SUMMARY 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.

Expression of the alpha-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose-mediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263 degrees on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.

A committee of the Mars Exploration Program Analysis Group (MEPAG) has reviewed and updated the description of Special Regions on Mars as places where terrestrial organisms might replicate (per the COSPAR Planetary Protection Policy). This review and update was conducted by an international team (SR-SAG2) drawn from both the biological science and Mars exploration communities, focused on understanding when and where Special Regions could occur. The study applied recently available data about martian environments and about terrestrial organisms, building on a previous analysis of Mars Special Regions (2006) undertaken by a similar team. Since then, a new body of highly relevant information has been generated from the Mars Reconnaissance Orbiter (launched in 2005) and Phoenix (2007) and data from Mars Express and the twin Mars Exploration Rovers (all 2003). Results have also been gleaned from the Mars Science Laboratory (launched in 2011). In addition to Mars data, there is a considerable body of new data regarding the known environmental limits to life on Earth-including the potential for terrestrial microbial life to survive and replicate under martian environmental conditions. The SR-SAG2 analysis has included an examination of new Mars models relevant to natural environmental variation in water activity and temperature; a review and reconsideration of the current parameters used to define Special Regions; and updated maps and descriptions of the martian environments recommended for treatment as "Uncertain" or "Special" as natural features or those potentially formed by the influence of future landed spacecraft. Significant changes in our knowledge of the capabilities of terrestrial organisms and the existence of possibly habitable martian environments have led to a new appreciation of where Mars Special Regions may be identified and protected. The SR-SAG also considered the impact of Special Regions on potential future human missions to Mars, both as locations of potential resources and as places that should not be inadvertently contaminated by human activity.

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H 2 O 2 ) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H 2 O 2 , as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.

The occurrence and diverse roles of Bacillus spp. and their endospores in the environment is reviewed, with particular emphasis on soil ecology, host-symbiont and host-parasite interactions, and human exploitation of spores as biological control agents and probiotics.

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.It has been shown that endospores of gram-positive bacteria can remain viable for at least thousands of years (5, 44, 54; reviewed in reference 31). Bacterial spores persist in a metabolically inactive state, and environmental damage to spore cellular components accumulates unrepaired until germination and outgrowth (32). However, Bacillus subtilis spores are highly resistant to different environmental stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and ionizing and UV radiation. The reason for this high resistance to environmental extremes lies partly in the spore structure itself: spores possess thick layers of highly cross-linked coat proteins (13), a modified peptidoglycan spore cortex, and abundant intracellular constituents such as the calcium chelate of dipicolinic acid and small, acidsoluble spore proteins (␣/␤-type SASP), as protectants of spore DNA (46, 50). Binding of ␣/␤-type SASP to spore DNA, coupled with spore core dehydration, appears to change the helical conformation of spore DNA from the B form to an A-like form (34, 48), which in turn alters its UV photochemistry to favor the production of 5-thyminyl-5,6-dihydrothymine, the unique spore-specific spore photoproduct (SP) (8,32,35,50). For the removal of the SP, spores possess an SP-specific repair enzyme called SP lyase, encoded by the splB gene, that monomerizes the SP dimer back to two thymine residues in an adenosyl-radical-dependent reaction (4,28,42).While the UV photochemistry of spore DNA and the repair of UV damage to DNA during germination are well described (12,32,33,47,50), there has been relatively little work on the nature of DNA damage in spores caused by ionizing radiation or extreme dryness and on the occurrence of a specific DNA repair system(s) for repair of this damage. It is assumed that DNA double-strand breaks (DSB), which are the most critical damage caused by ionizing radiation (57) and desiccation (9, 10, 39) in vegetative cells, are also induced in bacterial spores. Spores of B. subtilis contain a single chromosome arranged in a toroidal shape (16, 41); therefore, the homologous recombination pathway, which requires at least two homologous chromosomes, cannot operate on DSB during spore germination (55). An alternative repair pathway for DSB induced in spore DNA, nonhomologous-end joining (NHEJ) (3, 56), is considered here. This pathway as it occurs in eukaryotic cells requires a DNA end-binding component called Ku (Ku70 and Ku80) (58). The fir...

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UV irradiation of complexes of DNA and an a/fl-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were <0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m2; in the absence of SASP the yields were reversed-4.5% and 0.3%, respectively. Complexes of DNA with a/fl-type SASP from Bacilus cereus, Baciflus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of a/fl-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were signfcantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.Dormant spores of various Bacillus species are much more resistant to the cytotoxic effects of UV radiation than are their growing cell counterparts (1). This difference in UV resistance is rooted in a difference in the UV photochemistry of cell and spore DNA in vivo. The major photoproducts formed in DNA by UV irradiation of cells are cyclobutanetype pyrimidine dimers, primarily thymine-thymine dimers (TTs), with smaller amounts of cytosine-thymine dimers (CTs) and cytosine-cytosine dimers as well as the so called 6-4 photoproduct (2-4). In contrast, UV irradiation of dormant spores of various Bacillus species produces no detectable cyclobutane-type pyrimidine dimers but rather a series of other photoproducts, the most abundant being a 5-thyminyl-5,6-dihydrothymine adduct (2, 5) that has been termed spore photoproduct (SP). The contribution of this photochemistry to spore UV resistance is not that SP is formed in very low yields. Indeed, the yields for TT in vegetative cells and SP in spores are similar, and extremely high levels (>25% of total thymine) of SP can accumulate in DNA (2). Rather, the dormant spore has at least two mechanisms that efficiently eliminate SP in the early minutes of spore germination; one of these mechanisms is specific for SP (6, 7).Studies on the UV photochemistry of DNA under various conditions in vitro led to the conclusion that the production of pyrimidine dimers was characteristic of DNA in the B conformation, whereas SP formation was favored in DNA in the A conformation (8,9). This led to the suggestion that DNA in dormant spores might be in the A conformation (8). Subsequent...