The biochemical and physical organization of bacterial ice nuclei are examined in E. coli and Pseudomonas syringae, using methods developed during Phase I of this grant. Many envisioned uses of bacterial ice nuclei require the production of cell-free nuclei active above -5.C (warm-threshold nuclei). A detailed understanding of the structure of these nuclei is a prerequisite for such development. The program goals are a) Investigation of the reasons for the observed Mg+2- dependence of warm-threshold cell-free nuclei from E. coli and P. syringae. b) Visualization and localization of cell-bound bacterial ice nuclei in P. syringae cells and subcellular fractions, by immunogold electron microscopy. c) Elucidation of the steps involved in assembly and degradation of ice nuclei, in E. coli and P. syringae, using inducible expression systems, and drugs which block specific steps of protein synthesis and processing. The proposes research utilizes cloning and expression of bacterial genes in E. coli and P. syringae, immunoelectron microscopy, and temporal and spacial localization of bacterial proteins by established biochemical techniques. The planned investigations will demonstrate which structures must be synthesized and stabilized to produce warm-threshold, cell-free ice nuclei. Bacterial ice nuclei are of potential use to the oil exploration, ski slope snow making, food processing, clinical testing, and frost protection industries.
