Kansas State University Undergraduate Research Conference

DETERMINING THE ROLE OF CDR20291_0493 SPORULATION INITIATION IN CLOSTRIDIUM DIFFICILE

Our lab focuses on the gene regulatory networks of Clostridium difficilesporulation and toxins virulence factors. Spores are the major reason of the disease transmission; this is why it is important to understand how this spores are formed so drugs can be targeted to formation. Our objective is to identify the gene regulatory networks that control sporulation. The candidate target gene we are studying is CDR20291_0493 in C. difficileR20291 strain. We want to understand the role of this gene in sporulation initiation of C.difficile. We first created a mutant of CDR20291_0493 mutant R20291Dto look for sporulation phenotype and we found that the mutant was producing less spores compared to the parent R20291 strain. However, to confirm this phenotype was specific to CDR20291_0493, a complementation was performed, which means giving back CDR20291_0493 to mutant. After the complementation more sporulation was expected however, sporulation did not come back. So now, our question was why sporulation did not come back? It was predicted that CDR20291_0493 and the gene downstream of it CDR20291_0494 are part of an operon, and sporulation is associated to CDR20291_0494. So now we are trying to compliment both CDR20291_0493 and CDR20291_0494 by cloning them in a same vector. Our expectation is that complementation of CDR20291_0493 mutant with both CDR20291_0493 and CDR20291_0494 will rescue the sporulation phenotype.This would confirm our two predictions about them being in an operon and both CDR20291_0493and CDR20291_0494 having a role in sporulation initiation inC.difficile.