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Microbiology and Molecular Biology Reviews, September 2000, p. 548-572, Vol. 64, No. 3
1092-2172/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments

Wayne L. Nicholson,1,* Nobuo Munakata,2 Gerda Horneck,3 Henry J. Melosh,4 and Peter Setlow5

Department of Veterinary Science and Microbiology1 and Lunar and Planetary Laboratory,4 University of Arizona, Tucson, Arizona 85721; Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan 104-00452; Radiobiology Section, DLR, Institute of Aerospace Medicine, Cologne, Germany3; and Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 060325

SUMMARY
INTRODUCTION
SPORE RESISTANCE IN THE LABORATORY
    Parameters Contributing to Spore Resistance
        Genetic makeup of the sporulating species.
        Sporulation conditions.
        Spore coats.
        Core permeability.
        Core water content.
        Spore mineral content.
        alpha /beta -Type SASP.
        Repair of damage to macromolecules.
    Laboratory Spore Resistance Models
        Heat resistance.
        (i) Wet-heat resistance.
        (ii) Dry-heat resistance.
        Desiccation resistance.
        Chemical resistance.
        UV radiation resistance.
        gamma -Radiation resistance.
        Resistance to ultrahigh hydrostatic pressure.
SPORE DNA REPAIR MECHANISMS
    General DNA Repair Systems
        NER.
        Recombination-mediated repair.
        Other general repair systems.
    SP Lyase, an SP-Specific DNA Repair System
        Regulation of SP lyase expression during sporulation.
        Genetics and biochemistry of SP lyase.
SPORE RESISTANCE TO EXTREME TERRESTRIAL ENVIRONMENTS
    Differences between the Laboratory and the Environment
        Source of spores.
        Growth, sporulation, dormancy, and germination conditions.
    Solar Radiation as Primary Source
        Direct solar effects.
        Indirect solar effects.
    Protection of Spores from Lethal Solar UV Damage
    DNA Photochemistry of Spores Exposed to Sunlight
    B. subtilis Spores as Biological Solar Dosimeters
    Monitoring the Ozone Layer with Spore Dosimeters
SPORE RESISTANCE TO EXTRATERRESTRIAL ENVIRONMENTS
    Space
        Vacuum.
        Radiation.
        Temperature.
    Interplanetary Transfer of Spores
    Launching Spores into Space
        Heat.
        Pressure.
        Acceleration.
    Interplanetary Travel of Spores
        Effects of space vacuum.
        Effects of extraterrestrial solar UV radiation.
        Effects of cosmic ionizing radiation.
        Combined effects of space.
        Time scales of interplanetary or interstellar transport of life.
    Deposition of Spores from Space to a Planet
    Outlook for the Future
ACKNOWLEDGMENTS
REFERENCES


SUMMARY
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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.


INTRODUCTION
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In the latter half of the 19th century Tyndall, Cohn, and Koch (25, 84, 211a) independently discovered that certain species of bacteria spend at least part of their lives as dormant cellular structures now known as endospores (hereafter called simply spores for convenience). Spores have since been recognized as the hardiest known form of life on Earth, and considerable effort has been invested in understanding the molecular mechanisms responsible for the almost unbelievable resistance of spores to environments which exist at (and beyond) the physical extremes which can support terrestrial life. Examples of sporeforming bacteria are rather widespread within the low-G+C subdivision of the gram-positive bacteria and represent inhabitants of diverse habitats, such as aerobic heterotrophs (Bacillus and Sporosarcina spp.), halophiles (Sporosarcina halophila and the gram-negative Sporohalobacter spp.), microaerophilic lactate fermenters (Sporolactobacillus spp.), anaerobes (Clostridium and Anaerobacter spp.), sufate reducers (Desulfotomaculum spp.), and even phototrophs (Heliobacterium and Heliophilum spp.).

Despite the diversity exhibited by sporeforming bacterial species, the sporeformers about which we have gleaned the most detailed molecular information are common rod-shaped soil inhabitants belonging to the genera Bacillus and Clostridium, and among this restricted subset, most work has concentrated upon the descendants of a single strain of Bacillus subtilis called strain 168. Consequently, most of this review will concentrate upon spore resistance in B. subtilis 168 and its close relatives, from which we have gained several valuable (and hopefully universal) insights into spore resistance mechanisms. However, we can easily imagine that the spore resistance mechanisms uncovered through study of B. subtilis and closely related species may not be entirely applicable to sporeformers as phylogenetically and ecologically diverse as the gram-negative homoacetogen Sporomusa or to bacteria which do not form true endospores but form aerial spore-bearing mycelia (such as Streptomyces spp.) or fruiting structures (such as Myxobacter and Myxococcus spp.). This caveat has been most eloquently expressed by Slepecky and Leadbetter (200).

According to our current understanding, the developmental pathway leading from a vegetatively growing bacterial cell to a spore is triggered by depletion from the bacterium's local environment of a readily metabolized form of carbon, nitrogen, or phosphate. (For recent reviews of the molecular details of this surprisingly complex and fascinating differentiation process, see references 38, 46, 57, 65, 150, 190, 203, and 206.) In the dormant state, spores undergo no detectable metabolism and exhibit a higher degree of resistance to inactivation by various physical insults, including (but not limited to) wet and dry heat, UV and gamma radiation, extreme desiccation (including vacuum), and oxidizing agents. Despite their metabolic inactivity, however, spores are still capable of continually monitoring the nutritional status of their surroundings, and they respond rapidly to the presence of appropriate nutrients by germinating and resuming vegetative growth. Spore formation thus represents a strategy by which the bacterial cell escapes temporally from nutritionally unfavorable local conditions via dormancy. In addition to temporal escape, spores can also be relocated spatially via wind, water, living hosts, etc., to environments potentially favorable for germination and resumption of vegetative growth. As a result, bacterial spores can be found in environmental samples obtained from all parts of the Earth, both above and below the surface, and as such represent a highly successful strategy for the survival and widespread dispersal of microbial life.

Dormant spores exhibit incredible longevity and can be found in virtually every type of environment on Earth, even in geographical locations obviously removed spatially from their point of origin (for example, spores of strictly thermophilic Bacillus spp. can be isolated from cold lake sediments) (155, 156). Reliable reports exist of the recovery and revival of spores from environmental samples as old as 105 years (54, 81, 154), and there recently appeared a somewhat more controversial report that viable Bacillus sphaericus spores were recovered from the gut of a bee fossilized in Dominican amber for an estimated 25 to 40 million years (20)!

It becomes apparent from studying the process of spore formation, the ubiquitous global distribution of spores, and the environmental record of spore longevity that a sporulating bacterium cannot predict beforehand how long or in what environment it will spend its dormant state. Therefore the sporulating cell must "prepare for the worst" each time it undergoes differentiation. How does the spore achieve such hardiness? The molecular mechanisms underlying spore resistance properties were until recently relatively refractory to experimental dissection. However, as the result of decades of elegant genetic, molecular biological, and biochemical studies, molecular models have emerged which describe how spores of bacteria such as B. subtilis resist exposure to germicidal agents such as heat, UV, and oxidative damage in the laboratory (reviewed in references 52 and 190). From these laboratory studies it is clear that spore resistance mechanisms during dormancy rely on diverse physiological events which occur during all stages of the life cycle: growth, sporulation, and germination. However, in spite of the vast amount of data that has been collected in the laboratory, we have very little idea of the possible degree to which our experimental models accurately reflect the life history of sporeforming bacteria in their native habitats. In order to maintain the potential for viability, the dormant spore must either (i) prevent damage which would inactivate critical cellular components needed for successful germination and resumption of growth or (ii) repair or replace those damaged critical components during germination, before their inactivation results in cell death. In the terrestrial (soil) environment where many sporeforming bacteria are found, such potentially lethal extreme conditions can include cycles of heat and cold, including freezing-thawing, physical abrasion, extreme dessiccation, exposure to corrosive chemicals, attack by other organisms and their extracellular degradative enzymes, and prolonged exposure to solar radiation (133).

