AI-2 Mediated Quorum Sensing in Vibrio harveyi.

描述(申请人提供)：细菌通过分泌和检测小信号分子来相互交流和协调群落的行为，这种现象被称为群体感应。这项拟议的研究将调查来自细胞外信号分子的信息是如何通过膜传递来调节基因表达的。具体地说，将描述新发现的广泛存在的细菌AI-2自身诱导分子与其在哈维氏弧菌中的受体LuxPQ之间的相互作用。具体目的是：1.确定LuxP中影响与AI-2和LuxQ相互作用的突变。功能突变的增益将在模仿AI-2结合形式的LuxP中产生。还将发现不能与AI-2或LuxQ相互作用的显性LuxP突变。2.鉴定LUXQ基因周质结构域中影响与LUXP相互作用的突变。基于聚合酶链式反应的突变策略将被用于产生突变，以增强LuxQ与LuxP的相互作用。也将在LuxQ中分离到抑制在Aim 1.3中分离的LuxP的显性突变等位基因的突变。3.测试AI-2类似物的激动剂或拮抗剂活性。将测量每个合成类似物与纯化的LuxP的结合亲和力，并与体内AI-2活性进行比较。

DESCRIPTION (provided by applicant): Bacteria communicate with one another and coordinate the behavior of the community by means of secretion and detection of small signaling molecules, and this phenomenon is termed quorum sensing. The proposed research will investigate how the information from an extracellular signaling molecule is transduced across a membrane to regulate gene expression. Specifically, the interactions between the newly discovered and widespread bacterial AI-2 autoinducer molecule and its receptor LuxPQ in Vibrio harveyi will be characterized. The specific aims are: 1. Identify mutations in LuxP that affect interaction with AI-2 and with LuxQ. Gain of function mutations will be generated in LuxP that mimic the AI-2-bound form. Dominant LuxP mutations will also be identified that are unable to interact with AI-2 or with LuxQ. 2. Identify mutations in the periplasmic domain of LuxQ that affect interaction with LuxP. A PCR-based mutagenesis strategy will be used to generate mutations that enhance LuxQ's interaction with LuxP. Mutations will also be isolated in LuxQ that suppress dominant mutant alleles of LuxP isolated in Aim 1. 3. Test AI-2 analogs for agonist or antagonist activity. The binding affinities of each synthetic analog for purified LuxP will be measured and compared to AI-2 activity in vivo.