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