Repression of bacterial gene expression by antivitamin B₁₂ binding to a cobalamin riboswitch

The E. coli btuB riboswitch is a cobalamin-sensing RNA element that selectively binds coenzyme B₁₂ (adenosylcobalamin, AdoCbl) to downregulate the expression of the outer membrane B₁₂-transporter BtuB. Here, we examined adenosylrhodibalamin (AdoRhbl), the isostructural Rh-analogue of AdoCbl, as a surrogate effector ligand for this riboswitch. Two riboswitch-reporter systems were employed: an engineered E. coli strain with a fluorescent reporter for intracellular AdoCbl-sensing, and a plasmid-based construct for analogous in vitro transcription/translation assays. In the in-vitro system AdoRhbl closely mimicked AdoCbl in down-regulating reporter expression with apparent EC₅₀ values of 2.8 µM and 0.8 µM respectively. In contrast, the engineered E. coli strain revealed much higher effective sensitivities, with EC₅₀ values of 1.4 nM for AdoRhbl and of 6.9 nM for AdoCbl, reflecting strong intracellular accumulation of both corrinoids, and comparably efficient uptake. These findings uncover a previously undocumented gene-regulatory activity of an antivitamin, suggesting that AdoRhbl can repress bacterial B₁₂ uptake by binding to the btuB riboswitch. Together with its ability to inhibit AdoCbl-dependent enzymes, the designed antivitamin B₁₂AdoRhbl thus emerges as a multifunctional antibiotic candidate targeting B₁₂-utilizing microorganisms.