de Wet, Timothy J and Koch, Anastasia and Wiesner, Lubbe and Mizrahi, Valerie and Warner, Digby F},
doi = {10.1128/jb.00620-20},
file = {:C$\backslash$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Kipkorir et al. - 2021 - iDe novoi cobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine bi.pdf:pdf},
issn = {0021-9193},
journal = {Journal of Bacteriology},
keywords = {Digby F Warner,Gabriel T Mashabela,MEDLINE,NCBI,NIH,NLM,National Center for Biotechnology Information,National Institutes of Health,National Library of Medicine,OA,PubMed Abstract,Terry Kipkorir,doi:10.1128/JB.00620-20,fund{\_}not{\_}ack,original,pmid:33468593},
mendeley-tags = {OA,fund{\_}not{\_}ack,original},
month = {jan},
number = {7},
pages = {e00620--20},
pmid = {33468593},
publisher = {American Society for Microbiology},
title = {{De novo cobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis in Mycobacterium smegmatis}},
url = {https://pubmed.ncbi.nlm.nih.gov/33468593/},
volume = {203},
year = {2021}
}

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In addition to metH , M. smegmatis possesses a cobalamin-independent methionine synthase, metE , suggesting that enzyme use – 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 can transport and assimilate exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B 12 ) and its precursor, dicyanocobinamide ((CN) 2 Cbi). However, the uptake of CNCbl and (CN) 2 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 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 Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis as human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remain poorly understood. Using the non-pathogenic saprophyte, M. smegmatis , we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis , in contrast with M. tuberculosis , which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B 12 ) and its precursors is restricted in M. smegmatis , apparently depending on the co-factor requirements of the cobalamin-dependent methionine synthase. 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We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B 12 ) and its precursor, dicyanocobinamide ((CN) 2 Cbi). However, the uptake of CNCbl and (CN) 2 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 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. 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