使用荧光显微镜可视化中的细菌线虫
Summary
要研究之间的互利共生<em>嗜</em细菌和<em>斯氏线虫</em监测细菌的存在和位置内线虫线虫的方法被开发。实验的方法，它可以应用于其它系统，带来工程菌表达绿色荧光蛋白和可视化，使用荧光显微镜细菌内的透明的线虫。
Abstract
共生，生活在一起的两个或多个物种，广泛分布于所有的王国的生活。作为地球上的最普遍的生物，线虫和细菌形成共生广泛的范围从有利于致病1-3。一个这样的协会是互惠互利的关系致病杆菌的细菌和斯氏线虫，这已经成为了一个模型系统的共生4。 斯氏线虫，昆虫，细菌共生体，杀死昆虫5。昆虫宿主之间传输时，细菌定植于肠道线虫的感染青少年阶段6-8。最近，几个其他线虫种已被示出利用细菌杀死昆虫9-13，并调查已经开始研究在这些系统中的线虫和细菌之间的相互作用<SUP> 9。
我们描述了一种内共生菌或一种线虫主机上进行可视化的方法，利用通过显微镜观察时，光学透明度的线虫。这种细菌被设计来表达荧光蛋白，让他们通过荧光显微镜的可视化。许多质粒是可携带荧光在不同的波长（ 例如，绿色或红色），和共轭从一种供体到受体细菌共生体的大肠杆菌菌株的质粒编码的蛋白质的基因是成功的范围广泛的细菌。描述的方法进行调查之间的关联斯氏carpocapsae和嗜线虫致病杆菌 14。类似的方法已被用来调查其他线虫细菌协会9，15-18，因此，该方法是普遍适用的。
的METHOD允许的细菌内线虫的存在和定位的特性，在不同的发展阶段，深入了解协会的性质和过程的殖民统治14，16，19。微观分析揭示了两个殖民频率范围内的人口和本地化的细菌宿主组织，14，16，19-21。监测细菌内线虫的人群，如超声22或研磨23，可提供平均水平的殖民与其他方法相比，这是一个优势，但没有可能，例如，歧视的人群高频率的低共生体负载从人口低频率高的共生体负荷。判别的频率和负载的定殖菌筛选时，可能是特别重要或描述细菌殖民表型的突变体21，24。事实上，荧光显微镜被用于高通量筛选的细菌定植17，18中的缺陷的突变体，并且是不太费力的比其他的方法，包括超声处理22 25日至27日，和个别线虫夹层28，29。
Protocol
1。建设一个荧光的细菌菌株，通过共轭成长的受体菌（共生体进行审查）和供体菌过夜。供体菌株中，通常会大肠杆菌 ，应该是能够通过共轭捐赠DNA应转化的质粒（ 表2）的，携带编码荧光蛋白的基因。根据质粒上，共轭辅助菌株也可能是必需的。如果是这样的话，该菌株应还可以生长过夜。供体菌和辅助应变应种植与抗生素选择维护的质粒。 继代培养的?…
Discussion
这里所描述的协议提供了一种方法，一种线虫主机（ 图1）内的细菌的光学检测。此方法利用的光学透明度，线虫和荧光标记细菌的能力，使在体内分析的细菌内的线虫主机（ 图3）。具体而言，这种方法在其主机的标识细菌本地化的。横跨线虫种群的细菌定植的频率可以通过计数线虫种群和得分为细菌的存在，来确定（ 表1）。此方法是，可以用于研?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢欧金尼奥维瓦斯，库尔特Heungens，埃里克·马腾斯，查尔斯·考尔斯，美糖，埃里克Stabb，托德Ciche本协议和工具的发展所作出的贡献。 KEM，江铃支持由美国国立卫生研究院（NIH）国家研究服务奖T32（AI55397“微生物在健康和疾病”）。江铃汽车得到了美国国家科学基金会（NSF）研究生研究奖学金。这项工作是支持由美国国家科学基金会（IOS-0920631，IOS-0950873）的资助。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Lipid Agar (sterile) |
8 grams nutrient broth, 15 grams agar, 5 grams yeast extract, 890 ml water, 10 ml 0.2 g/ml MgCl2. 6H20, 96 ml corn syrup solution*, 4 ml corn oil* Stir media while pouring plates *add sterile ingredient after autoclaving |
||
| Corn Syrup Solution (sterile) |
7 ml corn syrup, 89 ml water mix and autoclave |
||
| Egg Solution | 16.6 ml 12% sodium hypochlorite, 5 ml 5M KOH, 80 ml water | ||
| Lysogeny Broth (sterile) |
5 grams yeast extract, 10 grams tryptone, 5 grams salt, 1 L water mix and autoclave |
||
| Microfuge | Fisher | 13-100-675 | Any microfuge that holds microfuge tubes will work |
| Centrifuge | Beckman | 366802 | Large table top centrifuge that holds 15 ml and 50 ml conical tubes |
| Sterile 60 mm X 15 mm Petri Dish | Fisher | 0875713 | |
| 50 ml centrifuge tubes | Fisher | 05-539-6 | |
| 15 ml centrifuge tubes | Fisher | 05-531-6 | |
| Sterile 100 mm X 20 mm Petri Dish | Fisher | 0875711Z | Deeper than standard Petri dishes |
| 24-well plate | Greiner Bio-One | 662000-06 | |
| Microscope | The microscope needs florescent capabilities compatible with your fluorophore | ||
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| PBS (sterile) |
8 g NaCL 0.2 g KCL 1.44 g Na2HPO4 0.24 g KH2PO4 1 L water Adjust to a pH of 7.4 and water to 1 L and autoclave |
||
| Microfuge tubes | Fisher | 05-408-138 | 2 ml or 1.5 ml tubes |
