Fungus: Nature’s Ice Machines

Originally published for MicroBites on January 15, 2024

Contrary to popular belief, water does not actually freeze at 0˚C. While ice formation is thermodynamically favorable below 0˚C, ice can only be formed when water molecules arrange themselves into crystalline formations. Pure water has to reach temperatures as low as -46˚C before crystallizing. However, crystallization can be induced at warmer temperatures with a bit of help. When there are impurities in the water, water molecules can build off a preexisting structure instead of needing to crystallize spontaneously. This process of kickstarting the crystal ice formation is called nucleation and abiotic ice nucleators can raise the freezing temperature up to -30˚C to -15˚C.

Ice nucleation (IN) is an ability that is very common in plant associated microbes. It is advantageous for plant pathogens when infecting frost-sensitive plants to induce the formation of ice crystals in and around the plant cell to rupture the cell walls, availing the nutrients inside the cells to extracellular bacteria, and establish a point of infection. Conversely, frost-resistant plants cultivate ice nucleating microbes as commensals since ice nucleation protects frost-resistant plants by inducing ice formation more evenly throughout the plant tissue, preventing damage.

The most well-characterized INs have been bacteria in the genera Pseudomonas, Pantoea, and Xanthomonas, with some producing proteins that induce ice nucleation at temperatures as warm as -2˚C. More recently, some species of fungus have been shown to produce their own versions of INs. This study focuses on INs produced by the fungus Fusarium acuminatum.

A microscopic view of water crystallizing around a nucleation site in Fusarium acuminatum cell extract after purifying for proteins that bind to ice.
Image source: Ralph Schwidetzky et al (2023)

Previous studies investigating this species of fungus determined that the IN proteins were likely less than 30kDa in size. This was determined by using filters with extremely small pore sizes, which can only provide a range and not an accurate measurement. This study aimed to characterize these proteins further and uncover how exactly these fungal proteins induce ice nucleation.

To do this, the authors purified out all the fungal proteins with ice nucleation activity and sorted them using size exclusion chromatography. This is a method that sorts a mixture of proteins by their size. Size exclusion chromatography involves passing the sample through a column filled with beads. The surface of these beads is covered in small pores of various sizes. The smaller proteins that pass through will get stuck in the pores while the larger proteins will pass by the beads without issue, so as the proteins get collected out the other end of the column, larger proteins will elute out first, and the smallest proteins will elute out last, having been slowed down by the beads.

Size exclusion chromatography. Smaller proteins get caught in the pores of the beads in the column, slowing them down, while larger proteins are able to pass by without getting stuck. Larger proteins pass through the columns and are collected first while smaller proteins pass through much slower and get collected at the end
Image source: Created with BioRender.com.

The authors found that the IN proteins eluted at a few different sizes: the first peak showed up at 660kDa, followed by an extremely large peak at 12.4kDa and a smaller peak at 5.3kDa. Interestingly, each isolated size retained ice nucleating activity. This suggested that the smaller protein sizes (12.4kDa and 5.3kDa) were most likely subunits that aggregated together to form the large 660kDa proteins.

While it is common for IN proteins in other species to form aggregates from smaller subunits, bacterial IN protein aggregates are often most effective when membrane bound. These fungal subunits are unique in their ability to form aggregates in a cell-free environment while retaining high IN activity. The authors estimated that, if the 5.3 kDa proteins isolated from the size exclusion chromatography are the smallest subunit made by the fungus, it would require at least 150 of these subunits to form an aggregate large enough to nucleate ice at -4˚C.

These findings have many implications in how we might be able to utilize ice nucleating proteins. IN proteins are useful to humans in many aspects. Being able to control how crystals form when freezing things improves the efficacy of cryopreservation. Many cells and tissues must be stored at very low temperatures, but freezing often risks damage. Inducing ice nucleation in a more even manner with IN proteins prevents large crystals from forming and rupturing cell membranes. Cell-free ice nucleation as shown in this study is useful since many cells are frozen for research or medical use, and would increase the rates of cell recovery and viability for procedures ranging from cell culturing in laboratories to IVF for patients.

The utility of IN proteins extend past the laboratory and medicine; INs are also implicated in cloud seeding and weather modification. IN bacteria are most well characterized in this context. Plant associated bacteria are emitted into the atmosphere and carried by up-drafts of air via storms and wind. Once in the upper atmosphere, the IN proteins on the bacterial membranes begin to catalyze ice crystallization, forming clouds and eventually precipitation. Artificial cloud seeding often uses the inorganic compound silver iodide, but some bacterial preparations are also used in fake snow.

Bioprecipitation, the specific phenomena of microbes inducing cloud formation and subsequent precipitation, is still an understudied field of research. The implications of cloud seeding and weather modifications and the long term effects on the climate are still not well characterized. In areas where drought is common, cloud seeding serves as a possible solution; however, it can also lead to more drought if clouds headed to one place are prematurely seeded in another. And repeated cloud seeding in an area with an agent like silver iodide may pose a risk to the environment as the silver accumulates in the soil. This is why finding alternatives, like the protein aggregates identified in this study, are important innovations in this space.

The researchers have only just begun to delve deep in the potential of fungal ice nucleators. The ability for these IN subunits to aggregate on their own without needing the fungus present promises potential in agriculture, cryopreservation, and cloud seeding. However, more research is necessary to ensure these can be implemented in a safe manner without long term climate effects.

Original Paper: Functional aggregation of cell-free proteins enables fungal ice nucleation. Ralph Schwidetzky, Ingrid de Almeida Ribeiro, Nadine Bothen, Anna T. Backes, Arthur L. DeVries, Mischa Bonn, Janine Fröhlich-Nowoisky, Valeria Molinero, and Konrad Meister. PNAS. November 9, 2023. DOI: 10.1073/pnas.2303243120