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

## TL;DR

A synthetic compound that mimics vitamin B12 can block bacterial gene expression and may serve as a new antibiotic.

## Contribution

The study reveals a new gene-regulatory activity of an antivitamin B12 analogue in repressing bacterial B12 uptake.

## Key findings

- AdoRhbl mimics AdoCbl in downregulating reporter expression in vitro with high affinity.
- AdoRhbl shows strong intracellular activity in E. coli with EC50 values as low as 1.4 nM.
- AdoRhbl inhibits B12-dependent enzymes and represses BtuB expression, suggesting antibiotic potential.

## 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 B12AdoRhbl thus emerges as a multifunctional antibiotic candidate targeting B12-utilizing microorganisms.

Coenzyme B12 and its Rh-analogue, the antivitamin B12AdoRhbl, bind both the btuB-riboswitch with exceptional affinity and inhibit expression of the bacterial B12 uptake protein BtuB, signifying AdoRhbl's high potential as multifunctional antibiotic.