Because of their notorious resistance and longevity, bacterial spores have also been studied as possible candidates for transfer of life between the planets as a result of impact processes (66, 107). The natural processes by which spores could be transported through interplanetary space would expose them to an entirely new set of environmental stresses, including extreme shock, acceleration, vacuum, and bombardment by UV and ionizing radiation as well as heavy high-energy atomic (HZE) particles (109). In this review, we will attempt for the first time to summarize recent advances in our understanding of how bacterial spores resist inactivation by stresses imposed by both terrestrial and extraterrestrial environments and to explore how the current laboratory models of spore resistance can serve as important intellectual scaffolds from which we can construct new environmental spore resistance models.


SPORE RESISTANCE IN THE LABORATORY
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Studies of the resistance of Bacillus and Clostridium spores to a variety of treatments in the laboratory have identified a number of factors important in determining the level of spore resistance. These factors include the genetic makeup of the sporulating species, the precise sporulation conditions, particularly the temperature, the spore coats, the relative impermeability of the spore core, the low water content of the hydrated spore's core, the high level of minerals in the spore core, the saturation of spore DNA with alpha /beta -type small, acid-soluble proteins (SASP), and repair of damage to macromolecules during spore germination and outgrowth. While all of these factors are important in at least one or more spore resistance property, their relative importance varies considerably both for the same resistance property in spores of different species and for different resistance properties within the same species (Table 1).

                              
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TABLE 1.   Role of various factors in resistance of spores of Bacillus species to different treatmentsa

Parameters Contributing to Spore Resistance

Genetic makeup of the sporulating species. While mutations in a number of individual genes alter specific factors involved in spore resistance (181, 190, 191), it is also clear that in wild-type organisms the overall genomic information is extremely important in determining levels of spore resistance (52). This is seen most notably with wet heat resistance, as spores of thermophiles are more resistant than spores of mesophiles, which in turn are more resistant than spores of psychrophiles (52). At least a part of the increased wet-heat resistance of spores of thermophiles is due to their decreased core water content (see below). However, this does not explain fully these spores' increased wet-heat resistance, which may be due simply to the generally increased thermostability of the proteins of thermophiles.

Sporulation conditions. Many studies have shown that within a single species, modulation of the sporulation conditions has a significant effect on spore resistance (26, 52, 169). Parameters that have been varied include metal ion concentrations and temperature, and most often only spore wet-heat resistance has been analyzed. In general, the interpretation of changes in spore resistance with variation of the mineral ion content of sporulation media has been difficult on a mechanistic basis. However, sporulation at an elevated temperature invariably results in spores with increased heat resistance. This effect is mediated at least in part by a decrease in core water content in spores prepared at higher temperature. However, the mechanism(s) controlling spore core water content is not known.

Spore coats. The spore coats appear to play some role in spore resistance, especially in preventing the access of peptidoglycan-lytic enzymes to the spore cortex, and also likely plays a role in spore resistance to some chemicals, such as hydrogen peroxide (38, 102, 165, 169, 191). A B. subtilis mutant completely lacking the spore coat layers has been constructed (38). Spores of this mutant are highly sensitive to lysozyme and 5% H2O2 but exhibit normal resistance to wet heat and 254-nm UV light (165), indicating that resistance to wet heat and laboratory-generated 254-nm UV-C are probably not functions of the spore coat. However, the precise role(s) of individual coat proteins in these resistance properties is not clear.

Core permeability. The spore core exhibits relatively low permeability to hydrophilic molecules greater than approximately 200 Da (53). Since the spore has two membranes, either or both could be the permeability barrier restricting entry into the spore core. Although it is not clear if the outer membrane is an intact membrane in the mature dormant spore, some data are consistent with its being a functional membrane. The inner membrane is clearly an intact membrane, and while the reason(s) for its decreased permeability is not clear, it is significantly compressed in the dormant spore (205).

Core water content. While the water content of the cortex, coat, and exosporium regions of a spore suspended in water is similar to that in growing cells (75 to 80% of wet weight), the water content of the spore core is much lower (28 to 50% of wet weight) (52). While the precise mechanism by which the core's water content is reduced during sporulation is not clear, this event does involve the function of the spore cortex (4, 39, 52, 151, 152, 191). There is abundant evidence that core water content is inversely related to spore wet-heat resistance (52).

Spore mineral content. Spores have very high levels of divalent ions, in particular Ca2+, with the great majority of these cations being present in the spore core (128, 189). Both the amounts and identities of the major cations in spores can be varied, either by alterations in the metal ion content of sporulation medium (199) or by removal of spore metal ions by titration to low pH and then back-titration to pH 7 with appropriate metal ion hydroxide (12). Using these procedures, spores with extremely low divalent cation levels can be generated (H+, Na+, or K+ spores), as can spores with high levels of any of a variety of divalent cations (Ca2+, Mg2+, and Mn2+). Upon analyses of spore wet-heat resistance, Ca2+ spores are the most resistant, with resistance similar to that of native spores, while H+ spores are the least resistant; in general, divalent cation-loaded spores are more resistant than monovalent cation-loaded spores (12, 52, 98, 99). While studies are not as extensive, increased spore core mineralization is also associated with increased resistance to oxidizing agents (98) and, at least in spores of Bacillus stearothermophilus, with increased resistance to dry heat (1). Not all the reasons for the effects of spore core mineral content on spore resistance are known. However, increased core mineralization is often associated with decreased core water content, and this may contribute to increased spore resistance to wet heat.

In contrast to spore core minerals, which play a clear role in spore resistance, the role of dipicolinic acid (DPA), with which much of the spore's divalent cations are likely chelated, in spore resistance is less clear. Studies in B. subtilis have shown that spores lacking DPA due to a specific mutation in the spoVFA or spoVFB locus (also called dpaA and dpaB), which encode the two subunits of DPA synthetase (33), have significantly increased spore core water and decreased heat and H2O2 resistance (7; M. Paidhungat, B. Setlow, and P. Setlow, unpublished).

