BIOMEX Experiment: Ultrastructural Alterations, Molecular Damage and Survival of the Fungus Cryomyces antarcticus after the Experiment Verification Tests.

The search for traces of extinct or extant life in extraterrestrial environments is one of the main goals for astrobiologists; due to their ability to withstand stress producing conditions, extremophiles are perfect candidates for astrobiological studies. The BIOMEX project aims to test the ability of biomolecules and cell components to preserve their stability under space and Mars-like conditions, while at the same time investigating the survival capability of microorganisms. The experiment has been launched into space and is being exposed on the EXPOSE-R2 payload, outside of the International Space Station (ISS) over a time-span of 1.5 years. Along with a number of other extremophilic microorganisms, the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515 has been included in the experiment. Before launch, dried colonies grown on Lunar and Martian regolith analogues were exposed to vacuum, irradiation and temperature cycles in ground based experiments (EVT1 and EVT2). Cultural and molecular tests revealed that the fungus survived on rock analogues under space and simulated Martian conditions, showing only slight ultra-structural and molecular damage.

LIFE experiment: isolation of cryptoendolithic organisms from Antarctic colonized sandstone exposed to space and simulated Mars conditions on the international space station.

Desiccated Antarctic rocks colonized by cryptoendolithic communities were exposed on the International Space Station (ISS) to space and simulated Mars conditions (LiFE-Lichens and Fungi Experiment). After 1.5 years in space samples were retrieved, rehydrated and spread on different culture media. Colonies of a green alga and a pink-coloured fungus developed on Malt-Agar medium; they were isolated from a sample exposed to simulated Mars conditions beneath a 0.1 % T Suprasil neutral density filter and from a sample exposed to space vacuum without solar radiation exposure, respectively. None of the other flight samples showed any growth after incubation. The two organisms able to grow were identified at genus level by Small SubUnit (SSU) and Internal Transcribed Spacer (ITS) rDNA sequencing as Stichococcus sp. (green alga) and Acarospora sp. (lichenized fungal genus) respectively. The data in the present study provide experimental information on the possibility of eukaryotic life transfer from one planet to another by means of rocks and of survival in Mars environment.

Genomic bipyrimidine nucleotide frequency and microbial reactions to germicidal UV radiation.

The role of the genomic bipyrimidine nucleotide frequency in pyrimidine dimer formation caused by germicidal UV radiation was studied in three microbial reference organisms (Escherichia coli K12, Deinococcus radiodurans R1, spores and cells of Bacillus subtilis 168). The sensitive HPLC tandem mass spectrometry assay was used to identify and quantify the different bipyrimidine photoproducts induced in the DNA of microorganisms by germicidal UV radiation. The yields of photoproducts per applied fluence were very similar among vegetative cells but twofold reduced in spores. This similarity in DNA photoreactivity greatly contrasted with the 11-fold range determined in the fluence causing a decimal reduction of survival. It was also found that the spectrum of UV-induced bipyrimidine lesions was species-specific and the formation rates of bi-thymine and bi-cytosine photoproducts correlated with the genomic frequencies of thymine and cytosine dinucleotides in the bacterial model systems.

EXPOSE, an astrobiological exposure facility on the international space station - from proposal to flight.

Following an European Space Agency announcement of opportunity in 1996 for "Externally mounted payloads for 1st utilization phase" on the International Space Station (ISS), scientists working in the fields of astrobiology proposed experiments aiming at longterm exposure of a variety of chemical compounds and extremely resistant microorganisms to the hostile space environment. The ESA exposure facility EXPOSE was built and an operations' concept was prepared. The EXPOSE experiments were developed through an intensive pre-flight experiment verification test program. 12 years later, two sets of astrobiological experiments in two EXPOSE facilities have been successfully launched to the ISS for external exposure for up to 1.5 years. EXPOSE-E, now installed at the balcony of the European Columbus module, was launched in February 2008, while EXPOSE-R took off to the ISS in November 2008 and was installed on the external URM-D platform of the Russian Zvezda module in March 2009.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment.

The role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination, spore photoproduct lyase, and DNA polymerase I and genome protection via alpha/beta-type small, acid-soluble spore proteins (SASP) in Bacillus subtilis spore resistance to accelerated heavy ions (high-energy charged [HZE] particles) and X rays has been studied. Spores deficient in NHEJ and alpha/beta-type SASP were significantly more sensitive to HZE particle bombardment and X-ray irradiation than were the recA, polA, and splB mutant and wild-type spores, indicating that NHEJ provides an efficient DNA double-strand break repair pathway during spore germination and that the loss of the alpha/beta-type SASP leads to a significant radiosensitivity to ionizing radiation, suggesting the essential function of these spore proteins as protectants of spore DNA against ionizing radiation.

