【作者】 程琴；

【导师】 陈丽梅； 黄庶识；

【作者基本信息】 昆明理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 芽孢杆菌休眠孢子的萌发是孢子恢复到营养生长的第一个决定性步骤。孢子被营养性萌发剂和各种非营养信号诱导而萌发恢复到营养细胞状态。芽孢萌发后就丧失了对外界胁迫的抵抗力。2,6-二羧酸吡啶(DPA-Ca2+)是细菌芽孢特有的成分,其在芽孢抗性和稳定性方面有很重要的作用,随着DPA-Ca2+释放,不含DPA-Ca2+的芽孢就失去对外界胁迫的抗性。研究芽孢DPA-Ca2+释放行为及其外界胁迫下芽孢成分变化,对于深入了解细菌芽孢抗性机制有重要意义。运用激光镊子拉曼光谱技术(LTRS)研究了单个枯草杆菌芽孢响应甲醛胁迫过程中动态的变化过程以及DPA-Ca2+释放方式,并结合多元统计分析方法统计分析芽孢用甲醛处理前后的成分差异。结果表明：甲醛胁迫下芽孢耐受甲醛能力比萌发后DPA-Ca2(?)完全释放的芽孢强500多倍；与萌发剂诱导或氯气、高温湿热胁迫不同,甲醛的胁迫不会引起芽孢DPA-Ca2+的释放,甲醛是直接穿透细菌孢子多层屏障进到孢子内部作用于孢子的蛋白质和DNA。多元统计方法分析1000mM甲醛胁迫芽孢后芽孢内生物大分子的光谱数据显示,导致芽孢致死原因是甲醛损伤核酸的嘌呤碱、G-C碱基对,破坏蛋白质的C-C主链,对膜脂也有一定的伤害。1.5mM甲醛胁迫萌发后芽孢(germinated spores)的致死原因除了破坏蛋白质的C-C主链以外,也伤害其侧链；对腺嘌呤和膜脂类有轻微的损伤。958cm-1的载荷绝对值最大,提示甲醛对萌发后芽孢伤害最大是它的蛋白质。氯气和湿热胁迫枯草芽孢杆菌孢子,其核心DPA-Ca2+(?)释放与孢子致死并不是同步的,孢子被胁迫致死时孢子内依然还保留有DPA-Ca2+,氯气和湿热处理孢子都能使核心DPA-Ca2+(?)释放,此过程非常迅速,一般在1-2分钟释放完,DPA-Ca2+完全释放后跟随着1655cm-1谱峰漂移到1665cm-1蛋白质主链有序结构(α-螺旋)减少,无规则卷曲增加,导致蛋白质分子空间结构发生变化。主成分分析(PCA)分析结果表明：氯气主要破坏芽孢的内膜,引起DPA-Ca2+释放,氯气进到孢子内部破坏孢子蛋白质的C-C主链,损伤核酸,导致芽孢致死；高温损伤了酪氨酸的对羟苯基环、色氨酸的吲哚环,蛋白质的侧链基团受到破坏甚至破裂,同时胁迫会引起脯氨酸的积累,表明湿热胁迫主要损伤其蛋白质或使酶失活。枯草芽孢杆菌是可以同化甲醛的非甲基营养菌,其营养细胞可以通过核酮糖单磷酸途径(RuMP)同化甲醛为自身细胞的组分。为了探究枯草芽孢杆菌萌发后孢子(germinated spores)响应甲醛胁迫过程的生理反应及其耐受甲醛机理,利用激光镊子拉曼光谱系统(LTRS)研究不同浓度甲醛胁迫萌发后孢子(g. spore) 2 h的响应过程。结果显示,营养细胞和萌发后的孢子(g. spores)都具有耐受甲醛的能力,但营养细胞的耐受能力远强于萌发后的孢子(g. spores),与营养细胞含有相同的遗传物质和功能性蛋白的萌发后孢子(g. spores)不能吸收、代谢甲醛。萌发后孢子(g. spores)响应甲醛胁迫的拉曼光谱特征峰的走势显示,0.4 mmol/L甲醛胁迫萌发后的孢子(g. spore),在0-1.5 h范围内代表蛋白质、脂类和核酸的特征峰变化峰强增加的幅度大于孢子在不含甲醛的培养基中变化趋势,这种变化趋势反映了萌发后孢子(g. spores)在受到甲醛胁迫初期做出较强烈的响应模式,很多基因的表达水平上升,细胞内代谢水平增加,大分子物质积累也跟着增加,在第1.5 h后体内各组分下降,表明细胞受到轻度的伤害；0.8 mmol/L甲醛胁迫萌发后孢子,细胞体内成分下降的临界点提前到了第1.0 h,归属核酸的谱峰一开始就慢慢下降,显示整个过程DNA不能进行复制,归属膜磷脂和蛋白质的C-S伸缩振动的谱峰在0.5 h后即呈明显下降趋势,提示0.8 mmol/L甲醛胁迫细胞后,甲醛导致膜脂碳氢链断裂；1.0 mmol/L甲醛胁迫下,谱峰下降的临界时间点是第0.5 h,表明细胞对该浓度甲醛产生了剧烈的胁迫效应,在0.5 h后就导致生物大分子含量严重下降,最终使细胞受到的伤害逐步加重。更多还原

