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.

May 26, 2024

Here I am, back again after a long hiatus talking about the effects of perchlorate salts on chymotrypsin, but this time it’s not bad news! The literature on the effects of chaotropic molecules (perchlorates included) on enzyme activity and stability overwhelmingly shows that they are deleterious to enzymes, something I have been able to show myself through my own research. But this time, I get to write about how perchlorate salts can actually increase enzyme activity at low temperatures.

So how did this research idea even come about? It all started when two of my research interests, extremophilic enzymes and perchlorates, basically collided into each other. All of my previous research has shown that perchlorate salts reduced the enzymatic activity and structural stability of my model enzyme, chymotrypsin. On the face of it, these effects are bad news for biochemistry and life in perchlorate rich environments. But is structural instability always a bad thing?

When might an enzyme being less stable, actually be beneficial for its activity? This brings us to the psychrophilic enzymes, which come from organisms that are uniquely adapted to life in cold environments. Psychrophilic enzymes are generally characterised by a few notable features; they exhibit greater enzymatic activity at low temperatures compared to their mesophilic and thermophilic counterparts, which is obviously beneficial for the organism expressing them. These psychrophilic enzymes also tend to lose their activity at lower temperatures and are more susceptible to unfolding by chaotropic molecules. An important point here is that the structural instability and low temperature activity of psychrophilic enzymes are not separate phenomena, they are linked, and wonderfully so. Because psychrophilic enzymes are less stable, they are routinely reported to have increased flexibility, and it is this flexibility which actually allows them to be active at low temperatures! You can even increase the low temperature activity of an enzyme by simply making it more flexible by introducing a single glycine substitution. Quick caveat: this doesn’t always work, but it works often enough that we can view it as a general rule. We can also look at the flexibility-activity relationship from a thermodynamic viewpoint, which really brings the whole picture together. The key to increasing low temperature enzyme activity is to make the reaction require less activation energy (make ΔH^‡ smaller). As psychrophilic enzymes have less stabilising interactions, fewer of these interactions need to be broken in order to facilitate catalysis and as a result you need less energy input to start the reaction. This isn’t a free meal, the reduced activation energy comes with a price. As there are less stabilising interactions, and the enzyme is more flexible as a result, it means that to form the activated enzyme-substrate complex, you must pay a larger entropic cost (ΔS^‡ becomes more negative). These are the thermodynamic hallmarks of psychrophilic enzymes, a lower energy of activation and a more negative entropy of activation.

After researching all this, I realised that I might be able to link the psychrophilic activity-flexibility relationship to my own work with perchlorates and chymotrypsin. My thought was, if perchlorate salts partially unfold my enzyme, thus reducing the number of stabilising interactions, would this make my enzyme more flexible and then potentially more active at low temperatures? I became convinced that this should be the case when I found a couple of rare examples of chaotropic molecules increasing enzyme activity at room temperature. All I had to do now was test the hypothesis.

The resulting data did indeed show that perchlorate salts (0.25M) could increase the activity of chymotrypsin at 5°C by ~10% despite perchlorates lowering chymotrypsin activity at ambient temperatures. The thermodynamic analysis suggested that this stemmed from a reduced enthalpy of activation, as had been predicted, but that the effect was diminished by an increasingly negative entropy of activation. These two facets suggested to me that perchlorate induced flexibility was probably the cause of this increased enzyme activity. However the picture wasn’t that simple. Higher concentrations of perchlorate salts (0.5M) did not increase the enzyme activity over the assayed temperature range, however the results suggested that by ~0°C these higher perchlorate concentrations should confer increased activity. So what emerged was this complex interaction between enzyme activity, temperature, and perchlorate concentrations. It’s worth noting in general that enzymes don’t work frozen in ice, and perchlorate salts depress the freezing point of water, so provided an enzyme can remain sufficiently folded to be active, perchlorate salts effectively offer an avenue for activity where there’d be no activity without them at subzero temperatures.

So perchlorate salts aren’t necessarily as bad for biology as we think, at least in colder environments. But that then raises the question of how cells behave at low temperatures in the presence of perchlorates. A question which will hopefully be answered and published soon.

Jul 8, 2023