Surface-Enhanced Raman Spectroscopy (SERS) for Identifying Traces of Adenine in Organic-Bearing Extraterrestrial Dust Analogs Captured in the Tanpopo Aerogel after Hypervelocity Impacts.

Raman spectroscopy is a non-destructive analytical technique for characterizing organic and inorganic materials with spatial resolution in the micrometer range. This makes it a method of choice for space-mission sample characterization, whether on return or in situ. To enhance its sensitivity, we use signal amplification via interaction with plasmonic silver-based colloids, which corresponds to surface-enhanced Raman scattering (SERS). In this study, we focus on the analysis of biomolecules of prebiotic interest on extraterrestrial dust trapped in silica aerogel, jointly with the Japanese Tanpopo mission. The aim is twofold: to prepare samples as close as possible to the real ones, and to optimize analysis by SERS for this specific context. Serpentinite was chosen as the inorganic matrix and adenine as the target biomolecule. The dust was projected at high velocity into the trapping aerogel and then mechanically extracted. A quantitative study shows effective detection even for adenine doping from a 5·10-9mol/L solution. After the dust has been expelled from the aerogel using a solvent, SERS mapping enables unambiguous adenine detection over the entire dust surface. This study shows the potential of SERS as a key technique not only for return samples, but also for upcoming new explorations.

Multidimensional vibrational circular dichroism for insect wings: Comparison of species.

This study reports the microscopic measurements of vibrational circular dichroism (VCD) on four different insect wings using a quantum cascade laser VCD system equipped with microscopic scanning capabilities (named multi-dimensional VCD [MultiD-VCD]). Wing samples, including (i) beetle, Anomala albopilosa (female), (ii) European hornet, Verspa crabro flavofasciata Cameron, 1903 (female), (iii) tiny dragonfly, Nannophya pygmae Rambur, 1842 (male), and (iv) dragonfly, Symetrum gracile Oguma, 1915 (male), were used in this study. Two-dimensional patterns of VCD signals (~10 mm × 10 mm) were obtained at a spatial resolution of 100 µm. Measurements covered the absorption peaks assigned to amides I and II in the range of 1500-1740 cm-1 . The measurements were based on the enhancement of VCD signals for the stereoregular linkage of peptide groups. The patterns were remarkably dependent on the species. In samples (i) and (ii), the wings comprised segregated domains of protein aggregates of different secondary structures. The size of each microdomain was approximately 100 µm. In contrast, no clear VCD spectra were detected in samples (iii) and (iv). One possible reason was that the chain of stereoregular polypeptides was too short to achieve VCD enhancement in samples (iii) and (iv). Notably, the unique features were only observed in the VCD spectra because the IR spectra were nearly the same among the species. The VCD results hinted at the connection of protein microscopic structures with the wing flapping mechanisms of each species.

Comparative analysis of reconstructed ancestral proteins with their extant counterparts suggests primitive life had an alkaline habitat.

To understand the origin and early evolution of life it is crucial to establish characteristics of the primordial environment that facilitated the emergence and evolution of life. One important environmental factor is the pH of the primordial environment. Here, we assessed the pH-dependent thermal stabilities of previously reconstructed ancestral nucleoside diphosphate kinases and ribosomal protein uS8s. The selected proteins were likely to be present in ancient organisms such as the last common ancestor of bacteria and that of archaea. We also assessed the thermal stability of homologous proteins from extant acidophilic, neutralophilic, and alkaliphilic microorganisms as a function of pH. Our results indicate that the reconstructed ancestral proteins are more akin to those of extant alkaliphilic bacteria, which display greater stability under alkaline conditions. These findings suggest that the common ancestors of bacterial and archaeal species thrived in an alkaline environment. Moreover, we demonstrate the reconstruction method employed in this study is a valuable technique for generating alkali-tolerant proteins that can be used in a variety of biotechnological and environmental applications.