Role of DNA protection and repair in resistance of Bacillus subtilis spores to ultrahigh shock pressures simulating hypervelocity impacts.

Impact-induced ejections of rocks from planetary surfaces are frequent events in the early history of the terrestrial planets and have been considered as a possible first step in the potential interplanetary transfer of microorganisms. Spores of Bacillus subtilis were used as a model system to study the effects of a simulated impact-caused ejection on rock-colonizing microorganisms using a high-explosive plane wave setup. Embedded in different types of rock material, spores were subjected to extremely high shock pressures (5 to 50 GPa) lasting for fractions of microseconds to seconds. Nearly exponential pressure response curves were obtained for spore survival and linear dependency for the induction of sporulation-defective mutants. Spores of strains defective in major small, acid-soluble spore proteins (SASP) (alpha/beta-type SASP) that largely protect the spore DNA and spores of strains deficient in nonhomologous-end-joining DNA repair were significantly more sensitive to the applied shock pressure than were wild-type spores. These results indicate that DNA may be the sensitive target of spores exposed to ultrahigh shock pressures. To assess the nature of the critical physical parameter responsible for spore inactivation by ultrahigh shock pressures, the resulting peak temperature was varied by lowering the preshock temperature, changing the rock composition and porosity, or increasing the water content of the samples. Increased peak temperatures led to increased spore inactivation and reduced mutation rates. The data suggested that besides the potential mechanical stress exerted by the shock pressure, the accompanying high peak temperatures were a critical stress parameter that spores had to cope with.

Microbial rock inhabitants survive hypervelocity impacts on Mars-like host planets: first phase of lithopanspermia experimentally tested.

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.

Ultraviolet radiation-induced limitation to epilithic microbial growth in arid deserts--dosimetric experiments in the hyperarid core of the Atacama Desert.

Experiments were conducted during November 2003 in the dry core of the Atacama Desert, Yungay, Chile to test the hypothesis that UV radiation, in environments where liquid water is not available, and thus enzymatic repair of UV-induced damage is inhibited, can prevent epilithic colonization. Novel dosimeters made from the cryptoendolithic, desiccation and radiation-resistant cyanobacterium Chroococcidiopsis sp. isolated from the dry Negev desert, Israel, showed that monolayers of this organism were killed within one day. The diurnal profile of microbial loss of viability was investigated with dosimeters of Bacillus subtilis, which similarly showed cell death within one day. Soil grains obtained from south of Yungay where liquid water is more abundant and transported to the hyperarid core showed killing of indigenous vegetative organisms within one day. Gypsum and mineral grain coverings of 1mm were sufficient to prevent measurable UV-induced damage of Chroococcidiopsis and B. subtilis after 8d exposure. These results show that under extreme desiccation and an ambient UV flux the surface of rocks can potentially be rendered sterile, but that millimetre thick mineral coverings can protect organisms from UV-induced killing, consistent with the observed patterns of lithophytic colonization in the Atacama Desert. These data further show that UV radiation can be an important limiting factor in surface biological rock weathering in arid regions.

Integrity of the DNA and Cellular Ultrastructure of Cryptoendolithic Fungi in Space or Mars Conditions: A 1.5-Year Study at the International Space Station.

The black fungi Cryomyces antarcticus and Cryomyces minteri are highly melanized and are resilient to cold, ultra-violet, ionizing radiation and other extreme conditions. These microorganisms were isolated from cryptoendolithic microbial communities in the McMurdo Dry Valleys (Antarctica) and studied in Low Earth Orbit (LEO), using the EXPOSE-E facility on the International Space Station (ISS). Previously, it was demonstrated that C. antarcticus and C. minteri survive the hostile conditions of space (vacuum, temperature fluctuations, and the full spectrum of extraterrestrial solar electromagnetic radiation), as well as Mars conditions that were simulated in space for a 1.5-year period. Here, we qualitatively and quantitatively characterize damage to DNA and cellular ultrastructure in desiccated cells of these two species, within the frame of the same experiment. The DNA and cells of C. antarcticus exhibited a higher resistance than those of C. minteri. This is presumably attributable to the thicker (melanized) cell wall of the former. Generally, DNA was readily detected (by PCR) regardless of exposure conditions or fungal species, but the C. minteri DNA had been more-extensively mutated. We discuss the implications for using DNA, when properly shielded, as a biosignature of recently extinct or extant life.

