btuB riboswitch from Klebsiella pneumoniae as a target for new antibiotics: in vivo study of riboswitch activation by coupling to a fluorescent protein

Abstract

Riboswitches are mRNA elements that bind metabolites or metal ions as ligands and regulate mRNA expression by forming alternative structures in response to ligand binding. The btuB riboswitch from Klebsiella pneumoniae, responsible for cobalamin uptake, could be an interesting target for new antibiotics, as this bacterium has become an important pathogen in nosocomial infections. A plasmid (pJP04) has been designed and prepared by site-directed mutagenesis in order to study this riboswitch in vivo. The original btuB gene was substituted by mCherry gene, a red fluorescent protein, the synthesis of which was modulated by two different cobalamin derivatives so that riboswitch regulation could be visualized. As in other bacteria, whose riboswitch sequences are partially evolutionary conserved, the interaction of cobalamin with the riboswitch was shown to inhibit protein synthesis.