Elke Rabbow

Tardigrades survive exposure to space in low Earth orbit

Summary Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space [1]. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation [2–4]. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.

Survival of Rock-Colonizing Organisms After 1.5 Years in Outer Space

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.

Resistance of Antarctic black fungi and cryptoendolithic communities to simulated space and Martian conditions.

Dried colonies of the Antarctic rock-inhabiting meristematic fungi Cryomyces antarcticus CCFEE 515, CCFEE 534 and C. minteri CCFEE 5187, as well as fragments of rocks colonized by the Antarctic cryptoendolithic community, were exposed to a set of ground-based experiment verification tests (EVTs) at the German Aerospace Center (DLR, Köln, Germany). These were carried out to test the tolerance of these organisms in view of their possible exposure to space conditions outside of the International Space Station (ISS). Tests included single or combined simulated space and Martian conditions. Responses were analysed both by cultural and microscopic methods. Thereby, colony formation capacities were measured and the cellular viability was assessed using live/dead dyes FUN 1 and SYTOX Green. The results clearly suggest a general good resistance of all the samples investigated. C. minteri CCFEE 5187, C. antarcticus CCFEE 515 and colonized rocks were selected as suitable candidates to withstand space flight and long-term permanence in space on the ISS in the framework of the LIchens and Fungi Experiments (LIFE programme, European Space Agency).

Biological dosimetry of solar radiation for different simulated ozone column thicknesses.

During the Spacelab mission D-2, in the experiment RD-UVRAD, precalibrated biofilms consisting of dry monolayers of immobilised spores of Bacillus subtilis (strain Marburg) were exposed, for defined intervals, to extraterrestrial solar radiation filtered through an optical filtering system, to simulate different ozone column thicknesses. After the mission, the biofilms were processed and optical densities indicative of any biological activity were determined for each exposure condition by image analysis. For the different simulated ozone column thicknesses, biologically effective irradiances were experimentally determined from the biofilm data and compared with calculated data using a radiative transfer model and the known biofilm action spectrum. The data show a strong increase in biologically effective solar UV irradiance with decreasing (simulated) ozone concentrations. The full spectrum of extraterrestrial solar radiation leads to an increment of the biologically effective irradiance by nearly three orders of magnitude compared with the solar spectrum at the surface of the Earth for average total ozone columns.

EXPOSE-E: an ESA astrobiology mission 1.5 years in space.

The multi-user facility EXPOSE-E was designed by the European Space Agency to enable astrobiology research in space (low-Earth orbit). On 7 February 2008, EXPOSE-E was carried to the International Space Station (ISS) on the European Technology Exposure Facility (EuTEF) platform in the cargo bay of Space Shuttle STS-122 Atlantis. The facility was installed at the starboard cone of the Columbus module by extravehicular activity, where it remained in space for 1.5 years. EXPOSE-E was returned to Earth with STS-128 Discovery on 12 September 2009 for subsequent sample analysis. EXPOSE-E provided accommodation in three exposure trays for a variety of astrobiological test samples that were exposed to selected space conditions: either to space vacuum, solar electromagnetic radiation at >110 nm and cosmic radiation (trays 1 and 3) or to simulated martian surface conditions (tray 2). Data on UV radiation, cosmic radiation, and temperature were measured every 10 s and downlinked by telemetry. A parallel mission ground reference (MGR) experiment was performed on ground with a parallel set of hardware and samples under simulated space conditions. EXPOSE-E performed a successful 1.5-year mission in space.

