The Pxp Complex Detoxifies 5‐Oxoproline and Promotes the Growth of <i>Clostridioides difficile</i>

Clostridioides difficile is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For C. difficile and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In Bacillus subtilis, one such mechanism for preventing sporulation is the prokaryotic 5-oxoprolinase, PxpB (KipI), which impedes the activation of the main sporulation kinase. In addition, PxpB functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid and its derivatives. In this study, we investigate the orthologous Pxp proteins in C. difficile to determine their roles in the regulation of sporulation and metabolism. Through deletion of the pxpAGBC operon, we show that, unlike in B. subtilis, the Pxp (Kip) proteins have no significant impact on sporulation. However, we found that the pxp operon encodes a functional oxoprolinase that facilitates detoxification of OP. Furthermore, our data demonstrate that PxpAGBC not only detoxifies OP but also allows OP to be used as a nutrient source that supports the growth of C. difficile, thereby facilitating the conversion of a toxic by-product of metabolism into an energy source.