Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999     

冷激蛋白的研究进展

冷激蛋白（CSPs）广泛存在于革兰氏阳性菌和阴性菌中,它是细胞在应对冷刺激时所产生的一系列7 ku左右的蛋白质,结构上富含芳香族氨基酸,起着重要的分子伴侣作用,能够增强细胞抵御冷激环境胁迫的能力.以大肠杆菌、枯草杆菌、嗜热链球菌、沙门氏杆菌等为例,介绍各种冷激蛋白在产生、结构、调控等方面的异同,以及它在生产、生活中的应用价值.     

Cold Shock Syndrome in Anacystis nidulans

The phenomenon of cold shock in Anacystis nidulans has been explored further in terms of loss of viability and immediate and subsequent metabolic effects. Cold shock was observed also in two closely related strains in which unsaturated fatty acid contents are also known to be low and temperature-dependent. Loss of viability was maximum for cells grown at temperatures above 40 C (<10⁻⁴ survivors after 5 min at 0 C) but became negligibly small for cells grown below 34 C. Development of the cold-sensitive condition after transfer 25 → 39 C was slow and comparable to rate of growth; development of the insensitive condition after transfer 39 → 25 C was rapid, implying rapid in situ alteration. An immediate metabolic effect, observed as a decrease in rate of photosynthetic O₂ evolution measured at growth temperature, was less severe than loss of viability. Continued light incubation under growth conditions led to slow decay in rate of O₂ evolution accompanied by loss of membrane chlorophyll. The multiple effects which comprise the cold shock syndrome appear to be membrane-related phenomena and thereby provide an experimental probe of normal membrane function.     

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. Received: 18 October 1996 / Accepted: 28 January 1997     

Cold Shock of Germinating Bacillus subtilis Spores

Susceptibility of germinating spores of Bacillus subtilis to a rapid chilling was examined by viable countings. Dormant spores were quite resistant to the cold shock but the spores, immediately upon germination, lose viability almost completely by the same treatment. The presence of divalent cation, magnesium, calcium or manganese, in a buffer to which the germinating spores were suspended, markedly protected the cells from the death by the cold shock effect. When the shocked cells were incubated in the buffer containing casein acid hydrolyzate, glucose and magnesium ion for short period of time, a remarkable increase in viable counts was observed. The existence of two critical temperature zones, which were determined by the initial temperature of cell suspension, was confirmed in the cold shock of germinating spores.     

微生物产生的冷休克蛋白研究进展

冷休克蛋白(cold shock protein,Csp)首先在大肠杆菌中发现,它与微生物对冷环境的适应及多种细胞功能有关。冷休克蛋白基因是一段编码70个左右氨基酸的DNA序列,在这段序列中有5′非翻译区(5′UTR)、冷盒及下游盒等特征。冷休克蛋白作为DNA或RNA结合蛋白在基因表达调控过程中起重要作用。冷休克蛋白在转录、mRNA稳定性及翻译等几个水平上被严格调控。     

Cold Shock Lethality and Injury in Clostridium perfringens

Several observations have been made in regard to cold shock lethality of Clostridium perfringens: (i) loss of viability was not consequence of exposure of the cells to air; (ii) stationary-phase cells were much more resistant to cold shock at 4 C than exponential-phase cells; (iii) at 4 C 96% of an initial population of exponential-phase cells was killed upon cold shock and 95% of the remaining population was killed within 90 min of continued exposure at 4 C; (iv) the minimal temperature differential for detectable cold shock lethality was between 17 and 23 C, and the maximum beyond which lethality was not appreciably increased was between 28 and 33 C. Up to 75% of viable cold-shocked cells were injured, as demonstrated by cold shocking late exponential-phase cells at 10 C and using differential plating procedure for recovery. Repair of injury was temperature dependent, and occurred in a complex medium and 0.1% peptone but not water. Nalidixic acid, chloramphenicol, and rifampin did not inhibit repair of injury.     

植物冷激蛋白的研究进展

冷激蛋白是存在于细菌、植物与动物中的一类高度保守的核酸结合蛋白,其通过RNA分子伴侣活性参与转录、翻译及生长发育和逆境胁迫应答等细胞生理活动。本文主要从植物冷激蛋白的结构、表达模式、生物学功能以及应用前景等几个方面介绍了植物冷激蛋白的研究进展。     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock

Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24°C to 4°C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24°C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before.     

微生物冷休克蛋白的生理功能及其潜在应用

冷休克反应是普遍存在于微生物体内的一种适应环境温度骤降的应答机制,而氨基酸序列高度保守的冷休克蛋白则是调控冷休克反应的重要因子。越来越多的研究表明,冷休克蛋白除了调控冷休克反应外,还参与调控了生物体多个性状,如宿主正常生长和分化、病原菌的侵袭力和致病性以及宿主对多种逆境（高渗透压、抗生素）的应答。综述了冷休克蛋白及其来源、其参与调控的生理性状和基于冷休克反应及冷休克蛋白的应用,以期为发酵条件优化、食品储藏、致病菌抑制以及作物性状改良等方面提供有益参考。     

离体培养细胞系冷休克敏感差异性的研究

应用台盼蓝活体染色方法、Hoechst332 5 8荧光探针技术研究低温冷休克 (4℃ )对人肝癌细胞系 (74 0 2 )、秋行军虫细胞系 (Sf9)、幼蚊细胞系 (C6 36 )及草鱼肾细胞系 (CIK)的影响。结果显示 :在冷休克处理 6天后 ,Sf9、C6 36、CIK、74 0 2细胞系的死亡率分别是 2 0 .0 3%、10 0 %、2 8.6 9%、10 0 % ;凋亡率分别为 2 .4 5 %、38.38%、8.2 5 %、96 .4 7% ,其细胞的死亡率远远大于凋亡率。可见冷休克导致细胞死亡过程中 ,应是细胞坏死和凋亡并存。但就其细胞凋亡的敏感性而言 ,4种细胞顺序应为 74 0 2 >C6 36 >CIK >Sf9。研究结果为在细胞水平、分子水平深入研究低体温生物离体细胞冷休克机理奠定基础。     

Cold Shock and Its Effect on Ribosomes and Thermal Tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0°C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 ± 0.1°C (mean ± standard deviation) to 72.1 ± 0.5°C (P ≤ 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 ± 0.4°C to 66.8 ± 0.5°C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

嗜温冷休克蛋白力致去折叠研究

嗜温冷休克蛋白拥有一个由五个β股形成的反平行β桶结构,目前已被用于蛋白质去折叠的研究。当使用机械力对嗜温冷休克蛋白进行拉伸研究时,发现嗜温冷休克蛋白的去折叠过程具有明显的中间态。在常速和常力两种情况下对嗜温冷休克蛋白进行拉伸分子动力学模拟,发现其在两种情况下具有相同的去折叠次序,即C端β片层首先去折叠,随后N端β片层去折叠;同时这两种模拟都表现出明确的中间态。研究结果表明,嗜温冷休克蛋白抵抗外力作用除了依赖链间氢键外,分子内的静电相互作用也发挥着重要的作用。