Exposure of phototrophs to 548 days in low earth orbit: microbial selection pressures in outer space and on early earth

An epilithic microbial community was launched into low Earth orbit, and exposed to conditions in outer space for 548 days on the European Space Agency EXPOSE-E facility outside the International Space Station. The natural phototroph biofilm was augmented with akinetes of Anabaena cylindrica and vegetative cells of Nostoc commune and Chroococcidiopsis. In space-exposed dark controls, two algae (Chlorella and Rosenvingiella spp.), a cyanobacterium (Gloeocapsa sp.) and two bacteria associated with the natural community survived. Of the augmented organisms, cells of A. cylindrica and Chroococcidiopsis survived, but no cells of N. commune. Only cells of Chroococcidiopsis were cultured from samples exposed to the unattenuated extraterrestrial ultraviolet (UV) spectrum (>110 nm or 200 nm). Raman spectroscopy and bright-field microscopy showed that under these conditions the surface cells were bleached and their carotenoids were destroyed, although cell morphology was preserved. These experiments demonstrate that outer space can act as a selection pressure on the composition of microbial communities. The results obtained from samples exposed to >200 nm UV (simulating the putative worst-case UV exposure on the early Earth) demonstrate the potential for epilithic colonization of land masses during that time, but that UV radiation on anoxic planets can act as a strong selection pressure on surface-dwelling organisms. Finally, these experiments have yielded new phototrophic organisms of potential use in biomass and oxygen production in space exploration.

Damage Escape and Repair in Dried Chroococcidiopsis spp. from Hot and Cold Deserts Exposed to Simulated Space and Martian Conditions

The cyanobacterium Chroococcidiopsis, overlain by 3 mm of Antarctic sandstone, was exposed as dried multilayers to simulated space and martian conditions. Ground-based experiments were conducted in the context of Lichens and Fungi Experiments (EXPOSE-E mission, European Space Agency), which were performed to evaluate, after 1.5 years on the International Space Station, the survival of cyanobacteria (Chroococcidiopsis), lichens, and fungi colonized on Antarctic rock. The survival potential and the role played by protection and repair mechanisms in the response of dried Chroococcidiopsis cells to ground-based experiments were both investigated. Different methods were employed, including evaluation of the colony-forming ability, single-cell analysis of subcellular integrities based on membrane integrity molecular and redox probes, evaluation of the photosynthetic pigment autofluorescence, and assessment of the genomic DNA integrity with a PCR-based assay. Desiccation survivors of strain CCMEE 123 (coastal desert, Chile) were better suited than CCMEE 134 (Beacon Valley, Antarctica) to withstand cellular damage imposed by simulated space and martian conditions. Exposed dried cells of strain CCMEE 123 formed colonies, maintained subcellular integrities, and, depending on the exposure conditions, also escaped DNA damage or repaired the induced damage upon rewetting.

Role of DNA Protection and Repair in Resistance of Bacillus subtilis Spores to Ultrahigh Shock Pressures Simulating Hypervelocity Impacts

ABSTRACT 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) (α/β-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.

SOS-LUX- and LAC-FLUORO-TEST for the quantification of genotoxic and/or cytotoxic effects of heavy metal salts ☆

Abstract The constantly increasing industrial, agricultural and domestic activities with their growing risk of contaminating fresh water and ground water by discharged compounds lead to an increasing concern focused on the quality of water. Because of their simplicity and sensitivity, bacterial tests play an important role in the detection and screening of genotoxins or cytotoxins in water. One of those bacterial tests is the SOS- LUX - and LAC- FLUORO -TEST, which is a combination of two bioassays, that simultaneously measures the genotoxicity (SOS- LUX -TEST) and the cytotoxicity (LAC- FLUORO -TEST) of substances and mixtures of substances. The SOS- LUX -TEST is based on genetically modified Salmonella typhimurium TA1535 bacteria, which have been transformed with the plasmid pPLS-1 carrying the promoterless lux operon of Photobacterium leiognathi as reporter element under the control of a DNA damage-dependent SOS promoter from ColD as sensing element. This system reacts in a dose-dependent manner to agents which induce DNA damages inside these bacterial cells with the production of bioluminescence that can easily be measured. The analogous LAC- FLUORO -TEST has been developed for the detection of cellular responses to cytotoxins. It is based on the constitutive expression of green fluorescent protein (GFP) mediated by the bacterial protein expression vector pGFPuv (Clontech, Palo Alto, USA). In response to cytotoxic agents, this system reacts with a dose-dependent reduction of GFP-fluorescence. A panel of recombinant S. typhimurium strains carrying either the SOS- LUX plasmid or the fluorescence-mediating lac-GFPuv plasmid was used to determine in parallel on one microplate the genotoxic and the cytotoxic potential of heavy metal salts like K 2 Cr 2 O 7 , CrCl 3 , ZnSO 4 , CuSO 4 , NiSO 4 , KH 2 AsO 4 and As 2 O 3 at the same time. Light and fluorescence emission of untreated and chemical-treated cells were measured in a microplate luminometer–fluorometer–photometer combination and the luminescence induction as well as the fluorescence reduction were used to determine the genotoxic and/or cytotoxic potential of the heavy metal salts.

