单细胞分析研究二氧化氯对杀蚊细菌球形赖氨酸芽胞杆菌芽胞的影响

doi:10.16409/j.cnki.2095-039x.2021.09.028

中国生物防治学报 ›› 2022, Vol. 38 ›› Issue (3): 565-572.DOI: 10.16409/j.cnki.2095-039x.2021.09.028

• 研究论文 • 上一篇

单细胞分析研究二氧化氯对杀蚊细菌球形赖氨酸芽胞杆菌芽胞的影响

李翠梅^1,2, 王晓春², 张秀娟^1,2, 刘军贤¹, 王桂文²

1. 广西师范大学物理科学与技术学院, 桂林 541004;
2. 广西科学院, 南宁 530007

收稿日期:2021-05-12 发布日期:2022-06-20
通讯作者: 王桂文,研究员,E-mail:wguiwen@gxas.cn;刘军贤,博士,教授,E-mail:jxliu@mail.gxun.edu.cn。
作者简介:李翠梅,硕士研究生,E-mail:2217895011@qq.com。
基金资助:
国家自然科学基金（11264004）；广西自然科学基金（2017GXNSFAA198029）

Investigation of the Effect of Chlorine Dioxide on Spores of the Mosquito-killing Bacterium Lysinibacillus sphaericus by Single-cell Analysis

LI Cuimei^1,2, WANG Xiaochun², ZhANG Xiujuan^1,2, LIU Junxian¹, WANG Guiwen²

1. School of Physical Science and Technology, Guangxi Normal University, Guilin 541004, China;
2. Guangxi Academy of Sciences, Nanning 530007, China

Received:2021-05-12 Published:2022-06-20

摘要/Abstract

摘要： 球形赖氨酸芽胞杆菌Lysinibacillus sphaericus（Ls）是应用广泛的蚊虫生物防治制剂，在实际应用时可能会遇到不利的环境因子或杀菌剂。本研究通过单细胞分析方法监测ClO₂处理后的单个Ls芽胞及其萌发过程，探究ClO₂对Ls芽胞结构和成分的影响及与芽胞萌发相关蛋白的影响，以及ClO₂的杀胞机制。经0.05%～0.3% ClO₂处理5 min后，36%～86%的芽胞不能形成单菌落。吡啶二羧酸钙（CaDPA）特征峰（1017 cm^-1）强度随处理程度增大有所降低，而蛋白质α-螺旋峰（1652 cm^-1）强度没有明显变化。ClO₂处理显著改变Ls芽胞的萌发动态，芽胞启动CaDPA快速释放的时间、完全释放的时间以及皮层水解完成的时间显著延长；而外源CaDPA仅能触发低浓度ClO₂（0.05%）处理的芽胞，且显著迟缓。表明ClO₂处理没有直接破坏芽胞内膜但严重损伤了与萌发相关蛋白的功能，特别是皮层水解酶容易失活。这可能就是被处理的芽胞可以启动芽胞萌发的最初步骤，而不能进一步发展为营养细胞的原因。

关键词: 球形赖氨酸芽胞杆菌, 二氧化氯, 拉曼光谱, 微分干涉差成像, 芽胞萌发, 单细胞分析

Abstract: Lysinibacillus sphaericus (Ls) is a widely used biological control agent that produces insecticidal proteins with high activity against mosquito larvae. In practical applications, Ls may encounter unfavorable environmental factors or different disinfectants; chlorine dioxide (ClO₂) is an efficient disinfectant. In this study, the individual chlorine dioxide-treated Ls spores were observed by single-cell Raman spectroscopy and the germination dynamics of spores was monitored by differential interference contrast microscopy to investigate the effects of chlorine dioxide on the structure and composition of Ls spores and proteins involved in spore germination, as well as the spore-killing mechanism of chlorine dioxide. Thirty six to eighty six percent of the spores were nonculturable but remained their CaDPA at 5 min post treatment with 0.05%-0.3% chlorine dioxide. The intensity of peak derives from pyridine-2, 6-dicarboxylic acid, DPA (1017 cm^-1) decreased with increasing concentration of the treatment solution, while the intensity of peak from α-helix of protein (1652 cm^-1) did not change significantly. Treatment remarkably altered the germination dynamics of individual spores triggered by L-alanine or dodecylamine, significantly prolonging the time to initiate rapid release of CaDPA, to complete CaDPA release, and to complete hydrolysis of spore cortex. In contrast, exogenous CaDPA was only able to trigger the germination of spores treated with low concentrations of chlorine dioxide (0.05%) and was significantly delayed. These results show that chlorine dioxide does not destroy the inner membrane of spores directly, but severely impairs the function of germination-related proteins, especially the cortex-lytic enzymes. This may be the reason that the chlorine dioxide- killed spores can undergo the initial steps of germination but cannot go further to grow into vegetative cells.

Key words: Lysinibacillus sphaericus, chlorine dioxide, Raman spectrum, differential interference contrast imaging, spore germination, single cell analysis

中图分类号:

TQ453.5

李翠梅, 王晓春, 张秀娟, 刘军贤, 王桂文. 单细胞分析研究二氧化氯对杀蚊细菌球形赖氨酸芽胞杆菌芽胞的影响[J]. 中国生物防治学报, 2022, 38(3): 565-572.

LI Cuimei, WANG Xiaochun, ZhANG Xiujuan, LIU Junxian, WANG Guiwen. Investigation of the Effect of Chlorine Dioxide on Spores of the Mosquito-killing Bacterium Lysinibacillus sphaericus by Single-cell Analysis[J]. Chinese Journal of Biological Control, 2022, 38(3): 565-572.

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单细胞分析研究二氧化氯对杀蚊细菌球形赖氨酸芽胞杆菌芽胞的影响

Investigation of the Effect of Chlorine Dioxide on Spores of the Mosquito-killing Bacterium Lysinibacillus sphaericus by Single-cell Analysis

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