PNNL

Abstract

Understanding the function of microbial communities and the numerous interactions that occur between species in microbiomes is a grand challenge. The abundance of cobalamin (vitamin B12), which is an important enzyme cofactor and regulator of gene expression, directly effects microbiome structure. Technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed to understand microbiomes. To meet this need, we demonstrate that B12 auxotrophic bacteria and archaea can be cultured on a probe mimic of B12. We show how the B12 activity-based probe (B12-ABP) is transported into Escherichia coli cells, and converted to adenosyl-B12-ABP. 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 is also shown to regulate the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Overall these results demonstrate that the B12-ABP is a vitamin B12 substitute that is functional, yet not disruptive, and, thus, provides a powerful tool to analyze B12 interactions in living cells. The results show that the B12-ABP can be used to discover the role of B12 in diverse microbial systems.