The search for biosignatures of past microbial life has promoted the interest in halophilic archaea trapped inside fluid inclusions of salt crystals. These hypersaline environments are promising targets for the preservation of microbial cell envelope biomolecules. In this study, we focused on the preservation of bacterioruberin, a carotenoid pigment found in the cell envelope of Halobacterium salinarum, within fluid inclusions of salt crystals mimicking early Mars environments and modern Earth. Halite (NaCl) and sylvite (KCl) crystals were subjected to Mars-like proton irradiation, and the preservation of carotenoids was assessed using in situ and ex situ Raman spectroscopy. Our findings demonstrate that Raman spectroscopy efficiently detected carotenoids within fluid inclusions in non-irradiated crystals. However, post-irradiation analyses posed great challenges due to fluorescence induced by the formation of colour centres in the crystal lattice, which suppressed the carotenoid signal. Cleavage of irradiated crystals revealed preserved carotenoid pigments beyond the radiation penetration depth, suggesting potential preservation of biomolecules in deeper inclusions within larger crystals. Furthermore, in some cases, carotenoids were detected even within fluorescent zones, suggesting extensive preservation. This study underscores the potential of Raman spectroscopy for the detection of carotenoids as biosignatures in planetary exploration contexts, particularly as a preliminary screening tool. However, it also highlights the need for optimized protocols to overcome fluorescence-related limitations. These findings contribute to the methodologies for detecting and interpreting biosignatures in salt deposits, advancing the search for possible traces of past microbial life beyond Earth.

The Martian regolith is known to contain a maximum of 0.5% (w/v) perchlorate (ClO4 −) that is toxic for most living organisms. With such high concentrations of perchlorates on Mars, is there any possibility of life? Here, in order to search and identify potential organisms on Earth, which could survive the perchlorate levels on Mars, we have isolated four perchlorate resistant, halophilic/halotolerant bacterial species from Big Soda Lake (BSL) in Nevada, USA. The 16S ribosomal RNA sequences revealed that these halophiles belong to the genera Bacillus, Alkalibacillus and Halomonas. Growth curves were obtained using a saline medium with different concentrations of magnesium, sodium and/or calcium perchlorate salt to simulate the Martian eutectic brine water. All four species, BSL1-4, grew in high saline media in the presence of perchlorates. This is the first growth experiment using multiple perchlorate salts. BSL3 relative to Halomonas salifodinae showed high maximum growth (Optical Density) comparing with other isolates in the presence of 1% perchlorate salts. Also, BSL1 relative to Bacillus licheniformis survived in the presence of 5% Na-perchlorate, but growth was slower in the absence of Na-perchlorate. The results revealed that these new model microbes are capable of tolerating the hypothesized hypersaline and perchlorate-rich Martian subsurface water environment. Perchlorate-resistant halophile would serve as a new model to understand the biochemistry that may occur on Mars.

Salt influences protein stability through electrostatic mechanisms as well as through nonpolar Hofmeister effects. In the present work, a continuum solvation based model is developed to explore the impact of salt on protein stability. This model relies on a traditional Poisson-Boltzmann (PB) term to describe the polar or electrostatic effects of salt, and a surface area dependent term containing a salt concentration dependent microscopic surface tension function to capture the non-polar Hofmeister effects. The model is first validated against a series of cold-shock protein variants whose salt-dependent protein fold stability profiles have been previously determined experimentally. The approach is then applied to HIV-1 protease in order to explain an experimentally observed enhancement in stability and activity at high (1M) NaCl concentration. The inclusion of the salt-dependent non-polar term brings the model into quantitative agreement with experiment, and provides the basis for further studies into the impact of ionic strength on protein structure, function, and evolution.