中国农业科学 ›› 2020, Vol. 53 ›› Issue (5): 1046-1057.doi: 10.3864/j.issn.0578-1752.2020.05.015

• 食品科学与工程 • 上一篇    下一篇

热胁迫对大肠杆菌细胞膜和膜蛋白的影响

张爱静,李琳琼,王鹏杰,高瑀珑()   

  1. 南京财经大学食品科学与工程学院/江苏省现代粮食流通与安全协同创新中心/江苏高校粮油质量安全控制及深加工重点实验室,南京 210023
  • 收稿日期:2019-07-09 接受日期:2019-10-22 出版日期:2020-03-01 发布日期:2020-03-14
  • 通讯作者: 高瑀珑
  • 作者简介:张爱静,E-mail:zhangaijing525902@163.com。
  • 基金资助:
    国家自然科学基金(31371864);江苏省自然科学基金(BK20131435);公益性行业粮食科研专项(201413007-05)

Effects of Heat Stress on Cell Membrane and Membrane Protein of Escherichia coli

ZHANG AiJing,LI LinQiong,WANG PengJie,GAO YuLong()   

  1. College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing 210023
  • Received:2019-07-09 Accepted:2019-10-22 Online:2020-03-01 Published:2020-03-14
  • Contact: YuLong GAO

摘要:

【目的】以大肠杆菌ATCC43889为试材,研究热胁迫处理对其细胞膜和膜蛋白的影响,为高温杀菌技术在食品工业中的应用奠定理论基础。【方法】研究ATCC43889分别经50℃、60℃和70℃热胁迫处理15 min并转接10次培养后,获得的3种抗热性ATCC43889菌株的细胞膜和膜蛋白变化。采用扫描电子显微镜分别观察原始对照菌株和3种抗热性菌株个体形态的变化;采用96孔微量酶标板法测定各菌株生物被膜生成能力的强弱;用气相色谱法测定各菌株细胞膜脂肪酸组成的变化及其差异;用差示扫描量热法测定各菌株细胞膜磷脂相变温度的变化;通过聚丙烯酰胺凝胶电泳(SDS-PAGE)法检测各菌株外膜蛋白表达的变化。【结果】大肠杆菌ATCC43889分别经50℃、60℃和70℃各10次热胁迫处理并转接10次培养后,其个体形态变化明显,经50℃热胁迫后,部分菌株由球状体变为长杆状;经60℃热胁迫后的菌株个体形态较50℃热胁迫处理的菌株细长;经70℃热胁迫处理后大部分菌株变成了更细长的杆状,大量的菌株聚集在一起,菌体表面呈凸凹不平的无规则形态。随着热胁迫温度的提高,ATCC43889抗热性菌株的生物被膜生成活力增大,其生物被膜生成能力增强,3种抗热性菌株的生物被膜生成能力与对照组相比差异显著(P<0.05),60℃和70℃下获得的2种抗热性菌株的生物被膜生成能力差异不显著(P>0.05)。与对照的原始菌株相比,经过50℃、60℃和70℃各10次热胁迫处理并转接10次培养的菌株,缺失了3种脂肪酸,分别为C18:1n9c、C18:3n3和C21:0,随着热胁迫温度的提高,热胁迫菌株细胞膜中C13:0、C16:0和C17:0等饱和脂肪酸(SFA)及其总含量升高,而C14:1、C16:1、C17:1、C18:1n9t和C18:2n6t等不饱和脂肪酸(USFA)及其总含量降低,总SFA/总USFA比值增大,SFA/USFA比值越大,菌体细胞膜流动性越弱。随着热胁迫温度的提高,ATCC43889抗热性菌株细胞膜磷脂的熔点(Tm)升高,表明细胞膜磷脂的相变温度升高,细胞膜流动性降低,热胁迫温度越高,细胞膜流动性越低;随着热胁迫温度的提高,分子质量在63 kD和75 kD附近的条带颜色逐渐变深,分别经60℃和70℃各10次热胁迫并转接10次培养获得的2种抗热性菌株,其细胞外膜蛋白分子质量在48—75 kD均增加了特异条带;胁迫温度越高,一些外膜蛋白表达量及其种类增加,ATCC43889的耐热性越强。【结论】随着热胁迫温度的升高,ATCC43889个体形态变长,生物被膜生成能力增强,饱和脂肪酸含量升高,不饱和脂肪酸含量下降,细胞膜磷脂的相变温度升高,细胞膜流动性降低,一些外膜蛋白表达量提高、表达种类增加,菌株耐热性增强,这些变化有利于其适应不利的热胁迫环境,提高生存能力。

