Rajamani Sathish, Sayre Richard
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH, USA.
Methods Mol Biol. 2011;692:31-46. doi: 10.1007/978-1-60761-971-0_3.
Intercellular small molecular weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum sensing molecules. These molecules mediate a variety of population-dependent responses, including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules has important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. A set of ligand-insensitive LuxP-mutant FRET protein sensor was also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.
细胞间小分子信号分子可调节细菌的多种生物学功能。由细胞间信号分子介导的更为复杂的行为之一是群体感应分子所调控的一系列活动。这些分子介导多种群体依赖性反应，包括调节生物发光、III型分泌、铁载体产生、菌落形态、生物膜形成和金属蛋白酶产生的基因的表达。鉴于它们在调节这些反应中的核心作用，群体感应分子的检测和定量具有重要的实际意义。直到最近，革兰氏阴性菌群体感应分子的检测主要依赖于细菌报告系统。这些生物测定虽然非常有用，但会受到影响细菌生长和代谢的化合物的干扰。此外，报告反应高度依赖于培养时间和细胞群体密度。为了克服这些限制，我们开发了一种基于蛋白质的体外检测系统，用于快速检测和定量自诱导物-2（AI-2）群体感应分子的呋喃糖硼酸二酯（BAI-2）亚类。该生物传感器基于BAI-2与哈维弧菌群体感应受体LuxP的相互作用。与BAI-2结合到LuxP受体相关的构象变化改变了分别融合到LuxP受体N端和C端的绿色荧光蛋白（GFP）的青色和黄色变体（CFP和YFP）的方向。LuxP-BAI2结合诱导CFP和YFP之间荧光共振能量转移（FRET）的变化，其变化幅度取决于配体浓度。还开发了一组对配体不敏感的LuxP突变FRET蛋白传感器用作对照生物传感器。基于FRET的BAI-2生物传感器对合成的和生物来源的BAI-2化合物都有选择性响应。本报告描述了LuxP-FRET生物传感器用于检测和定量BAI-2的用途。
