The Extraordinary Bacterial Proteins That Make Snow

Crisp bluebird skies, fresh powder that makes it feel like you’re floating through air and burning wobbly legs at the end of a long and challenging run; there’s no doubt about it – skiing is the best!

Unfortunately, the reality of skiing as we know it is changing rapidly as Earth’s climate warms. The average ski season during the years 2000 to 2019 was roughly a week shorter compared to 1960-1979. A week doesn’t seem like that long, but this represents an average loss of $250 million for the skiing industry each year in the United States alone (Igini, 2024).

One way that ski resorts are mitigating shorter seasons and economic losses is by making artificial snow. Water is pumped into these artificial snowmakers that blow snow out onto the ski slopes (Figure 1). But, did you know that bacterial proteins play a crucial role in this process?

Ice Formation

At the molecular level, water is very chaotic. Water molecules rapidly flow and do not have a fixed orientation relative to neighboring water molecules (Figure 1). This is why we can drink water out of a cup, but not out of our hands without water spilling out all over the place.

But ice, solid water, is very different. Water molecules take on a fixed orientation relative to their surrounding molecules and form a crystal lattice (Figure 1). This is why you can easily slide your hand through water, but not through a big block of ice.

Figure 1. Water molecules (pink circles) move rapidly and do not have a fixed position relative to one another in the liquid phase (left). When water freezes to form ice, those molecules form a lattice and take on fixed positions relative to the other water molecules.

Depending on which temperature scale you’re using, water freezes at 0 degrees Celsius or 32 degrees Fahrenheit. These are the numbers we’re all familiar with in our everyday life, but technically this is not correct. The true freezing temperature of pure water is -40 degrees Celsius. In real life, however, water freezes at warmer temperatures due to a couple of reasons. First, water in our environment is not pure water – there are salts, metal ions, other small molecules, and even biological organisms intermixed with the water. Second, water in our environment is not in a vacuum, it interfaces with different surfaces that can help it freeze at relatively higher temperatures (Figure 2). This second point is how bacterial INPs help water freeze – they position water molecules into the right pattern to facilitate ice lattice formation, as we’ll dive into deeper in a couple of sections.

Figure 2. Bacterial

INP (green) positions water molecules (pink circles) at the right spacing to

form the first row of an ice lattice (faded pink circles and gray lines).

Bacterial INPs in Nature

But before we get to the atomic details, why do bacteria even freeze water anyway? These bacteria are plant pathogens that use INPs to nucleate ice formation to rupture plant cells (Lindow et al, 1982). In this context, nucleate means that the protein initiates or “seeds” ice formation by serving as a surface for ice crystals to grow off of (Figure 2). We’ll discuss more below about how exactly these bacterial proteins stimulate ice formation. Once these pathogens used their ice spears to disrupt the plant’s cell wall, they can then siphon off nutrients and further infiltrate the plant to enhance their own growth and survival.

These bacteria cause blight in crops such as corn and wheat, but for a long time it was difficult to tell where exactly these bacteria were coming from. In one example in Montana,

farmers had taken great care to remove all of the disease-causing bacteria from their fields and only use seeds that were bacteria free, and yet the crops still suffered blight.

Dr. David Sands, a plant pathology professor at Montana State University, figured that the infectious agent must be coming through the air. Indeed, in samples collected by sticking a petri dish out of the window of an airplane, Sands was able to show that the bacterium Pseudomonas syringae was falling down on these fields out of the sky (Christner et al, 2008; Tsang 2019).

We now know that the INPs from bacteria such as P. syringae play an important and natural role in nucleating clouds with ice crystals which leads to precipitation – rain or snow – on the land below. That precipitation carries P. syringae down to the crops and other plants that it will pillage, which is what made it impossible for the Montana farmers to completely clear their fields of this bacterium. After feeding and replicating on (agricultural or native) plants for some time, it’s proposed that wind kicks some of the P. syringae back into the air where it accumulates into clouds and starts the whole cycle over again. So, as you can see there is interesting synergy here between P. syringae’s life cycle and Earth’s water cycle (Morris et al 2008; Tsang, 2019).

Note that minerals such as silver iodide are sometimes intentionally used to “seed” clouds and manipulate where and how much precipitation falls – such as preventing rain during the opening ceremony of the Olympics, for instance (Coonan, 2008). To be clear, the process I’m referring to above is different. It’s the natural interplay of bacteria and weather in the absence of intentional human influence.

Use of INPs in Snowmaking

P. syringae’s ice-nucleating activities have long been used to make artificial snow. Products including Snomax® use the proteins derived from the outside of bacteria to enhance the snow generated by snow blowers. One study showed that Snomax® increases the amount of snow made by a snow blower by as much as 90% (Snomax® International, 2013).

However, not all locations prefer, or are even allowed to use Snomax®.