A Comparative Study of Methods for Detecting Extraterrestrial Life in Exploration Missions to Mars and the Solar System II: Targeted Characteristics, Detection Techniques, and Their Combination for Survey, Detection, and Analysis.

We present a comparative study of the methods used in the search for extraterrestrial microorganism life, including a summary table where different life-detection techniques can be easily compared as an aid to mission and instrument design aimed at life detection. This is an extension of previous study, where detection techniques for a series of target characteristics and molecules that could constitute a positive life detection were evaluated. This comparison has been extended with a particular consideration to sources of false positives, the causes of negative detection, the results of detection techniques when presented regarding terrestrial life, and additional science objectives that could be achieved outside the primary aim of detecting life. These additions address both the scientific and programmatic side of exploration mission design, where a successful proposal must demonstrate probable outcomes and be able to return valuable results even if no life is found. The applicability of the life detection techniques is considered for Earth life, Earth-independent life (life emerging independently from that on Earth,) and Earth-kin life (sharing a common ancestor with life on Earth), and techniques effective in detecting Earth life should also be useful in the detection of Earth-kin life. However, their applicability is not guaranteed for Earth-independent life. As found in our previous study, there exists no realistic single detection method that can conclusively determine the discovery of extraterrestrial life, and no method is superior to all others. In this study, we further consider combinations of detection techniques and identify imaging as a valuable addition to molecule detection methods, even in cases where there is insufficient resolution to observe the detailed morphology of a microbial cell. The search for extraterrestrial life is further divided into a survey-and-detection and analysis-and-conclusion step. These steps benefit from different detection techniques, but imaging is necessary for both parts.

Complete Genome Sequence of the Radioresistant Bacterium Deinococcus aetherius ST0316, Isolated from Air Dust Collected in the Lower Stratosphere above Japan.

Deinococcus aetherius ST0316 is a radioresistant bacterium that possess proficient DNA repair capacity. Here, we report the complete genome sequence of D. aetherius, which was obtained by hybrid assembly using short- and long-read sequencing. This sequence will be important information for elucidating the unique DNA repair mechanism of Deinococcus bacteria.

Comparative study of methods for detecting extraterrestrial life in exploration mission of Mars and the solar system.

The detection and analysis of extraterrestrial life are important issues of space science. Mars is among the most important planets to explore for extraterrestrial life, owing both to its physical properties and to its ancient and present environments as revealed by previous exploration missions. In this paper, we present a comparative study of methods for detecting extraterrestrial life and life-related substances. To this end, we have classified and summarized the characteristics targeted for the detection of extraterrestrial life in solar system exploration mission and the methods used to evaluate them. A summary table is presented. We conclude that at this moment (i) there is no realistic single detection method capable of concluding the discovery of extraterrestrial life, (ii) no single method has an advantage over the others in all respects, and (iii) there is no single method capable of distinguishing extraterrestrial life from terrestrial life. Therefore, a combination of complementary methods is essential. We emphasize the importance of endeavoring to detect extraterrestrial life without overlooking possible alien life forms, even at the cost of tolerating false positives. Summaries of both the targets and the detection methods should be updated continuously, and comparative studies of both should be pursued. Although this study assumes Mars to be a model site for the primary environment for life searches, both the targets and detection methods described herein will also be useful for searching for extraterrestrial life in any celestial environment and for the initial inspection of returned samples.

In situ biochemical characterization of Venus cloud particles using a life-signature detection microscope.

Much of the information about the size and shape of aerosols forming haze and the cloud layer of Venus is obtained from indirect inferences from nephelometers on probes and from the analysis of the variation of polarization with the phase angle and the glory feature from images of Venus. The microscopic imaging of Venus' aerosols has recently been advocated. Direct measurements from a fluorescence microscope can provide information on the morphology, density, and biochemical characteristics of the particles; thus, fluorescence microscopy is attractive for in situ particle characterization of the Venus cloud layer. Fluorescence imaging of Venus cloud particles presents several challenges owing to the sulfuric acid composition and corrosive effects. In this article, we identify the challenges and describe our approach to overcoming them for a fluorescence microscope based on an in situ biochemical and physical characterization instrument for use in the clouds of Venus from a suitable aerial platform. We report that pH adjustment using alkali was effective for obtaining fluorescence images and that fluorescence attenuation was observed after the adjustment, even when the acidophile suspension in concentrated sulfuric acid was used as a sample.

