Preparation of active cell-free ice nuclei fromPseudomonas syringae

Abstract

Ice-nucleating bacteria have attracted attention because of their ability to cause frost damage to crops, their possible role in the rain cycle and their potential use in the freeze-texturing of materials. They are also being used to investigate atomic interactions during the initial stages of crystal formation. A method for preparing cell-free type I ice nuclei (active at -5°C and above) from Pseudomonas syringae is described. Five different isolates of P. syringae were grown under optimal conditions for expression of the ice nucleation phenotype and their individual nucleation frequencies determined. Bacteria were treated with a combination of Na 2 . EDTA and lysozyme and the nature of the material released from the cells assessed by assaying the supernatant for biochemical markers specific for either the inner or outer membrane. A procedure was identified that released an outer membrane marker but not one from the inner membrane, and provided a supernatant that contained cell-free type I ice nuclei. Up to 3.6% of the type I nuclei present in the starting culture could be obtained in cell-free form, a substantially higher recovery level than previously achieved.