Strains and plasmids used in this study.

<div><p><i>Clostridioides difficile</i> is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that <i>C</i>. <i>difficile</i> SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that <i>C</i>. <i>difficile sspA</i> and <i>sspB</i> are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of <i>C</i>. <i>difficile</i> SASP mutants. Many of these strains contained mutations in <i>CDR20291_0714</i> (<i>spoIVB2</i>) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression.</p></div>