However, these DPA-less spores exhibit no decrease in UV resistance and are actually more UV resistant than spores of their wild-type parents (Paidhungat et al., unpublished). This finding is not unexpected, as DPA has been shown to be a photosensitizer in spores (190, 191). Spores of Bacillus cereus that lack DPA have also been isolated, although the specific mutation causing loss of DPA has not been identified; as expected, these DPA-less spores are heat sensitive (60, 231). However, secondary mutations which restored much of the spore's heat resistance but which did not restore DPA production were identified in the DPA-less strain (60), suggesting that DPA is not essential for full spore heat resistance. Unfortunately, the elevated heat resistance phenotype of these DPA-less B. cereus spores was extremely unstable, and these strains have never been studied further. A B. subtilis mutant that produces DPA-less spores that retain heat resistance has also been isolated (231). However, the identity of the mutation(s) generating spores with the DPA-less yet heat-resistant phenotype has not been determined, and again these mutant spores have not been studied further.

alpha /beta -Type SASP. Spore DNA is saturated with a group of unique proteins called alpha /beta -type SASP (184, 186, 189-191). These proteins are synthesized only during sporulation in the developing spore and are degraded beginning early in spore germination. These proteins bind to DNA largely on the outside of the DNA helix and straighten and stiffen the DNA while changing the DNA to an A-like helix. DNA properties in vitro are also dramatically changed when DNA is bound by alpha /beta -type SASP, and the DNA's reactivity with a variety of chemicals decreases dramatically. The properties of a DNA-alpha /beta -type SASP complex in vitro appear to be duplicated in vivo, so much so that DNA does not appear to be the target for spore killing by wet heat or a number of potentially genotoxic or mutagenic chemicals. However, spore killing by dry heat and radiation occurs in large part (and possibly completely) through DNA damage, and any deficiency in alpha /beta -type SASP in the spore results in spores that are more sensitive to a variety of treatments than are the corresponding wild-type parental spores and killed in large part by damage to DNA, even by treatments (e.g., wet heat) that do not kill wild-type spores by DNA damage.

Repair of damage to macromolecules. As noted above, spores are killed by some treatments at least in part by damage to DNA. Consequently, it is not surprising that DNA repair during spore germination and outgrowth plays a role in resistance of spores to these treatments (133, 190, 191). Spores appear to contain at least some of the enzymes found in growing cells for repair of DNA damage, and DNA damage accumulated in the dormant spore will also often induce synthesis of DNA repair proteins upon subsequent spore germination (182). In addition, at least one protein is uniquely present in spores which is dedicated to the repair during germination and outgrowth of the major lesion caused by UV irradiation of spores, the thyminyl-thymine adduct termed the spore photoproduct (SP) (45, 185, 191). Not surprisingly, spores of species with mutations eliminating particular DNA repair pathways are invariably more sensitive to killing by agents which generate DNA damage repaired by the eliminated pathway than are spores of their wild-type parents (182, 185).

Since some of the treatments used to kill spores (e.g., wet heat and oxidizing agents) can damage proteins as well as DNA, it is possible that repair of protein damage during spore germination and outgrowth might also be important in determining spore survival after various treatments. Enzymes that might be involved in "repairing" protein damage include aspartate-O-methyltransferase, methionine sulfoxide reductase, and various heat shock proteins. However, aspartate-O-methyltransferase appears to be absent from at least B. subtilis (63), and methionine sulfoxide reductase plays no role in spore resistance to wet heat or oxidizing agents (62). Many workers have noted that spore recovery after a killing treatment (usually wet heat) is often much greater on rich media than on poor media, and this difference has been ascribed to the need for some type of protein "repair" in order for spore outgrowth and eventual colony formation on a poor medium (168, 169). Indeed, the heat shock proteins, which play a major role in the resistance of growing cells to heat stress (64a), have been suggested to play a role in spore heat resistance (174), but more recent work has indicated that this is not the case (112a; E. Melly and P. Setlow, unpublished).

Laboratory Spore Resistance Models

There have been an enormous number of studies of resistance of spores of Bacillus and Clostridium spp. under many different conditions. However, many fewer studies have analyzed the effect of one or more key variables on spore heat resistance. The following discussion will focus on these studies, as these have given us the best insight into specific factors contributing to particular spore resistance properties. As mentioned above, many of these studies have been carried out with spores of B. subtilis, as the ease of genetic manipulation as well as the availability of the complete genomic sequence (86) have greatly facilitated studies with this organism. However, analyses of spores of other species have also provided valuable information. Several general conclusions that have come from these studies are that the causes of spore resistance are multifactorial and that the importance of these multiple factors varies both between species and for different resistance properties. The current state of our understanding of the importance of various factors in spore resistance to different treatments, primarily in B. subtilis, is summarized in Table 1.

Heat resistance. The hallmark property of bacterial spores is their remarkable resistance to heat. B. subtilis spores can survive moist heat (100°C at atmospheric pressure) with a D value (decimal reduction time, the time required to lower viability by a factor of 10) of 20 to 30 min (47) (Fig. 1). Moreover, spores survive approximately 1,000-fold longer in dry heat than in moist heat (47) (Fig. 1).


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FIG. 1.   Survival times (D values; decimal reduction time) for spores of B. subtilis strain 5230 exposed to wet and dry heat. (Data are modified from reference 47.)

(i) Wet-heat resistance. Wet-heat resistance is one of the most striking properties of spores of Bacillus and Clostridium species, as these spores require incubation at temperatures 30 to 40°C higher to achieve inactivation equivalent to that of growing cells of the same organisms (52). The target for spore killing by wet heat is not clear but is almost certainly not spore DNA (190, 191), and there is evidence that the target is a spore protein (11, 52, 191). However, the identity of this protein(s) is by no means clear. Multiple factors cause spore resistance to wet heat, with at least four factors identified to date, including sporulation temperature, protection of spore DNA by alpha /beta -type SASP, spore core mineralization, and spore core dehydration (52, 191). However, the proteins of the heat shock response, which can play a major role in protecting growing cells of Bacillus species from heat stress (64a), play no significant role in spore heat resistance (112a; Melly and Setlow, unpublished).

Sporulation will take place at temperatures above those optimum for growth, although for an individual strain the maximum temperature at which sporulation will take place is generally a few degrees below the maximum temperature for growth. A number of studies have shown that both within and across species, spores prepared at higher temperatures are generally more heat resistant than spores prepared at lower temperatures (26, 52). A major reason for this effect is that in general spore core water content goes down as the sporulation temperature increases, and as discussed below, there is a good inverse correlation between core dehydration and spore wet-heat resistance. However, it also seems likely that other factors are important in this effect, in particular the greater heat stability of proteins of thermophilic spore formers than of those of mesophiles.

As noted above, spore killing by wet heat does not occur through DNA damage, such as depurination, that might be expected at elevated temperatures. Indeed, spore DNA is well protected against DNA damage caused by wet heat, including depurination, by the saturation of the spore DNA with alpha /beta -type SASP (180, 191). However, in mutant B. subtilis strains lacking the majority of alpha /beta -type SASP due to deletion of appropriate coding genes, the resultant alpha -beta - spores are significantly more sensitive to wet heat and are killed in large part (if not completely) by DNA damage, including abasic sites presumably generated as a result of depurination (180, 191).

As noted above, the spore core contains an extremely high level of divalent mineral ions, predominantly Ca2+, Mg2+, and, to a lesser extent, Mn2+ (128). While the majority (>= 75%) of these ions are associated with the spore's depot of DPA, some are also associated with other core anions. In general, the higher the level of core mineral ions, the more wet-heat resistant are the spores (52). This effect appears to be due in part to a decrease in core water with increasing core mineralization, but core minerals are also likely to have more specific effects on protein stability. The ability to remove core minerals by exchange with H+ and then back-titration with metal ion hydroxides has also allowed demonstration that the identity of the core mineral ion influences spore wet-heat resistance, as Ca2+ and Mg2+ spores are most resistant, with K+ and Na+ spores being less resistant and H+ spores being the least resistant (12, 52). However, the precise reason for these effects is not at present clear.

