Abstract
The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.
Highlights
Clostridium sporogenes is a Gram-positive, spore-forming, anaerobic bacterium and a significant agent of food spoilage, unlike its close relative C. botulinum, C. sporogenes does not produce the neurotoxins responsible for botulism, a severe and fatal neuro-paralytic disease of humans and animals (Brown et al, 2012; Taylor et al, 2013)
Spore germination is essential for C. botulinum spores to cause botulism, and bioinformatics analyses strongly suggest that C. sporogenes spore germination is very similar to that of spores of Group I C. botulinum
Laser tweezers Raman spectroscopy showed that the bands from Ca-dipicolinic acid (CaDPA) dominate the Raman spectra of individual C. sporogenes spores (Figure 1), just as with spores of Bacillus species, C. perfringens and C. difficile, as spores without CaDPA lack significant bands at 1017, 1395, and 1572 cm−1 (Kong et al, 2011; Wang et al, 2011, 2015c)
Summary
Clostridium sporogenes is a Gram-positive, spore-forming, anaerobic bacterium and a significant agent of food spoilage, unlike its close relative C. botulinum, C. sporogenes does not produce the neurotoxins responsible for botulism, a severe and fatal neuro-paralytic disease of humans and animals (Brown et al, 2012; Taylor et al, 2013). C. sporogenes spores are being investigated as vectors to deliver cancer-treating drugs to patients with tumor cells, where a nominal oxygen concentration enables spores to germinate, outgrow and locally produce the cancer drug (Nuyts et al, 2002). Understanding mechanisms of C. sporogenes spore germination may have practical applications in the management of Clostridium contamination and lead to the development of new drug vectors. This knowledge may lead to new methods for preventing spore germination and thereby subsequent growth, or efficiently promoting spore germination to facilitate inactivation of emergent sensitive vegetative cells or activation of desired drugs under appropriate conditions
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