Future space experiment platforms for astrobiology and astrochemistry research.

Space experiments are a technically challenging but a scientifically important part of astrobiology and astrochemistry research. The International Space Station (ISS) is an excellent example of a highly successful and long-lasting research platform for experiments in space, that has provided a wealth of scientific data over the last two decades. However, future space platforms present new opportunities to conduct experiments with the potential to address key topics in astrobiology and astrochemistry. In this perspective, the European Space Agency (ESA) Topical Team Astrobiology and Astrochemistry (with feedback from the wider scientific community) identifies a number of key topics and summarizes the 2021 "ESA SciSpacE Science Community White Paper" for astrobiology and astrochemistry. We highlight recommendations for the development and implementation of future experiments, discuss types of in situ measurements, experimental parameters, exposure scenarios and orbits, and identify knowledge gaps and how to advance scientific utilization of future space-exposure platforms that are either currently under development or in an advanced planning stage. In addition to the ISS, these platforms include CubeSats and SmallSats, as well as larger platforms such as the Lunar Orbital Gateway. We also provide an outlook for in situ experiments on the Moon and Mars, and welcome new possibilities to support the search for exoplanets and potential biosignatures within and beyond our solar system.

Membrane lipid adaptation of soil Bacteroidetes isolates to temperature and pH.

3-hydroxy fatty acids (3-OH FAs) are characteristic components of the Gram-negative bacterial membrane, recently proposed as promising temperature and pH (paleo) proxies in soil. Nevertheless, to date, the relationships between the 3-OH FA distribution and temperature/pH are only based on empirical studies, with no ground truthing work at the microbial level. This work investigated the influence of growth temperature and pH on the lipid composition of three strains of soil Gram-negative bacteria belonging to the Bacteroidetes phylum. Even though non-hydroxy FAs were more abundant than 3-OH FAs in the investigated strains, our results suggest that 3-OH FAs are involved in the membrane adaptation of these bacteria to temperature. The strains shared a common adaptation mechanism to temperature, with a significant increase in the ratio of anteiso vs. iso or normal 3-OH FAs at lower temperature. In contrast with temperature, no common adaptation mechanism to pH was observed, as the variations in the FA lipid profiles differed from one strain to another. We suggest that models reconstructing environmental changes in soils should include the whole suite of 3-OH FAs present in the membrane of Gram-negative bacteria, as all of them could be influenced by temperature or pH at the microbial level.

Molecular acclimation of Halobacterium salinarum to halite brine inclusions.

Halophilic microorganisms have long been known to survive within the brine inclusions of salt crystals, as evidenced by the change in color for salt crystals containing pigmented halophiles. However, the molecular mechanisms allowing this survival has remained an open question for decades. While protocols for the surface sterilization of halite (NaCl) have enabled isolation of cells and DNA from within halite brine inclusions, "-omics" based approaches have faced two main technical challenges: (1) removal of all contaminating organic biomolecules (including proteins) from halite surfaces, and (2) performing selective biomolecule extractions directly from cells contained within halite brine inclusions with sufficient speed to avoid modifications in gene expression during extraction. In this study, we tested different methods to resolve these two technical challenges. Following this method development, we then applied the optimized methods to perform the first examination of the early acclimation of a model haloarchaeon (Halobacterium salinarum NRC-1) to halite brine inclusions. Examinations of the proteome of Halobacterium cells two months post-evaporation revealed a high degree of similarity with stationary phase liquid cultures, but with a sharp down-regulation of ribosomal proteins. While proteins for central metabolism were part of the shared proteome between liquid cultures and halite brine inclusions, proteins involved in cell mobility (archaellum, gas vesicles) were either absent or less abundant in halite samples. Proteins unique to cells within brine inclusions included transporters, suggesting modified interactions between cells and the surrounding brine inclusion microenvironment. The methods and hypotheses presented here enable future studies of the survival of halophiles in both culture model and natural halite systems.

Improved Isolation of SlaA and SlaB S-layer proteins in Sulfolobus acidocaldarius.

The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer into the tetraether lipid membrane. While SlaA has previously been purified by exploiting its thermotolerance and high resistance to detergents, SlaB has resisted isolation, particularly from the cell membrane. Removal of proteins other than those of the S-layer is especially difficult if large batch-scale culture volumes are unavailable. Here, we describe a benchtop-scale protocol for the purification of SlaA from S. acidocaldarius, enabling isolation of SlaB using size exclusion chromatography (gel filtration). Using this protocol, we were able to identify for the first time tetraether lipids strongly attached to SlaB via heat- and detergent-resistant interactions.

Exploring the microbial biotransformation of extraterrestrial material on nanometer scale.