While the factors cited above have significant effects on spore wet-heat resistance, several of these factors exert their effects indirectly through modulation of spore core water content. This is clearly the major factor determining spore wet-heat resistance, as over a rather wide range of core water contents in spores of different species, there is a good inverse correlation between spore wet-heat resistance and core water content (10, 52). However, it is not precisely clear how a lower core water content results in increased spore wet-heat resistance. It is thought that reduced core water reduces the amount of water associated with spore proteins, thus stabilizing them to thermal denaturation. Unfortunately, there are no good data on the precise level of free water in the spore core, as this knowledge would allow detailed examination of the effects of this level of free water on protein heat resistance in vitro. In addition, although it is clear that achievement of a reduced spore core water content requires the action of the developing spore's peptidoglycan cortex, exactly how this structure functions in modulating spore core water content is not clear (39).

(ii) Dry-heat resistance. In contrast to the situation with wet heat, the killing of spores by dry heat does appear to proceed in large part via DNA damage, as spores exposed to dry heat acquire both DNA damage and mutations (140, 181, 231). In addition, spores of DNA repair mutants are more sensitive to dry heat than are spores of their wild-type parents, and during germination of dry-heat-treated spores, genes encoding DNA repair proteins are greatly induced (182). Consequently, DNA repair capacity is an important parameter in determining spore dry-heat resistance. Again in contrast to the situation with spore wet-heat resistance, spores of thermophiles do not have higher resistance to dry heat than spores of mesophiles (1). In addition to DNA repair capacity, two other factors have been identified which affect spore dry heat resistance, spore core mineralization and DNA protection by alpha /beta -type SASP.

Although there have been studies with spores of only a few species, in at least one species (B. stearothermophilus), variations in spore core mineralization affect spore resistance to dry heat, with demineralized spores having significantly lower resistance (1). However, the mechanism of this effect is not understood, and it has not been seen with spores of all species examined.

As noted above, spore killing by dry heat is accompanied by DNA damage which is likely to be due to base loss, largely through depurination (181). B. subtilis spores lacking alpha /beta -type SASP are much less resistant than wild-type spores to dry heat, and killing of alpha -beta - spores is also accompanied by DNA damage (181). Since alpha /beta -type SASP protect DNA in vitro against depurination caused by dry heat, these data suggest that alpha /beta -type SASP binding to DNA in spores is a major factor in spore dry-heat resistance.

Desiccation resistance. Spores are clearly much more resistant than their growing counterparts to extended desiccation and multiple cycles of freeze-drying with freezing at -78°C. When typical laboratory vacuum systems are used for desiccation or freeze-drying, wild-type spores often exhibit no detectable killing after extended desiccation or multiple cycles of freeze-drying and rehydration (56, 167, 191). A major reason for spore resistance to these processes is protection of spore DNA by alpha /beta -type SASP, as alpha -beta - spores are much more sensitive to freeze-drying, and possibly extended desiccation, and killing by these processes is accompanied by DNA damage (190, 191). It has been reported that extreme desiccation (10-6 Pa at 77 K for 24 h) resulted in complete killing of Escherichia coli and Halobacterium halobium cells, but spores of Clostridium mangenoti and B. subtilis survived to 55 and 75%, respectively (85). Under prolonged desiccation in high vacuum (<10-4 Pa for 80 h), spores of a repair-deficient (rec uvr spl) B. subtilis strain were inactivated to less than 10-4 survival. Lethality for wild-type spores was not observed under this condition, but the survivors exhibited significant mutagenesis (127). However, neither the mechanism for this DNA protection by alpha /beta -type SASP nor the DNA damage caused by desiccation is known.

Chemical resistance. Spores are generally significantly more resistant than growing cells to a wide variety of toxic chemicals, including acids, bases, phenols, aldehydes, alkylating agents, and oxidizing agents (15, 102, 168, 169, 191, 209a). In many cases the reasons for spore resistance to these types of agents are not known, and for many chemicals (e.g., acids, bases, aldehydes, and oxidizing agents), the target for spore killing is not known, although there are some data implicating protein damage in killing by oxidizing agents (145, 146). However, for other agents (e.g., alkylating agents), it is clear that the target for spore killing is spore DNA (183).

Four factors important in spore resistance to at least some chemicals have been identified and/or suggested, including the presence of spore coats, the impermeability of the spore core to hydrophilic chemicals, low spore core water content, and protection of spore DNA by alpha /beta -type SASP (15, 169, 191, 209a). However, in contrast to the situation in growing cells, in which specific enzymes sometimes detoxify chemical poisons, this appears not to be a factor in spore resistance to chemicals, presumably because of the inactivity of enzymes in the spore core (6, 21, 189).

The various layers of proteinaceous spore coats (and possibly the outer spore membrane) which surround the spore cortex certainly protect the spore from attack by very large molecules such as lytic enzymes that can hydrolyze the spore cortex. There are also data indicating that the coat and outer spore membrane protect spores against killing by some smaller chemical agents, including glutaraldehyde, iodine, and some oxidizing agents (15, 38, 102, 165, 169, 209a). The mechanism of this effect is not clear; possibly the coat or outer membrane is a permeability barrier to some chemicals, or the toxic chemicals may simply react with the spore coats, thus reducing the amount of toxic agent which can attack more-central spore molecules such as enzymes or DNA in the spore core. However, for some toxic chemicals (e.g., alkylating agents), the spore coats appear to play no role in spore resistance (38, 183).

Pioneering work by Gerhardt and his coworkers showed that the spore, in particular the spore core, is relatively impermeable to small hydrophilic molecules larger than about 200 Da (53), and even smaller molecules may penetrate the spore core only very slowly (178). It seems very likely that this low spore core permeability must play a significant role in spore resistance to toxic chemicals. However, there are no data bearing directly on this point, as there is no way known to readily modulate spore core permeability.

Since most of the toxic chemicals used to kill spores are water soluble and carry out reactions in water, it is reasonable to suppose that a low spore core water content might slow reactions of toxic chemicals with targets in the spore core. While one study found a decrease in spore resistance to H2O2 with increasing core water content (153), further study on the importance of this factor is needed.

For several types of toxic chemicals (e.g., formaldehyde and peroxides), there is strong evidence that one factor in spore resistance is protection of spore DNA from attack by the binding of alpha /beta -type SASP (93, 175, 191). Oxidizing agents do not appear to kill wild-type spores by DNA damage, while alpha -beta - spores lose resistance to these agents and are killed by DNA damage (175, 190, 191). Formaldehyde does kill wild-type spores by DNA damage, and alpha -beta - spores are much more sensitive to formaldehyde killing (93). However, for some other chemicals such as alkylating agents, DNA protection by alpha /beta -type SASP binding plays no role in spore resistance, even though these chemicals kill spores by DNA damage (183, 191).

UV radiation resistance. Depending on the species analyzed, spores are 10 to 50 times more resistant than growing cells to UV radiation at 254 nm in water (185). However, the magnitude of the difference in UV resistance between spores and growing cells can be different at different wavelengths. There are two main reasons for the increased UV resistance of spores: a difference in the UV photochemistry of DNA in spores and the efficient and relatively error-free repair of the novel photoproduct formed by UV light in spore DNA. While the major UV photoproduct formed in DNA of growing cells is a cis,syn-cyclobutane-type thymine dimer, this photoproduct is not generated by 254-nm UV irradiation of spores in water (34, 185, 190, 191). Rather, the major UV photoproduct formed in spores is the unique thymine adduct 5-thyminyl-5,6-dihydrothymine (130, 215), which is called SP (Fig. 2).


