Cobalamin activity-based probe enables microbial cell growth and finds new cobalamin-protein interactions across domains.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B12 ). Our knowledge of the roles of vitamin B12 is limited because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need we show that a synthetic probe mimic of B12 supports growth of B12 auxotrophic bacteria and archaea. We demonstrate that a B12 activity-based probe (B12 -ABP) is actively transported into Escherichia coli cells, and converted to adenosyl-B12 -ABP akin to native B12 Identification of the proteins that bind the B12 -ABP in vivo in E. coli , a Rhodobacteraceae sp. and Haloferax volcanii , demonstrate the specificity for known and novel B12 protein targets. The B12 -ABP also regulates the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B12 in microbe functions. Our approach provides a powerful, non-disruptive tool to analyze B12 interactions in living cells, and can be used to discover the role of B12 in diverse microbial systems. Importance We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B12 in microbial growth and regulation, by supporting growth of B12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B12 -protein interactions. The B12 -ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B12 The application of the B12 -ABP at biologically relevant concentrations facilitates a unique way to measure B12 microbial dynamics and identify new B12 protein targets in bacteria and archaea. We demonstrate that the B12 -ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms, to the salt-tolerant archaea Haloferax volcanii .