In our latest paper we demonstrated that magnesium perchlorate can depress the onset of intracellular vitrification in Bacillus subtilis cells. But why did we do that and why does it matter?
Well it comes back to the fact that Mars is a very cold place, and if you want to know whether there’s life on Mars, you need to know whether life could be there. For Mars, temperatures of -70°C are common, both on the surface and in the subsurface. Therefore, one of the central questions in Martian astrobiology is, can you have active life at such low temperatures? To answer this question you have to investigate the mechanisms which are believed to set the limits to life at low temperatures. But what are they?
Well first off, all life needs liquid water in order to survive, and at ambient pressure water freezes at 0°C. So there’s our first constraint. We also know that ice formation inside of cells is a considerable problem for life as ice can easily pierce membranes and rupture cells. But let’s assume you can prevent ice forming, either from the presence of ions in the environment or from the production of molecules such as glycerol, what sets the next low temperature limit for life?
Answering this is where things have gotten a bit more tricky. The current low temperature record holder is Planococcus halocryophilus, a bacterium isolated from the Canadian high arctic. It’s capable of replicating at -15°C and is metabolically active down to -25°C. Beyond that, kinetic data from enzymatic studies suggested that life may be able to maintain itself at any deep subzero temperature, as enzymatic rates of repair always exceeded those of spontaneous molecular damage. Note however that maintaining cellular structure is a world away from growth and division. But beyond P. halocryophilus and the kinetic arguments, it was exceedingly hard to find life much below -20°C. Therefore, in 2013 Andrew Clarke and his co-authors argued that it was the onset of intracellular vitrification occurring at ~-20°C which was setting a physical limit to life at low temperatures. But what is intracellular vitrification?
Intracellular vitrification is the process by which the interior of a cell transitions from being liquid and dynamic, to becoming an incredibly viscous glass-like substance. As biomolecular diffusion ceases beyond the glass transition, so does biochemical processes and as such life is suspended in a cryopreserved state. It’s worth making a distinction here about what is meant exactly by vitrification and glass transitions as these terms are used by multiple fields to mean slightly different things. In the field of cryopreservation, vitrification is used to mean the process by which biological material (cells, seeds, tissue) are preserved without ice formation i.e. your entire sample vitrifies, water and all. This is commonly achieved by using cryoprotectants to depress ice formation, and instead induce a glassy state which lacks sharp ice crystals which cause cellular damage. In the environmental context vitrification is slightly different. In the environment, intracellular vitrification occurs as the extracellular environment begins to freeze, thus concentrating solutes which generates an osmotic gradient across cell membranes. This osmotic gradient forces water out of cells and the cell shrinks. This process of ice formation, solute concentration, and intracellular water loss continues until you reach a point at which there is no longer sufficient intracellular water to maintain biomolecular diffusion, and consequently the intracellular space undergoes a glass transition. Or so goes the theory. Also a quick apology to the physicists and material scientists, we know that “glasses” and “glass transitions” mean very precise and detailed events in your fields, but we’ve simply stolen them as they’re the closest terms to what we’re trying to describe. But now that we know what vitrification is, what does it have to do with life on Mars?
Well if vitrification occurs at roughly -20°C for most unicellular organisms, and Mars is far colder than that, then we’d have to conclude that without some mechanism to depress vitrification, there can be no active life on Mars. Sad. But wait, vitrification requires liquid water to freeze, and as luck would have it Mars is covered in salts which are exceptionally good at depressing the freezing point of water. Here enters perchlorate salts to this story, and the genesis of my research question. If perchlorate salts can depress the freezing point of water, and vitrification only occurs after water has frozen, do perchlorate salts also then depress the onset of intracellular vitrification?
This is the research question which took me to Fernanda Fonseca’s lab in Paris, one of the original co-authors in the 2013 paper on the low temperature limit for life. The plan was simple, grow some Bacillus subtilis, incubate it in various concentrations of magnesium perchlorate, then measure it’s intracellular vitrification, easy. Why B. subtilis? Practicality. I was on a travelling fellowship so only had two months to complete this work. As such, I needed a workhorse model organism that would grow rapidly to large amounts, and was also well characterised in the literature. B. subtilis ticked both these boxes, and it also helps that we already knew exactly how it behaved in response to perchlorate salts. How do you measure intracellular vitrification? In our case we used a differential scanning calorimeter, or DSC for short. A DSC is a piece of equipment which can measure the difference in energy required to heat or cool a sample compared to a reference, hence the differential part of the name. Briefly, if you heat your sample and it melts, it takes energy to break all those bonds, and a DSC “sees” this by having to put more energy into the sample to keep it at the same temperature as the reference. On the other hand if your sample starts to give out heat, then the DSC works less hard and can then record this as requiring less energy to heat your sample. In our case, we were looking for thermal signals during the heating ramp, and so technically we were measuring de-vitrification. As de-vitrification is going from glass to liquid, an energy requiring process, we were looking for endothermic events in our DSC traces. For B. subtilis, its intracellular vitrification without perchlorate occurred at ~-23°C, so fairly consistent with what Clarke et al. had reported for other microbes. But, as we added magnesium perchlorate, its intracellular vitrification began to depress, so much so that by 2.5 M magnesium perchlorate, the vitrification peak was depressed to -83°C. Therefore, we demonstrated that not only do perchlorate salts depress the freezing point of water, but that in doing so they also depress the onset of intracellular vitrification, the process believed to set the low temperature limit for life.
Does this mean there’s active life on Mars? Of course not. We simply demonstrated that the physical process of vitrification can be depressed by perchlorate salts, which unfortunately bring with them a host of other problems. Chief among these is the fact that there’s no known organism capable of tolerating eutectic concentrations of magnesium perchlorate. Then we have other issues such as protein unfolding at subzero temperatures and membrane stiffness. As ever, these issues will be explored in future research, as will other aspects of intracellular vitrification. But what’s coming out of this research is hopefully an altered view of how we think of perchlorate salts with regards to biology. As an archetype for chaotropicity they’ve always been seen as deleterious towards life, but this is a simplification of a complex web of interactions which change across temperature, pressure, salt concentration and cation species. So going forward we need to determine how life can utilise the benefits of perchlorate salts, while also mitigating their negative effects. Do that, and we might get close to making a Martian.
