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Abstract

Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation more efficiently than any other material. The details of their working mechanism remain elusive, but their high activity has been shown to involve the formation of functional INP aggregates. Here we reveal the importance of electrostatic interactions for the activity of INPs from the bacterium Pseudomonas syringae by combining a high-throughput ice nucleation assay with surface-specific sum-frequency generation spectroscopy. We determined the charge state of nonviable P. syringae as a function of pH by monitoring the degree of alignment of the interfacial water molecules and the corresponding ice nucleation activity. The net charge correlates with the ice nucleation activity of the INP aggregates, which is minimal at the isoelectric point. In contrast, the activity of INP monomers is less affected by pH changes. We conclude that electrostatic interactions play an essential role in the formation of the highly efficient functionally aligned INP aggregates, providing a mechanism for promoting aggregation under conditions of stress that prompt the bacteria to nucleate ice.