Characterization of a hypersporulating strain derivative of Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive and obligate anaerobe pathogen capable of forming spores, which are essential for the transmission and recurrence of C. difficile infections. A major challenge in sporulation studies in C. difficile is the low yield and asynchronous nature of this process. In this work, a hypersporulating strain, derivative of R20291, with an early sporulation onset and enhanced sporulation efficiency was isolated by serendipity. It showed up to a 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs reveal that spores of both strains have similar hair-like projections, electron-dense outer exosporium layer features. Whole genome sequencing and genomic analyses revealed that the hypersporulating strain had a deletion of 2356 bp spanning three ORF, including a non-essential proC1 involved in proline metabolism, and a missense mutation in rsbV, an anti-anti-SigB factor of RsbW. These observations suggest that RsbV-variant might contribute to constitutive repression of the SigB-dependent general stress response, and therefore, derepressing sporulation.