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bioRxiv · 10.1101/2025.11.25.690273

Characterization of a hypersporulating strain derivative of Clostridioides difficile R20291

Cid-Rojas, F.·Paredes-Sabja, D.
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Abstract

Clostridioides difficile is a Gram-positive and obligate anaerobic spore-former pathogen. C. difficile spores are essential for the transmission and recurrence of C. difficile infections (CDI). 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. This strain had 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs revealed 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 2356 bp-deletion 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 this RsbV-variant might contribute to constitutive repression of the SigB-dependent general stress response, and therefore, derepressing sporulation in this hypersporulating strain.

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Cid-Rojas, F., Paredes-Sabja, D.. 2025-11-25. Characterization of a hypersporulating strain derivative of Clostridioides difficile R20291. https://doi.org/10.1101/2025.11.25.690273

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