ISOLATION OF ICE-NUCLEATING ACTIVE BACTERIA FROM THE FREEZE-TOLERANT FROG, RANA-SYLVATICA

Ice-nucleating active (INA) bacteria were isolated from the gut of field-collected freeze-tolerant wood frogs (Rana sylvatica) collected in winter. Thirteen strains of Pseudomonas fluorescens, four strains of Pseudomonas putida and two strains of Enterobacter agglomerans had ice-nucleating activity. Each of the INA pseudomonad strains was psychrophilic. P. putida strains were differentiated from P. fluorescens strains by gelatinase, lecithinase, and lipase production. The maximum nucleation temperatures (T-max) of aqueous suspensions (10(9) bacteria/ml) of the four INAP. putida strains ranged from -1.6 to -3.0 degrees C, which places this INA species among the most potent known biological nucleators. Ingestion of INAP. putida isolated from R. sylvatica by another freeze-tolerant frog, Pseudacris crucifer, decreased the capacity of this frog to supercool acid remain unfrozen at -2 degrees C. This is the first report of INA bacteria isolated from a vertebrate, and suggests that, as part of the gut flora in some posthibernation freeze-tolerant wood frogs, these bacteria may play a role in enhancing winter survival by promoting ice nucleation at high subzero temperatures (ca. -2 degrees C). (C) 1995 Academic Press, Inc.