| Shaker | Any shaker that causes the liquid to gently move will work | ||
| Diaminopimelic acid | Sigma | D-1377 | If needed, supplement media to a concentration or 1 mM |
References
- Holterman, M., van der Wurff, A. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Mol. Biol. Evol. 23, 1792-1800 (2006).
- Lambshead, P. J. D., Boucher, G. Marine nematode deep-sea biodiversity – hyperdiverse or hype. J. Biogeogr. 30, 475-485 (2003).
- Poinar, G. O. J., Thomas, G. M. Significance of Achromobacter nematophilus Poinar and Thomas (Achromobacteraceae: Eubacteriales) in the development of the nematode, DD-136 (Neoaplectana sp. Steinernematidae). Parasitol. 56, 385-390 (1966).
- Herbert, E. E., Goodrich-Blair, H. Friend and foe: the two faces of Xenorhabdus nematophila. Nat. Rev. Microbiol. 5, 634-646 (2007).
- Kaya, H. K., Gaugler, R. Entomopathogenic nematodes. Annu. Rev. Entomol. 38, 181-206 (1993).
- Bird, A. F., Akhurst, R. J. The nature of the intestinal vesicle in nematodes of the family Steinernematidae. Int. J. Parasitol. 13, 599-606 (1983).
- Martens, E. C., Goodrich-Blair, H. The Steinernema carpocapsae intestinal vesicle contains a subcellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cell. Microbiol. 7, 1723-1735 (2005).
- Snyder, H. A., Stock, S. P., Kim, S. K., Flores-Lara, Y., Forst, S. New insights into the colonization and release process of Xenorhabdus nematophila and the morphology and ultrastructure of the bacterial receptacle of its nematode host, Steinernema carpocapsae. Appl. Environ. Microbiol. 73, 5338-5346 (2007).
- Abebe, E., Abebe-Akele, F., Morrison, J., Cooper, V., Thomas, W. K. An insect pathogenic symbiosis between a Caenorhabditis and Serratia. Virulence. 2, 158-161 (2011).
- Abebe, E., Jumba, M. An entomopathogenic Caenorhabditis briggsae. J. Exp. Biol. 213, 3223-3229 (2010).
- Torres-Barragan, A., Suazo, A., Buhler, W. G., Cardoza, Y. J. Studies on the entomopathogenicity and bacterial associates of the nematode Oscheius carolinensis. Biol. Control. 59, 123-129 (2011).
- Ye, W. M., Torres-Barragan, A., Cardoza, Y. Oscheius carolinensis n. sp (Nematoda: Rhabditidae), a potential entomopathogenic nematode from vermicompost. Nematology. 12, 121-135 (2010).
- Zhang, C., Liu, J. Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes. J. Invertebr. Pathol. 98, 153-168 (2008).
- Martens, E. C., Heungens, K., Goodrich-Blair, H. Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria. J. Bacteriol. 185, 3147-3154 (2003).
- Ciche, T. A., Ensign, J. C. For the insect pathogen, Photorhabdus luminescens, which end of a nematode is out. Appl. Environ. Microbiol. 69, 1890-1897 (2003).
- Ciche, T. A., Kim, K. S., Kaufmann-Daszczuk, B., Nguyen, K. C., Hall, D. H. Cell invasion and matricide during Photorhabdus luminescens transmission by Heterorhabditis bacteriophora nematodes. Appl. Environ. Microbiol. 74, 2275-2287 (2008).
