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Ice-nucleating proteins (INPs) catalyze ice formation at high subzero temperatures, with major biological and environmental implications. While bacterial INPs have been structurally characterized, their counterparts in other organisms remain unknown. Here, we identify a new class of efficient INPs in fungi. These proteins are membrane-free, adopt β-solenoid folds, and multimerize to form large ice-binding surfaces, showing mechanistic parallels with bacterial INPs. Structural modeling, sequence analysis, and functional assays show they are encoded by orthologs of the bacterial InaZ gene, likely acquired via horizontal gene transfer. Our results demonstrate that distinct lineages have independently converged on a common molecular strategy to overcome the energetic barriers of ice formation. The discovery of cell-free INPs provides tools for freezing applications and reveals biophysical constraints on nucleation across life. 

Abstract. From extracellular freezing to cloud glaciation, the crystallization of water is ubiquitous and shapes life as we know it. Efficient biological ice nucleators (INs) are crucial for organism survival in cold environments and, when aerosolized, serve as a significant source of atmospheric ice nuclei. Several lichen species have been identified as potent INs capable of inducing freezing at high subzero temperatures. Despite their importance, the abundance and diversity of lichen INs are still not well understood. Here, we investigate ice nucleation activity in the cyanolichen-forming genus Peltigera from across a range of ecosystems in the Arctic, the northwestern United States, and Central and South America. We find strong IN activity in all tested Peltigera species, with ice nucleation temperatures above −12 °C and 35 % of the samples initiating freezing at temperatures at or above −6.2 °C. The Peltigera INs in aqueous extract appear to be resistant to freeze–thaw cycles, suggesting that they can survive dispersal through the atmosphere and thereby potentially influence precipitation patterns. An axenic fungal culture termed L01-tf-B03, from the lichen Peltigera britannica JNU22, displays an ice nucleation temperature of −5.6 °C at 1 mg mL−1 and retains remarkably high IN activity at concentrations as low as 0.1 ng mL−1. Our analysis suggests that the INs released from this fungus in culture are 1000 times more potent than the most active bacterial INs from Pseudomonas syringae. The global distribution of Peltigera lichens, in combination with the IN activity, emphasizes their potential to act as powerful ice-nucleating agents in the atmosphere. 

Bacterial ice nucleating proteins (INPs) are exceptionally effective in promoting the kinetically hindered transition of water to ice. Their efficiency relies on the assembly of INPs into large functional aggregates, with the size of ice nucleation sites determining activity. Experimental freezing spectra have revealed two distinct, defined aggregate sizes, typically classified as class A and C ice nucleators (INs). Despite the importance of INPs and years of extensive research, the precise number of INPs forming the two aggregate classes, and their assembly mechanism have remained enigmatic. Here, we report that bacterial ice nucleation activity emerges from more than two prevailing aggregate species and identify the specific number of INPs responsible for distinct crystallization temperatures. We find that INP dimers constitute class C INs, tetramers class B INs, and hexamers and larger multimers are responsible for the most efficient class A activity. We propose a hierarchical assembly mechanism based on tyrosine interactions for dimers, and electrostatic interactions between INP dimers to produce larger aggregates. This assembly is membrane-assisted: Increasing the bacterial outer membrane fluidity decreases the population of the larger aggregates, while preserving the dimers. Inversely, Dulbecco’s Phosphate-Buffered Saline buffer increases the population of multimeric class A and B aggregates 200-fold and endows the bacteria with enhanced stability toward repeated freeze-thaw cycles. Our analysis suggests that the enhancement results from the better alignment of dimers in the negatively charged outer membrane, due to screening of their electrostatic repulsion. This demonstrates significant enhancement of the most potent bacterial INs.