Rare variants of the FMN riboswitch class in Clostridium difficile and other bacteria exhibit altered ligand specificity

Abstract

Many bacteria use flavin mononucleotide (FMN) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor. Representatives of one of the two variant subtypes were found to bind the FMN precursor riboflavin and the FMN degradation products lumiflavin and lumichrome. Although the biologically relevant ligand sensed by these variant FMN riboswitches remains uncertain, our findings suggest that many strains of C. difficile might use rare riboswitches to sense flavin degradation products and activate transporters for their detoxification.

Regulatory context drives conservation of glycine riboswitch aptamers

“…Variations in homologous riboswitch aptamers have demonstrated functional consequences. There are a range of variant riboswitch classes that interact with differing ligands [53,63,64], the most compelling of which are the homologous ykkC riboswitches [57,65] which include at least five subclasses each of which binds a distinct ligand involved in guanidine degradation and export [39,[66][67][68]. There are also examples where structurally distinct riboswitches interact with the same or very similar ligands, such as the seven riboswitch classes involved in regulating S-adenosylmethionine concentration [69,70].…”

A bacterial riboswitch class for the thiamin precursor HMP-PP employs a terminator-embedded aptamer

“…Liquid media (LB or GMM) was supplemented with X-gal (200 μg mL −1 ) to allow visual detection of reporter gene expression. Similarly, reporter expression analysis using 4-methylumbelliferyl β-D-galactopyranoside was conducted as described previously (Nelson et al, 2015; Atilho et al, 2019) to establish fluorescence units.…”

Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD+

“…Riboswitch reporter assays were performed in a manner similar to that described previously (Atilho et al 2019b). Briefly, bacterial cultures were grown overnight at 37°C in Lysogeny Broth (LB) with the appropriate antibiotic(s).…”

“…To establish liquid-based β-galactosidase assays for visual interpretation, cells were diluted to an OD 600 of 0.05 or 0.1 in LB supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 100 μg mL −1 ) and the appropriate antibiotic(s) before incubation at 37°C with shaking. Similarly, for reporter expression quantification by spectrophotometer, LB media containing antibiotic(s) were inoculated with cells to an OD 600 of 0.05 or 0.1 and quantified at the desired time point (4-7 h) using MUG (4-methylumbelliferyl β-D-galactopyranoside) as previously described (Atilho et al 2019b). Fluorescence units were calculated as described previously (Vidal-Aroca et al 2006).…”