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首页> 外文期刊>Biochemistry >A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds.
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A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds.

机译：基于LuxP-FRET的报告基因，用于检测和定量AI-2细菌群体感应信号化合物。

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摘要

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 Kd = 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.
机译：各种细菌产生并监测以种群密度依赖性方式调节特定基因组的低分子量信号分子。此过程称为定额感应（QS）。迄今为止，从革兰氏阴性细菌中检测QS信号分子主要依赖于细菌报告菌株。这些生物测定受到化合物的实质性干扰，这些化合物会影响报告菌株的生长和代谢。另外，报告菌株对QS信号分子的敏感性是种群密度依赖性的。在这里，我们描述了快速检测和量化呋喃糖基硼酸酯二酯（BAI-2）亚类的自动诱导剂2（AI-2），QS分子的体外测定系统的开发。该传感器基于与BAI-2受体LuxP末端融合的GFP的青绿色和黄色变体之间的配体结合诱导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BAI-2会导致末端荧光团之间FRET的水平降低。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生物传感器对野生型哈维弧菌的培养物滤液的添加有反应，但对BAI-2合成中有缺陷的哈维弧菌突变体的培养物没有反应。生物传感器对BAI-2的敏感性（表观Kd = 270 nM）类似于BAI-2生物测定系统。讨论了微生物生物测定系统的局限性以及基于FRET的BAI-2生物传感器的优势和潜在应用。

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