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王桂文,张鹏飞,王晓春,陈欢君,Peter Setlow,LI Yongqing.溶菌酶触发的芽孢杆菌芽孢萌发及其异质性研究[J].广西科学,2016,23(2):102-110. [点击复制]
- WANG Guiwen,ZHANG Pengfei,WANG Xiaochun,CHEN Huanjun,Peter Setlow,LI Yongqing.Kinetics of Germination of Individual Bacillus Spores and its Heterogeneity Triggered by Lysozyme[J].Guangxi Sciences,2016,23(2):102-110. [点击复制]
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| 溶菌酶触发的芽孢杆菌芽孢萌发及其异质性研究 |
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王桂文1, 张鹏飞2, 王晓春1, 陈欢君3, Peter Setlow4, LI Yongqing2
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| (1.广西科学院, 广西南宁 530007;2.东卡罗来纳大学物理系, 美国北卡罗来纳州格林维尔市, NC 27858;3.广西大学生命科学与技术学院, 广西南宁 530004;4.康涅狄格大学健康中心分子、微生物与结构生物学系, 美国康涅狄格州法明顿, CT 06030) |
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| 摘要: |
| [目的]了解溶菌酶触发芽孢杆菌孢子萌发的异质性及其机制,为认识芽孢(Spore)萌发机制和杀灭芽孢提供参考。[方法]应用拉曼光谱和微分干涉差(DIC)显微镜成像技术高通量分析大量单个Bacillus subtilis (Bs)和B.megaterium (Bm)芽孢经溶菌酶触发的萌发动态。[结果]溶菌酶浓度和温度越高,芽孢萌发越快,孢内CaDPA开始快速释放时间(Tlag)、快速释放所需时间(ΔTrelease)和芽孢皮层水解所需时间(ΔTlys)越短;低于20℃,Bs芽孢萌发的ΔTrelease值是25℃时的4倍以上。SpoVA蛋白高表达菌株的ΔTrelease值和普通菌株基本相同,而缺少皮层水解酶的菌株ΔTrelease值高于普通菌株。95℃处理15min的孢子,其Tlag、ΔTrelease和ΔTlys值是对照的2倍以上。Bs芽孢萌发的异质性明显,不仅表现在芽孢间,也表现在菌株间。Bm芽孢对溶菌酶更敏感,芽孢间的异质性显著低于Bs。[结论]溶菌酶触发的芽孢萌发在物种、菌株和单细胞层面都存在显著的异质性;溶菌酶浓度和温度对芽孢萌发有重大影响;皮层水解酶也可能参与溶菌酶触发的芽孢萌发进程。 |
| 关键词: 拉曼光谱 微分干涉差成像 芽孢杆菌 芽孢萌发 溶菌酶 单细胞分析 |
| DOI：10.13656/j.cnki.gxkx.20160512.008 |
| 投稿时间：2016-02-19修订日期：2016-04-01 |
| 基金项目:国家自然科学基金项目(11264004,31460035)资助。 |
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| Kinetics of Germination of Individual Bacillus Spores and its Heterogeneity Triggered by Lysozyme |
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WANG Guiwen1, ZHANG Pengfei2, WANG Xiaochun1, CHEN Huanjun3, Peter Setlow4, LI Yongqing2
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| (1.Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China;2.Department of Physics, East Carolina University, Greenville, NC 27858, USA;3.College of Life Sciences and Technology, Guangxi University, Nanning, Guangxi, 530004, China;4.Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA) |
| Abstract: |
| [Objective] The heterogeneous germination and its mechanism of Baillus spores trig-gered by lysozyme were studied in order to look insight into the mechanism of spore germination in general and provide new knowledge of inactivating bacterial spores.[Methods] The ki-netic of germinations of multiple individual decoated spores of Bacillus subtilis (Bs)and B.megaterium (Bm)triggered by lysozyme were followed by Raman spectroscopy and differentail interference contrast (DIC)microscopy.[Results] Higher concentrations of lysozyme and temperatures significantly speeded the germination of Bs decaoted spores and reduced the time Tlag, at which spores began release of the great majority of spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA), the period time of completing the release of CaDPA (named ΔTrelease)and the time period of hydrolysis of the spore's peptidoglycan cortex (named ΔTlys).The ΔTrelease values of germination below 20℃ were 4 times more than that at 25℃.The ΔTrelease values of Bs spores with 4-fold-elevated level of SpoVA protein were basically similar to control strains, while spores lacking cortex-lytic enzymes exhibited notably highe ΔTrelease values than wildtype spores.The values of T lag, ΔTrelease and ΔTlys of spores treated by wet heat at 95℃ for 15 min were two times more than the untreated spores.Heterogeneous germinations were observed not only among individual decoated Bs spores, but also among Bs strains.The decoated Bm spores were more sensitive to lysozyme and its germinations of individual spores were less heterogeneous.[Conclusion] The germinations of decoated Bacillus spores triggered by lysozyme, on which the lysozyme concentration and temperature have a significant effect, are significantly heterogeneous at the levels of single spores and strains, as well as of species.The cortexlytic enzymes may be involved in the initiation of germination. |
| Key words: Raman spectroscopy DIC imaging Baillus spore germination lysozyme single-cell analysis |
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