Description:
<jats:title>Summary</jats:title><jats:p><jats:italic>Clostridium difficile</jats:italic> is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which <jats:italic>C. difficile</jats:italic> initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore‐forming bacteria. In this study, we show that disruption of the gene <jats:italic>CD3668</jats:italic> reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus <jats:italic>rstA</jats:italic>, for <jats:styled-content>r</jats:styled-content>egulator of <jats:styled-content>s</jats:styled-content>porulation and <jats:styled-content>t</jats:styled-content>oxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing <jats:italic>sigD</jats:italic> transcription. Conserved RstA orthologs are present in other pathogenic and industrial <jats:italic>Clostridium</jats:italic> species and may represent a key regulatory protein controlling clostridial sporulation.</jats:p>