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In the past year we have been witnessing several important missions to Mars, including Mars 2020 Perseverance rover that landed to Jezero Crater to search for signs of ancient life. Multiple lines of evidence indicate an active hydrogeological history of Mars and chemolithoautotrophy-suited environments within its Noachian terrains. As a result, one of the primary aims of Mars missions is to search for signs of ancient life and collect a suite of samples to be returned to Earth via a Mars Sample Return mission. Being a few steps away from retrieving and returning the first Mars samples, we need to gain extensive knowledge how to access their potential biogenicity. In this connection, a valuable source of information can be extracted from microbial fingerprints of chemolithotrophic life based on Martian materials. Chemolithoautotrophy is the most ancient microbial form of life, which enables the transition of energy from a stone to the energy of a living entity. In our project, we investigate interactions of a wide variety of chemolithoautotrophs with Martian mineral materials (Martian meteorites and regolith simulants). Our recent research on the genuine Noachian Martian breccia “Black Beauty” permitted visualization and nanometer-scale imaging of microbial life designed and cultivated on Martian materials(1). Here we report on laboratory-scaled microbially assisted chemolithoautotrophic biotransformation(1) of the Noachian Martian breccia Northwest Africa (NWA) 7034 composed of ancient (~4.5 Gyr old) crustal materials from Mars. Nanoanalytical hyperspectral analysis provides clues for the trafficking and distribution of meteorite inorganic constituents in the microbial cell(1). We decipher biomineralization patterns associated with the biotransformation and reveal microbial nanometer-sized lithologies located inside the cell and on its outer surface layer(1). These investigations provide an opportunity to trace the putative bioalteration processes of the Martian crust and to assess the potential biogenicity of Martian materials. Our study on the Noachian Martian breccia composed of ~4.5 Gyr old crustal materials from Mars, delivered a prototype of microbial life experimentally designed on a real Martian material(1). This life of a pure Martian design is a rich source of Mars relevant biosignatures. 1. Milojevic, T., Albu, M., Kölbl, D. et al. Chemolithotrophy on the Noachian Martian breccia NWA 7034 via experimental microbial biotransformation. Commun Earth Environ 2, 39 (2021). https://doi.org/10.1038/s43247-021-00105-x

Aerosol phase elements such as phosphorus (P), sulfur (S), and metals including iron (Fe) are essential nutrients that could help sustain potential biodiversity in the cloud deck of Venus. While the presence of S and Fe in the venusian cloud deck has been broadly discussed (Zasova

With future long-term space exploration and life detection missions on Mars, understanding the microbial survival beyond Earth as well as the identification of past life traces on other planetary bodies becomes increasingly important. The series of the Tanpopo space mission experiments target a long-term exposure (one to three years) of microorganisms on the KIBO Module of the International Space Station (ISS) in the low Earth orbit (LEO) (Kawaguchi et al., 2020; Ott et al., 2020). In the search for possible past and/or present microbial life on Mars, metallophilic archaeal species are of a special interest due to their inherent extraordinary characteristics. Chemolithotrophic archaea (e.g., from the order Sulfolobales) employ a number of ancient metabolic pathways to extract energy from diverse inorganic electron donors and acceptors. Metallosphaera sedula, an iron- and sulfur-oxidizing chemolithotrophic archaeon, which flourishes under hot and acidic conditions (optimal growth at 74°C and pH 2.0), was cultivated on genuine extraterrestrial minerals (Milojevic et al., 2019; Milojevic et al., 2021) as well as synthetic Martian materials (Kölbl et al., 2017). In all cases, M. sedula cells were able to utilize given mineral materials as the sole energy source for cellular growth and proliferation. During the growth of M. sedula cells on these materials, a natural mineral impregnation and encrustation of microbial cells was achieved, followed by their preservation under the conditions of desiccation (Kölbl et al. 2020). Our studies indicate that this archaeon, when impregnated and encrusted with minerals, withstand long-term desiccation and can be even recovered from the dried samples to the liquid cultures (Kölbl et al., 2020). The achieved preservation of desiccated M. sedula cells facilitated our further survivability studies with this desiccated microorganism under simulated Mars-like environmental conditions and during the Tanpopo-4 space exposure experiment.Consequently, M. sedula cells grown on various mineral materials were exposed at the Astrobiology Space Simulation facilities (DLR, Cologne) to a simulated Martian environment for one month, to ensure their suitability for a long-term experiment on the ISS within the Tanpopo-4 mission. Recovery of cells after exposure to a simulated Martian environment shows an insignificant impairment in cellular growth compared to ground control cells.A set of desiccated M. sedula cells grown on Martian breccia NWA 7034 (Milojevic et al., 2021) has already been launched for 1 year outside the ISS to Mars-like conditions in frames of the Tanpopo-4 mission (2022-2023). Upon retrieval of cells from the ISS, we will evaluate survivability of this chemolithoautotroph protected by various mineral materials in Mars-like environment. Furthermore, high-resolution electron microscopy and (nano)-spectroscopy techniques will be applied to identify preservable biosignatures of chemolithotrophic life on Martian materials.Resolving the interface of microbial interactions with Mars minerals under the influence of destructive Martian environmental constrains can bring us closer to identify traces of unicellular life for current and future Mars exploration missions. We will also reveal a role of mineral encapsulation as a natural shielding crust for cell protection during exposure to simulated Martian conditions.References:Kawaguchi Y. et al., Front. Microbiol. 11:2050. (2020) doi: 10.3389/fmicb.2020.02050Kölbl D. et al., Front. Microbiol. 8, 1918 (2017) doi: 10.3389/fmicb.2017.01918Kölbl D. et al., Front. Astron. Space Sci. 7:41 (2020) doi: 10.3389/fspas.2020.00041Milojevic T. et al., Sci. Rep. 9, 18028 (2019). doi: 10.1038/s41598-019-54482-7Milojevic T. et al., Commun. Earth Environ. 2, 39 (2021) doi: 10.1038/s43247-021-00105-xOtt E. et al., Microbiome 8, 150 (2020) doi: 10.1186/s40168-020-00927-5

