Molecular Plant Sciences Section, Department of Biology, University of Oxford, Oxford, OX1 3RB, UK.
Microbiome. 2024 May 7;12(1):81. doi: 10.1186/s40168-024-01792-2.
After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom.
To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots.
DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly. Video Abstract.
经过二十年的广泛微生物组研究，目前科学探索的前沿领域涉及超越描述和分类，以揭示微生物群落融合背后的复杂机制。由于微生物多样性非常广泛，因此解析微生物组组装在技术上具有挑战性，但建立合成群落（SynCom）是揭示这一过程的关键策略。实现绝对定量对于确定组装动力学中的因果关系至关重要。然而，现有的方法主要用于区分特定 SynCom 中特定的微生物群体。
为了解决这个问题，我们开发了差异荧光标记（DFM）策略，在单个和双组合中使用三种可区分的荧光蛋白。DFM 基于 mini-Tn7 转座子，利用增强的稳定性和在广泛的 Proteobacteria 物种中的广泛适用性。各种 DFM 构建体使用 pTn7-SCOUT 质粒家族构建，实现了模块化组装，并在单个反应中方便地交换表达和抗生素盒。DFM 对适应性或群落组装动力学没有不利影响，通过流式细胞术的应用，我们成功地对不同的六个成员的 SynCom 进行了区分、定量和跟踪，在各种复杂条件下，如根际，显示出豌豆和大麦根之间不同的定植组装动态。
DFM 代表了一种强大的资源，它消除了对测序和/或培养的依赖，从而为研究微生物组组装开辟了新的途径。视频摘要。
