Detecting Cortex Fragments During Bacterial Spore Germination

Abstract

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model sporeforming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination. Video LinkThe video component of this article can be found at

Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism

“…To detect these fragments, we made use of a colorimetric assay that we, and others, have used to quantify the presence of reducing sugars (27, 28, 30, 40). This assay detects the presence N -acetylglucosamine (NAG) and N -acetylmuramic acid residues (reducing sugars) released during the degradation of cortex peptidoglycan (40). Purified spores were germinated in the presence of TA and glycine and were sampled every 2 min for the presence of both cortex fragments and DPA (Fig.…”

“…3 and 4). By using an assay that detects the presence of reducing sugars (i.e., cortex fragments) (40), we were able to compare when the cortex was degraded relative to DPA release under conditions where the osmotic strength of the medium was increased. Under these conditions, DPA release was delayed though cortex degradation was unaffected (Fig.…”

“…Cortex fragments were detected according to a method previously reported (30, 40). Briefly, C. difficile spores were heat activated, as described above, and stored on ice until use.…”

Imaging Clostridioides difficile Spore Germination and Germination Proteins

“…In prior work, we found that DPA release is a quick event, with the majority of DPA being released within 20 minutes of germination initiation (10,12,16). Because the DPA release from the spore core is dependent on the activation of the SpoVAC mechanosensing protein embedded in the inner spore membrane (16), the cortex degradation must be a quick event; indeed the cortex fragments are quickly detected after initiation of germination (58). In order to observe the structural changes that occur in the germinating spore (from the early stage of germination to the development of vegetative cell), we induced germination in spores derived from the wild-type C. difficile R20291 strain by incubating them with taurocholate (TA) and glycine in BHIS liquid medium.…”

The selenophosphate synthetase,selD, is important forClostridioides difficilephysiology

“…Spores were purified from C. difficile R20291, KNM6, and KNM9 strains as previously described (46, 50, 51). Briefly, spores were streaked onto BHIS agar medium (20 – 30 plates) and allowed to sporulate for 5 to 6 days before scraping each into microcentrifuge tubes containing 1 mL of sterile dH 2 O and kept at 4 °C overnight.…”