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.

SUMMARY The responses of microorganisms (viruses, bacterial cells, bacterial and fungal spores, and lichens) to selected factors of space (microgravity, galactic cosmic radiation, solar UV radiation, and space vacuum) were determined in space and laboratory simulation experiments. In general, microorganisms tend to thrive in the space flight environment in terms of enhanced growth parameters and a demonstrated ability to proliferate in the presence of normally inhibitory levels of antibiotics. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. A hypothesized interaction of microgravity with radiation-induced DNA repair processes was experimentally refuted. The survival of microorganisms in outer space was investigated to tackle questions on the upper boundary of the biosphere and on the likelihood of interplanetary transport of microorganisms. It was found that extraterrestrial solar UV radiation was the most deleterious factor of space. Among all organisms tested, only lichens (Rhizocarpon geographicum and Xanthoria elegans) maintained full viability after 2 weeks in outer space, whereas all other test systems were inactivated by orders of magnitude. Using optical filters and spores of Bacillus subtilis as a biological UV dosimeter, it was found that the current ozone layer reduces the biological effectiveness of solar UV by 3 orders of magnitude. If shielded against solar UV, spores of B. subtilis were capable of surviving in space for up to 6 years, especially if embedded in clay or meteorite powder (artificial meteorites). The data support the likelihood of interplanetary transfer of microorganisms within meteorites, the so-called lithopanspermia hypothesis.

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Cryptoendolithic microbial communities and epilithic lichens have been considered as appropriate candidates for the scenario of lithopanspermia, which proposes a natural interplanetary exchange of organisms by means of rocks that have been impact ejected from their planet of origin. So far, the hardiness of these terrestrial organisms in the severe and hostile conditions of space has not been tested over extended periods of time. A first long-term (1.5 years) exposure experiment in space was performed with a variety of rock-colonizing eukaryotic organisms at the International Space Station on board the European EXPOSE-E facility. Organisms were selected that are especially adapted to cope with the environmental extremes of their natural habitats. It was found that some-but not all-of those most robust microbial communities from extremely hostile regions on Earth are also partially resistant to the even more hostile environment of outer space, including high vacuum, temperature fluctuation, the full spectrum of extraterrestrial solar electromagnetic radiation, and cosmic ionizing radiation. Although the reported experimental period of 1.5 years in space is not comparable with the time spans of thousands or millions of years believed to be required for lithopanspermia, our data provide first evidence of the differential hardiness of cryptoendolithic communities in space.

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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...

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

The scenario of lithopanspermia describes the viable transport of microorganisms via meteorites. To test the first step of lithopanspermia, i.e., the impact ejection from a planet, systematic shock recovery experiments within a pressure range observed in martian meteorites (5-50 GPa) were performed with dry layers of microorganisms (spores of Bacillus subtilis, cells of the endolithic cyanobacterium Chroococcidiopsis, and thalli and ascocarps of the lichen Xanthoria elegans) sandwiched between gabbro discs (martian analogue rock). Actual shock pressures were determined by refractive index measurements and Raman spectroscopy, and shock temperature profiles were calculated. Pressure-effect curves were constructed for survival of B. subtilis spores and Chroococcidiopsis cells from the number of colony-forming units, and for vitality of the photobiont and mycobiont of Xanthoria elegans from confocal laser scanning microscopy after live/dead staining (FUN-I). A vital launch window for the transport of rock-colonizing microorganisms from a Mars-like planet was inferred, which encompasses shock pressures in the range of 5 to about 40 GPa for the bacterial endospores and the lichens, and a more limited shock pressure range for the cyanobacterium (from 5-10 GPa). The results support concepts of viable impact ejections from Mars-like planets and the possibility of reseeding early Earth after asteroid cataclysms.