The impact of YabG mutations onC. difficilespore germination and processing of spore substrates

Abstract

AbstractYabG is a sporulation-specific protease that is conserved among sporulating bacteria.C. difficileYabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations inyabGwere found which altered the co-germinants required to initiate spore germination. To understand how these mutations in theyabGlocus contribute toC. difficilespore germination, we introduced these mutations into an isogenic background. Spores derived fromC. difficile yabGC207A(catalytically inactive),C. difficile yabGA46D,C. difficile yabGG37E,andC. difficile yabGP153Lstrains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated withE. colilysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46Dshowed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes inyabGexpression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.