Amino Acid Specificity of Ancestral Aminoacyl-tRNA Synthetase Prior to the Last Universal Common Ancestor Commonote commonote.

Extant organisms commonly use 20 amino acids in protein synthesis. In the translation system, aminoacyl-tRNA synthetase (ARS) selectively binds an amino acid and transfers it to the cognate tRNA. It is postulated that the amino acid repertoire of ARS expanded during the development of the translation system. In this study we generated composite phylogenetic trees for seven ARSs (SerRS, ProRS, ThrRS, GlyRS-1, HisRS, AspRS, and LysRS) which are thought to have diverged by gene duplication followed by mutation, before the evolution of the last universal common ancestor. The composite phylogenetic tree shows that the AspRS/LysRS branch diverged from the other five ARSs at the deepest node, with the GlyRS/HisRS branch and the other three ARSs (ThrRS, ProRS and SerRS) diverging at the second deepest node. ThrRS diverged next, and finally ProRS and SerRS diverged from each other. Based on the phylogenetic tree, sequences of the ancestral ARSs prior to the evolution of the last universal common ancestor were predicted. The amino acid specificity of each ancestral ARS was then postulated by comparison with amino acid recognition sites of ARSs of extant organisms. Our predictions demonstrate that ancestral ARSs had substantial specificity and that the number of amino acid types amino-acylated by proteinaceous ARSs was limited before the appearance of a fuller range of proteinaceous ARS species. From an assumption that 10 amino acid species are required for folding and function, proteinaceous ARS possibly evolved in a translation system composed of preexisting ribozyme ARSs, before the evolution of the last universal common ancestor.

Space Exposure of Amino Acids and Their Precursors during the Tanpopo Mission.

Amino acids have been detected in extraterrestrial bodies such as carbonaceous chondrites (CCs), which suggests that extraterrestrial organics could be the source of the first life on Earth, and interplanetary dust particles (IDPs) or micrometeorites (MMs) are promising carriers of extraterrestrial organic carbon. Some amino acids found in CCs are amino acid precursors, but these have not been well characterized. The Tanpopo mission was conducted in Earth orbit from 2015 to 2019, and the stability of glycine (Gly), hydantoin (Hyd), isovaline (Ival), 5-ethyl-5-methylhydantoin (EMHyd), and complex organics formed by proton irradiation from CO, NH3, and H2O (CAW) in space were analyzed by high-performance liquid chromatography and/or gas chromatography/mass spectrometry. The target substances showed a logarithmic decomposition over 1-3 years upon space exposure. Recoveries of Gly and CAW were higher than those of Hyd, Ival, and EMHyd. Ground simulation experiments showed different results: Hyd was more stable than Gly. Solar ultraviolet light was fatal to all organics, and they required protection when carried by IDPs/MMs. Thus, complex amino acid precursors (such as CAW) were possibly more robust than simple precursors during transportation to primitive Earth. The Tanpopo 2 mission is currently being conducted to expose organics to more probable space conditions.

Mutation Analysis of the rpoB Gene in the Radiation-Resistant Bacterium Deinococcus radiodurans R1 Exposed to Space during the Tanpopo Experiment at the International Space Station.

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase ß-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase ß-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins.

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

Mapping of Supramolecular Chirality in Insect Wings by Microscopic Vibrational Circular Dichroism Spectroscopy: Heterogeneity in Protein Distribution.

