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. The high ice nucleation activity of INpro isolated from the bacteria Pseudomonas syringae has been suggested to occur by aggregation of proteins at the bacterial membrane or 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 P. syringae were enriched at the AWI of the droplets, in comparison to birch pollen extracts, which nucleated randomly distributed in the bulk. Removing cellular fragments by filtration increased the frequency of nucleation events at the AWI, whereas intact bacteria cells nucleated in the bulk with no AWI affinity. Overall, we provide visual evidence that INpro from P. syringae trigger freezing at hydrophobic interfaces.