Hierarchical assembly and environmental enhancement of bacterial ice nucleators

Bacterial ice nucleating proteins (INPs) are exceptionally effective in promoting the kinetically hindered transition of water to ice. Their efficiency relies on the assembly of INPs into large functional aggregates, with the size of ice nucleation sites determining activity. Experimental freezing spectra have revealed two distinct, defined aggregate sizes, typically classified as class A and C ice nucleators (INs). Despite the importance of INPs and years of extensive research, the precise number of INPs forming the two aggregate classes, and their assembly mechanism have remained enigmatic. Here, we report that bacterial ice nucleation activity emerges from more than two prevailing aggregate species and identify the specific number of INPs responsible for distinct crystallization temperatures. We find that INP dimers constitute class C INs, tetramers class B INs, and hexamers and larger multimers are responsible for the most efficient class A activity. We propose a hierarchical assembly mechanism based on tyrosine interactions for dimers, and electrostatic interactions between INP dimers to produce larger aggregates. This assembly is membrane-assisted: Increasing the bacterial outer membrane fluidity decreases the population of the larger aggregates, while preserving the dimers. Inversely, Dulbecco's Phosphate-Buffered Saline buffer increases the population of multimeric class A and B aggregates 200-fold and endows the bacteria with enhanced stability toward repeated freeze-thaw cycles. Our analysis suggests that the enhancement results from the better alignment of dimers in the negatively charged outer membrane, due to screening of their electrostatic repulsion. This demonstrates significant enhancement of the most potent bacterial INs.