Gene expression control by Bacillus anthracis purine riboswitches

Abstract

In all kingdoms of life, cellular replication relies on the presence of nucleosides and nucleotides, the building blocks of nucleic acids and the main source of energy. In bacteria, the availability of metabolites sometimes directly regulates the expression of enzymes and proteins involved in purine salvage, biosynthesis, and uptake through riboswitches. Riboswitches are located in bacterial mRNAs and can control gene expression by conformational changes in response to ligand binding. We have established an inverse reporter gene system in that allows us to monitor riboswitch-controlled gene expression. We used it to investigate the activity of five potential purine riboswitches from in response to different purines and pyrimidines. Furthermore, in vitro studies on the aptamer domains of the riboswitches reveal their variation in guanine binding affinity ranging from namomolar to micromolar. These data do not only provide insight into metabolite sensing but can also aid in engineering artificial cell regulatory systems.

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

“…To identify novel ligands for the B. anthracis xpt riboswitch from large compound libraries, our reverse reporter gene system established previously in B. subtilis 20 (Fig. 1A ) needed to be adapted for high-throughput screening.…”

“…To revert the reporter readout, we have previously established a reverse reporter gene system in the gram-positive model organism B. subtilis that enabled us to elucidate OFF riboswitch function 20 . The system consists of two parts which are integrated into two different loci in the B. subtilis W168 genome (Fig.…”

“…Additionally, a high signal/noise ratio can be expected due to a beneficial concentration ratio between parts integrated in the genomic DNA and RNA/proteins. With this reverse reporter gene system we have characterised the response and ligand specificity of five different guanine riboswitches from the pathogen B. anthracis , the causative agent of anthrax 20 . We could show that the xpt riboswitch from B. anthracis directly binds guanine and regulates reporter gene expression in response to guanine and hypoxanthine.…”

Ureide metabolism in plant-associated bacteria: purine plant-bacteria interactive scenarios under nitrogen deficiency

“…The first step, described in Bacillus subtilis, is the deamination of guanine to hypoxanthine catalyzed by the enzyme guanine deaminase (Rivas et al 2018). For this step, guanine riboswitches (Gong et al 2018) regulate the transcription of xpt-pbuX operon in B. subtilis, Staphylococcus aureus and in the vast majorities of Firmicutes (Kofoed et al 2016;Laney and Morse 2017;Kirchner and Schneider 2017). The second step is the conversion of hypoxanthine into xanthine and then to uric acid by the enzyme xanthine oxidoreductase (XOR) that occurs in two isoforms: the xanthine dehydrogenase (XDH) and the xanthine oxidase (XO).…”

“…Therefore, it can be assumed that bacteria residing in xylem neighboring parenchyma, protoxylem, and/or phloem of underground and aboveground organs will deplete the xylem sap of specific plant-produced purine intermediaries to attenuate bacterial stress as result of seasonal drought or salinity-incited N deficiencies. Greater rates of purine scavenging are expected in rhizospheric and endophytic PABs lacking an active ureide metabolism, but harboring the genes related to the purine pathways modulated by riboswitches (Kirchner and Schneider 2017). In general, microbial populations harboring reversible genotypes of amino acid transporter Gap1 genes, flanked by two direct repeats that can lead to GAP1 deletions (Δgap1) and a self-replicating GAP1 circle, have a selective advantage as purine or NH 3 scavengers and thus, higher stress tolerance (Møller et al 2013).…”