A regulatory protein that represses sporulation in Clostridioides difficile

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50 to 60% of those bacteria have the ability to produce resilient spores, important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile, and contains several genes of unknown function. We report on the characterization of a signature gene, csiA, which, as we show, is involved in the control of sporulation initiation in C. difficile. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of csiA results in increased sporulation, and increased expression of spo0A per cell. Spo0A also drives transcription of the spoIIA and spoIIG operons, coding for the first forespore-({sigma}F) and mother cell-specific ({sigma}E) RNA polymerase sigma factors. Strikingly, deletion of csiA increases expression of the spoIIG operon, but not that of the spoIIA operon. Increased expression of spoIIG results in increased production and proteolytic activation of pro-{sigma}E, suggesting that normally, the levels of active {sigma}E are limiting for sporulation. While other regulatory proteins affect both sporulation and several processes during the transition phase of growth, including toxin production or motility, deletion of the csiA gene does not alter the expression of the genes coding for the TcdA and TcdB cytotoxins or the genes involved in motility. Thus, our results establish that CsiA acts to modulate sporulation by reducing expression of the spo0A gene.