Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.

中文翻译：

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SecMet-FISH：产生次级代谢产物的微生物的标记、可视化和计数

我们对微生物群落中次生代谢物作用的理解受到实验室条件下培养细菌的内在局限性的挑战，因此需要独立于培养的方法。在这里，我们提出了一种称为次级代谢物 FISH (SecMet-FISH) 的方案，结合了基因靶向荧光原位杂交 (geneFISH) 与溶液内方法 (溶液内 FISH) 的优点，根据细胞的遗传能力来检测和量化细胞产生次级代谢产物。该方法利用编码腺苷酸化（AD）和酮合酶（KS）结构域的生物合成基因簇（BGC）的保守性，从而选择性地靶向非核糖体肽和聚酮化合物生物合成的遗传基础。该概念依赖于使用广泛靶向 AD 和 KS 结构域的简并引物生成扩增子池，然后进行荧光标记、检测和定量。最初，我们从 Pseuodoalteromonas rubra 中获得了 AD 和 KS 扩增子，这使我们能够成功标记和可视化 P. rubra 细胞内的 BGC，证明了 SecMet-FISH 的可行性。接下来，我们调整了该方案并优化了其在革兰氏阴性和革兰氏阳性细菌细胞悬浮液中的杂交，从而能够通过流式细胞术进行高通量单细胞分析。最终，我们使用 SecMet-FISH 成功区分了五人合成群落中的次级代谢产物生产者和非生产者。