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
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在 B 枯草。
我们使用荧光记者可视化和量化亚矩阵生产者B 的生物膜枯草 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 的荧光记者枯草 。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 |
References
- Costerton, J. W. Overview of microbial biofilms. J. Ind. Microbiol. 15, 137-140 (1995).
- Davey, M. E., O’Toole, G. A. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847-867 (2000).
- Kolenbrander, P. E. Oral microbial communities: biofilms, interactions, and genetic systems. Annu. Rev. Microbiol. 54, 413-437 (2000).
- O’Toole, G., Kaplan, H. B., Kolter, R. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54, 49-79 (2000).
- Donlan, R. M. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8, 881-890 (2002).
- Lopez, D., Vlamakis, H., Kolter, R. Biofilms. Cold Spring Harb. Perspect. Biol. 2, a000398-a000398 (2010).
- Branda, S. S., Vik, S., Friedman, L., Kolter, R. Biofilms: the matrix revisited. Trends Microbiol. 13, 20-26 (2005).
- Branda, S. S., Chu, F., Kearns, D. B., Losick, R., Kolter, R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59, 1229-1238 (2006).
- Latasa, C., Solano, C., Penades, J. R., Lasa, I. Biofilm-associated proteins. C. R. Biol. 329, 849-857 (2006).
- O’Gara, J. P. ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus. FEMS Microbiol Lett. 270, 179-188 (2007).
- Chai, Y., Chu, F., Kolter, R., Losick, R. Bistability and biofilm formation in Bacillus subtilis. Mol. Microbiol. 67, 254-263 (2008).
- Chen, R., Guttenplan, S. B., Blair, K. M., Kearns, D. B. Role of the sigmaD-dependent autolysins in Bacillus subtilis population heterogeneity. J. Bacteriol. 191, 5775-5784 (2009).
- Guttenplan, S. B., Blair, K. M., Kearns, D. B. The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation. PLoS Genet. 6, e1001243-e1001243 (2010).
- Kearns, D. B., Losick, R. Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev. 19, 3083-3094 (2005).
- Lopez, D., Vlamakis, H., Losick, R., Kolter, R. Paracrine signaling in a bacterium. Genes Dev. 23, 1631-1638 (2009).
- Veening, J. W., Smits, W. K., Hamoen, L. W., Jongbloed, J. D., Kuipers, O. P. Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis. Appl. Environ. Microbiol. 70, 6809-6815 (2004).
- Veening, J. W., Smits, W. K., Hamoen, L. W., Kuipers, O. P. Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media. J. Appl. Microbiol. 101, 531-541 (2006).
- Veening, J. W., Kuipers, O. P., Brul, S., Hellingwerf, K. J., Kort, R. Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis. J. Bacteriol. 188, 3099-3109 (2006).
- Vlamakis, H., Aguilar, C., Losick, R., Kolter, R. Control of cell fate by the formation of an architecturally complex bacterial community. Genes Dev. 22, 945-953 (2008).
- Stewart, P. S., Franklin, M. J. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 6, 199-210 (2008).
- Veening, J. W., Smits, W. K., Kuipers, O. P. Bistability, epigenetics, and bet-hedging in bacteria. Annu. Rev. Microbiol. 62, 193-210 (2008).
- Aguilar, C., Vlamakis, H., Guzman, A., Losick, R., Kolter, R. KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms. MBio. 1, (2010).
- Lopez, D., Fischbach, M. A., Chu, F., Losick, R., Kolter, R. Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 106, 280-285 (2009).
- Lopez, D., Vlamakis, H., Losick, R., Kolter, R. Cannibalism enhances biofilm development in Bacillus subtilis. Mol. Microbiol. 74, 609-618 (2009).
- Arima, K., Kakinuma, A., Tamura, G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31, 488-494 (1968).
- Romero, D., Vlamakis, H., Losick, R., Kolter, R. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms. Mol. Microbiol. 80, 1155-1168 (2011).
- Hardwood, C. R., Cutting, S. M. . Molecular Biological Methods for Bacillus. , (1990).
- Novick, R. P. Genetic systems in staphylococci. Methods Enzymol. 204, 587-636 (1991).
- Yasbin, R. E., Young, F. E. Transduction in Bacillus subtilis by bacteriophage SPP1. J. Virol. 14, 1343-1348 (1974).
- Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., Kolter, R. Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11626 (2001).
- Nakano, M. M. srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis. J. Bacteriol. 173, 1770-1778 (1991).
- Gonzalez-Pastor, J. E., Hobbs, E. C., Losick, R. Cannibalism by sporulating bacteria. Science. 301, 510-513 (2003).
- Aguilar, C., Vlamakis, H., Losick, R., Kolter, R. Thinking about Bacillus subtilis as a multicellular organism. Curr. Opin. Microbiol. 10, 638-643 (2007).
- Shapiro, J. A. Thinking about bacterial populations as multicellular organisms. Annu. Rev. Microbiol. 52, 81-104 (1998).
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).