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.

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 /-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).

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.

/-Type SASP. Spore DNA is saturated with a group of unique proteins called /-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 /-type SASP, and the DNA's reactivity with a variety of chemicals decreases dramatically. The properties of a DNA-/-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 /-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).

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).

View larger version (23K): [in this window] [in a new window]   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 /-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).

(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 /-type SASP.

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 /-type SASP, as 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 (10⁶ 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 (<10⁴ Pa for 80 h), spores of a repair-deficient (rec uvr spl) B. subtilis strain were inactivated to less than 10⁴ 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 /-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).

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).

View larger version (16K): [in this window] [in a new window]   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.

-Radiation resistance. In addition to UV resistance, spores are often significantly more resistant than growing cells to -radiation (56, 167). It has been suggested that one factor which may result in increased spore -radiation resistance is the decreased level of spore core water, which may reduce the amount of hydroxyl radicals formed by -irradiation. However, this suggestion has not yet been tested directly. In contrast to the situation with spore UV resistance, in which /-type SASP play a predominant role, these proteins appear to play no significant role in spore -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 -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 -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).

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.

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).

View larger version (60K): [in this window] [in a new window]   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.

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 E^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 sigG1 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 E^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 E^G regulon (138, 208; reviewed in reference 188).

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).

View larger version (56K): [in this window] [in a new window]   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.

SPORE RESISTANCE TO EXTREME TERRESTRIAL ENVIRONMENTS

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Differences between the Laboratory and the Environment

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 /-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.

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.

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.

View larger version (34K): [in this window] [in a new window]   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).