Physical and functional repetition in a bacterial ice nucleation gene

Abstract

Nucleation of a physical process is distinct from catalysis, and as the function of a protein it is highly unusual. The ability to nucleate ice formation in supercooled water is a property of some members of the bacterial genera Erwinia, Pseudomonas and Xanthomonas^1–3. This property is implicated in the ability of bacteria to cause frost injury to plants. Orser et al.⁴ have demonstrated that a single small region of DNA encodes this phenotype, and there is evidence that phosphatidylinositol is involved⁵. Biological ice nuclei are also found in the haemolymph of certain freeze-tolerant insects⁶. It is thought that an ice nucleation site must bind water molecules in an orderly array resembling an ice crystal. We have now determined the sequence of an ice nucleation gene from Pseudomonas syringae. It predicts a translation product whose structure is consistent with the role of template, containing 122 imperfect repeats of the consensus octapeptide Ala-Gly-Tyr-Gly-Ser-Thr-Leu-Thr. All the repeats are contiguous and higher-order periodicities are superimposed on the octapeptide pattern throughout. Deletion analysis showed that most regions of the repeating structure are not essential for function. Up to 68 octapeptides could be deleted without abolishing ice nucleation, but all deletions showed reduced activity. This indicates that the repeated peptides contribute individually to the nucleation process, as expected if they act as individual units of a water-binding array.