SecMet-FISH:对产生次级代谢物的微生物进行标记、可视化和计数。

IF 3.5 3区 生物学 Q2 MICROBIOLOGY
Yannick Buijs, Aileen Ute Geers, Iuliana Nita, Mikael Lenz Strube, Mikkel Bentzon-Tilia
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引用次数: 0

摘要

我们对次生代谢物在微生物群落中的作用的理解受到了实验室条件下培养细菌的内在限制的挑战,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生代谢物 FISH(SecMet-FISH)的方案,它结合了基因靶向荧光原位杂交(geneFISH)和溶液内方法(in-solution FISH)的优势,根据细胞产生次生代谢物的基因能力来检测和量化细胞。这种方法利用了编码腺苷酸化(AD)和酮合成酶(KS)结构域的生物合成基因簇(BGCs)的保守性,从而有选择地针对非核糖体肽和多酮生物合成的遗传基础。这一概念依赖于使用广泛针对 AD 和 KS 结构域的变性引物生成扩增子池,然后进行荧光标记、检测和定量。最初,我们从红掌藻(Pseuodoalteromonas rubra)中获得了AD和KS扩增子,从而成功地标记和观察了红掌藻细胞内的BGCs,证明了SecMet-FISH的可行性。接下来,我们对该方案进行了调整和优化,以便在革兰氏阴性和革兰氏阳性细菌细胞悬浮液中进行杂交,从而能够通过流式细胞仪进行高通量单细胞分析。最终,我们利用 SecMet-FISH 成功地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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
SecMet-FISH: labeling, visualization, and enumeration of secondary metabolite producing microorganisms.

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.

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来源期刊
FEMS microbiology ecology
FEMS microbiology ecology 生物-微生物学
CiteScore
7.50
自引率
2.40%
发文量
132
审稿时长
3 months
期刊介绍: FEMS Microbiology Ecology aims to ensure efficient publication of high-quality papers that are original and provide a significant contribution to the understanding of microbial ecology. The journal contains Research Articles and MiniReviews on fundamental aspects of the ecology of microorganisms in natural soil, aquatic and atmospheric habitats, including extreme environments, and in artificial or managed environments. Research papers on pure cultures and in the areas of plant pathology and medical, food or veterinary microbiology will be published where they provide valuable generic information on microbial ecology. Papers can deal with culturable and non-culturable forms of any type of microorganism: bacteria, archaea, filamentous fungi, yeasts, protozoa, cyanobacteria, algae or viruses. In addition, the journal will publish Perspectives, Current Opinion and Controversy Articles, Commentaries and Letters to the Editor on topical issues in microbial ecology. - Application of ecological theory to microbial ecology - Interactions and signalling between microorganisms and with plants and animals - Interactions between microorganisms and their physicochemical enviornment - Microbial aspects of biogeochemical cycles and processes - Microbial community ecology - Phylogenetic and functional diversity of microbial communities - Evolutionary biology of microorganisms
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