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FIG. 2.   Structures of a single pair of adjacent thymines on the same DNA strand (left), a cis-syn cyclobutyl thymine-thymine dimer (center), and SP (right). dRib, D-ribose.

The major and possibly only cause of the altered UV photochemistry of DNA in spores is the saturation of spore DNA with alpha /beta -type SASP. Indeed, alpha /beta -type SASP binding to DNA in vitro suppresses formation of all cyclobutane pyrimidine dimers upon UV irradiation and promotes SP formation and also blocks formation of various 6-4 addition products (135, 190, 191). Repair of SP and other types of DNA damage will be discussed below.

Spores are also more resistant than growing cells to UV irradiation in the dry state, and wild-type B. subtilis spores treated with 254-nm UV in the lab show similar inactivation rates whether irradiated as air-dried monolayers (90% lethal dose [LD90] = 114 J/m2) (227) or in aqueous suspension (LD90 = 156 J/m2) (C. Salazar and W. L. Nicholson, unpublished). However, UV irradiation of spores rendered severely anhydrous under high vacuum generates significant amounts of photoproducts other than SP (89); as yet, the identities of these additional photoproducts are not known, nor is the reason for their generation.

In addition to the elevated resistance of dormant spores to 254-nm UV, germinating spores undergo a transient period of UV resistance even higher than that of spores before they return to the UV resistance characteristic of vegetative cells (207; reviewed in reference 185). An explanation for the transient elevated UV resistance seen in germinating spores comes from the observation that this period coincides with an overall lowering of the photoreactivity of DNA within the germinating spore (78, 177, 204). It is thought that during return of spore DNA from the A to the B conformation during germination, it passes through a transitional conformation which is geometrically unfavorable to the production of either SP or cyclobutane-type dimers. Support for this hypothesis comes from the observation that germinating spores of mutants lacking alpha /beta -type SASP, whose DNA does not appear to be in an A-like conformation within the dormant spore (136, 176), do not exhibit this transient period of ultrahigh UV resistance (177). Another related but separate developmental phenomenon is high UV resistance exhibited by the fully germinated spores of repair-deficient (splB uvr) mutants. This phenomenon has been attributed to a specific germinative excision repair of pyrimidine dimers distinct from the nucleotide excision repair (114, 218), although direct genetic or biochemical evidence for this postulated pathway is lacking.

gamma -Radiation resistance. In addition to UV resistance, spores are often significantly more resistant than growing cells to gamma -radiation (56, 167). It has been suggested that one factor which may result in increased spore gamma -radiation resistance is the decreased level of spore core water, which may reduce the amount of hydroxyl radicals formed by gamma -irradiation. However, this suggestion has not yet been tested directly. In contrast to the situation with spore UV resistance, in which alpha /beta -type SASP play a predominant role, these proteins appear to play no significant role in spore gamma -radiation resistance (190, 191). Although it appears certain that DNA repair during spore germination will be an important element in determining the level of spore gamma -radiation resistance, there again has been relatively little work on the role of specific DNA repair systems or on the nature of the DNA damage caused by gamma -irradiation of spores.

Resistance to ultrahigh hydrostatic pressure. Extremophilic archaea and bacteria which have been isolated near deep-sea thermal vents from greater than 2 km below the ocean surface not only survive but grow at in situ pressures of 200 atm or greater (79) (1 atm = 1.013 bar or 101.3 kPa). Application of even higher hydrostatic pressure is currently being explored as a method for decreasing the numbers of vegetative bacterial and spore contaminants in a number of different types of food (94). Destruction of vegetative bacteria by pressure consists of two apparently distinct behaviors: (i) a step change in the number of survivors with the application of a pressure pulse and (ii) a first-order rate drop in the number of survivors during the ensuing pressure hold period (129, 160). The rate and degree of bacterial cell inactivation by ultrahigh pressure vary widely in different experiments and depend on a number of variables, including (i) the magnitude, rate, and duration of compression, (ii) the rate of decompression, (iii) the particular bacterium tested, (iv) the medium in which the bacteria are suspended, (v) the temperature, and (vi) the degree to which cells are allowed to resuscitate before viability is tested (196). Various mesophilic bacteria and fungi treated with 3,000 to 4,000 atm at 5°C demonstrated D values of 7.5 to 15 min (3). The effect of temperature can be quite dramatic; cells of Listeria innocua survived 4,400 atm at 25 to 26°C with a D value of 7.4 min (160), while Listeria monocytogenes cells were inactivated much more rapidly by application of less pressure (3,700 atm) but applied at a higher temperature of 45°C, exhibiting a D value of 1.1 min under these conditions (198).

Spores are extremely resistant to killing by ultrahigh hydrostatic pressures, generally more so than the corresponding growing cells (29, 168). Analysis of spore killing by hydrostatic pressure has shown that spore killing rises as the pressure increases to some maximum level of killing and then decreases as the pressure increases further (168, 224). Not all the reasons for the latter observation are known. However, it appears most likely that spore killing by hydrostatic pressure is due to the induction of spore germination by this treatment, with the germinated spores then being killed rapidly by pressure (139, 143, 161, 168, 224). With B. subtilis spores, the mechanism by which spore germination is triggered by moderate pressure (100 MPa or 987 atm) appears to involve the normal germinant receptors, but at higher pressures (600 MPa or 5,923 atm), triggering of spore germination does not require the germinant receptors (168, 225). In addition, spores germinated at lower pressures (100 MPa) become sensitive to UV and oxidizing agents, while spores germinated at higher pressures (600 MPa) are much more resistant (224). Thus, while spores are germinated by both high and lower pressures, spores germinated at high pressures remain significantly resistant, presumably even to killing by pressure itself. The sensitivity of lower-pressure (100 MPa)-germinated B. subtilis spores to UV and oxidizing agents appears to be due to the degradation of alpha /beta -type SASP accompanying the spore germination induced by these pressures; in contrast, while higher pressures induce spore germination, degradation of alpha /beta -type SASP is not induced (224). Although the reason for the lack of alpha /beta -type SASP degradation accompanying spore germination induced by high pressure is not known, this observation as well as other data indicate that alpha /beta -type SASP play an important role in the resistance of B. subtilis spores to extremely high pressure (224).


