How moist heat kills spores of Bacillus subtilis
William H Coleman1, De Chen, Yong-Qing Li
1Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305, USA.
Abstract:
Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but >/=98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.
Insights
Bacillus subtilis spores killed by heat release dipicolinic acid (DPA) only after death. Spore killing is likely due to protein damage, not DPA loss.
Area of Science:
- Microbiology
- Bacterial Spore Research
- Heat Resistance Studies
Background:
- Bacillus subtilis spores are highly resistant to environmental stresses.
- Dipicolinic acid (DPA) is a key component of bacterial spores, contributing to their heat resistance.
- Understanding the mechanisms of spore killing by heat is crucial for sterilization processes.
Purpose of the Study:
- To investigate the relationship between spore killing by moist heat and dipicolinic acid (DPA) release.
- To determine the primary cause of spore death under moist heat treatment.
- To elucidate the role of DPA and protein integrity in spore viability after heat stress.
Main Methods:
- Equilibrium density gradient centrifugation of heat-treated Bacillus subtilis spore populations.
- Analysis of DPA content in different spore fractions.
- Assessment of spore viability, germination, and outgrowth after heat treatment.
Main Results:
- Two distinct spore fractions were observed after heat treatment: DPA-less, less dense spores and DPA-containing, denser spores.
- Heat-killed spores retaining DPA showed compromised outgrowth, suggesting protein damage.
- DPA release was an all-or-nothing event occurring after spore killing.
Conclusions:
- Spore killing by moist heat precedes DPA release.
- Damage to essential spore proteins is the likely cause of spore death.
- DPA release is not the primary event leading to spore inactivation by heat.
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