The Clostridia are anaerobic, Gram-positive bacteria that sporulate when stressed and produce type-IV-pili that play a key role in biofilm development.
Spores allow bacteria to survive extreme conditions and play a key role in human infection. In this work, we investigated the dynamics of spore germination in Clostridium sporogenes, a non-pathogenic
model of the botulism-causing species Clostridium botulinum (Group I). Using a combination of maleimide-based dyes, time-lapse fluorescent/phase microscopy in anaerobic conditions, and automated image analysis, we resolved the phenotypes of mutants lacking key spore proteins by growing them in co-culture with wild-type cells. We found that spores lacking CsxB, a protein thought to be associated with the exosporium (the outermost layer of the spore), exhibited a transient stall during rehydration, suggesting that CsxB regulates the entry of water into the spore core. Furthermore, we found that in mutants lacking CsxC, which forms a layered structure inside the exosporium, the spore body tends to reside closer to one end of the exosporium compared to wild-type cells. Importantly, this asymmetry plays a key role in germination of vegetative cells, which preferentially emerge from the distal pole of the exosporium in all the strains we tested. These findings provide new insights into regulatory and morphological processes that control spore germination in Clostridia.
We also investigated pili-based motility in Clostridioides difficile, a human pathogen that causes diarrhea and life threatening colitis. On agar surfaces, C. difficile colonies form branch-like structures on their periphery that previous studies have indicated are driven by the extension and retraction of pili. In this work, we sought to directly visualize pili-based movement of both solitary C. difficile cells and those in densely packed colonies using high-resolution, time lapse microscopy.
As many species of Clostridia are pathogenic, elucidating the fundamental processes that underlie spore germination and colonization is critical for developing new treatment strategies.