The supramolecular chirality of the hindwing of Anomala albopilosa (male) was investigated using a microscopic vibrational circular dichroism (VCD) system, denoted as MultiD-VCD. The source of intense infrared (IR) light for the system was a quantum cascade laser. Two-dimensional maps of IR and VCD spectra were taken by scanning the surface area (ca. 2 mm × 2 mm) of the insect hindwing tissue. The spectra ranged from 1500 to 1700 cm-1, and the maps have a spatial resolution of 100 µm. The distribution of proteins, including their supramolecular structures, was analyzed from the location-dependent spectral shape of the VCD bands assigned to amides I and II. The results revealed that the hindwing consists of segregated domains of proteins with different secondary structures: an α-helix (in one part of the membrane), a hybrid of α-helix and ß-sheet (in another part of the membrane), and a coil (in a vein).

Four-Year Operation of Tanpopo: Astrobiology Exposure and Micrometeoroid Capture Experiments on the JEM Exposed Facility of the International Space Station.

The Tanpopo experiment was the first Japanese astrobiology mission on board the International Space Station. It included exposure experiments of microbes and organic compounds as well as a capture experiment of hypervelocity impacting microparticles. We deployed three Exposure Panels, each consisting of 20 Exposure Units that contained microbes, organic compounds, an alanine UV dosimeter or an ionizing radiation dosimeter. The three Exposure Panels were situated on the zenith face of the Exposed Experiment Handrail Attachment Mechanism (ExHAM) that was pointing in zenith direction toward space, which was attached on a handrail of the Japanese Experiment Module (Kibo) Exposed Facility (JEM-EF) outside the International Space Station. The three Exposure Panels were one by one retrieved and returned to the ground after approximately 1, 2, and 3 years of exposure to the space environment. Capture Panels, each of which contained one or two blocks of amorphous silica aerogel, were exposed to collect hypervelocity impact microparticles. Possible captured particles may include micrometeoroids, human-made orbital debris, and natural terrestrial particles. Each year, Capture Panels containing from 11 to 12 aerogel blocks were attached to the three faces of the ExHAM (pointing to zenith, ram, and port); they remained in place for about 1 year and were then returned to the laboratory. This process was repeated three times, in total, during 2015-2018. Additional exposure of a Capture Panel facing ram was conducted between 2018 and 2019. Once the aerogel blocks were returned to the laboratory, they were encapsulated in dedicated transparent plastic cases and optically inspected by a specially designed microscopic system. Once located and recorded, hypervelocity impact signatures were excavated one by one and distributed for further detailed analyses. The apparatus, operation, and environmental factors of all the Tanpopo experiments are summarized in this article.

Scientific Targets of Tanpopo: Astrobiology Exposure and Micrometeoroid Capture Experiments at the Japanese Experiment Module Exposed Facility of the International Space Station.

The Tanpopo experiment was the first Japanese astrobiology mission on board the Japanese Experiment Module Exposed Facility on the International Space Station (ISS). The experiments were designed to address two important astrobiological topics, panspermia and the chemical evolution process toward the generation of life. These experiments also tested low-density aerogel and monitored the microdebris environment around low Earth orbit. The following six subthemes were identified to address these goals: (1) Capture of microbes in space: Estimation of the upper limit of microbe density in low Earth orbit; (2) Exposure of microbes in space: Estimation of the survival time course of microbes in the space environment; (3) Capture of cosmic dust on the ISS and analysis of organics: Detection of the possible presence of organic compounds in cosmic dust; (4) Alteration of organic compounds in space environments: Evaluation of decomposition time courses of organic compounds in space; (5) Space verification of the Tanpopo hyper-low-density aerogel: Durability and particle-capturing capability of aerogel; (6) Monitoring of the number of space debris: Time-dependent change in space debris environment. Subthemes 1 and 2 address the panspermia hypothesis, whereas 3 and 4 address the chemical evolution. The last two subthemes contribute to space technology development. Some of the results have been published previously or are included in this issue. This article summarizes the current status of the Tanpopo experiments.

