枯草芽孢杆菌萌发后孢子响应甲醛胁迫过程的拉曼光谱研究

Raman Spectrum of Germinated Spores of Bacillus Subtilis in Response to Formaldehyde Stress

为了探究枯草芽孢杆菌萌发后孢子响应甲醛胁迫过程的生理反应及其耐受甲醛机理,利用激光光镊拉曼光谱（LTRS）系统研究不同浓度甲醛胁迫萌发后孢子2 h的响应过程。结果显示,营养细胞和萌发后孢子对甲醛都具有耐受能力,但萌发后孢子对不同浓度甲醛胁迫产生的生理响应效应不同;当萌发后孢子在不含甲醛的培养基中生长时,与生物大分子相应的特征峰强度增加先经历0~0.5 h的延缓期,接着1.5~2.0 h是一个快速上升的过程;当分别用0.4,0.8和1.0 mmol/L甲醛胁迫萌发后孢子时,相应特征峰强度都有一个先上升后下降的过程,峰强下降的时间拐点分别是1.5,1.0和0.5 h。光谱分析结果表明,0.4 mmol/L甲醛的胁迫会对细胞造成轻度伤害;0.8 mmol/L甲醛胁迫会抑制孢子DNA的复制,造成膜脂碳氢链断裂;1.0 mmol/L甲醛胁迫时,细胞在0.5 h内对甲醛产生了剧烈的胁迫响应,导致生物大分子含量在0.5~2.0 h内严重下降,细胞受到严重伤害。

In order to explore the physiological response and tolerance mechanism of Bacillus subtilis after exposure to formaldehyde,laser tweezers Raman spectroscopy（LTRS） is employed to perform the experimental process in which germinated spores（g.spores） are stressed with different concentrations of formaldehyde for 2 h.The results show that both vegetative cells and g.spores have tolerance to formaldehyde,but the growth and physiological effect of g.spores stressed by different concentrations of formaldehyde are dissimilar.A lag phase of characteristic spectrum involved with various biomacromolecules is observed while g.spores are exposed to 0 mmol/L of formaldehyde medium from 0 to 0.5 h,subsequently,the Raman spectra peaks tend to rapidly ascend from 0.5 to 2.0 h.Characteristics and trend of Raman spectra variation are all from ascending to descending while g.spores are exposed to 0.4,0.8,and 1.0 mmol/L of formaldehyde medium respectively for 2 h,and the corresponding inflexion of Raman spectra causes various components in vivo to decline where the time points are 1.5,1.0,and 0.5 h,respectively.The analytic results of Raman spectra display that various components in vivo decrease gradually after 1.5 h while g.spores are stressed by 0.4 mmol/L of formaldehyde,implying that g.spores are harmed mildly.While g.spores are in 0.8 mmol/L formaldehyde medium,the band of nucleic acid decreases slowly at the very start,indicating that DNA cannot duplicate in the whole process.The bands of membrane phospholipids and C-S（protein） stretching mode descend obviously after formaldehyde stress for 0.5 h,which suggested formaldehyde could break down the membrane lipid hydrocarbon chains.While g.spores are in 1.0 mmol/L of formaldehyde medium,the high formaldehyde concentration makes a severe stress effect on cells,the content of biological macromolecules drops significantly after 0.5 h leading to cells deterioration gradually.