Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS
Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS
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DOI：
10.1128/aem.00191-08
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发表时间：
2008-05-01
影响因子：
4.4
通讯作者：
Spormann, Alfred M.
中科院分区：
文献类型：
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作者：
Behrens, Sebastian;Loesekann, Tina;Spormann, Alfred M.
为了研究复杂微生物群落中单个细胞的系统发育特征和代谢活性，我们开发了一种方法，该方法将基于rRNA的原位杂交与基于纳米尺度二次离子质谱（NanoSIMS）的稳定同位素成像相结合。通过16 S rRNA靶向探针将氟或溴原子引入细胞，从而能够通过NanoSIMS成像对单个细胞进行系统发育鉴定。为了克服天然的氟和溴的背景，我们修改了目前的催化报告沉积荧光原位杂交（FISH）技术，通过使用含卤素的荧光标记的酪胺作为底物的酶酪胺沉积。由此，我们获得了通过FISH的微生物细胞的增强的元素标记（EL-FISH）。EL-FISH后氟或溴的相对细胞丰度超过天然背景浓度高达180倍，使我们能够在NanoSIMS氟或溴图像中区分靶细胞和非靶细胞。对大肠杆菌和霍乱弧菌的单细胞培养物进行了优化。EL-FISH/NanoSIMS，然后应用于研究在一个双物种财团组成的丝状蓝藻和异养alphaproteobacterium的相互关系。我们还评估了从人类口腔生物膜获得的复杂微生物聚集体的方法。在这两个样品中，我们通过观察用C-13-碳和N-15-氮标记的底物的命运发现了代谢相互作用的证据，同时通过EL-FISH通过卤素标记同时鉴定了单个细胞。我们的新方法将有助于进一步研究复杂环境和群落中已知和未培养微生物的生理生态学。
To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with C-13-carbon and N-15-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.