Charles T. Campbell Laboratory of Ophthalmic Microbiology, Department of Ophthalmology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania, USA.
Department of Ophthalmology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania, USA.
Appl Environ Microbiol. 2020 Jul 2;86(14). doi: 10.1128/AEM.00250-20.
Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both and The promoter, derived from the operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the -derived promoter using as a reporter. Green fluorescent protein (GFP) fluorescence from the promoter was inducible in both species, but not in , which may facilitate the cloning of genes toxic to to generate plasmids. The promoter was activated at a lower inducer concentration than in , and higher levels were achieved using Flow cytometry analysis indicated that was leakier than in the species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of or and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the type II secretion system gene in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in species. species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in and , and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from , XcpQ, is important for host-pathogen interactions in a corneal wound closure model.
基因表达的可调控制是生物实验中非常有价值的工具。在本研究中，我们描述了一种新的木糖诱导启动子系统，并在 和 中对其进行了评估。该启动子源自 操纵子，被整合到一个广泛宿主范围的 pBBR1 基础质粒中，并与基于 的 启动子进行了比较，以 作为报告基因。在 和 物种中，来自 启动子的绿色荧光蛋白 (GFP) 荧光均可诱导，但在 中不可诱导，这可能有助于将对 有毒的基因克隆到质粒中。与 在 中相比， 启动子在较低的诱导剂浓度下被激活，并且使用 可以获得更高的 水平。流式细胞术分析表明，与 在测试的 物种中， 启动子的泄漏率更高，但在诱导时表达的细胞比例更高。作为唯一碳源的 d-木糖不能支持 或 的生长，而且比许多其他常用的诱导剂更便宜，这可能有利于大规模应用。该系统的功效通过其在揭示 型 II 分泌系统基因 在细菌抑制角膜上皮细胞伤口闭合中的作用的研究中得到了证明。本研究介绍了一种用于 物种基因表达的新型诱导启动子系统。 物种在人类感染、生物技术和作为研究基础科学问题的模型系统方面都具有极其重要的意义。在本研究中，我们开发了一种木糖诱导启动子系统，在 和 中对其进行了评估，并发现它适合于强烈诱导基因表达。此外，我们还证明了其在控制基因表达中的功效，表明来自 的 II 型分泌系统蛋白 XcpQ 在角膜伤口闭合模型中对宿主-病原体相互作用很重要。