Two missions are on Mars looking for traces of life (MSL and Mars 2020) while the ExoMars mission, slated for launch later this year, is postponed. All missions are equipped with a powerful array of instrumentation for in situ analyses. In addition, the ExoMars rover, Rosalind Franklin, has the advantage of a 2 m drill to penetrate beneath the oxidised and UV-irradiated surface [1]. However, unless phototrophic life forms were able to develop on a planet that was never permanently habitable and that was characterised by punctuated and likely unconnected habitable niches [2, 3], martian life likely remained in a very primitive state of evolution, potentially similar to terrestrial chemotrophs, i.e. discrete, very small cells and colonies that leave even more subtle fossil remains. Indeed, only in the vicinity of nutrient-rich hydrothermal environments can chemotrophic biomass develop to create potentially visible morphological signatures in the form of a clotted texture in the associated sediments – a texture that is not immediately identifiable as a biosignature but that would warrant further analysis of such rocks by other instruments, such as SAM on Curiosity, SHERLOC on Perseverance, or MOMA on Rosalind Franklin. Taking the early terrestrial environment and early life as analogues for habitable environments and primitive life forms on early Mars, it is becoming clearer that the most primitive of life forms known on Earth, chemolithotrophs that obtain energy from oxidation of inorganic substrates (e.g. ferrous iron, hydrogen sulfide, elemental sulfur, thiosulfate, or ammonia), were widely distributed but, because of their very small size (< 1 µm diameter) and very small, heterogeneously distributed colonies (5 µm to some tens of microns), are typically undetectable through bulk analyses (bulk carbon contents are generally 0.01-0.05 %.) (Fig. 1). To compound the challenge, morphologically-recognisable cells are rarely observed, while degraded cells mixed with generic extracellular polymeric substances (EPS) are more frequent. Chemolithotrophs can inhabit sediment particle surfaces and pore space close to the surface or at depth, as long as they have access to nutrients and water. But how to identify them in situ on Mars? Even on Earth with well-equipped, state of the art laboratories, detection and identification of fossil chemolithotrophs is difficult and, often, controversial. The solution requires seeking to simultaneously detect structural information of the organisms’ presence and analyse well-preserved organogeochemical signature with spatial precisions on the order of a micron and detection limits as low as possible. This would be a challenge for Mars Sample Return in the near future. Figure 1. (A-C) distribution of tiny colonies and EPS clumps (red circles) in a silicified, volcanic sediment [3] at increasing magnifications. Individual dividing cells visible only at maximum resolution in (C). References: [1] Vago, J.L., et al. (2017) Astrobiology, 17: 471–510. [2] Cockell et al., 2012. Icarus, 217:184–193. [3] Westall et al., (2015) Astrobiology, 17: 998-1029. [4] Author J. et al. (2002) LPS XXXIII, Abstract #2110.

BackgroundThe extraordinarily resistant bacteriumDeinococcus radioduranswithstands harsh environmental conditions present in outer space.Deinococcus radioduranswas 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.MethodsWe monitoredDeinococcus radioduranscells 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 ofDeinococcus radioduransin low Earth orbit.ResultsD. radioduranscells 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.ConclusionLow 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 bacteriumDeinococcus radioduranssurvived long-term low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2effectively 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.