Lichens survive in space: results from the 2005 LICHENS experiment.

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 combined bacterial Lux-Fluoro test for the detection and quantification of genotoxic and cytotoxic agents in surface water: results from the "Technical Workshop on Genotoxicity Biosensing".

The bioassay Lux-Fluoro test was developed for the rapid detection and quantification of environmental pollutants with genotoxic and/or cytotoxic potential. This bacterial test system uses two different reporter genes whose gene products and their reactions, respectively, can be measured easily and simultaneously by optical methods. Genotoxicity is measured by the increase of bioluminescence in genetically modified bacteria which carry a plasmid with a complete lux operon for the enzyme luciferase from the marine photobacterium P. leiognathi under the control of a DNA-damage dependent so-called SOS promoter. If the deoxyribonucleic acid in these bacteria is damaged by a genotoxic chemical, the SOS promoter is turned on and the lux operon is expressed. The newly synthesized luciferase reacts immediately with its substrate thereby producing bioluminescence in a damage-proportional manner. In the second part of the system, genetically modified bacteria carry the gene for the green fluorescent protein (gfp) from the jellyfish Aequora victoria downstream from a constitutively expressed promoter. These bacteria are fluorescent under common growth conditions. If their cellular metabolism is disturbed by the action of cytotoxic chemicals, the fluorescence decreases in a dose-proportional manner. The combined Lux-Fluoro test is shown to be well suited for the biological assessment of the geno- and cytotoxicity of a series of model agents and environmental samples at the Technical Workshop on Genotoxicity Biosensing (TECHNOTOX).

STARLIFE-An International Campaign to Study the Role of Galactic Cosmic Radiation in Astrobiological Model Systems.

In-depth knowledge regarding the biological effects of the radiation field in space is required for assessing the radiation risks in space. To obtain this knowledge, a set of different astrobiological model systems has been studied within the STARLIFE radiation campaign during six irradiation campaigns (2013-2015). The STARLIFE group is an international consortium with the aim to investigate the responses of different astrobiological model systems to the different types of ionizing radiation (X-rays, γ rays, heavy ions) representing major parts of the galactic cosmic radiation spectrum. Low- and high-energy charged particle radiation experiments have been conducted at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. X-rays or γ rays were used as reference radiation at the German Aerospace Center (DLR, Cologne, Germany) or Beta-Gamma-Service GmbH (BGS, Wiehl, Germany) to derive the biological efficiency of different radiation qualities. All samples were exposed under identical conditions to the same dose and qualities of ionizing radiation (i) allowing a direct comparison between the tested specimens and (ii) providing information on the impact of the space radiation environment on currently used astrobiological model organisms. Key Words: Space radiation environment-Sparsely ionizing radiation-Densely ionizing radiation-Heavy ions-Gamma radiation-Astrobiological model systems. Astrobiology 17, 101-109.

AstRoMap European Astrobiology Roadmap.

The European AstRoMap project (supported by the European Commission Seventh Framework Programme) surveyed the state of the art of astrobiology in Europe and beyond and produced the first European roadmap for astrobiology research. In the context of this roadmap, astrobiology is understood as the study of the origin, evolution, and distribution of life in the context of cosmic evolution; this includes habitability in the Solar System and beyond. The AstRoMap Roadmap identifies five research topics, specifies several key scientific objectives for each topic, and suggests ways to achieve all the objectives. The five AstRoMap Research Topics are • Research Topic 1: Origin and Evolution of Planetary Systems • Research Topic 2: Origins of Organic Compounds in Space • Research Topic 3: Rock-Water-Carbon Interactions, Organic Synthesis on Earth, and Steps to Life • Research Topic 4: Life and Habitability • Research Topic 5: Biosignatures as Facilitating Life Detection It is strongly recommended that steps be taken towards the definition and implementation of a European Astrobiology Platform (or Institute) to streamline and optimize the scientific return by using a coordinated infrastructure and funding system.

Role of pigmentation in protecting Bacillus sp. endospores against environmental UV radiation.

Bacillus endospores show different kinds of pigmentation. Red-pigmented spores of Bacillus atrophaeus DSM 675, dark-gray spores of B. atrophaeus(T) DSM 7264 and light-gray spores of B. subtilis DSM 5611 were used to study the protective role of the pigments in their resistance to defined ranges of environmental UV radiation. Spores of B. atrophaeus DSM 675 possessing a dark-red pigment were 10 times more resistant to UV-A radiation than those of the other two investigated strains, whereas the responses to the more energetic UV-B and UV-C radiation were identical in all three strains. The methanol fraction of the extracted pigment from the spores absorbs in the associated wavelength area. These results indicate that the carotene-like pigment of spores of B. atrophaeus DSM 675 affects the resistance of spores to environmental UV-A radiation.

