The fungi that can freeze water better than bacteria
by Sanjukta Mondal  /  January 19, 2024

“Water doesn’t always freeze at 0 degrees C. Unlike what we have been taught in school, the transformation of liquid water into ice is more nuanced than dropping the temperature down to its freezing point. Supercooled water found in clouds remains in a liquid state at temperatures as low as minus 40 degrees C. Scientists have even found that completely pure water can remain unfrozen until it’s cooled to temperatures below minus 46 degrees C. To freeze, water molecules need to arrange themselves in an ordered way and form a crystalline structure. But ice formation is also kinetically hindered, meaning it requires a bit of extra energy – especially for the first step, called ice nucleation. This energy demand is not small.

To correctly orient themselves to create a crystalline structure, water molecules also need an initiation point, or a nucleus – a place that can serve as a surface on which the ice crystals can grow. This nucleus could be an ice particle or an impurity like dust, minerals, or microorganisms commonly found in water. The lack of these nucleators prevents pure water from freezing, whereas less-pure tap water readily freezes at minus 5 degrees C in our refrigerators. So pure water struggles to freeze, and this could pose dangers to species adapted to living in cold environs. Then again, life always finds a way.

Several microorganisms like bacteria, lichen, and fungi have evolved to manipulate water so that it forms ice more easily. They achieve this with the help of efficient molecular strategies that trigger the nucleation process. This phenomenon is called biological ice nucleation. In a study published in the Proceedings of the National Academy of Sciences, scientists have explored how fungi can start biological ice nucleation to produce ice on demand. Biological ice-nucleators are among the most efficient nucleation initiators in nature. However, scientists are yet to fully understand the molecular foundations of this ability. In the study, a team of scientists from Germany and the U.S. took a closer look at Fusarium acuminatum, a fungal plant pathogen and a known ice-nucleator.

“P. syringae’s ice nucleation ability helps make frost on plants”

Scientists first spotted biological ice nucleation in the 1970s when studying the bacteria Pseudomonas syringae, a plant pathogen that causes multiple diseases in crops. Along with other species of bacteria, P. syringae can start ice formation at temperatures just below the melting point of water (0 degrees C). These bacteria produce special ice nucleation proteins (INPs) near their cell membranes, which become anchor points for water molecules to start forming ice crystals. Water freezes around the INPs so well it nearly mimics natural ice. A 2016 study found that interactions in certain amino acid sequences within the INPs of P. syringae lead to stronger hydrogen bonds and better structural ordering in water networks. This process moves heat from the water into the bacteria, resulting in quick aggregation of water molecules.

Bacterial INPs are so good at making ice that ski resorts often use a commercial snowmaking product, called Snowmax, which consists of INPs bound to inactivated or dead P. syringae to start the crystallisation process. In F. acuminatum, however, the scientists found a different mechanism at work. Fungi produce highly efficient ice nucleators that can cause water to start crystallising at temperatures as warm as minus 2 degrees C. Their presence in the soil, the atmosphere, and cloud water-samples has led scientists to suggest they may be able to influence both local and regional weather patterns, if not global. But unlike bacterial ice-nucleators, the macromolecular structures and interactions in fungal nucleators were still a mystery at the time the study was conducted. Here, the scientists combined nucleation theory and numerical modelling studies with ice nucleation measurements and physicochemical tests to decipher the chemistry and ice shaping abilities of F. acuminatum.

“A cryomicroscopic image of a hexagonal ice crystal
grown in a Fusarium acuminatum ice-nucleating extract”

The team found its ice-nucleators to be small extracellular protein subunits made of around 50 amino acids each. F. acuminatum possesses more than a hundred such ice-nucleation proteins that can form functional aggregates outside the fungus’s cells, triggering ice formation. “In bacteria, the proteins are basically anchored to the cell membrane. You basically can’t extract the proteins without ripping them out of their natural environment, which sometimes makes them unstable,” Konrad Meister, a biochemist at the Max Planck Institute for Polymer Research, Mainz, and Boise State University, Idaho, told this author. “On the other hand, these fungi are extremely stable because [the proteins are] basically released into the environment and not bound to any membrane.” Dr. Meister was the study’s corresponding author.