Rapidly evolving space exploration makes understanding the short- and long- term effects of microgravity on humans, plants, and microorganisms an important task. The ubiquitous presence of the gravitational force has had an influence on the development of all living entities on Earth, and short- and long-term changes in perceived gravitational force can induce notable changes within cells. Deinococcus radiodurans is the Gram-positive bacterium that is best known for its extreme resistance to UV-C and gamma radiation, oxidation stress, and desiccation. Thus increased interest has been placed on this species in the context of space research. The present study aims to elucidate the short-term proteomic response of this species to real microgravity during parabolic flight. Overnight cultures of D. radiodurans were subjected to microgravity during a single parabola, and metabolic activity was quenched using methanol. Proteins were extracted and subsequently measured using HPLC nESI MS/MS. The results, such as the enrichment of the peptidoglycan biosynthesis pathway with differentially abundant proteins and altered S-layer protein abundance, suggested molecular rearrangements in the cell envelope of D. radiodurans. Altered abundance of proteins involved in energy metabolism and DNA repair could be linked with increased endogenous ROS production that contributes to the stress response. Moreover, changes in protein abundance in response to microgravity show similarities with previously reported stress responses. Thus, the present results could be used to further investigate the complex regulation of the remarkable stress management of this bacterium.

The search for life beyond Earth has focused on Mars and the icy moons Europa and Enceladus, all of which are considered a safe haven for life due to evidence of current or past water. The surface of Venus, on the other hand, has extreme conditions that make it a nonhabitable environment to life as we know it. This is in contrast, however, to its cloud layer, which, while still an extreme environment, may prove to be a safe haven for some extreme forms of life similar to extremophiles on Earth. We consider the venusian clouds a habitable environment based on the presence of (1) a solvent for biochemical reactions, (2) appropriate physicochemical conditions, (3) available energy, and (4) biologically relevant elements. The diversity of extreme microbial ecosystems on Earth has allowed us to identify terrestrial chemolithoautotrophic microorganisms that may be analogs to putative venusian organisms. Here, we hypothesize and describe biological processes that may be performed by such organisms in the venusian clouds. To detect putative venusian organisms, we describe potential biosignature detection methods, which include metal-microbial interactions and optical methods. Finally, we describe currently available technology that can potentially be used for modeling and simulation experiments.

The interest in the possibility of life on Venus is driven not just by curiosity about life originating in another Earth-like environment, but because of the possibility that life may be playing a critical role in the planet’s present, and possibly its past, atmospheric state. The brilliance of Venus in the night sky (as viewed from Earth) is due to its highly reflective cloud cover, about 28 km thick at the equator. Its spectral albedo is about 90% at wavelengths > 500 nm, but it drops gradually to about 40% around 370 nm before rising slightly at shorter wavelengths. This albedo drop is due to the presence of several absorbers in the atmosphere and the cloud cover. A very large fraction of the energy absorbed by Venus is at ultraviolet wavelengths with sulfur dioxide above the clouds contributing to the absorption below 330 nm; however, the identities of the other absorbers remain unknown. The inability to identify the absorbers that are responsible for determining the radiative energy balance of Venus over the last century is a major impediment to understanding how the planet “works”, a major component of NASA’s efforts in planetary exploration. Limaye et al. (Astrobiology 18, 1181-1198, 2018) presented a hypothesis suggesting that cloud-based microbial life could be contributors to the spectral signatures of Venus’ clouds, building upon previous suggestions of the possibility of life in the clouds of Venus.Four interconnected themes for the exploration of Venus as an astrobiology target are: – (i) investigations focused on the likelihood that liquid water existed on the surface in the past leading to the potential for the origin and evolution of life, (ii) investigations into the potential for habitable zones within Venus’ clouds and Venus-like atmospheres, (iii) theoretical investigations into how active aerobiology may impact the radiative energy balance of Venus’ clouds and Venus-like atmospheres, and (iv) application of these investigative themes towards better understanding the atmospheric dynamics and habitability of exoplanets. These themes can serve as a basis for proposed Venus Astrobiology Objectives and suggestions for measurements for future missions, as per the goals and objectives developed by the Venus Exploration Analysis Group (VEXAG), which is sponsored by NASA to plan for the future exploration of Venus. A Venus Collection to be published in Astrobiology journal in 2021 will include papers from the “Habitability of the Venus Cloud Layer”, Moscow (October 2019) workshop.

