Limits of life in MgCl₂‐containing environments: chaotropicity defines the window

<jats:title>Summary</jats:title><jats:p>The biosphere of planet Earth is delineated by physico‐chemical conditions that are too harsh for, or inconsistent with, life processes and maintenance of the structure and function of biomolecules. To define the window of life on Earth (and perhaps gain insights into the limits that life could tolerate elsewhere), and hence understand some of the most unusual biological activities that operate at such extremes, it is necessary to understand the causes and cellular basis of systems failure beyond these windows. Because water plays such a central role in biomolecules and bioprocesses, its availability, properties and behaviour are among the key life‐limiting parameters. Saline waters dominate the Earth, with the oceans holding 96.5% of the planet's water. Saline groundwater, inland seas or saltwater lakes hold another 1%, a quantity that exceeds the world's available freshwater. About one quarter of Earth's land mass is underlain by salt, often more than 100 m thick. Evaporite deposits contain hypersaline waters within and between their salt crystals, and even contain large subterranean salt lakes, and therefore represent significant microbial habitats. Salts have a major impact on the nature and extent of the biosphere, because solutes radically influence water's availability (water activity) and exert other activities that also affect biological systems (e.g. ionic, kosmotropic, chaotropic and those that affect cell turgor), and as a consequence can be major stressors of cellular systems. Despite the stressor effects of salts, hypersaline environments can be heavily populated with salt‐tolerant or ‐dependent microbes, the halophiles. The most common salt in hypersaline environments is NaCl, but many evaporite deposits and brines are also rich in other salts, including MgCl<jats:sub>2</jats:sub> (several hundred million tonnes of bischofite, MgCl<jats:sub>2</jats:sub>·6H<jats:sub>2</jats:sub>O, occur in one formation alone). Magnesium (Mg) is the third most abundant element dissolved in seawater and is ubiquitous in the Earth's crust, and throughout the Solar System, where it exists in association with a variety of anions. Magnesium chloride is exceptionally soluble in water, so can achieve high concentrations (&gt; 5 M) in brines. However, while NaCl‐dominated hypersaline environments are habitats for a rich variety of salt‐adapted microbes, there are contradictory indications of life in MgCl<jats:sub>2</jats:sub>‐rich environments. In this work, we have sought to obtain new insights into how MgCl<jats:sub>2</jats:sub> affects cellular systems, to assess whether MgCl<jats:sub>2</jats:sub> can determine the window of life, and, if so, to derive a value for this window. We have dissected two relevant cellular stress‐related activities of MgCl<jats:sub>2</jats:sub> solutions, namely water activity reduction and chaotropicity, and analysed signatures of life at different concentrations of MgCl<jats:sub>2</jats:sub> in a natural environment, namely the 0.05–5.05 M MgCl<jats:sub>2</jats:sub> gradient of the seawater : hypersaline brine interface of Discovery Basin – a large, stable brine lake almost saturated with MgCl<jats:sub>2</jats:sub>, located on the Mediterranean Sea floor. We document here the exceptional chaotropicity of MgCl<jats:sub>2</jats:sub>, and show that this property, rather than water activity reduction, inhibits life by denaturing biological macromolecules. <jats:italic>In vitro</jats:italic>, a test enzyme was totally inhibited by MgCl<jats:sub>2</jats:sub> at concentrations below 1 M; and culture medium with MgCl<jats:sub>2</jats:sub> concentrations above 1.26 M inhibited the growth of microbes in samples taken from all parts of the Discovery interface. Although DNA and rRNA from key microbial groups (sulfate reducers and methanogens) were detected along the entire MgCl<jats:sub>2</jats:sub> gradient of the seawater : Discovery brine interface, mRNA, a highly labile indicator of active microbes, was recovered only from the upper part of the chemocline at MgCl<jats:sub>2</jats:sub> concentrations of less than 2.3 M. We also show that the extreme chaotropicity of MgCl<jats:sub>2</jats:sub> at high concentrations not only denatures macromolecules, but also preserves the more stable ones: such indicator molecules, hitherto regarded as evidence of life, may thus be misleading signatures in chaotropic environments. Thus, the chaotropicity of MgCl<jats:sub>2</jats:sub> would appear to be a window‐of‐life‐determining parameter, and the results obtained here suggest that the upper MgCl<jats:sub>2</jats:sub> concentration for life, in the absence of compensating (e.g. kosmotropic) solutes, is about 2.3 M.</jats:p>