The fungus’s nucleators were also some 25-times smaller than those of the bacteria but still comparably efficient. And because they’re released into the environment, scientists have an opportunity to use the fungal proteins without having to kill the fungi, unlike artificial snow-makers that currently use dead bacteria. The study concluded that despite the differences in molecular structures of ice-nucleating proteins, nature uses a common strategy to promote high-subzero ice-nucleation temperatures: by assembling the proteins into large, functional aggregates. “What’s interesting is that fungi and bacteria are very different organisms, right? But somehow, they came up with the same idea and independently evolved to make ice,” Dr. Meister told this author.

The researchers also said they need to further investigate the interplay of water molecules and fungal ice-nucleating proteins, and explore potential applications, especially to make snow-making, cloud-seeding, and cryo-preservation techniques more efficient. If done right, the scientists believe these advancements could save large amounts of power currently required to turn water into ice. The team is also curious about the ecological advantages of organisms that produce ice-nucleating proteins and their role in cloud formation or rainfall. “There is a possibility,” Dr. Meister said, “they might be responsible for the beautiful snowfall we experience.”

Fungi join the list of organisms that can control when ice forms
by   / 12/7/2023

“While it may be the reason behind tires skidding, pipes bursting, and closed roads making traffic a nightmare, ice doesn’t always form as easily as it seems. It often gets an assist from proteins made by fungi. Never mind the common thinking that ice forms at 0° C (32° F). Though this is water’s freezing point, pure water will only freeze when temperatures plummet as low as minus 46° C (minus 50.8° F). So why does it usually freeze at zero anyway? Organisms such as bacteria, insects, and fungi produce proteins known as ice nucleators (non-protein nucleators can also be of abiotic origin). These proteins can kick-start the formation, or nucleation, of ice at higher temperatures than pure water would freeze at.

While the exact reason fungi make these proteins remains unknown, researchers Valeria Molinero of the University of Utah and Konrad Meister of Boise State University led a study that has revealed more about how fungal ice nucleators can both promote and hold back ice formation more efficiently than those of many other life-forms. Organisms capable of producing ice nucleators belong to different biological kingdoms but are thought to have evolved the same ability independently—a phenomenon known as convergent evolution. Fungal ice nucleators had been something of an enigma until Molinero, Meister, and their team studied the nucleators produced by the fungus Fusarium acuminatum. “We find ice-binding and ice-shaping activity of Fusarium [ice nucleators], suggesting a potential connection between ice growth and inhibition,” the scientists said in a study recently published in PNAS.

Ice nucleators help freeze water by fast-forwarding nucleation, which is the process by which water molecules initiate the formation of ice crystals. Forming a crystal means molecules of H2O must align themselves in specific orientations to form a rigid lattice structure. This starts as a small group of latticed molecules, a “nucleus” in liquid water. If this nucleus is large enough, more water molecules will aggregate on it until an ice crystal forms. This is difficult for pure water to do on its own because the motion of its molecules makes it difficult to form a nucleus. Ice nucleator proteins help water molecules aggregate into a nucleus. Some bacterial nucleators are so effective that the bacteria that produce them are used to make snow at ski resorts. Fusarium nucleators are also highly efficient and effective.

Meister and Molinero’s team found that these fungal proteins are much smaller than many of those synthesized by bacteria and other organisms, and large numbers of them join together into complex structures that help ice nucleate. Even when the levels of Fusarium proteins were reduced in the lab, they could still trigger the nucleation process. The scientists have a hypothesis as to why Fusarium and some other organisms evolved such small molecules that aggregate so efficiently: “We expect that the energetic benefit for the organism in producing smaller proteins, rather than a single large one, contributes to the success and adoption of [this] strategy across species that are not evolutionary-related,” they said in the same study. Another question—one where the answer has not crystallized yet—is why fungi use ice nucleators. Whether they are meant to promote ice formation or whether it is an incidental side effect of another benefit they provide to the fungus still needs to be researched.

But creatures like frogs might give us a clue. Many frog species have a natural antifreeze that keeps them alive while they fall into a state of torpor during the colder months. It is not uncommon for them to end up covered in ice, but ice nucleators produced in their cells help them control where ice forms, keeping them alive (along with an antifreeze made of urea and glucose). Some species also ingest bacteria that help with the process. Could ice nucleators in fungi be part of a similar antifreeze system? At this point, nobody knows. As more continues to be demystified about biological and abiotic ice nucleators, they could eventually be used for more efficient methods of freezing food, making snow, creating clouds, and possibly the cryogenic freezing of human cells, which was recently successful. The future is frozen.”

PNAS, 2023.  DOI: 10.1073/pnas.2303243120



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