Space Radiation Dosimetry at the Exposure Facility of the International Space Station for the Tanpopo Mission.

Radiation dosimetry was carried out at the exposure facility (EF) and the pressurized module (PM) of the Japanese Kibo module installed in the International Space Station as one study on environmental monitoring for the Tanpopo mission. Three exposure panels and three references including biological and organic samples and luminescence dosimeters were launched to obtain data for different exposure durations during 3 years from May 2015 to July 2018. The dosimeters were equipped with additional shielding materials (0.55, 2.95, and 6.23 g/cm2 mass thickness). The relative dose variation, as a function of shielding mass thickness, was observed and compared with Monte Carlo simulations with respect to galactic cosmic rays (GCRs) and typical solar energetic particles (SEPs). The mean annual dose rates were DEF = 231 ± 5 mGy/year at the EF and DPM = 82 ± 1 mGy/year at the PM during the 3 years. The PM is well shielded, and the GCR simulation indicated that the measured mean dose reduction ratio inside the module (DPM/DEF = 0.35) required ∼26 g/cm2 additional shielding mass thickness. Observed points of the dose reduction tendency could be explained by the energy ranges of protons (10-100 MeV), where the protons passed through, or were absorbed in, the shielding materials of different mass thickness that surrounded dosimeters.

Evolution of Superoxide Dismutases and Catalases in Cyanobacteria: Occurrence of the Antioxidant Enzyme Genes before the Rise of Atmospheric Oxygen.

Knowledge on the evolution of antioxidant systems in cyanobacteria is crucial for elucidating the cause and consequence of the rise of atmospheric oxygen in the Earth's history. In this study, to elucidate the origin and evolution of cyanobacterial antioxidant enzymes, we analyzed the occurrence of genes encoding four types of superoxide dismutases and three types of catalases in 85 complete cyanobacterial genomes, followed by phylogenetic analyses. We found that Fe superoxide dismutase (FeSOD), Mn superoxide dismutase (MnSOD), and Mn catalase (MnCat) are widely distributed among modern cyanobacteria, whereas CuZn superoxide dismutase (CuZnSOD), bifunctional catalase (KatG), and monofunctional catalase (KatE) are less common. Ni superoxide dismutase (NiSOD) is distributed among marine Prochlorococcus and Synechococcus species. Phylogenetic analyses suggested that bacterial MnSOD evolved from cambialistic Fe/MnSOD before the diversification of major bacterial lineages. The analyses suggested that FeSOD evolved from MnSOD before the origin of cyanobacteria. MnCat also evolved in the early stages of bacterial evolution, predating the emergence of cyanobacteria. KatG, KatE, and NiSOD appeared 2.3-2.5 billion years ago. Thus, almost all cyanobacterial antioxidant enzymes emerged before or during the rise of atmospheric oxygen. The loss and appearance of these enzymes in marine cyanobacteria may be also related to the change in the metal concentration induced by the increased oxygen concentration in the ocean.

Molecular repertoire of Deinococcus radiodurans after 1 year of exposure outside the International Space Station within the Tanpopo mission.

