High-speed cryo-microscopy proves that ice-nucleating proteins of Pseudomonas syringae trigger freezing at hydrophobic interfaces

Ice-nucleating proteins (INpro) trigger the freezing of supercooled water droplets relevant to atmospheric, biological, and technological applications. Ice nucleation caused by INpro from the bacteria Pseudomonas syringae has been suggested to initiate at the bacterial membrane or at the air-water interface (AWI) of droplets. Here, we show direct evidence of this proposed mechanism. We monitored the freezing of droplets, between two hydrophobic glass slides, with a high-speed camera on a cryo-microscope. Onset nucleation sites of INpro from sterilized P. syringae (Snomax) were enriched at the AWI of the droplets. Removing cellular fragments by filtration or adding surfactants increased the frequency of nucleation events at the AWI. On the other hand, cultivated intact bacteria cells or lipid-free droplets nucleated ice without an affinity to the AWI. Overall, we provide visual evidence that INpro from P. syringae trigger freezing at hydrophobic interfaces, with important mechanistic implications for applications of INpro.