A Cobalamin Activity-Based Probe Enables Microbial Cell Growth and Finds New Cobalamin-Protein Interactions across...

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 the 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 nondisruptive 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 the 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.