Isolation and Characterization of a Novel Ice-nucleating Bacterium, Pseudomonas sp. KUIN-4, Which Has Stable Activity in Acidic Solution

Abstract

A novel ice-nucleating bacterium, KUIN-4 was isolated from a cherry leaf, which was unsusceptible to frost injury. Strain KUIN-4 was identified as Pseudomonas sp. from its characteristics and taxonomics ; the optimum temperature and pH for its growth were 18°C and 4.5, respectively. When strain KUIN-4 was cultured aerobically in CYE medium (pH 4.5) for 48h at 18°C, the ice-nucleating activities of strain KUIN-4 cells and culture broth (extracellular ice-nucleating matter) after removal of the cell were obtained. The ice-nucleating temperature, T₅₀(°C), was indicated to be -2.8°C in cell suspensions (4.25×10⁷ cell/ml) of strain KUIN-4. Also, it had become apparent that the ice-nucleating activity involved class A, B, and C structures as judged by its freezing difference spectrum in D₂O versus H₂O. The ice-nucleating activity of this strain as well as other ice-nucleating bacteria was significantly decreased by heat treatment (40°C, 30min). The ice-nucleating activity from this strain had unique features, which was stable under acidic conditions (pH 3.5-5.0) and was weakly inhibited by denaturants and protein-modifying reagents.