An in vivo high-throughput screening for riboswitch ligands using a reverse reporter gene system

Abstract

Riboswitches are bacterial RNA elements that regulate gene expression in response to metabolite or ion abundance and are considered as potential drug targets. In recent years a number of methods to find non-natural riboswitch ligands have been described. Here we report a high-throughput in vivo screening system that allows identifying OFF-riboswitch modulators in a 384 well bioluminescence assay format. We use a reverse reporter gene setup in Bacillus subtilis, consisting of a primary screening assay, a secondary assay as well as counter assays to detect compounds in a library of 1,280 molecules that act on the guanine-responsive xpt riboswitch from B. anthracis. With this in vivo high-throughput approach we identified several hit compounds and could validate the impact of one of them on riboswitch-mediated gene regulation, albeit this might not be due to direct binding to the riboswitch. However, our data demonstrate the capability of our screening assay for bigger high-throughput screening campaigns. Furthermore, the screening system described here can not only be generally employed to detect non-natural ligands or compounds influencing riboswitches acting as genetic OFF switches, but it can also be used to investigate natural ligands of orphan OFF-riboswitches.

Employing a ZTP Riboswitch to Detect Bacterial Folate Biosynthesis Inhibitors in a Small Molecule High-Throughput Screen

“…If the hit compound is bound by the riboswitch aptamer and activates reporter gene expression, then the output of the reporter construct will appear the same as for a compound that inhibits folate metabolism. Such riboswitch-targeting screens have been described previously. ,− In addition, the M6 riboswitch variant would likely remain in the OFF state when exposed to such compounds, thereby confirming the hit.…”

Overview on Strategies and Assays for Antibiotic Discovery

“…As natural or synthetic ligand analogues (small molecules) can bind the riboswitches and stop their regulatory functions, they represent a promising target for antibiotics [ 175 ]. To screen for antibiotics that bind to bacterial riboswitches, assays that utilize reporter-based systems have been developed [ 169 , 176 , 177 ]. For example, Lee et al, used the lacZ reporter system in B. subtilis and demonstrated that roseoflavin (naturally produced by Streptomyces davawensis ), a chemical analog of flavin mononucleotide FMN and riboflavin [ 178 , 179 , 180 ], binds to the FMN riboswitch and downregulates the expression of the FMN riboswitch- lacZ reporter gene [ 177 ].…”

Identification of Efflux Substrates Using a Riboswitch-Based Reporter in Pseudomonas aeruginosa

“…These natural biosensors allow bacteria to appropriately regulate gene expression in the setting of fluctuating concentrations of metabolites, metals, or salts. Riboswitch-based reporters have been used to characterize novel antibacterial drugs and targets ( 31 , 32 ), to identify riboswitch ligands ( 33 , 34 ), to describe biosynthetic pathways ( 35 ), and to screen for high-yield vitamin-producing strains ( 36 ). To date, nearly 40 classes of riboswitches have been characterized, many of them monitoring essential metabolic pathways and controlling expression of virulence factors ( 37 ).…”