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.
Walsh et al. (Wed,) studied this question.