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.

40Adaption to environmental conditions is reflected by protein adaptation. In particular, 41 proteins of extremophiles display distinctive traits ensuring functional, structural and 42 dynamical properties under permanently extreme physical and chemical conditions. While it 43 has mostly been studied with approaches focusing on specific proteins, biophysical 44 approaches have also confirmed this link between environmental and protein adaptation at 45 the more complex and diverse scale of the proteome. However, studies of this type remain 46 challenging and often require large amounts of biological material. 47We report here the use of nanoDSF as a tool to study proteome stability and solubility 48 in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results 49 show that, as with single halophilic protein studies, proteome stability was correlated to the 50 concentration of NaCl or KCl under which the cells were lysed and hence the proteome 51 exposed. This work highlights that adaptation to environmental conditions can be 52 experimentally observed at the scale of the proteome. Still, we show that the biochemical 53 properties of single halophilic proteins can only be partially extrapolated to the whole 54 proteome. 55Page 2 of 23 Extremophiles 9 310 observed between H. marismortui proteome and MDH. Nonetheless, this stresses that the 311 behaviour of the proteome can only partially be extrapolated from that of a given enzyme. 312Our experimental setup allowed little to no observation of peaks of first derivative of 313 ratio of 350/330nm fluorescence during the cooling step following the heating. When 314 observed, these renaturation peaks were less distinctive than those associated with 315 denaturation. Moreover, no peak of first derivative of light scattering could be observed at all, 316 showing that aggregation process was also mostly irreversible. Since aggregation peaks 317 happened after denaturation peaks during the heating step, irreversible aggregation may be 318 the reason why renaturation was very limited, even with the pure MDH. In the case of the 319 proteome, the limited renaturation could be associated with a specific subset of proteins 320 whose denaturation was reversible, rather than a partial renaturation of the whole proteome. 321 This was unlike the ß-lactamase from the moderately halophilic bacterium Chromohalobacter 322 sp. 560 which has been shown to refold spontaneously after thermal denaturation in 0.2M 323 NaCl (Tokunaga et al. 2004). However, after being expressed in non-halophilic Escherichia coli 324 cells where it has undergone low salt denaturation, H. marismortui MDH can be refolded in 325 vitro by increasing the salinity (Cendrin et al. 1993;Franzetti et al. 2001). Similarly, a 326 nucleoside diphosphate kinase from the extremely halophilic archaea H. salinarum, which has 327 been expressed in E. coli, has also been shown to require high salt concentration for refolding 328 while being stable and active in low salinity conditions (Ishibashi et...

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 MgCl2, CaCl2 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 MgCl2 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.