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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 importantenvironmental 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.

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 Expo...

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

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.

This paper presents a review of the space exploration for life signature search with a special focus on the fluorescence microscope we developed for the life signature search on Mars and in other sites. Considering where, what, and how to search for life signature is essential. Life signature search exploration can be performed on the Mars surface and underground, on Venus’ cloud, moon, asteroids, icy bodies (e.g., moons of Jupiter and Saturn), and so on. It is a useful strategy to consider the targeted characteristics that may be similar to those of terrestrial microorganisms, which are microorganisms with uniform spherical or rod structures with approximately 1 μm diameter surrounded by a membrane having a metabolic activity and mainly made of carbon-based molecules. These characteristics can be analyzed by using a fluorescence microscope and a combination of fluorescence pigments with specific staining characteristics to distinguish the microorganism characteristics. Section 1 introduces the space exploration for life signature search. Section 2 reviews the scientific instruments and achievements of past and ongoing Mars exploration missions closely related to astrobiology. Section 3 presents the search targets and analysis of astrobiology. Section 4 discusses the extraterrestrial life exploration methods that use a microscope together with other methods (based on mass spectrometry, morphology, detection of growth, movement, and death, etc. for microscopic and macroscopic organism). Section 5 expounds on the life signature detection fluorescence microscope, for which we have manufactured a bread board model and tested for extraterrestrial life exploration.

Surface pressure versus molecular area (π-A) curves were measured on pure water as a subphase for a series of N,N′-diperfluoroalkanoyl-1,2(R,R)-diaminocyclohexanes. A molecule is denoted as RR-CFn,...

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.

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.

An ultrathin film was prepared by hybridizing a cationic Ir(III) complex with exfoliated nanosheets of clay minerals such as natural montmorillonite (denoted by MON) or synthetic saponite (denoted ...

Solid-state vibrational circular dichroism (SD-VCD) spectra were measured on the ion-exchange adducts of sodium montmorillonite and [Cu(RR- or SS-oxa)]2+ as prepared in the presence of a substrate ...

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, NH

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium

This work reviews the search for extraterrestrial life signatures with a special focus on the fluorescence microscope that we have been developing for the life-signature search on Mars and other sites. The surface and subterranean Mars, clouds of Venus, the Moon, asteroids, icy bodies, such as the moons of Jupiter and Saturn, and so on are important sites for life-signature exploration in the solar system. One possible exploration strategy is to target characteristics similar to those in terrestrial life, such as microorganisms with metabolic activity and similar uniform small structures microbes surrounded by a membrane that primarily comprise carbon-based molecules. These characteristics can be analyzed with fluorescence microscopy, which has a high spatial resolution and employs a combination of fluorescent pigments to distinguish microbial properties. Following an introduction, the life signature search and astrobiological analysis of the targeted characteristics are discussed. The extraterrestrial life exploration methods using a microscope are described. Also, other methods, including mass spectrometry, the sterilization-and-comparison method (detection of ability to die), proliferation, and analysis of shape, color, growth, or movement, are discussed. Lastly, we overview the life-signature detection fluorescence microscope that we have been developing, and present the Bread Board Model of it.

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.

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.

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 β-sheet (in another part of the membrane), and a coil (in a vein).

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/cm

The optimum growth temperature of ancestral cyanobacteria inhabiting the sea surface in the Neoproterozoic was estimated based on the thermal stability of experimentally reconstructed ancestral NDK enzymes. Ancestral NDKs of cyanobacteria that diversified ∼1.7, ∼1.0, ∼0.9, ∼0.7, ∼0.6, and ∼0.5 billion years ago were reconstructed and analyzed, and the unfolding midpoint temperatures ( T m s) ranged from ∼65 °C to ∼70 °C. Among the host of analyzed NDKs, the ancestors of marine, planktonic α-cyanobacteria diversified ≤ ∼1.0 Ga are highly likely to have inhabited marine environments during the Neoproterozoic, while ancestral cyanobacteria diversified ∼1.7 billion years ago were possibly marine but the habitat is less constrained compared to the others. According to the calibration curves derived from extant organisms, the obtained T m s of α-cyanobacteria diversified ≤ ∼1.0 Ga correspond to the range of optimum growth temperatures of around ∼33–48 °C. The temperature range agrees well with the long-term sea temperature trend during Neoproterozoic suggested by δ 18 O and δ 30 Si records from marine cherts. Adaptation to the low temperature during the snowball glaciations in the late Neoproterozoic was not observed, implying that adaptation of optimum growth conditions to the episodic low temperature may not have been necessary. Therefore, ancestral marine plankton must have consistently adapted to the interglacial sea surface temperature in the Neoproterozoic, which was approximately 5–20 °C higher than that is today. They may have survived the glaciations by acquiring cold tolerance and/or by suppressing growth rate.

(S)-3-O-Geranylgeranylglyceryl phosphate synthase (GGGPS) catalyzes the initial ether-bond formation betweensn-glycerol 1-phosphate (G1P) and geranylgeranyl pyrophosphate to synthesize (S)-3-O-geranylgeranylglyceryl phosphate in the production of an archaeal cell-membrane lipid molecule. Archaeal GGGPS proteins are divided into two groups (group I and group II). In this study, the crystal structure of the archaeal group II GGGPS fromThermoplasma acidophilum(TaGGGPS) was determined at 2.35 Å resolution. The structure of TaGGGPS showed that it has a TIM-barrel fold, the third helix of which is disordered (α3*), and that it forms a homodimer, although a pre-existing structure of an archaeal group II GGGPS (fromMethanothermobacter thermautotrophicus) showed a hexameric form. The structure of TaGGGPS showed the precise G1P-recognition mechanism of an archaeal group II GGGPS. The structure of TaGGGPS and molecular-dynamics simulation analysis showed fluctuation of the β2–α2, α3* and α5a regions, which is predicted to be important for substrate uptake and/or product release by TaGGGPS.

Vibrational circular dichroism (VCD) spectra were recorded for the intercalation compounds of sodium montmorillonite co-adsorbing two kinds of metal complexes, Δ- (or Λ-)[Ru(phen)3]2+ and Λ- (or Δ-...

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.

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.

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 β-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.

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.

Due to circumstances beyond the presenter's control, an audio recording was not possible for this presentation.

Proteins that specifically bind to metals have been used for research on development of new organic-inorganic hybrid materials. Several peptides and proteins that bind to metals have been reported; this property can be attributed to their structures. In this paper, we assessed the binding of a sequence of the protein called LARFH to a metal surface. We performed simulation with several models of LARFH protein with different loop sequences binding to the metal surface. It was found that the angle between the LARFH protein and the metal surface varied for different loop sequences. We also performed simulations of the detachment process of LARFH. It was concluded that the stronger binding to metal is established by two gold-binding loops of the LARFH sequences.