cpr0186 contributes to promoting sporulation in Clostridium perfringens SM101

Although the precise mechanism underlying activation of Spo0A needs to be clarified to fully understand C. perfringens sporulation, the molecules directly interacting with Spo0A have not been fully elucidated. This study aimed to identify proteins interacting with Spo0A during C. perfringens sporulation by a yeast two-hybrid (Y2H) assay.

Prey libraries constructed using the genomes of C. perfringens SM101 and str. 13 and a bait plasmid encoding the receiver domain of Spo0A in C. perfringens SM101 were used for Y2H assay. The deletion mutant and its complemented strain were constructed by homologous recombination. Phenotypic analyses included measurements of bacterial growth, sporulation efficiency, cell morphology, mRNA expression of sporulation-related genes, and the amount of phosphorylated Spo0A. Potential protein interactions were evaluated using AlphaFold 3.

Y2H screening identified CPR0186, a hypothetical protein in C. perfringens SM101, as a Spo0A-interacting candidate, and AlphaFold 3 modeling supported the possibility of the direct interaction with the receiver domain of Spo0A. The cpr0186-null mutant exhibited growth comparable to that of the wild-type strain, but showed significantly reduced sporulation. The mutant also showed significantly decreased mRNA expression of spo0A, sigF, sigE, sigG, and sigK. No apparent difference in the ratio of Spo0A∼P to unphosphorylated Spo0A was observed among the strains under the conditions tested.

cpr0186 promotes sporulation in C. perfringens SM101 and is suggested to contribute to the expression of Spo0A-regulated genes. CPR0186 may interact directly with Spo0A although the functional significance of this interaction remains to be determined.