of where the organisms present live and act. For example, sea otters reside on the surface of kelp beds and feed on sea urchins that consume kelp from the ocean floor. An organism’s location in an ecosystem reflects its physiology and function. This is particularly true for microbes, which live in and respond to highly dynamic and diverse habitats. Micrometre-scale gradients of nutrients or pH, for instance, are a typical feature of microbial habitats that drive bacterial spatial organization and behaviour. Yet, despite the incredible range of functions that different bacteria perform independently and in association with other living systems, most microbes are similar in shape and indistinguishable under the microscope. On page 676, Shi et al. present a method to tackle the major challenge of differentiating between the hundreds to thousands of bacterial species found in microbes’ natural habitats. A key tool used in biogeographical studies to assess the spatial location of components of interest is called fluorescence in situ hybridization (FISH). This technique relies on the use of fluorescently labelled nucleic-acid sequences called probes to locate matching specific sequences of DNA or RNA in a sample that has been immobilized by a process termed fixation. When applied to samples containing bacteria, a DNA probe can be used to identify a target species in the context of its native environment, and a fluorescent molecule (a fluorophore) attached to the probe enables the location of the species to be observed under the microscope. Designing the DNA component of FISH probes is now relatively easy. However, the maximum number of bacterial species identifiable by FISH within a single sample has been constrained by the limited number of fluorophores available for simultaneous visualization. Methods that exceed the limits of traditional fluorescence microscopy, using spectral imaging and combinations of fluorophores, can distinguish between 15 and 120 different types of microbe captured in the same image. But a drawback of these techniques is the cost of the large number of fluorescently tagged probes needed. Shi and colleagues introduce a method that exceeds previous FISH benchmarks by combining a new type of probe design with custom image analysis. Their technique (Fig. 1), named high phylogenetic resolution microbiome mapping by fluorescence in situ hybridization (HiPR-FISH), builds on a combinatorial strategy in which bacteria are targeted and labelled in two steps. First, a DNA probe, described as an encoding probe, is designed to match a species-specific sequence (16S ribosomal RNA) for the targeted bacteria. This encoding probe is flanked on either side by integral parts of the probe described as readout sequences. Each of these two readout sequences in an encoding probe can be one of ten possible readout sequences. Then, each readout sequence is targeted by another DNA probe fused to a fluorophore that is specific for the particular readout sequence. Bacterial cells contain hundreds of copies of 16S rRNA, and so each bacterial species can be targeted by an array of encoding probes: each targeting the same RNA sequence, yet flanked by different pairs of readout sequences, enabling a variety of readout sequences to be associated with a particular species. Choosing the readout sequences for each targeted bacterial species allows the assignment of a unique combination of readout sequences (and therefore fluorophores) to correspond to each species. Depending on the encoding probes used, each bacterial species can bind up to ten of the possible fluorophores. Thus, this system can generate 1,023 unique visual Microbiology

中文翻译：

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从多样化的人群中挑选出的细菌物种

存在的生物在哪里生活和行动。例如，海獭栖息在海藻床的表面，以海胆为食，海胆从海底吃海带。生物体在生态系统中的位置反映了其生理机能和功能。对于生活在高度动态和多样化的栖息地并对其做出反应的微生物来说尤其如此。例如，营养物质或 pH 值的微米级梯度是微生物栖息地的典型特征，可驱动细菌空间组织和行为。然而，尽管不同细菌独立执行并与其他生命系统相关联的功能范围令人难以置信，但大多数微生物的形状相似，在显微镜下无法区分。在第 676 页，Shi 等人。提出了一种方法来解决区分微生物自然栖息地中发现的数百到数千种细菌的主要挑战。生物地理学研究中用于评估感兴趣成分的空间位置的关键工具称为荧光原位杂交 (FISH)。该技术依赖于使用称为探针的荧光标记核酸序列来定位已通过称为固定的过程固定的样本中匹配的特定 DNA 或 RNA 序列。当应用于含有细菌的样品时，DNA 探针可用于在其天然环境的背景下识别目标物种，并且附着在探针上的荧光分子（荧光团）可以在显微镜下观察物种的位置. 现在设计 FISH 探针的 DNA 组件相对容易。然而，单个样品中 FISH 可识别的细菌种类的最大数量受到可用于同时可视化的有限数量的荧光团的限制。超越传统荧光显微镜限制的方法，使用光谱成像和荧光团的组合，可以区分同一图像中捕获的 15 到 120 种不同类型的微生物。但这些技术的一个缺点是需要大量荧光标记探针的成本。Shi 及其同事通过将新型探针设计与自定义图像分析相结合，推出了一种超越先前 FISH 基准的方法。他们的技术（图 1），通过荧光原位杂交（HiPR-FISH）命名的高系统发育分辨率微生物组图谱，建立在组合策略的基础上，在该策略中，分两步定位和标记细菌。首先，被描述为编码探针的 DNA 探针被设计为匹配目标细菌的物种特异性序列（16S 核糖体 RNA）。该编码探针的两侧是被描述为读出序列的探针的组成部分。编码探针中的这两个读出序列中的每一个都可以是十个可能的读出序列之一。然后，每个读出序列被另一个 DNA 探针靶向，该探针融合到对特定读出序列具有特异性的荧光团。细菌细胞含有数百个 16S rRNA 拷贝，因此每个细菌物种都可以被一系列编码探针靶向：每个探针都靶向相同的 RNA 序列，但两侧有不同的读出序列对，使各种读出序列与特定物种相关联。为每个目标细菌物种选择读出序列允许分配一个独特的读出序列组合（以及荧光团）以对应于每个物种。根据所使用的编码探针，每种细菌种类最多可以结合十种可能的荧光团。因此，该系统可以生成 1,023 个独特的视觉微生物学