SPORE DNA REPAIR MECHANISMS
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Dormant but viable spores can persist in the environment over millennial time spans (see above) in a metabolically inactive state. Therefore, environmental damage accumulates unrepaired in spore cellular components during dormancy. As discussed in the preceding section, the cellular target of many sporicidal treatments is DNA, and spore resistance to DNA-damaging treatments is due in part to protective mechanisms which either (i) prevent or dramatically slow the rate of formation of certain types of DNA damage (e.g., damage induced by oxidizing agents, dry heat, or desiccation) or (ii) alter the type of damage formed in spore DNA (e.g., 254-nm UV light inducing formation in spore DNA of SP rather than cyclobutane pyrimidine dimers) (187, 190, 191). Despite these protective mechanisms, potentially lethal and mutagenic damage nonetheless does accumulate in spore DNA during long-term storage of spores in the laboratory (190) and during exposure of spores to environmental stresses, particularly solar radiation (201, 212) (see below). Therefore, another major determinant of the degree of spore resistance to extreme environments is the speed and accuracy with which spore DNA damage can be repaired during germination. In the laboratory, the critical time frame for DNA repair during germination can be quite short. For example, in germinating spores of Bacillus megaterium, de novo RNA and protein synthesis begins well within the first 5 min of germination, using entirely endogenous reserves of precursors and energy (192-194). Abasic sites, helix-distorting lesions such as SP, or breaks in the phosphodiester backbone of spore DNA could exert potentially lethal effects early in germination by blocking the progress of RNA polymerase, thus halting expression of any number of critical pathways leading to replicative DNA synthesis and outgrowth. In addition, unrepaired lesions in spore DNA itself would physically block replication, leading to both lethal and mutagenic consequences (reviewed in reference 51). We have not yet obtained a complete catalog either of the identities of all types of damage incurred in the DNA of spores exposed to extreme environments or of all DNA repair systems involved in spore DNA repair. Below is a summary of what we know to date from laboratory studies.

General DNA Repair Systems

NER. Nucleotide excision repair (NER) in B. subtilis closely resembles the analogous system in Escherichia coli, which has been extremely well characterized (87; reviewed in references 50, 51, and 88). Although the molecular details of NER in B. subtilis have not been elucidated to the same degree as NER in E. coli, several lines of evidence suggest that the two processes are essentially similar. First, the B. subtilis homologs of the genes encoding the UvrB and UvrC subunits of the E. coli excinuclease have been identified, mapped (115), cloned, and sequenced (22, 23) for some time, and the B. subtilis homolog of the UvrA protein has recently been identified as part of the B. subtilis genomic sequencing project (86). The deduced amino acid sequences of all three of these proteins show a high degree of similarity to their E. coli counterparts. Second, functional Uvr(A)BC excinuclease activity was obtained in vitro by mixing the purified B. subtilis homolog of UvrC protein with purified E. coli UvrA and UvrB proteins (87).

Regulation of expression of some genes for the B. subtilis NER pathway has been studied using uvr-lacZ fusions. The NER genes are expressed constitutively at a low level during vegetative growth (23) and during sporulation (P. J. Riesenman and W. L. Nicholson, unpublished). Expression of uvr-lacZ fusions is inducible by DNA damage both during vegetative growth (23) and during the outgrowth phase of germination of spores treated with 254-nm UV or dry heat (182). B. subtilis strains carrying mutations of genes in the NER pathway make UV-sensitive vegetative cells, but their spores are only slightly more UV sensitive than wild-type spores (115, 122) (Fig. 3).


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FIG. 3.   UV resistance of spores and vegetative cells of B. subtilis DNA repair mutants. All strains were derivatives of the prototype laboratory strain 168. The average UV dose required to kill 90% of the population (LD90 value) is given for vegetative cells (open bars) and spores (solid bars). LD90 values are averages of values reported in the literature; the number of reports from which each value was derived is listed on the top of each bar. Data are from references 45, 58, 59, 113, 122, 126, 131, 132, 134, 179, 182, 185, 186 and 227 and from P. Fajardo-Cavazos (unpublished data).

Recombination-mediated repair. Munakata and Rupert (126) reported that in B. subtilis, spores of a recA single mutant were no more sensitive to 254-nm UV than were wild-type spores. However, when combined with mutations inactivating either NER (uvrB) or SP lyase (splB), mutation in recA significantly enhanced spore sensitivity to 254-nm UV, thus implicating the Rec pathway in spore UV resistance (126). More recently, it has been reported that recA single mutants of B. subtilis produce spores which are about twofold more UV sensitive than wild-type spores (59, 182). Expression of a recA-lacZ fusion is inducible by DNA damage and entrance of cells into the competent state (reviewed in reference 228). Levels of RecA protein were found to be quite low in dormant spores, and expression of a recA-lacZ fusion was induced during the outgrowth phase of germination of spores previously treated with 254-nm UV or dry heat (182). Thus, it appears that expression of the NER and Rec pathways in response to DNA damage is controlled by classic DNA damage-inducible (Din) circuitry (228).

Other general repair systems. In the recently completed sequence of the B. subtilis genome, a number of open reading frames presumably encoding components of various base excision repair and mismatch repair systems have been identified (86). To date, however, virtually no information exists regarding the role of these additional general DNA repair systems in spore resistance to extreme environmental conditions.

SP Lyase, an SP-Specific DNA Repair System

Unlike the generalized NER and Rec systems, which operate in both vegetative cells and outgrowing spores on a variety of types of DNA damages (182, 190), SP lyase is specifically dedicated to the repair of SP which has accumulated in dormant spores exposed to UV. Elegant genetic, biochemical, and physiological experiments performed in the late 1960s and 1970s indicated that SP lyase was produced during sporulation, packaged in the dormant spore, and activated during early germination to monomerize SP in situ back to two thymines (124, 125, 219). This broad scheme of repair has in large part been substantiated and explored in greater detail during the 1990s using a number of molecular approaches. DNA from B. subtilis strain 168 which could rescue a mutant lacking SP lyase activity (the original splB1 mutation isolated by Munakata [113]) was cloned, mapped genetically to the pts-kinA region of the chromosome, and sequenced (45). From the nucleotide sequence of the region, it was observed that the SP lyase (spl) locus was organized as a bicistronic operon, consisting of splA, encoding a protein of 79 amino acids (a.a.) and 9.2 kDa of unknown function, and splB, encoding a 40-kDa protein which exhibited limited regional homology to the DNA photolyase/6-4 photolyase/blue-light photoreceptor family of proteins (45, 133). Mutational inactivation and complementation experiments indicated that the splB cistron encoded the information missing in the SP lyase-deficient splB1 mutant (43, 45, 132).

Regulation of SP lyase expression during sporulation. Expression of the splAB operon during B. subtilis growth and development has been studied by constructing a translational fusion between splB and the E. coli lacZ gene (148). In these experiments it was observed that the splAB operon is expressed as part of the Esigma G regulon of forespore-specific genes, by the following criteria: (i) the splB-lacZ fusion was expressed in the forespore at stage III of sporulation, (ii) expression of the splB-lacZ fusion was abolished in a sigGDelta 1 mutant strain lacking sigma-G, (iii) expression of the splB-lacZ fusion could be activated during vegetative growth by artificially inducing expression in trans of the sigG gene encoded on an extrachromosomal plasmid, and (iv) the splAB operon was efficiently transcribed in vitro by purified Esigma G RNA polymerase from a major sigma G-type promoter preceding the operon called P1 (148). Unlike DNA repair genes such as uvr and rec (182, 228), expression of the splB-lacZ fusion was not DNA damage inducible during vegetative growth, nor was expression induced by growing cells under conditions which induce genetic competence (148). Interestingly, the SASP themselves are also expressed in the developing forespore at stage III of sporulation as part of the Esigma G regulon (138, 208; reviewed in reference 188).