第35回宇宙環境利用シンポジウム(2021年1月19日-20日. オンライン開催), Space Utilization Research (January 19-20, 2021. Online Meeting), 著者人数: 15名, 資料番号: SA6000156027, レポート番号: S-03

第21回宇宙科学シンポジウム (2021年1月6日-7日. オンライン開催), 21st Space Science Symposium (January 6-7, 2021. Online Meeting), 著者人数: 27名 (所属. 宇宙航空研究開発機構宇宙科学研究所(JAXA)(ISAS): 矢野, 創), Number of authors: 27 (Affiliation. Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency(JAXA)(ISAS): YANO, Hajime), 資料番号: SA6000163204, レポート番号: Pc.13

We present a case for the exploration of Venus as an astrobiology target—(1) investigations focused on the likelihood that liquid water existed on the surface in the past, leading to the potential for the origin and evolution of life, (2) investigations into the potential for habitable zones within Venus’ present-day clouds and Venus-like exo atmospheres, (3) theoretical investigations into how active aerobiology may impact the radiative energy balance of Venus’ clouds and Venus-like atmospheres, and (4) application of these investigative approaches toward better understanding the atmospheric dynamics and habitability of exoplanets. The proximity of Venus to Earth, guidance for exoplanet habitability investigations, and access to the potential cloud habitable layer and surface for prolonged in situ extended measurements together make the planet a very attractive target for near term astrobiological exploration.

The tungsten-microbial interactions and microbial bioprocessing of tungsten ores, which are still underexplored, are the focus of the current study. Here we show that the biotransformation of tungsten mineral scheelite performed by the extreme thermoacidophile Metallosphaera sedula leads to the breakage of scheelite structure and subsequent tungsten solubilization. Total soluble tungsten is significantly higher in cultures containing M. sedula grown on scheelite than the abiotic control, indicating active bioleaching. Advanced analytical electron microscopy was used in order to achieve nanoscale resolution ultrastructural studies of M. sedula grown on tungsten bearing scheelite. In particular, we describe that M. sedula mediated the biotransformation of scheelite, which was accompanied by the release of tungsten into solution and tungsten biomineralization of the cell surface. Furthermore, we observed intracellular incorporation of redox heterogenous Mn- and Fe-containing nano-clusters. Our results highlight unique metallophilic life in hostile environments extending the knowledge of tungsten biogeochemistry. Based on these findings biohydrometallurgical processing of tungsten ores can be further explored. Importantly, biogenic tungsten carbide-like nanolayers described herein are potential targets for developing nanomaterial biotechnology.

Regarding future space exploration missions and long-term exposure experiments, a detailed investigation of all factors present in the outer space environment and their effects on organisms of all life kingdoms is advantageous. Influenced by the multiple factors of outer space, the extremophilic bacterium Deinococcus radiodurans has been long-termly exposed outside the International Space Station in frames of the Tanpopo orbital mission. The study presented here aims to elucidate molecular key components in D. radiodurans, which are responsible for recognition and adaptation to simulated microgravity. D. radiodurans cultures were grown for two days on plates in a fast-rotating 2-D clinostat to minimize sedimentation, thus simulating reduced gravity conditions. Subsequently, metabolites and proteins were extracted and measured with mass spectrometry-based techniques. Our results emphasize the importance of certain signal transducer proteins, which showed higher abundances in cells grown under reduced gravity. These proteins activate a cellular signal cascade, which leads to differences in gene expressions. Proteins involved in stress response, repair mechanisms and proteins connected to the extracellular milieu and the cell envelope showed an increased abundance under simulated microgravity. Focusing on the expression of these proteins might present a strategy of cells to adapt to microgravity conditions.

This protocol describes a procedure how primary metabolites and proteins can be extracted from Deinococcus radiodurans simultaneously. Polar metabolites are purified and derivatized for GC-MS (TOF) analysis. Proteins are tryptically digested in-gel and peptides are prepared for bottom-up proteomics by LC-MS (orbitrap).

The multiple extremes resistant bacterium Deinococcus radiodurans is able to withstand harsh conditions of simulated outer space environment. The Tanpopo orbital mission performs a long-term space exposure of D. radiodurans aiming to investigate the possibility of interplanetary transfer of life. The revealing of molecular machinery responsible for survivability of D. radiodurans in the outer space environment can improve our understanding of underlying stress response mechanisms. In this paper, we have evaluated the molecular response of D. radiodurans after the exposure to space-related conditions of UVC irradiation and vacuum. Notably, scanning electron microscopy investigations showed that neither morphology nor cellular integrity of irradiated cells was affected, while integrated proteomic and metabolomic analysis revealed numerous molecular alterations in metabolic and stress response pathways. Several molecular key mechanisms of D. radiodurans, including the tricarboxylic acid cycle, the DNA damage response systems, ROS scavenging systems and transcriptional regulators responded in order to cope with the stressful situation caused by UVC irradiation under vacuum conditions. These results reveal the effectiveness of the integrative proteometabolomic approach as a tool in molecular analysis of microbial stress response caused by space-related factors.

A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A2a adenosine receptor, a Gs-coupled receptor. Usp4 binds to the carboxyl terminus of the A2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.