Palmer Gregory C, Schertzer Jeffrey W, Mashburn-Warren Lauren, Whiteley Marvin
Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX, USA.
Methods Mol Biol. 2011;692:207-17. doi: 10.1007/978-1-60761-971-0_15.
Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.g. cell cultures) is described. Separation of PQS from extracts by Thin-Layer Chromatography (TLC) is used in combination with the natural fluorescence of the molecule for quantification. A second separation technique for the PQS precursor HHQ using High-Performance Liquid Chromatography (HPLC) is also described, and this assay exploits the molecule's characteristic absorbance for quantification. A third method for quantification of PQS from simple mixtures (e.g. enzyme assays) using fluorescence is outlined. Finally, a protocol for determining PQS interactions with membrane lipids through Fluorescence Resonance Energy Transfer (FRET) is presented. These techniques allow for quantification and characterization of PQS from diverse environments, a prerequisite to understanding the biological functions of QS molecules.
铜绿假单胞菌产生一种群体感应分子，称为铜绿假单胞菌喹诺酮信号（2-庚基-3-羟基-4-喹诺酮；PQS），它调控一系列与毒力相关的基因。本章介绍了四种用于检测和定量PQS的相关技术。首先，描述了从复杂混合物（如细胞培养物）中提取PQS的方法。通过薄层色谱（TLC）从提取物中分离PQS，并结合该分子的天然荧光进行定量。还介绍了使用高效液相色谱（HPLC）对PQS前体HHQ进行分离的第二种技术，该测定利用该分子的特征吸光度进行定量。概述了使用荧光从简单混合物（如酶测定）中定量PQS的第三种方法。最后，介绍了一种通过荧光共振能量转移（FRET）确定PQS与膜脂相互作用的方案。这些技术能够对来自不同环境的PQS进行定量和表征，这是理解QS分子生物学功能的先决条件。
