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

Abstract

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 B12 AdoRhbl thus emerges as a multifunctional antibiotic candidate targeting B12-utilizing microorganisms.TBC