【Abstract】 The germination of Bacillus spores is the first step to return the vegetative growth, and is induced by nutrients and a variety of non-nutrient agents, and spores completely loss resistance to outer stress.As a characteristic composition of bacteria spores, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plays an important role in spores resistance as well as spores stability to many environmental stresses. Following DPA-Ca2+ released entirely, spores lose their refraction and resistance completely. It is significant to explore the behavior of DPA-Ca2+ release and the rule of compositional changes in spores under environmental stresses for understanding the resistance mechanism of bacterial spores. We reported the application of laser tweezers Raman spectroscopy (LTRS) to research the character of DPA-Ca2+ release, the dynamics of an individual spore and the component difference combined with Principal Components Analysis (PCA) under formaldehyde stress. The results indicated that the toleration ability of spores to formaldehyde is 500 times stronger than germinated spores (DPA-Ca2+ released completely); Formaldehyde treatment didn’t cause DPA-Ca2+ release of spores, which was different with germinants induction or chlorine/high temperature and wet heat stress:Formaldehyde was to penetrate multilayer barriers of the bacterial spore into the spores internal and interact with proteins and DNA directly. The PCA results showed that killing of spores by 1000mM of formaldehyde is due to damaging purine and G-C bases of nucleic acid, C-C backbone, and also harming to membrane lipids partly; oppositely, killing of germinated spores treated with 1.5mM of formaldehyde is due to C-C main chain and side chain damage, which adenine and membrane lipid have been damaged slightly, the maximum absolute value of PC1(principal component1) loading of 958cm-1 exhibited that the max damage to germinated spores by formaldehyde was spores proteins.the spores of bacillus subtitles in response to bleaching water and wet heat, the release of DPA-Ca+ from its core is not couple with spores’death, Ca2+-DPA retain when Spores were dead. bleaching water and wet heat make DPA-Ca2+ release; this process is very fast, generally in 1-2 minutes. after DPA-Ca2+ releasing, the band of 1655cm-1 shifts to 1665cm-1,α-helix of protein decreasing, no regular coil increasing, so space structure of Protein changes. Principal Components Analysis (PCA) analysis results indicate:bleaching mainly damage the spore’s inner membrane, causing DPA-Ca2+ release, Chlorine damage C-C Lord chain of protein breaking into the spores, Damaging nucleic acid, making g.spores dead; high temperature damage the hydroxyl phenyl rings of Tyrosine、the indoles rings of tryptophan, damage side chain of Protein, even rupture, following accumulation of proline,suggesting that damage protein or make enzyme deactivation in wet heat stress.Bacillus subtilis is non-methylotrophic bacteria which can assimilate formaldehyde, whose vegetative cell can transform formaldehyde to one’s own components by RuMP (Ribulose monophosphate) pathway. In order to explore germinated spores’(g.spore) of bacillus subtitles physical reaction and tolerance mechanism in response to formaldehyde, researching on the process of different concentrations formaldehyde stress g. spore 2 hours by laser tweezers Raman spectra system (LTRS).The result shows, g.spores contain the same genetic material and functional proteins with vegetative cell, which have the ability of tolerating formaldehyde, but the ability of vegetative cell is stronger than g.spore, and g.spore can’t absorb and metabolize formaldehyde. The Raman spectrum band changes of g.spores in response to formaldehyde stress displays:g.spores in 0.4mmol/L formaldehyde medium, whose Change intensity of protein, Lipid and nucleic acid is stronger than g.spores in no formaldehyde medium, The trends reflect that g.spores makes a strong response pattern early in formaldehyde stress, many gene expression level rise, cells metabolism level increases, macromolecular substances accumulation also follow increase, After the 1.5 hours, various components in vivo decrease, showing cells are mild hurt; g.spores in 0.8mmol/L formaldehyde medium, various components in vivo decrease at 1.0 hour, the band of nucleic acid decreases slowly at the very start, indicating DNA can’t duplicate in the whole process, the bands of membrane phospholipids and C-S(protein) stretching mode decline obviously after 0.5 hours, which suggests formaldehyde Causing the membrane lipid hydrocarbon chain breakedown;g.spores in 1.0mmol/L formaldehyde medium, the bands decrease at 0.5 hour, suggesting the concentration formaldehyde makes a severe stress effect on cells, the content of biological macromolecules drops significantly after 0.5 hour, making cell hurts gradually deteriorated.更多还原