- Easom, C. A., Joyce, S. A., Clarke, D. J. Identification of genes involved in the mutualistic colonization of the nematode Heterorhabditis bacteriophora by the bacterium Photorhabdus luminescens. BMC Microbiol. 10, 45 (2010).
- Somvanshi, V. S., Kaufmann-Daszczuk, B., Kim, K. S., Mallon, S., Ciche, T. A. Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis. Mol. Microbiol. 77, 1021-1038 (2010).
- Martens, E. C., Russell, F. M., Goodrich-Blair, H. Analysis of Xenorhabdus nematophila metabolic mutants yields insight into stages of Steinernema carpocapsae nematode intestinal colonization. Mol. Microbiol. 51, 28-45 (2005).
- Sugar, D. R., Murfin, K. E. Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes. Env. Microbiol. 14, 924-939 (2012).
- Cowles, C. E., Goodrich-Blair, H. The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes. J. Bacteriol. 190, 4121-4128 (2008).
- Heungens, K., Cowles, C. E., Goodrich-Blair, H. Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes. Mol. Microbiol. 45, 1337-1353 (2002).
- Goetsch, M., Owen, H., Goldman, B., Forst, S. Analysis of the PixA inclusion body protein of Xenorhabdus nematophila. J. Bacteriol. 188, 2706-2710 (2006).
- Bhasin, A., Chaston, J. M., Goodrich-Blair, H. Mutational Analyses Reveal Overall Topology and Functional Regions of NilB, a Bacterial Outer Membrane Protein Required for Host Association in a Model of Animal-Microbe Mutualism. J. Bacteriol. 194, 1763-1776 (2012).
- Ciche, T. A., Goffredi, S. K., Reddy, C. A. . Methods in General and Molecular Microbiology. , 394-419 (2007).
- Goodrich-Blair, H., Clarke, D., Grewal, P. S., Ciche, T. A., Stock, S. P., Boemare, N., Vandenberg, J., Glazar, I. . Insect Pathogens, Molecular Approaches and Techniques. , 239-269 (2009).
- Stock, S. P., Goodrich-Blair, H., Lacey, L. A. . Manual of Techniques in Invertebrate Pathology. , 375-425 (2012).
- Martens, E. C., Gawronski-Salerno, J. Xenorhabdus nematophila requires an intact iscRSUA-hscBA-fdx locus to colonize Steinernema carpocapsae nematodes. J. Bacteriol. 185, 3678-3682 (2003).
- Vivas, E. I., Goodrich-Blair, H. Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes. J. Bacteriol. 183, 4687-4693 (2001).
- White, G. F. A method for obtaining infective nematode larvae from cultures. Science. 66, 302-303 (1927).
- Ciche, T. A., Ensign, J. C. For the insect pathogen Photorhabdus luminescens, which end of a nematode is out. Appl. Environ. Microbiol. 69, 1890-1897 (2003).
- Sicard, M., Brun, N. L. e. Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction. Parasitol. Res. 91, 520-524 (2003).
- Lambertsen, L., Sternberg, C., Molin, S. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ. Microbiol. 6, 726-732 (2004).
- Miller, W. G., Leveau, J. H., Lindow, S. E. Improved gfp and inaZ broad-host-range promoter-probe vectors. Mol. Plant Microbe Int. 13, 1243-1250 (2000).
- Teal, T. K., Lies, D. P., Wold, B. J., Newman, D. K. Spatiometabolic stratification of Shewanella oneidensis biofilms. Appl. Environ. Microbiol. 72, 7324-7330 (2006).
- Bao, Y., Lies, D. P., Fu, H., Roberts, G. P. An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of Gram-negative bacteria. Gene. 109, 167-168 (1991).
- Woodring, J. L., Kaya, H. K. Steinernematid and Heterorhabditid Nematodes: A Handbook of Biology and Techniques. Southern Cooperative Series Bulletin 331. , (1988).
Tags
VisualizingBacteriaNematodesFluorescent MicroscopySymbiotic AssociationsXenorhabdus BacteriaSteinernema NematodesModel SystemEntomopathogenicBacterial SymbiontIntestineInfective Juvenile StageNematode SpeciesInteractionsVisualization MethodOptical TransparencyMicroscopyFluorescent ProteinPlasmidsEscherichia Coli Strain
Cite This Article
Murfin, K. E., Chaston, J., Goodrich-Blair, H. Visualizing Bacteria in Nematodes using Fluorescent Microscopy. J. Vis. Exp. (68), e4298, doi:10.3791/4298 (2012).