Exploration of microbial-meteorite redox interactions highlights the possibility of bioprocessing of extraterrestrial metal resources and reveals specific microbial fingerprints left on extraterrestrial material. In the present study, we provide our observations on a microbial-meteorite nanoscale interface of the metal respiring thermoacidophile Metallosphaera sedula. M. sedula colonizes the stony meteorite Northwest Africa 1172 (NWA 1172; an H5 ordinary chondrite) and releases free soluble metals, with Ni ions as the most solubilized. We show the redox route of Ni ions, originating from the metallic Ni° of the meteorite grains and leading to released soluble Ni2+. Nanoscale resolution ultrastructural studies of meteorite grown M. sedula coupled to electron energy loss spectroscopy (EELS) points to the redox processing of Fe-bearing meteorite material. Our investigations validate the ability of M. sedula to perform the biotransformation of meteorite minerals, unravel microbial fingerprints left on meteorite material, and provide the next step towards an understanding of meteorite biogeochemistry. Our findings will serve in defining mineralogical and morphological criteria for the identification of metal-containing microfossils.

Novel Mechanism for Surface Layer Shedding and Regenerating in Bacteria Exposed to Metal-Contaminated Conditions.

Surface layers (S-layers) are components of the cell walls throughout the Bacteria and the Archaea that provide protection for microorganisms against diverse environmental stresses, including metal stress. We have previously characterized the process by which S-layers serve as a nucleation site for metal mineralization in an archaeon for which the S-layer represents the only cell wall component. Here, we test the hypothesis originally proposed in cyanobacteria that a "shedding" mechanism exists for replacing S-layers that have become mineral-encrusted, using Lysinibacillus sp. TchIII 20n38, metallotolerant gram-positive bacterium, as a model organism. We characterize for the first time a mechanism for resistance to metals through S-layer shedding and regeneration. S-layers nucleate the formation of Fe-mineral on the cell surface, depending on physiological state of the cells and metal exposure times, leading to the encrustation of the S-layer and changes in the cell morphology as observed by scanning electron microscopy. Using Nanoscale Secondary Ion Mass Spectrometry, we show that mineral-encrusted S-layers are shed by the bacterial cells after a period of latency (2 days under the conditions tested) in a heterogeneous fashion likely reflecting natural variations in metal stress resistance. The emerging cells regenerate new S-layers as part of their cell wall structure. Given the wide diversity of S-layer bearing prokaryotes, S-layer shedding may represent an important mechanism for microbial survival in metal-contaminated environments.

Preservation of Archaeal Surface Layer Structure During Mineralization.

Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer "ghosts" during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

Post-translational methylations of the archaeal Mre11:Rad50 complex throughout the DNA damage response.

The Mre11:Rad50 complex is central to DNA double strand break repair in the Archaea and Eukarya, and acts through mechanical and nuclease activities regulated by conformational changes induced by ATP binding and hydrolysis. Despite the widespread use of Mre11 and Rad50 from hyperthermophilic archaea for structural studies, little is known in the regulation of these proteins in the Archaea. Using purification and mass spectrometry approaches allowing nearly full sequence coverage of both proteins from the species Sulfolobus acidocaldarius, we show for the first time post-translational methylation of the archaeal Mre11:Rad50 complex. Under basal growth conditions, extensive lysine methylations were identified in Mre11 and Rad50 dynamic domains, as well as methylation of a few aspartates and glutamates, including a key Mre11 aspartate involved in nuclease activity. Upon γ-irradiation induced DNA damage, additional methylated residues were identified in Rad50, notably methylation of Walker B aspartate and glutamate residues involved in ATP hydrolysis. These findings strongly suggest a key role for post-translational methylation in the regulation of the archaeal Mre11:Rad50 complex and in the DNA damage response.

High-pressure tolerance in Halobacterium salinarum NRC-1 and other non-piezophilic prokaryotes.

In this study, we examined the high-pressure survival of a range of prokaryotes not found in high-pressure environments to determine the effects of adaptations to osmotic and oxidative stresses on piezo-resistance. The pressure survivals of Halobacterium salinarum NRC-1, Deinococcus radiodurans R1, and Chromohalobacter salexigens were compared to that of Escherichia coli MG1655. C. salexigens, which uses the compatible solute ectoine as an osmolyte, was as piezo-sensitive as E. coli MG1655, suggesting that ectoine is not a piezolyte. D. radiodurans R1 and H. salinarum NRC-1, both resistant to oxidative stress, were found to be highly piezo-resistant. H. salinarum NRC-1 showed nearly full survival after pressurization up to 400 MPa; a survival 3.5 log units higher than E. coli MG1655. This piezo-resistance was maintained in H. salinarum NRC-1 for pressurizations up to 1 h. We hypothesize that the high-pressure resistance of H. salinarum NRC-1 is due to a combination of factors including cell envelope structure and the presence of intracellular salts.

An integrated systems approach for understanding cellular responses to gamma radiation.

Cellular response to stress entails complex mRNA and protein abundance changes, which translate into physiological adjustments to maintain homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to (60)Co ionizing gamma radiation in an effort to understand the correlation between genetic information processing and physiological change. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein-DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation among several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady-state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation.

Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation.

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and 60Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and 60Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.