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Extreme halophiles are microorganisms adapted to low water activity living at the upper salt concentration that life can tolerate. We review here recent data that specify the main factors, which determine their peculiar salt-dependent biochemistry. The data suggested that evolution proceeds by stage to modify the molecular dynamics properties of the entire proteome. Extreme halophiles therefore represent tractable models to understand how fast and to what extent microorganisms adapt to environmental changes. Halophiles are also robust organisms, capable to resist multiple stressors. Preliminary studies indicated that they have developed a cellular response specifically aimed to survive when the salt condition fluctuates. Because of these properties halophilic organisms deserve special attention in the search for traces of life on other planets.

Incoherent neutron scattering experiments have produced important insights into intracellular molecular dynamics in vivo. Selected results highlight the role of water dynamics in cancer and brain cells, as well as cellular adaptation through the evolution of appropriate molecular dynamics, in order to respond to environmental challenges.

Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contrast variation (H2O/D2O exchange) allows focussing selectively on the signal of specific proteins in multi-protein complexes or mixtures of isolated proteins. We illustrate this unique capacity by the example of a functional protein-degradation system in solution, the PAN-20S proteasome complex in the presence of a protein substrate, ssrA-tagged GFP. By comparing experimental SANS data with synthetic SAXS (small-angle X-ray scattering) data, predicted for the same system under identical conditions, we show that SANS, when combined with macromolecular deuteration and solvent contrast variation, can specifically focus on the conformation of the PAN unfoldase, even in the presence of very large GFP aggregates. Likewise, structural information of native GFP states can be visualized in detail, even in the presence of the much larger PAN-20S unfoldase-protease oligomers, which would dominate the overall scattering signal when using X-rays instead of neutrons.

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human-wildlife interactions along gradients of human influence., (© 2024. The Author(s).)

Evidence of stable liquid water oceans beneath the ice crust of moons within the Solar System is of great interest for astrobiology. In particular, subglacial oceans may present hydrothermal processes in their abysses, similarly to terrestrial hydrothermal vents. Therefore, terrestrial extremophilic deep life can be considered a model for putative icy moon extraterrestrial life. However, the comparison between putative extraterrestrial abysses and their terrestrial counterparts suffers from a potentially determinant difference. Indeed, some icy moons oceans may be so deep that the hydrostatic pressure would exceed the maximal pressure at which hydrothermal vent organisms have been isolated. While terrestrial microorganisms that are able to survive in such conditions are known, the effect of high pressure on fundamental biochemical processes is still unclear. In this study, the effects of high hydrostatic pressure on DNA synthesis catalyzed by DNA polymerases are investigated for the first time. The effect on both strand displacement and primer extension activities is measured, and pressure tolerance is compared between enzymes of various thermophilic organisms isolated at different depths.

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl 2 , CaCl 2 and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl 2 for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C 2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C 2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN-20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The recent discovery of extrasolar Earth-like planets that orbit in their habitable zone of their system, and the latest clues of the presence of liquid water in the subsurface of Mars and in the subglacial ocean of Jupiter's and Saturn's moons, has reopened debates about habitability and limits of life. Although liquid water, widely accepted as an absolute requirement for terrestrial life, may be present in other bodies of the solar system or elsewhere, physical and chemical conditions, such as temperature, pressure, and salinity, may limit this habitability. However, extremophilic microorganisms found in various extreme terrestrial environments are adapted to thrive in permanently extreme ranges of physicochemical conditions. This review first describes promising environments for life in the Solar System and the microorganisms that inhabit similar environments on the Earth. The effects of extreme temperatures, salt, and hydrostatic pressure conditions on biomolecules will be explained in some detail, and recent advances in understanding biophysical and structural adaptation strategies allowing microorganisms to cope with extreme physicochemical conditions are reviewed to discuss promising environments for life in the Solar System in terms of habitability.

Adaption to environmental conditions is reflected by protein adaptation. In particular, proteins of extremophiles display distinctive traits ensuring functional, structural and dynamical properties under permanently extreme physical and chemical conditions. While it has mostly been studied with approaches focusing on specific proteins, biophysical approaches have also confirmed this link between environmental and protein adaptation at the more complex and diverse scale of the proteome. However, studies of this type remain challenging and often require large amounts of biological material. We report here the use of nanoDSF as a tool to study proteome stability and solubility in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results show that, as with single halophilic protein studies, proteome stability was correlated to the concentration of NaCl or KCl under which the cells were lysed and hence the proteome exposed. This work highlights that adaptation to environmental conditions can be experimentally observed at the scale of the proteome. Still, we show that the biochemical properties of single halophilic proteins can only be partially extrapolated to the whole proteome., (© 2021. The Author(s), under exclusive licence to Springer Japan KK, part of Springer Nature.)

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Wild boar and feral swine number and range are increasing worldwide in parallel with their impact on biodiversity and human activities. The ecological and economic impact of this species include spread of diseases, vehicle collisions, damage to crops, amenities and infrastructures and reduction in plant and animal abundance and richness. As traditional methods such as culling have not contained the growth and spread of wild boar and feral pigs, alternative methods such as fertility control are now advocated. We used empirical data on two isolated wild boar populations to model and compare the effects of different regimes of culling and fertility control on population trends. We built a Bayesian population model and applied it to explore the implications for population control of various management options combining culling and/or contraception. The results showed that, whilst fertility control on its own was not sufficient to achieve the target reduction in wild boar number, adding fertility control to culling was more effective than culling alone. In particular, using contraceptives on 40% of the population to complement the culling of 60% of the animals, halved the time to achieve our target reduction compared with culling only. We conclude that, assuming the effort of adding fertility control to culling was found to be cost-effective in terms of population reduction, these two methods should be used simultaneously if a rapid decrease in wild boar number is required for a closed population., Competing Interests: The authors have declared that no competing interests exist.

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome by the crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating and phasing properties. This article presents examples of new crystallization conditions induced by the presence of Tb-Xo4. These new crystalline forms bypass crystal defects often encountered by crystallographers, such as low-resolution diffracting samples or crystals with twinning. Thanks to Tb-Xo4's high phasing power, the structure determination process is greatly facilitated and can be extended to serial crystallography approaches.

Hepatitis E virus (HEV) is an RNA virus causing an acute generally self-limited disease in humans. An increasing number of autochthonous cases linked to zoonotic transmission of HEV genotype 3 have been reported over the last 10 years in Europe. Pigs and wild boars are considered the main reservoirs. The principal route of transmission in Europe is food-borne, linked by direct or indirect evidence to the consumption of raw or undercooked pork products and wild boar meat. In this study, we sampled 92 wild boar (Sus scrofa) livers during active surveillance in five municipalities in Central Italy throughout the hunting season 2016-2017. HEV RNA was detected in 52.2% of liver sampled with prevalence ranging from 0.0% to 65.7%. HEV-positive wild boars were detected in all but one area of hunting. Phylogenetic analysis showed that strains clustered within the two subtypes HEV-3c and HEV-3f and displayed a wide range of phylogenetic diversity. Several strains were circulating in the areas investigated; animals possibly belonging to the same family group hunted by the same team were infected with a unique strain (100% nucleotide identity). As wild animals are a proven source of HEV transmission to humans and pigs, the high prevalence observed (mean 52.2%) poses a question on the risk of consuming raw or undercooked wild boar meat, and thus, this subject deserves further investigations., (© 2018 Blackwell Verlag GmbH.)