流式细胞术揭示出枯草芽孢杆菌多态异质性

Multiple States of Bacillus subtilis Revealed by Flow Cytometry

新近的研究发现,微生物群体异质性现象普遍存在,与微生物群体许多关键功能密切相关.微生物群体中的多种异质性状态需要单细胞水平的分析技术才能被揭示,流式细胞术是获取异质性状态精确分布的重要工具.但微生物细胞尺寸微小、生物分子含量少、常常缺乏特异性试剂等都限制着传统流式细胞技术在微生物研究领域的应用.本论文采用新型的低背景、高灵敏度和高分辨率流式细胞仪,以增强的前向散射光、侧向散射光以及紫外光激发的细菌自发荧光水平这三个无需任何荧光标记就可以检测的信号为参数,首次揭示出不同生长状态的枯草芽孢杆菌具有复杂、动态的异质性状态分布.这一方法鉴定出的枯草芽孢杆菌多种状态及其与生理功能相关的、高度关联的变化,可能对该菌的生理变化规律及其分子机理的认识提供新的机遇.本论文也讨论了这一采用新型高灵敏度、高分辨率流式细胞仪测量非标记细胞参数的方法对于广泛开展各种微生物多态性研究具有巨大潜力.

Recent studies revealed that heterogeneity is a general phenomenon among microbes with identical genomes, and it is closely related to physiological functions of microbes. The multiple heterogeneous states of microbes requires single-cell level tools to uncover. Flow cytometry is one of the most important methods to obtain accurate information on quantitative distribution of heterogeneous states of microbes. However, the small size, much less biomolecule contents and general lack of specific reagents limit the application of conventional flow cytometry to investigations of the heterogeneity of microbes. In this study, we applied a newly developed flow cytometer with increased resolution and sensitivity on three label-free parameters which include FSC,SSC and autofluorescence level of NAD（P）H to analyse Bacillus subtilis. And for the first time we revealed that the bacterial culture exhibited complicated and dynamically distributed multiple heterogeneous subgroups depended on its growth stage. This method has low background noise, high sensitivity and great resolution. The newly identified multiple subpopulations of Bacillus subtilis and their changes dynamically associated with the biological functions are likely to provide new opportunities to understand physiological change patterns and their underlying molecule mechanisms. We also discussed that the new method based on flow cytometric measurements of non-label parameters with high sensitivity and resolution had great potency for studies on the heterogeneity of various microbes.