The SOS-LUX-LAC-FLUORO-Toxicity-test on the International Space Station (ISS)

In the 21st century, an increasing number of astronauts will visit the International Space Station (ISS) for prolonged times. Therefore it is of utmost importance to provide necessary basic knowledge concerning risks to their health and their ability to work on the station and during extravehicular activities (EVA) in free space. It is the aim of one experiment of the German project TRIPLE-LUX (to be flown on the ISS) to provide an estimation of health risk resulting from exposure of the astronauts to the radiation in space inside the station as well as during extravehicular activities on one hand, and of exposure of astronauts to unavoidable or as yet unknown ISS-environmental genotoxic substances on the other. The project will (i) provide increased knowledge of the biological action of space radiation and enzymatic repair of DNA damage, (ii) uncover cellular mechanisms of synergistic interaction of microgravity and space radiation and (iii) examine the space craft milieu with highly specific biosensors. For these investigations, the bacterial biosensor SOS-LUX-LAC-FLUORO-Toxicity-test will be used, combining the SOS-LUX-Test invented at DLR Germany (Patent) with the commercially available LAC-FLUORO-Test. The SOS-LUX-Test comprises genetically modified bacteria transformed with the pBR322-derived plasmid pPLS-1. This plasmid carries the promoterless lux operon of Photobacterium leiognathi as a reporter element under control of the DNA-damage dependent SOS promoter of ColD as sensor element. This system reacts to radiation and other agents that induce DNA damages with a dose dependent measurable emission of bioluminescence of the transformed bacteria. The analogous LAC-FLUORO-Test has been developed for the detection of cellular responses to cytotoxins. It is based on the constitutive expression of green fluorescent protein (GFP) mediated by the bacterial protein expression vector pGFPuv (Clontech, Palo Alto, USA). In response to cytotoxic agents, this system reacts with a dose-dependent reduction of GFP-fluorescence. Currently, a fully automated miniaturized hardware system for the bacterial set up, which includes measurements of luminescence and fluorescence or absorption and the image analysis based evaluation is under development. During the first mission of the SOS-LUX-LAC-FLUORO-Toxicity-Test on the ISS, a standardized, DNA-damaging radiation source still to be determined will be used as a genotoxic inducer. A panel of recombinant Salmonella typhimurium strains carrying either the SOS-LUX plasmid or the fluorescence-mediating lac-GFPuv plasmid will be used to determine in parallel on one microplate the genotoxic and the cytotoxic action of the applied radiation in combination with microgravity. Either in addition to or in place of the fluorometric measurements of the cytotoxic agents, photometric measurements will simultaneously monitor cell growth, giving additional data on survival of the cells. The obtained data will be available on line during the TRIPLE-LUX mission time. Though it is the main goal during the TRIPLE-LUX mission to measure the radiation effect in microgravity, the SOS-LUX-LAC-FLUORO-Toxicity-test in principle is also applicable as a biomonitor for the detection and measurement of genotoxic substances in air or in the (recycled) water system on the ISS or on earth in general.