A database search was at first unable to identify a protein homolog for the putative 79-a.a. protein encoded by the splA cistron preceding splB. However, genetic experiments hinted that the splAB operon contained unexplored regulatory features. First, deletions extending from upstream removing P1 and part of splA did not inactivate operon expression (45). Second, primer extension mapping of in vivo splAB mRNA revealed a second transcript arising from the intercistronic region between splA and splB, apparently from another Esigma G-dependent promoter called P3 (148). Third, in vitro mutations which inactivated the major P1 promoter and an in-frame deletion of splA had the effect of increasing the expression levels but not the timing, forespore compartmentalization, or Esigma G dependence of expression of splB-lacZ fusions integrated at the prophage SPbeta locus (149). cis/trans analysis of partial diploids for splA indicated that the SplA gene product is a trans-acting negative regulator of splB-lacZ expression and apparently acts by modulating the level of transcription initiating from P1 versus P3 (44). Although neither the molecular mechanism of this regulatory circuit nor its potential function during sporulation has yet been elucidated, it is interesting to note that the deduced SplA amino acid sequence is similar to that of another small regulatory protein, the trp RNA-binding attenuation protein (TRAP) of B. subtilis. By analogy, this observation opens the possibility that SplA may operate at the level of splAB mRNA by a TRAP-like mechanism (44; reviewed recently in reference 5).

Genetics and biochemistry of SP lyase. Soon after cloning of the splAB operon, it was demonstrated that the splB cistron encoded SP lyase activity, from evidence that (i) only subclones of splAB containing wild-type splB DNA could rescue Munakata's splB1 mutation (43, 45) and (ii) knockout mutations of the splB cistron but not the splA cistron resulted in spore UV sensitivity (45, 132). The first clue to the enzymatic mechanism of SP lyase came from examination of the deduced amino acid sequence of the B. subtilis SplB protein. The 342-a.a. sequence of SplB was observed to contain only four cysteines, three of which were tightly clustered at residues 91, 95, and 98 and the fourth at residue 141 (45). The SplB sequence surrounding residues C91, C95, and C98 was found through sequence database searching to be highly similar to the amino acid signature for the [4Fe-4S] clusters of a family of S-adenosylmethionine (SAM)-dependent, radical-utilizing enzymes represented by anaerobic (type III) ribonucleotide reductase, pyruvate-formate lyase, lysine-2,3-aminomutase, biotin synthases (BioB), and lipoic acid synthetase (LipA) (132) (Fig. 4). All members of the family of SAM-dependent, radical-utilizing enzymes have three features in common: (i) they contain oxygen-labile [4Fe-4S] clusters and hence require anaerobic conditions for their activity; (ii) they utilize SAM as a cofactor; and (iii) they operate by a radical mechanism, in which the [4Fe-4S] cluster splits SAM to generate methionine and a 5'-adenosyl radical, which then proceeds to participate either directly or indirectly in catalysis (reviewed in references 163 and 223).


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FIG. 4.   Comparison of amino acid regions forming [4Fe-4S] clusters in the SplB amino acid sequences from Bacillus anthracis (B.an), B. amyloliquefaciens (B.am), B. stearothermophilus (B.st), B. subtilis (B.su), Clostridium acetobutylicum (C.ac), and C. difficile (C.di) with the [4Fe-4S] clusters of: the activating subunits of ribonucleotide reductase (NrdG) and pyruvate-formate lyase (Act) from Escherichia coli (E.co), phage T4 (T4), and Haemophilus influenzae (H.in) and the [4Fe-4S] clusters from lysine-2,3-aminomutase (KamA) from Clostridium subterminale (C.su), biotin synthase (BioB) from B. subtilis, and the probable lipoic acid synthase (YutB) from B. subtilis. Highly conserved residues are in bold, and invariant cysteines are marked with an asterisk.

The evidence is mounting that SP lyase belongs to the family of SAM-dependent, radical-utilizing enzymes. First, iron was shown to be associated with SP lyase activity, as survival of UV-irradiated spores of a B. subtilis strain relying only upon SP lyase for DNA repair was lower when the spores were germinated on solid growth medium lacking iron (132). Second, SplB protein carrying an amino-terminal histidine tag was overexpressed and purified from E. coli; the purified protein exhibited a reddish-brown color characteristic of iron-containing proteins (162). Third, the presence of a [4Fe-4S] cluster in the active form of SP lyase was inferred by chemical assay for iron and acid-labile sulfide and by UV-visible spectroscopy (162) and most recently has been confirmed by electron paramagnetic resonance spectroscopy (R. Rebeil and W. L. Nicholson, unpublished observations). Fourth, in vitro SP lyase activity was obtained from purified SplB protein only when reactions were carried out under anaerobic conditions, and activity was shown to be absolutely dependent on SAM (162). Whereas many of the molecular details of the SP cleavage reaction remain to be elucidated, it is clear from the evidence gathered to date that SP lyase is indeed a member of the SAM-dependent, radical-utilizing enzyme family and is unique in being the first and only DNA repair protein discovered to date which utilizes this novel mechanism of catalysis.

As noted above for spore protective mechanisms, the evidence collected to date indicates that repair of DNA damage in the spore during germination is also the result of a number of general and specific DNA repair mechanisms acting in concert. The net result of the relative contributions to the known DNA repair systems in determining the resistance of B. subtilis spores versus vegetative cells to 254-nm UV is summarized in Fig. 3.


SPORE RESISTANCE TO EXTREME TERRESTRIAL ENVIRONMENTS
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Differences between the Laboratory and the Environment

As discussed in the preceding sections, a fairly detailed molecular picture of spore resistance properties in the laboratory is emerging, which appears to be a combination of (i) mechanisms for protection of critical spore components, particularly DNA, during dormancy and (ii) mechanisms for rapid and accurate repair of cellular damage during germination. A fundamental biological question inevitably arises from these studies: Do the models which have been developed to explain spore resistance in the laboratory provide an adequate description of the phenomenon as it occurs in the environment? Although considerable effort has been directed towards investigation of spore UV resistance and DNA repair under a number of environmental conditions (see below), the gulf between the laboratory and the environment is only beginning to be bridged. In an attempt to answer the question of biological relevance, it is perhaps instructive to first compare some important parameters which differ between the laboratory and the field. We will use the spore UV resistance model as an example, as it is this model which has been advanced from laboratory studies in the greatest detail.

Source of spores. Most but not all of the laboratory model of spore UV resistance has been derived directly from studies on a single organism, Bacillus subtilis. This situation has arisen for the sensible reason that B. subtilis is by far the best-characterized spore-forming microorganism because it is amenable to highly refined genetic and molecular biological manipulation (61, 86, 203). In addition, there is a body of experimental evidence gathered from other spore-forming microorganisms which supports the B. subtilis model describing the role of alpha /beta -type SASP in spore DNA protection and UV photochemistry (reviewed in references 184 and 190). Studies on spore DNA repair mechanisms, in contrast, have focused almost exclusively on B. subtilis because of the early isolation of mutations affecting NER and SP lyase in this species (113, 122); the sole exception in this case is the Bacillus amyloliquefaciens homolog of SP lyase, which has been cloned, completely sequenced, and shown to function in B. subtilis (133). In addition, SP lyase homologs have been cloned and partially sequenced from Clostridium perfringens (211) and identified in the sequenced genomes of Bacillus anthracis, B. stearothermophilus, Clostridium acetobutylicum, C. difficile, and Streptomyces coelicolor. Therefore, the evidence to date indicates that SP lyases, like SASP, are widely conserved among the spore-forming eubacteria.

