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

嗜冷性冰核杆菌荧光假单胞菌Kuin-1通过诱导被归类为冷休克蛋白(CSPs)的蛋白质来响应温度的下降。在菌株Kuin-1中，从18到4ﾟC的冷休克诱导了26 kDa蛋白质的合成。经4ﾟC处理后，经(NH_4)_2SO_4沉淀和柱层析纯化得到明显均一的26-kDa蛋白。纯化的26 kDa蛋白由6个亚基组成，经凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，其相对分子质量约为159,000，N端序列为Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-GIn-Gln-Val-Gln-Glu-His-Trp-Gly-His-His-.具体地说，菌株Kuin-1的CSPs的26 kDa蛋白对低温不稳定酶乳酸脱氢酶具有很好的保护作用。…的特点更多的26 kDa蛋白类似于植物的冷调节蛋白。此外，一株冰核细菌库因-3的胞外冰核物质(EIM)和冰核蛋白(INP)的分泌量与冰核培养液中酵母提取物的浓度成比例增加。这种分泌物的水平可以用成冰温度T_&lt；50&gt；(ﾟC)来表示，用抗Ina A抗衰老的Eliza方法来表示。添加酵母膏后，所有A、B、C级结构榆树细胞表面的分泌物和产物均被激活。此外，根据涉及各种胎盘成分缺失的检查，发现Lys与A、B类结构中EIM的分泌密切相关。Lys的加入可以影响ELM中的多胺含量，从而使EIM的表面电荷从5.2变为4.9。此外，加入身体碱可促进EIM的分泌。根据这些结果，INP聚集体的表面电荷对INP的转运和IN的分泌是必不可少的。我们发现这种分泌，特别是B类结构的分泌，被N，N‘-二半己基碳酰亚胺抑制，这是一种H^+-ATPase抑制剂，而不是正常生长。我们还发现，这种分泌需要ATP和ELKI表面的正电荷。较少

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