BACKGROUND: The extraordinarily resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space. Deinococcus radiodurans was exposed for 1 year outside the International Space Station within Tanpopo orbital mission to investigate microbial survival and space travel. In addition, a ground-based simulation experiment with conditions, mirroring those from low Earth orbit, was performed. METHODS: We monitored Deinococcus radiodurans cells during early stage of recovery after low Earth orbit exposure using electron microscopy tools. Furthermore, proteomic, transcriptomic and metabolomic analyses were performed to identify molecular mechanisms responsible for the survival of Deinococcus radiodurans in low Earth orbit. RESULTS: D. radiodurans cells exposed to low Earth orbit conditions do not exhibit any morphological damage. However, an accumulation of numerous outer-membrane-associated vesicles was observed. On levels of proteins and transcripts, a multi-faceted response was detected to alleviate cell stress. The UvrABC endonuclease excision repair mechanism was triggered to cope with DNA damage. Defense against reactive oxygen species is mirrored by the increased abundance of catalases and is accompanied by the increased abundance of putrescine, which works as reactive oxygen species scavenging molecule. In addition, several proteins and mRNAs, responsible for regulatory and transporting functions showed increased abundances. The decrease in primary metabolites indicates alternations in the energy status, which is needed to repair damaged molecules. CONCLUSION: Low Earth orbit induced molecular rearrangements trigger multiple components of metabolic stress response and regulatory networks in exposed microbial cells. Presented results show that the non-sporulating bacterium Deinococcus radiodurans survived long-term low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2 effectively provides a shield below 190 nm. These results should be considered in the context of planetary protection concerns and the development of new sterilization techniques for future space missions. Video Abstract.

Vibrational circular dichroism towards asymmetric catalysis: chiral induction in substrates coordinated with copper(II) ions.

Cu(ii) complexes containing RR- or SS-2,2'-isopropylidene-bis(4-phenyl-2-oxazoline) (denoted as [Cu(RR- or SS-oxa)]2+) are known to catalyse many asymmetric organic reactions. Herein, the source of enantioselectivity was investigated by vibrational circular dichroism (VCD) spectroscopy. An achiral ß-diketonato ligand (denoted as LH), such as 1-phenyl-1,3-butanedione and dibenzoylmethane, was added to form [Cu(RR- or SS-oxa)L]+. Clear VCD peaks were obtained from a CDCl3 solution of [Cu(RR- or SS-oxa)]2+ or [Cu(RR- or SS-oxa)L]+ at 1000-1800 cm-1. It is to be noted that when LH was coordinated, a new VCD peak appeared at ∼1380 cm-1, which was assigned to the C-O asymmetric stretching vibration of L-. Theoretical simulation helped rationalise the results in terms of the transformation of coordinated L- into a twisted chiral form. The extent of steric control within the coordination sphere was demonstrated, revealing the first step for enantioselectivity during catalysis.

DNA Damage and Survival Time Course of Deinococcal Cell Pellets During 3 Years of Exposure to Outer Space.

The hypothesis called "panspermia" proposes an interplanetary transfer of life. Experiments have exposed extremophilic organisms to outer space to test microbe survivability and the panspermia hypothesis. Microbes inside shielding material with sufficient thickness to protect them from UV-irradiation can survive in space. This process has been called "lithopanspermia," meaning rocky panspermia. We previously proposed sub-millimeter cell pellets (aggregates) could survive in the harsh space environment based on an on-ground laboratory experiment. To test our hypothesis, we placed dried cell pellets of the radioresistant bacteria Deinococcus spp. in aluminum plate wells in exposure panels attached to the outside of the International Space Station (ISS). We exposed microbial cell pellets with different thickness to space environments. The results indicated the importance of the aggregated form of cells for surviving in harsh space environment. We also analyzed the samples exposed to space from 1 to 3 years. The experimental design enabled us to get and extrapolate the survival time course to predict the survival time of Deinococcus radiodurans. Dried deinococcal cell pellets of 500 µm thickness were alive after 3 years of space exposure and repaired DNA damage at cultivation. Thus, cell pellets 1 mm in diameter have sufficient protection from UV and are estimated to endure the space environment for 2-8 years, extrapolating the survival curve and considering the illumination efficiency of the space experiment. Comparison of the survival of different DNA repair-deficient mutants suggested that cell aggregates exposed in space for 3 years suffered DNA damage, which is most efficiently repaired by the uvrA gene and uvdE gene products, which are responsible for nucleotide excision repair and UV-damage excision repair. Collectively, these results support the possibility of microbial cell aggregates (pellets) as an ark for interplanetary transfer of microbes within several years.