Analysis of Formation Mechanisms of Ice-nucleating Matter from an Ice-nucleating Bacteria

冰核细菌冰核物质形成机制分析

• 批准号: 09660105
• 负责人: OBATA Hitoshi
• 金额: $ 1.92万
• 依托单位: KANSAI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660105/
• 关键词: Pseudomonas; cold shock protein; cold-regulated protein; extracellular ice-nucleating material; Erwinia; ice nucleation protein; 菌体外氷核物質; 氷核形成温度; 酵母エキス; DCCD; 氷核活性細菌; 氷核活性物質; 低温誘導物質; 乳酸デヒドロゲナーゼ; 凍結保護活性

The psychrotrophic ice-nucleating bactenum, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs). In strain KUIN-1, a cold shock from 18 to 4ﾟC induced the synthesis of the 26-kDa protein. By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4ﾟC.The 26-kDa protein was purified to apparent homogeneity by (NH_4)_2SO_4 precipitation and some chromatographies. The purified 26-kDa protein is composed of 6 subunits of 26,500 with a molecular mass of approximately 159,000 according to gel filtration and SDS-PAGE.The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-GIn-Gln-Val-Gln-Glu-His-Trp-Gly-His-His-. Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing. The characteristics of … More 26-kDa protein are analogous to the cold-regulated protein of the plants.Also, the secretion of extracellular ice-nucleating material (EIM) with ice nucleation proteins (INP) from an ice-nucleating bacterium, Erwinia urehvora KUIN-3, increased in proportion to the yeast extract concentration in the ice-nucleating medium. The level of this secretion could be exhibited in terms of the ice-nucleating temperature, T_<50>(ﾟC), using the ELIZA method with the anti-Ina A antisenim. The secretions and the production on the cell surface of all ElM containing the class A, B and C stmctures were activated by the addition of yeast extract. Furthermore, based on the examination involving the deletion of various mecium components, it was found that Lys was closely associated with the secretions of EIM in the class A and B stnictures. The addition of Lys could affect the polyamine content in the ELM, thereby changing the surface charge of EIM from 5.2 to 4.9. Also, the addition of cadaverine could enhance the EIM secretion. Based on these results, the surface charge of the INP aggregate was shown to be essential for the translocation of INP and the secretion of IN.We found that this secretion, especially the secretion of a class B stnicture, was inhibited by N,N'-dicydohexylcarbodlimide, which was an H^+-ATPase inhibitor regardless of nomial growth. We also found that this secretion required ATP and the positive charge on the ElkI surface. Less

低温冰核细菌荧光假单胞菌KUIN-1对温度降低的反应是诱导一种被归类为冷休克蛋白（CSP）的蛋白质。在菌株KUIN-1中，从18 ℃到4 ℃的冷休克诱导了26 kDa蛋白的合成。SDS-PAGE分析表明，4 ℃处理后的菌体产生了26-kDa蛋白。纯化的26-kDa蛋白质由6个亚基组成，分子量为26，500，根据凝胶过滤和SDS-PAGE，分子量约为159，000，26-kDa蛋白质的N-末端序列为Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-Gln-Gln-Val-Gln-Glu-His-Trp-Gly-His-His-。具体而言，26 kDa的蛋白质的CSP菌株KUIN-1是非常有效的保护冷不稳定的酶，乳酸脱氢酶对变性冷冻。的特点 ...更多信息 26-此外，冰核细菌欧文氏菌（Erwiniurehvora KUIN-3）分泌的含冰核蛋白（INP）的胞外冰核物质（EIM）与冰核培养基中的酵母抽提物浓度成比例增加。用抗InaA抗体ELISA法测定冰核温度T_（100 ℃），可反映分泌量的高低<50>。添加酵母抽提物可激活所有含有A、B和C类结构的ElM的分泌和细胞表面的生产。此外，在A类和B类结构中，赖氨酸与EIM的分泌物密切相关。赖氨酸的加入可以影响ELM中多胺的含量，从而改变EIM的表面电荷从5.2到4.9。同时，尸胺的加入可促进EIM的分泌。结果表明，INP聚集体的表面电荷对INP的转运和IN的分泌起着重要作用。N，N '-二环己基碳二亚胺抑制INP的分泌，尤其是B类结构的分泌，而与正常生长无关。我们还发现，这种分泌需要ATP和ElkI表面上的正电荷。少

项目成果

Hidehisa Kawahara, Mariko Matsushita, Kazuhiro Yamade and Hitoshi Obata: "The Control of the Production and Secretion of Extracellular Ice-nucleating Materials of Erwinia uredovora KUIN-3" Biocontrol Science. in press. (1999)

Hidehisa Kawahara、Mariko Matsushita、Kazuhiro Yamade 和 Hitoshi Obata：“Erwinia uredovora KUIN-3 细胞外冰核物质的产生和分泌的控制”生物防治科学。

Hitoshi Obata: "Purification and Characterization of a Novel Cold-regulated Protein from an Ice-nucleating Bacterium, Pseudomonas fluorescens KUIN-1" Bioscience, Biotechnology and Biochemistry. 62. 2091-2097 (1998)

Hitoshi Obata：“冰核细菌荧光假单胞菌 KUIN-1 中新型冷调节蛋白的纯化和表征”生物科学、生物技术和生物化学。

Hitoshi Obata, Hitoshi Ishigaki, Hidehisa Kawahara and Kazuhiro Yamade: "Purification and Characterization of a Novel Cold-regulated Protein from an Ice-nucleating Bacterium, Pseudomonas fluorescens KUIN-1" Bioscience, Biotechnology and Biochemistry. 62・1

Hitoshi Obata、Hitoshi Ishigaki、Hidehisa Kawahara 和 Kazuhiro Yamade：“冰核细菌荧光假单胞菌 KUIN-1 中新型冷调节蛋白的纯化和表征”生物科学、生物技术和生物化学 62・1。

Hidehisa Kawahara: "The Control of the Production and Secretion of Extracellular Ice-nucleating Material of Erwinia uredovora KUIN-3" Biocontrol Science. (in press). (1999)

Hidehisa Kawahara：“欧文氏菌 KUIN-3 细胞外冰核物质的产生和分泌的控制”生物防治科学。

Hidehisa Kawahara, Noriko Koda, Mika Oshio, Kazuhiro Yamade and Hitoshi Obata: "Functional Analyses of High Cryotolerance and Cold Shock Protein,Hsc 25" Applied Environment and Microbiology. (in press). (1998)

Hidehisa Kawahara、Noriko Koda、Mika Oshio、Kazuhiro Yamade 和 Hitoshi Obata：“高低温耐受性和冷休克蛋白的功能分析，Hsc 25”应用环境和微生物学。