Department of Plant Sciences, University of Oxford, Oxford, UK.
Department of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium.
Environ Microbiol. 2021 Apr;23(4):2070-2085. doi: 10.1111/1462-2920.15296. Epub 2021 Jan 22.
Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.
细菌生物发光被广泛用于在群体水平上研究细菌种群和基因表达的时空动态，但很难用于研究单个细胞水平上的细菌活性。在这项研究中，我们描述了一个新的 mini-Tn7-lux 和 lux::eyfp 报告基因构建体文库的开发，该文库提供了广泛的 lux 表达水平，并结合了细菌生物发光和荧光蛋白的优势，从而弥合了宏观和微观成像技术之间的差距。我们证明，使用 lux 操纵子和 eYFP 的双生物发光-荧光方法可用于宏观和微观监测植物中的细菌运动，并证明丁香假单胞菌 pv 菜豆可以定殖于叶维管束系统并系统感染普通菜豆（Phaseolus vulgaris）的叶片。我们还表明，细菌生物发光可用于研究植物免疫反应对细菌增殖、活力和在植物组织内传播的影响。本研究中描述的构建体和方法可用于研究细菌定殖的时空动态，并将群体动态和细胞相互作用联系起来，应用于广泛的生物学背景。
