Summary

单细胞分析枯草芽孢杆菌生物膜,用荧光显微镜和流式细胞仪

Published: February 15, 2012
doi:

Summary

微生物生物膜一般由专门的细胞不同亚群构成。这些亚群的单细胞分析需要使用荧光记者。在这里,我们描述了一个协议,以可视化和监视几个subpopulationswithin<em> B枯草</em>生物膜,用荧光显微镜和流式细胞仪。

Abstract

几乎所有的细菌1-6生物膜的形成是一个普遍的属性。当细菌形成生物膜,包裹在细胞外基质,大多在7-10的其他因素所构成的蛋白质和胞外多糖,。包裹内生物膜的微生物群落,往往显示11-17专门的细胞不同亚群的分化。这些亚群共存,并常常表现出时间和空间内的生物膜18-21组织。

在模式生物的枯草芽孢杆菌的生物膜的形成需要专门的细胞不同亚群的分化。其中,亚矩阵生产商,负责产生和分泌的生物膜的细胞外基质是必不可少的生物膜形成11,19。因此,矩阵生产者的分化是生物膜形成的标志 B 枯草。

我们使用荧光记者可视化和量化亚矩阵生产者B 生物膜枯草 15,19,22-24。具体来说,我们观察到亚矩阵生产者自产外信号表面活性25存在区别。有趣的是,表面活性是由亚矩阵生产者15不同的专门细胞亚群。

我们已详列于本报告必要的技术方法,可视化和量化矩阵生产者和表面活性生产者在枯草杆菌生物膜亚群。要做到这一点,基质生产和生产表面活性所需的基因插入染色体B 荧光记者枯草 。记者们表示,只有在一个专门的细胞亚群。然后,亚能监测用荧光显微镜和流式细胞仪(见图1)。

专门的细胞不同亚群内的细菌多细胞群落共存的事实给了我们一个不同的角度对原核生物的基因表达调控。该协议解决了这一实验现象和它可以很容易地适应任何其他的工作模式,以澄清在微生物群落的基本表型异质性的分子机制。

Protocol

1。标签B。枯草和生物膜形成实验通过PCR扩增感兴趣的基因的启动子区域。我们的P TAPA,负责生产的的TASA基质蛋白26基因启动子的克隆为例。克隆带够TAPA到pkm008向量(Rudner实验室,哈佛医学院,波士顿,美国)(图2)。 线性质粒,通过消化酶(酶建议,XhoI位)。 在 B诱导自然能力枯草芽孢杆菌菌株按照协议,先前?…

Discussion

事实上,细菌群落表明细胞亚群的表达一组特定的基因证据33,34微生物群落的复杂性。本议定书应帮助,以确定是否对任何感兴趣的基因的表达仅限于微生物群落,特别是一个专门的细胞亚群。这些亚群的可视化需要新技术的发展,因为传统的方法来监测基因表达微阵列基因表达分析率水平,整个微生物群落内的微生物群落的基因表达的波动一般失手。

荧光显微镜和?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由青年研究者的研究计划,从维尔茨堡大学传染病研究中心(ZINF)。胡安·Ç加西亚贝坦库尔是从维尔茨堡大学生命科学院研究生院(GSLS)博士研究员。

Materials

Technique Name of the reagent Company Catalog number
MSgg composition potassium phosphate 5mM Roth 6878
MOPS 100mM Sigma-Aldrich M1254
Magnesium chloride 2mM Roth 2189.1
Calcium chloride 700μM Roth A119.1
Ferric chloride 50μM Sigma-Aldrich 157740
Zinc chloride 1μM Applichem A2076
Thiamine 2μM Sigma-Aldrich 74625
Glycerol 0.5% Roth 7533
Glutamate 0.5% Sigma-Aldrich 49621
Tryptophan 50μg/ml Sigma-Aldrich T0254
Phenylalanine 50μg/ml Sigma-Aldrich P2126
Cell fixation Paraformaldehyde Roth 0335
Name of the equipment Company Catalog number
Sonication Cell Sonicator Bandelin D-1000
Fluorescence Microscopy Fluorescence Microscope Leica DMI6000B
Name of the software Company Catalog Number
Fluorescence Microscopy AsaF Leica
Flow cytometry FCASDiva BD
Flow cytometry FlowJo Treestar

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Cite This Article
Garcia-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry. J. Vis. Exp. (60), e3796, doi:10.3791/3796 (2012).

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