Both laboratory and environmental studies of spore resistance mechanisms have until recently been conducted almost exclusively using "tame" laboratory strains which have not experienced either growth or sporulation under natural environmental conditions in several decades. Conversely, until very recently we had no data regarding the UV resistance properties of spores isolated from natural environmental sources or from extreme environments. Recently it was shown that populations of dormant Bacillus spp. spores isolated and purified directly from soils in the Sonoran desert were significantly more UV resistant than the benchmark laboratory B. subtilis strain, 168 (134). One round of growth and sporulation of these Sonoran desert isolates on standard laboratory media lowered the UV resistance of their spores to levels essentially identical to that of B. subtilis 168, suggesting that spore UV resistance is determined at least in part by the environment in which a bacterium sporulates (134).

Growth, sporulation, dormancy, and germination conditions. In the laboratory, B. subtilis spores are usually prepared by cultivating the bacterium at 37°C in a liquid nutrient broth-based sporulation medium at a high growth rate and to high cell density until some essential nutrient (usually the carbon source) is exhausted from the medium (171). After 1 to 2 days of aerobic incubation, spores are purified from vegetative cells, the pasteurized spore suspension is air dried or diluted in buffer and exposed to the extreme condition being studied, and survival is quantitated by plating the spores on nutrient medium and counting colonies arising after incubation at 37°C (reviewed in reference 137). Although very little is known about the growth or sporulation of bacteria in their natural habitats, it is not difficult to envision that this process probably bears little resemblance to the manner in which spores are prepared and assayed in the laboratory. Growth of spore-forming bacteria in their natural environments (e.g., soil, decaying organic matter, plant surfaces, and insect and mammalian guts) (i) is almost certainly slower, (ii) probably takes place in microcolonies on and within a solid substrate (aggregated soil particles, e.g.), (iii) at the very least is subject to wide variations in temperature, UV flux, nutrient, water, and oxygen availability, and (iv) probably occurs in direct competition with other micro- and macroorganisms. To date, we know almost nothing about how spore-forming bacteria grow and sporulate under these diverse conditions or of the properties of the resulting spores.

Solar Radiation as Primary Source

Historically, most laboratory studies of spore UV photochemistry and DNA repair have been performed using monochromatic 254-nm UV light (UV-C) due to two technical expedients: (i) 254-nm UV is relatively cheap and simple to generate using a low-pressure mercury arc lamp, and (ii) 254 nm coincides well with the absorption maximum of DNA, so that biological and photochemical effects can be observed at relatively low fluences. However, sunlight reaching the Earth's surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, the UV portion spanning approximately 290 to 400 nm (the so-called UV-B and UV-A portions of the UV spectrum) (214) (Fig. 5). The laboratory model of spore UV resistance has therefore been constructed largely using a wavelength of UV radiation not normally experienced on the Earth's surface, even though ample evidence exists that both DNA photochemistry (195) and cellular responses to UV (213) are strongly wavelength dependent.


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FIG. 5.   Solar spectrum in space (thin line) and on Earth's surface (thick line). Below the spectrum is an expansion of the UV portion, showing the approximate boundaries between UV-C, UV-B, and UV-A. Also shown is the UV wavelength commonly used in the laboratory (254 nm) and the UV wavelengths incident on the Earth's surface (290 nm and longer). vis, visible; IR, infrared.

Radiation from the sun drives essentially all life processes at the Earth's surface, but paradoxically is also a major source of lethal damage to spore cellular components and both lethal and mutagenic damage to spore DNA. The physical effects of solar radiation can be divided into direct and indirect effects.

Direct solar effects. Solar UV radiation can cause lethal and mutagenic damage to spores by direct interaction between photons and DNA (116, 212). As mentioned above, the best-characterized UV damage in spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly called SP, as well as a number of less-abundant unidentified photoproducts (34), one of which may be the (6-4) photoproduct (90). SP has been detected in spore DNA irradiated at a number of UV wavelengths extending through the UV-C, UV-B, and UV-A portions of the spectrum, using either artificial UV sources or sunlight (212). The presence of additional photoproducts in spore DNA exposed to solar UV has long been inferred (212, 227), but the identity of some of these photoproducts has only recently begun to be elucidated (201) (see below). Although much attention has focused on the UV-B portion of sunlight, since it is a major cause of DNA damage, recent research has clearly demonstrated that UV-A, especially wavelengths in the range from 320 to 365 nm, can also induce production of pyrimidine dimers besides producing other kinds of DNA and cellular damage (201, 212, 213).

Indirect solar effects. Solar radiation exerts a number of indirect, albeit important secondary effects which have in some cases been mimicked in the laboratory, including heating of spores, desiccation of spores through evaporation, and generation of reactive oxygen species in spores (213).

As noted above from laboratory studies (Fig. 1), spores can withstand heating for extended periods at temperatures well above those normally prevailing in most environments at the Earth's surface. This observation has been confirmed experimentally using spores of several different B. subtilis strains exposed as air-dried films to solar heating at temperatures exceeding 70°C but shielded from primary UV effects (165, 201, 226, 227). The net result of these studies is that spores suffered little or no detectable loss in viability after exposures of up to 30 h under these conditions.

The effects of extreme desiccation on B. subtilis spores and spore DNA have been studied using vacuum generated either in the laboratory (13, 36, 37, 42, 127) (see above) or in space orbit (66) (see below). Current evidence indicates that extreme desiccation under vacuum leads to predominantly single-strand breaks, DNA-protein cross-links, and other uncharacterized DNA damage in spores (13, 42); to date, vacuum-induced damage to non-DNA spore components has not been reported.

It is almost certain that the desiccation induced by extreme vacuum in the laboratory is far greater than the levels of desiccation found in terrestrial environments. Although no systematic studies of desiccation resistance of spores in terrestrial environments have been undertaken, it is interesting to note that viable spores have been recovered from 300-year-old air-dried herbarium soils and from 10-meter-deep permafrost soils at least 10,000 years old (reviewed in reference 154).

Several lines of evidence indicate that absorption of solar UV-B and UV-A by a variety of compounds within the cell can result in the generation of reactive oxygen intermediates, such as hydrogen peroxide and superoxide anion. These activated oxygen species target several cellular components in addition to DNA, causing enzyme photoinactivation and lipid peroxidation (reviewed in reference 213). These additional types of solar damage to non-DNA spore cellular components have not been examined to date.

We have some idea of how each of the above secondary solar effects impacts spores in the laboratory, but how do each of these physical parameters affect spore DNA in the environment? Spores in the environment are not exposed to single, isolated stresses as studied in the laboratory. Rather, solar exposure creates some or all of these effects simultaneously. Factors such as the constantly shifting solar angle resulting from Earth's rotation and tilt combined with ever-changing atmospheric conditions cause complex cyclic variations in total radiation flux and spectrum, particularly at the UV-B extreme (214). In addition, spores can undergo untold numbers of cycles of desiccation-hydration, heating-cooling, and freezing-thawing during dormancy. Even spores buried in soil, whose DNA is well shielded from the primary effects of solar UV, are subjected to these indirect stresses.

Protection of Spores from Lethal Solar UV Damage

What structures or features of the spore have been identified in laboratory studies which could protect the spore from lethal solar UV damage? As mentioned above, SASP binding to spore DNA in the spore core protects it both from 254-nm UV-C damage and from damage due to oxidizing agents (see above). These results imply that SASP may also protect spore DNA from the direct