Nemecek-Marshall M, LaDuca R, Fall R
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
J Bacteriol. 1993 Jul;175(13):4062-70. doi: 10.1128/jb.175.13.4062-4070.1993.
Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).
人们尝试使从番茄叶片中分离出的丁香假单胞菌T1中的冰核表达最大化。在32℃下对氮、磷、硫或铁而非碳进行营养饥饿处理，并将温度转变为14至18℃，会导致1型冰核（即，在高于-5℃的温度下具有活性的冰核）的快速诱导。诱导在以山梨醇作为碳源生长并快速冷却的稳定期细胞中最为明显，在最佳条件下，1型冰核的表达在2至3小时内从每10(7)个细胞中少于1个（即，不可检测）增加到每个细胞中有1个。该诱导被蛋白质和RNA合成抑制剂阻断，这表明有新基因表达。在转移至低温后用[35S]甲硫氨酸对非生长培养物进行脉冲标记表明，诱导合成了一组新的“低温”蛋白质。诱导产生的冰核在低温下稳定，在4℃下8天后活性无损失，但在转移回32℃后，1型冰核完全消失，半衰期约为1小时。用相同的非生长细胞可以证明这些冰核的低温诱导和高温周转的重复循环。并非来自番茄或其他植物的所有丁香假单胞菌菌株在与T1菌株相同的培养条件下都能被完全诱导，但在转移至低温后，所有菌株的1型冰核表达均增加。支持这一观点的是，对inaZ基因翻译起始位点之前已发表的DNA序列进行分析（R.L.格林和G.沃伦，《自然》[伦敦]317:645 - 648，1985）表明存在一种齿轮箱型启动子（M.文森特、S.R.库什纳、T.加里多和M.阿尔德亚，《分子微生物学》5:2085 - 2091，1991）。
