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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 Earth’s radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens (Lp) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in “pearl chain”-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes.