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 B-12). Our knowledge of the roles of vitamin B-12 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 B-12 supports the growth of B-12-auxotrophic bacteria and archaea. We demonstrate that a B-12 activity-based probe (B-12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B-12-ABP akin to native B-12. Identification of the proteins that bind the B-12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B-12 protein targets. The B-12-ABP also regulates the B-12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B-12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B-12 interactions in living cells and can be used to discover the role of B-12 in diverse microbial systems. IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B-12 in microbial growth and regulation by supporting the growth of B-12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B-12-protein interactions. The B-12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B-12. The application of the B-12-ABP at biologically relevant concentrations facilitates a unique way to measure B-12 microbial dynamics and identify new B-12 protein targets in bacteria and archaea. We demonstrate that the B-12-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 salttolerant archaea Haloferax volcanii.