Biochemical characterization of a fungal ice nucleation protein from Podila clonocystis

Biological ice nucleation plays a pivotal role in atmospheric processes, yet the molecular basis of fungal ice nucleation remains poorly understood. We report the biochemical characterization of an ice nucleation protein (PcINP) from the soil-dwelling fungus Podila clonocystis, an organism that has been implicated in ice nuclei production; however, its ice nucleation activity has not been demonstrated. Using sequence similarity networks, we identified PcINP as a putative fungal homolog of bacterial INPs and confirmed its function through recombinant expression in Escherichia coli. We probed the function of PcINP structure through domain truncations and demonstrated that the N-terminal region is not necessary for ice nucleation activity and can be replaced with an expression-enhancing tag that significantly increases the number of active ice nuclei. Finally, we observed that a solution that contains monomeric PcINP has identical activity to solutions containing oligomerized INP, suggesting PcINP utilizes an in vitro aggregation mechanism to generate active oligomers. Our findings establish PcINP as a member of the emerging class of fungal INPs, expanding the known diversity of biological ice nucleators and highlighting their potential for environmental and biotechnological applications.