Enhanced production of extracellular ice nucleators from Erwinia herbicola

The effects of growth conditions and chemical or physical treatments on the production of extracellular ice nucleators (ECINs) by Erwinia herbicola cells were investigated. The spontaneous release of ECINs, active at temperatures higher than -4 degrees C, into the environment depended on culture conditions, with optimal production when cells were grown in yeast extract to an early stationary phase at temperatures below 22 degrees C. ECINs were vesicular, released from cell surfaces with sizes ranging from 0.1 to 0.3 &mgr;m as determined by ultrafiltration and transmission electron microscopy. Protein profiles of ECIN fractions during bacterial growth were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Ina proteins were detected by Western blotting. ECIN production was enhanced 5-fold when cells were treated with EDTA and 20- to 30-fold when subjected to sonication. These conditions provide a means for large-scale preparationage> ECINs by E. herbicola.