Investigating the “Outside In” Mechanism of Germination in Clostridia

The vegetative cells of Gram-positive, endospore-forming bacteria form dipicolinic acid (DPA)-rich, metabolically dormant spores upon exposure to nutrient or environmental stressors. These spores identify environmental signals (germinants) using germinant receptor proteins in the spore to return to the vegetative state. For the pathogenic bacterium Clostridioides difficile, the germination process is an attractive target for the rational design of novel therapeutics. Prior research on germination by C. difficile spores has shown that there are significant differences, when compared to what is observed in the model organism, Bacillus subtilis, in the germinant receptors used to initiate the germination process. Moreover, there are significant differences in the order of events that occur post-germinant recognition. Here, I describe a newly identified function for the C. difficile spore bile acid germinant receptor, CspC. Using semi-quantitative western blot analyses on several C. difficile strains, I show that this receptor likely has an inhibitor role for C. difficile spore germination. Building upon this work, I also demonstrate that the mechanism of germination by C. difficile spores is conserved in another organism, Paraclostridium bifermentans. Finally, I developed a reliable and reproducible protocol for transformation C. difficile R20291 using electroporation. This protocol also works in another commonly utilized strain, C. difficile CD630