关键词: 热胁迫, 大肠杆菌, 生物被膜, 膜脂肪酸, SDS-PAGE

Abstract:

【Objective】Effects of heat stress on cell membrane and membrane protein of Escherichia coli were investigated with ATCC43889 as the test microorganism in this work, which would provide a theoretical basis for the application of high temperature sterilization technology in the food industry. 【Method】 The changes of the cell membrane and membrane protein for three heat-resistant ATCC43889 strains which were treated with 10 times of heat stress treatments at 50℃, 60℃, and 70℃ for 15 min, transferred into TSB and incubated again were studied, respectively. The morphologic changes of original control strain and the three heat-resistant strains of ATCC43889 were observed using scanning electron microscopy. The biofilm-forming ability for each strain was determined using a 96-well microplate method. The changes and differences of cell membrane fatty acid composition for each strain were monitored using gas chromatography. The changes of phase transition temperature of cell membrane phospholipid for each strain were measured using differential scanning calorimetry (DSC). The changes of outer membrane protein expression for each strain were examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 The experimental results showed that the individual morphology of ATCC43889 changed evidently after 10 times of heat stress treatments at 50℃, 60℃, and 70℃, transfers and incubation again, respectively. The individual morphology for a part of the cells changed from spheroids to long rods after heat stress treatment at 50℃. The individual morphology of the cells upon heat stress treatment at 60℃ was thinner and longer than that of the cells upon heat stress treatment at 50℃. Upon heat stress treatment at 70℃, most of the cells became longer rods, a large number of cells gathered together, and the surface of the cells was irregular and uneven. The vitality and ability of biofilm-forming for the three heat-resistant ATCC43889 strains enhanced with increasing heat stress temperature. The ability of biofilm-forming for the three ATCC43889 heat-resistant strains significantly (P<0.05) differ from that of the control group, while the ability of biofilm-forming for the heat-resistant strain at 60℃ was not significantly (P>0.05) different from that of the heat-resistant strain at 70℃. Among the fatty acids identified, three fatty acids were absent after 10 times of heat stress treatments at 50℃, 60℃ and 70℃ compared to the original control strain, which were C18:1n9c, C18:3n3, and C21:0, respectively. The amount of saturated fatty acid such as C13:0, C16:0, C17:0 and their total amounts increased, while the amount of unsaturated fatty acid such as C14:1, C16:1, C17:1, C18:1n9t, C18:2n6t and their total amounts in the cell membrane for the three heat-resistant strains decreased with the increase of heat stress temperature. The ratio of saturated to unsaturated fatty acids (SFA/USFA), the melting point (Tm) and the phase transition temperature of cell membrane phospholipids increased as the heat stress temperature increased. These changes in membrane fatty acid composition and the phase transition temperature of cell membrane phospholipids resulted in decreased membrane fluidity of the three heat-resistant strains. The protein band color of molecular weight about 63 and 75 kD deepened with the increase of heat stress temperature. The increase of heat-resistance for the two heat-resistant strains after 10 times of heat stress treatments at 60℃ and 70℃ was accompanied by synthesis of specific outer membrane proteins of molecular weight about 48 to 75 kD. These results indicated that synthesis and increased amounts of some specific outer membrane proteins could induce heat-resistance for E. coli ATCC43889. 【Conclusion】The longer individual morphology, the enhanced ability of biofilm-forming, the increased ratio of saturated to unsaturated fatty acids, the increased phase transition temperature of cell membrane phospholipid and the increased expression of some outer membrane proteins correlated with the greater heat-resistance of E. coli ATCC43889. These changes contributed to the adaptation of ATCC43889 to heat stress environment and improvement of cellular survival viability.

Key words: heat stress, Escherichia coli, biofilm, membrane fatty acid, SDS-PAGE