Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes.

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Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments.

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2005.02736.x

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

A REVIEW Tailing of Survival Curves of Bacterial Spores

Glutathione and soluble thiol content were examined in a broad spectrum of bacteria. Significant soluble thiol was present in all cases. The thiol compound was glutathione in most of the gram-negative bacteria but not in most of the gram-positive bacteria studied. Glutathione was absent in four anerobes and one microaerophile but was present in a blue-green bacterium. The glutathione content of Escherichia coli increased significantly during transition from exponential to stationary phase.

Occurrence of glutathione in bacteria.

The chemistry and use of lysozyme as a food preservative and a pharmaceutical are reviewed. Lysozyme inhibits the growth of deleterious organisms, thus prolonging shelf life. Chemicals used to improve the preservative effect of lysozyme and those that inhibit the enzyme are discussed, along with the stability of lysozyme in various chemical environments. Lysozyme has been used to preserve fresh fruits and vegetables, tofu bean curd, seafoods, meats and sausages, potato salad, cooked burdock with soy sauce, and varieties of semihard cheeses such as Edam, Gouda, and some Italian cheeses. Lysozyme added to infant‐feeding formulas makes them more closely resemble human milk. Lysozyme has been used clinically in the treatment of periodontitis, administered in chewing gum, and implemented to prevent tooth decay. It has also been administered to patients suffering from cancer for its analgesic effect and has been used as a potentiating agent in antibiotic therapy.

The chemistry of lysozyme and its use as a food preservative and a pharmaceutical.

Sacks, L E (Western Regional Research Laboratory, Albany, California), and Gordon Alderton Behavior of bacterial spores in aqueous polymer two-phase systems J Bacteriol 82:331–341 1961—Free spores of various species of the Bacillaceae enter the upper phase in a two-phase aqueous polymer system containing polyethylene glycol 4000 and potassium phosphate Vegetative cells appear in the lower phase and subcellular debris is concentrated chiefly at the interface The high selectivity of the upper phase for spores makes possible the preparation of greatly enriched concentrates of spores directly from complex natural materials, such as soil and feces Attraction of spores for the upper phase is not diminished by autoclaving and the release of dipicolinic acid Loss of attraction of spores for the upper phase is associated in time with the shedding of the spore coat, upon outgrowth The partition behavior of one preparation of Bacillus megaterium showed the presence of two classes of spores

Behavior of bacterial spores in aqueous polymer two-phase systems.

Base exchange and heat resistance in bacterial spores.

Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is prominently nonlogarithmic for both chemical forms of the spore. Log survival varies approximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, Ea, and Q10 cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed.

Chemical states of bacterial spores: heat resistance and its kinetics at intermediate water activity.

A new spore phenomenon is described in which induction of increased heat resistance occurs during the process of heating. In buffered environments the acid form of dormant mature bacterial spores shows a temperature dependent adaptation to heat which is correlated with the rate of uptake of Ca(OH)(2) by titration at constant p(H) in neutral salt solution.

Heat adaptation and ion exchange in bacillus megaterium spores.

Lysozyme in the recovery medium increased the recovery of heated spores, thereby raising the measured heat resistance of type E Clostridium botulinum spores about 1,800-fold and type A spores up to 3-fold.

Gordon Alderton

Papers

Behavior of bacterial spores in aqueous polymer two-phase systems.

Base exchange and heat resistance in bacterial spores.

Chemical states of bacterial spores: heat resistance and its kinetics at intermediate water activity.

Heat adaptation and ion exchange in bacillus megaterium spores.

Effect of Lysozyme on the Recovery of Heated Clostridium botulinum Spores