A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds
A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds
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DOI：
10.1021/bi602479e
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发表时间：
2007-04-03
期刊：
影响因子：
2.9
通讯作者：
Sayre, Richard
中科院分区：
文献类型：
--
作者：
Rajamani, Sathish;Zhu, Jinge;Sayre, Richard
各种细菌产生并监测低分子量信号分子，这些信号分子以种群密度依赖性方式调节特定基因组。此过程称为群体感应 (QS)。迄今为止，革兰氏阴性菌 QS 信号分子的检测主要依赖于细菌报告菌株。这些生物测定受到影响报告菌株生长和代谢的化合物的严重干扰。此外，报告菌株对 QS 信号分子的敏感性取决于种群密度。在这里，我们描述了一种体外测定系统的开发，用于快速检测和定量自诱导剂 2 (AI-2) QS 分子的呋喃糖硼酸二酯 (BAI-2) 亚类。该传感器基于融合到 BAI-2 受体 LuxP 末端的 GFP 青色和黄色变体之间的配体结合引起的荧光共振能量转移 (FRET) 变化。出乎意料的是，向纯化的生物传感器中添加合成的 BAI-2 会导致末端荧光团之间的 FRET 水平降低。包括配体结合位点突变在内的几条证据表明观察到的 FRET 变化是 BAI-2 依赖性的。基于FRET的BAI-2生物传感器对添加来自野生型哈维氏弧菌的培养滤液有反应，但对来自BAI-2合成缺陷的哈维氏弧菌突变体的培养滤液没有表现出反应。生物传感器对 BAI-2 的灵敏度（表观 K-d = 270 nM）与 BAI-2 生物测定系统相似。讨论了微生物生物测定系统的局限性以及基于 FRET 的 BAI-2 生物传感器的优点和潜在应用。
Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2-dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wild-type Vibrio harveyi but exhibited no response to culture filtrates from V. harveyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent K-d = 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed.