Study of extracellular ice-nucleating matter from ice-nucleating bacteria

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The extracellular ice-nucleating matter (EM) produced by the ice-nucleating bacterium, Erwinia uredovora KUIN-3, was purified to apparent homogeneity by ultrafiltration, sucrose density-gradientultracentrifugation, and gel filtration. The purified EM was sphercial matter (0.2-0.4mum) by examination using a transmission electron microscope. The ice nucleating activity from the EM was equal to that of the cells in this strain. It had become apparent that the EM had class A (--5ﾟC) , B ( -5--8ﾟC) , and C (-8ﾟC) structures as judged by its freezin differencespectrum in D_2O versus H_2O.The enzyme treatments by protease K,alpha-mannosidase, beta-galactosidase, and phospholipase C decreased the acyivity of the EM as well as of the cells. Further, the activities of class A and B from the EM were decreased by treatment with diamine oxidase. Also the activity of the purified EM was stable grom pH 5.0 to 10 and at temperatures below 25ﾟC.It was demonstrated that the components of the EM were lipid (10%) , protein (43%) , saccharide (35%) , and polyamine (12%) .The effects of polyamines on the ice-nuceating activity of an ice-nuceating bacterium, Erwinia uredovora KUIN-3, were investigated. The addition of methylglyoxal bis (guanylhydrazone) {MGBG} which inhibited the activity of S-adenosylmethionine decaboxylase, inhibited the ice-nucleating activity of the cell of this bacterium. The amounts of polyamines in the cells and outer membranes after MGBG treatment decreased. The results suggested that polyamine was the most important component of the ice-nucleating material in this bacterium.