One concern is that since Snomax® is derived from bacteria, it could potentially make humans sick (Boyle, 2015; The Local, 2015). This is not likely a valid concern for a couple of reasons. First, as the story about P. syringae’s discovery showed, this bacterium is everywhere (Christner et al, 2008)! On plants, in clouds, in rain and snow. While the ski resort personnel handling concentrated Snomax® should take proper precautions, any trace amount of P. syringae debris added from the snow blower is not likely to add a significant amount to the bacterium already on the slopes.

The second concern is ecological. Snow made from Snomax® melts slower compared to naturally occurring snow, and there is concern that this will mess up the natural snow melting process and the plants that rely on this water for growth (Xu, 2023).

These concerns have led to the ban of Snomax® in Austria and France (Boyle, 2015). As we will discuss below, new variants of INPs may enable a new version of snow enhancers that mitigate these concerns.

Recent Advances in Understanding INP Activity

So, now that we’ve covered INP’s native function and their use in snowmaking – how do they actually go about nucleating ice? The overall point is that there are a lot of weak interactions between specific amino acid motifs in INPs and water molecules. These motifs are repeated a bunch in a single INP, however individual proteins are still pretty poor at nucleating ice. But when several INPs bundle together, there is a sufficient local concentration of the water-organizing motifs that drive ice formation.

A useful analogy for ice nucleation is tiling a floor. Tile floors have a repeating pattern, just like an ice lattice. And after you get enough tiles in place – usually one or two rows worth – the rest of the tiles basically position themselves just by following the existing present pattern. All of this is assuming the person tiling the floor is skilled enough, of course!

Ok, now back to how INPs position the first row of ice formation. The water-binding motif is quite simple. It is two threonine amino acids flanking a spacer amino acid. For convenience we denote this TxT, where T stands for threonine, and x means any amino acid. However, in reality, the amino acids between the threonine residues in INPs are relatively small and flexible ones, most often glutamine, glycine, and serine (Hansen et al, 2023). By the way, if you want to brush up on amino acids and their characteristics, check out this article!

Those two threonine residues coordinate, or bind, to a water molecule. So just one of these TxT motifs would bind a single water molecule, which really isn’t enough to nucleate ice formation (Pandey et al, 2016). The P. syringae INP has a little more than 50 of these TxT motifs repeated in close proximity to one another, and INPs from related species can have over 70 TxT motifs (Hansen et al, 2023).

These TxT motifs aren’t randomly incorporated throughout the protein though – rather, they repeat in a particular pattern. This section of the INP is a b-solenoid, which is where pairs of antiparallel b-strands stack right next to one another. Each slice of the b-solenoid has one TxT water-binding motif that all aligns on the same side of the protein (Figure 3).

Figure 3. b-solenoid structure of P. syringae INP (top). The b-solenoid is a stack of antiparallel b-strands with one water-binding TxT motif each (bottom). Structure prediction from AlphaFold (Jumper et al, 2021; Varadi et al, 2022).

So, arranging a bunch of water-binding motifs enables INPs to bind many water molecules at the same time. Yet as it turns out, a single INP, even with all of its repeating water-binding motifs, it is still quite lousy at nucleating ice formation. By bundling many INPs together, the collective group gets much better at nucleating ice (Renzer et al, 2024). Tyrosine residues line up on another side of the b-solenoid and these so-called tyrosine ladders oligomerize INPs to potently nucleate ice formation (Figure 4).

Figure 4. INPs have weak freezing activity as monomers (left) but strong freezing activity as oligomers (right). INPs are green with TxT motifs reaching out to bind water molecules (small salmon-colored circles).

The Future of Custom INPs

Knowing how exactly INPs nucleate ice formation may enable attempts at engineering custom snowmaking INPs with desired properties. For example, using the purified protein itself - rather than inactivated bacterial extraction would eliminate the perceived health risk associated with Snomax®.

Furthermore, replacing the tyrosine ladders with a temperature-sensitive oligomerization component would enable potent ice nucleation when it is cold, but allow the nucleation activity to dissipate when the temperature rises, and it is time for the snow to melt (Figure 5). While this version of INPs doesn’t exist yet, temperature-sensitive oligomerization units have been observed and understood in other types of proteins, indicating they could be transferred over to and optimized in INPs (Varanko et al, 2020). Alternatively, an entirely novel oligomerization unit could be developed using saturated mutagenesis and a high-throughput phenotypic screen at different temperatures (Pak et al, 2024; Pines et al, 2022). Saturated mutagenesis is where you mutate a protein sequence to see which amino acid changes provide the characteristic – in this case temperature-sensitive oligomerization – that you’re looking for. With either method, development of new forms of INPs would enable efficient snowmaking for the ski slopes while easing health concerns and ecological impact.

Figure 3. Hypothetical engineered INP with temperature sensitive oligomerizing element (purple triangle) would oligomerize to freeze water at cold temperatures (right) but depolymerize at warmer temperatures to facilitate snowmelt (right).

So that’s how a bacterial protein nucleates artificial snowmaking. Next time you’re sitting on the skilift while the snowmakers whir away underneath you – you’ll know exactly what’s going on down there at the molecular level!

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