Survival of Antarctic Cryptoendolithic Fungi in Simulated Martian Conditions On Board the International Space Station.

UNLABELLED: Dehydrated Antarctic cryptoendolithic communities and colonies of the rock inhabitant black fungi Cryomyces antarcticus (CCFEE 515) and Cryomyces minteri (CCFEE 5187) were exposed as part of the Lichens and Fungi Experiment (LIFE) for 18 months in the European Space Agency's EXPOSE-E facility to simulated martian conditions aboard the International Space Station (ISS). Upon sample retrieval, survival was proved by testing colony-forming ability, and viability of cells (as integrity of cell membrane) was determined by the propidium monoazide (PMA) assay coupled with quantitative PCR tests. Although less than 10% of the samples exposed to simulated martian conditions were able to proliferate and form colonies, the PMA assay indicated that more than 60% of the cells and rock communities had remained intact after the "Mars exposure." Furthermore, a high stability of the DNA in the cells was demonstrated. The results contribute to assessing the stability of resistant microorganisms and biosignatures on the surface of Mars, data that are valuable information for further search-for-life experiments on Mars. KEY WORDS: Endoliths-Eukaryotes-Extremophilic microorganisms-Mars-Radiation resistance.

Generation of stably transfected Mammalian cell lines as fluorescent screening assay for NF-kappaB activation-dependent gene expression.

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB-dependent gene expression. The time course of NF-kappaB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kappaB-ELISA. NF-kappaB binding in-creased after 15-min incubation with TNF-alpha. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kappaB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-alpha to be caused by the activation of NF-kappaB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

Influence of ice and snow covers on the UV exposure of terrestrial microbial communities: dosimetric studies.

Bacillus subtilis spore biological dosimeters and electronic dosimeters were used to investigate the exposure of terrestrial microbial communities in micro-habitats covered by snow and ice in Antarctica. The melting of snow covers of between 5- and 15-cm thickness, depending on age and heterogeneity, could increase B. subtilis spore inactivation by up to an order of magnitude, a relative increase twice that caused by a 50% ozone depletion. Within the snow-pack at depths of less than approximately 3 cm snow algae could receive two to three times the DNA-weighted irradiance they would receive on bare ground. At the edge of the snow-pack, warming of low albedo soils resulted in the formation of overhangs that provided transient UV protection to thawed and growing microbial communities on the soils underneath. In shallow aquatic habitats, thin layers of heterogeneous ice of a few millimetres thickness were found to reduce DNA-weighted irradiances by up to 55% compared to full-sky values with equivalent DNA-weighted diffuse attenuation coefficients (K(DNA)) of >200 m(-1). A 2-mm snow-encrusted ice cover on a pond was equivalent to 10 cm of ice on a perennially ice covered lake. Ice covers also had the effect of stabilizing the UV exposure, which was often subject to rapid variations of up to 33% of the mean value caused by wind-rippling of the water surface. These data show that changing ice and snow covers cause relative changes in microbial UV exposure at least as great as those caused by changing ozone column abundance.

Microbial existence in controlled habitats and their resistance to space conditions.

The National Research Council (NRC) has recently recognized the International Space Station (ISS) as uniquely suitable for furthering the study of microbial species in closed habitats. Answering the NRC's call for the study, in particular, of uncommon microbial species in the ISS, and/or of those that have significantly increased or decreased in number, space microbiologists have begun capitalizing on the maturity, speed, and cost-effectiveness of molecular/genomic microbiological technologies to elucidate changes in microbial populations in the ISS and other closed habitats. Since investigators can only collect samples infrequently from the ISS itself due to logistical reasons, Earth analogs, such as spacecraft-assembly clean rooms, are used and extensively characterized for the presence of microbes. Microbiologists identify the predominant, problematic, and extremophilic microbial species in these closed habitats and use the ISS as a testbed to study their resistance to extreme extraterrestrial environmental conditions. Investigators monitor the microbes exposed to the real space conditions in order to track their genomic changes in response to the selective pressures present in outer space (external to the ISS) and the spaceflight (in the interior of the ISS). In this review, we discussed the presence of microbes in space research-related closed habitats and the resistance of some microbial species to the extreme environmental conditions of space.