氢氧化钠对苏云金芽孢杆菌芽孢及其萌发的影响

Effect of NaOH on the Bacillus thuringiensis Spores and Its Germination

关键词:  苏云金芽孢杆菌芽孢  拉曼光谱  DIC成像  碱  芽孢萌发

Key words:  Bacillus thuringiensis  Raman spectroscopy  DIC microscopy imaging  alkali  spore germination

摘要:

[目的]了解碱对细菌芽孢及萌发的影响及机制,进一步认识芽孢的抗性,为寻找更好的杀灭芽孢的方法提供理论参考。[方法]应用单细胞拉曼光谱采集经NaOH处理后的单个苏云金芽孢杆菌(Bt)芽孢的拉曼光谱,并应用微分干涉差显微镜成像术分析其萌发动态。[结果]1.0 mol/L NaOH处理30 min、45 min和60 min后,Bt芽孢的存活率分别为25.0%、3.6%和1.8%左右;芽孢吡啶二羧酸(DPA)和蛋白质的特征峰有微弱的降低,蛋白质二级结构α螺旋特征峰(1 280/1 652 cm^-1)的位置和信号强度与对照没有明显的差异。在10 mmol/L丙氨酸下,单个芽胞开始大量释放CaDPA的时间(T_lag)、快速释放CaDPA所需的时间(ΔT_release)和芽胞皮层水解所需的时间(ΔT_lys)分别是对照的3.5~5倍、12~13倍和1.5~2倍;在月桂胺触发下,T_lag值和ΔT_release值分别是对照的4倍和11倍左右。外源CaDPA无法触发处理30 min后的芽孢的萌发,而对照则萌发极快。[结论]NaOH处理没有破坏Bt芽孢的内膜,也没有引起成孢内CaDPA泄漏,但令与芽孢萌发相关的蛋白质受损,受损最严重的是皮层水解酶,对芽胞萌发产生严重的影响。

Abstract:

[Objective] To determine the effect and mechanism of alkali on Bacillus thuringiensis (Bt) spores and its germination and to further understand the resistance of spores can provide a theoretical reference for seeking better ways to kill bacterial spores.[Methods] Single-cell Raman spectroscopy was used to analysis the spores treated by alkali at room temperature, and differential interference contrast (DIC) microscopy imaging was used to monitor the kinetics of spore germination.[Results] The viability of Bt spores was 25.0%, 3.6% and 1.8%, respectively, when treated with 1.0 mol/L NaOH for 30, 45 and 60 min. There was slightly decreased intensity at Raman bands from dipicolinic acid (DPA) and proteins, while there was no evident difference on the positions and intensities of α-helical structures (1 280, 1 652 cm^-1) between treated and untreated spors.When treated spores germinated in L-alanine, the time, Tlag, at which spores began release of the great majority of their 1:1 chelate of Ca²⁺ and DPA (CaDPA), the time periods, ΔT_release, at which a spore germinating completed the release of the great majority of its CaDPA, and the time periods, ΔT_lys, at which a spore completed the hydrolysis of peptidoglycan cortex, was about 3.5~5, 12~13 and 1.5~2 times of the untreated spores, respectively.The T_lag and ΔT_release values were about 4 and 11 times of untreated spores when germinated with dodecylamine. However, treated spores did not germinate in exogenous CaDPA.[Conclusion] These results indicate that alkali does not disrupt the Bt spores permeability barrier and the spore does not lose its DPA, but some proteins important in spore germination are damaged by alkali, and the cortex-lytic enzymes are the target of alkali damage which has a great effect on the germination of Bt spores.