<i>In Vivo</i> Behavior of the Tandem Glycine Riboswitch in <i>Bacillus subtilis</i>

Abstract

In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous <i>in vitro</i> studies, the <i>in vivo</i> behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in <i>Bacillus subtilis</i> using β-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the <i>B. subtilis</i> genome. Our results indicate that glycine does not need to bind both aptamers for regulation <i>in vivo</i> and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in <i>B. subtilis</i>, the glycine riboswitch-regulated <i>gcvT</i> operon is important for glycine detoxification.<b>IMPORTANCE</b> The glycine riboswitch is a unique <i>cis</i>-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many <i>in vitro</i> experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch <i>in vivo</i> In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the <i>Bacillus subtilis</i> genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function <i>in vivo</i> and reinforces the potential of riboswitches as novel antimicrobial targets.