长双歧杆菌新的LuxS/AI-2依赖的QS系统及其调控机制的研究

Bifidobacteria are one group of the most important human intestinal physiological bacteria. Athough the molecular mechanism of interactions between Bifidobacteria and their host remains unclear, study of the molecular mechanisms of bifidobacterial LuxS/AI-2 dependent quorum sensing (QS) system can reveal their intestinal adaptation. In an early experiement conducted by our group member, QS system was first discovered in the type strain B. longum NCC2705 in a rabbit intestinal culture model, and recognized that the receptor of signal molecule autoinducer-2 (AI-2) is an ATP-binding cassette (ABC) transporter system. Therefore, we propose to knockout some releated genes at first. Then, with these deletion mutants and complement strains, we will test the specificity of substrates binding. While the Bifidobacterium LuxS/AI-2 dependent QS system can be validated through the establishment of the SPN model, which can disclose the transcriptional regulation mechanism of LuxS as well. Thirdly, experiments of GST pull-down, co-immunoprecipitation can identify the interactions of AI-2 receptors in vivo. Research of B.longum ABC transporter proteins specific interactions can annotate the function and their regulation net of these primarily unknown genes. In conclusion, after a systemitic analysis of results gained from above studies, the molecular pathway of Bifidobacteria AI-2 transport in vivo can be modeled, and some unknown proteins or regulators will be assigned new functions. The work can explain the adaptability of bifidobacteria in the intestinal environment, and show some new gene regulatory networks. All of the work are backstones for the further development Bifidobacterium as a carrier of food-grade expression system to improve the quality of human life.

双歧杆菌是人体肠道中最重要的生理性细菌，LuxS/AI-2依赖的QS系统分子调控机制可以揭示其在肠道中的适应性。本课题组前期利用家兔肠培养模型首次发现在双歧杆菌中存在QS系统，自诱导子AI-2的识别受体是一个ABC转运系统。因此，拟通过基因敲除进行缺失突变体的构建和回复试验以及底物特异性结合试验等，建立SPN模型，验证双歧杆菌的LuxS/AI-2 QS系统，确定LuxS的转录调控机制；采用GST沉降实验、免疫共沉淀实验等，研究长双歧杆菌AI-2的体内识别受体- - -ABC转运系统中蛋白两两的特异性相互作用，同时对这些未知功能基因（在基因组注释中它们的功能为理论推测）进行功能和调控的研究。结合以上结果，建立双歧杆菌AI-2体内转运的分子模型，注释一些未知功能蛋白和调控子，从而解释双歧杆菌在肠道环境中的适应性，建立新的基因调控网络，为进一步开发以双歧杆菌为载体的食品级表达系统奠定坚实的理论基础

双歧杆菌是人体肠道中最重要的生理性细菌，LuxS/AI-2依赖的QS系统分子调控机制可以.揭示其在肠道中的适应性。本课题组前期利用家兔肠培养模型首次发现在双歧杆菌中存在.QS系统，自诱导子AI-2的识别受体是一个ABC转运系统。因此，拟通过基因敲除进行缺失.突变体的构建和回复试验以及底物特异性结合试验等，建立SPN模型，验证双歧杆菌的Lux.S/AI-2 QS系统，确定LuxS的转录调控机制；采用GST沉降实验、免疫共沉淀实验等，研究.长双歧杆菌AI-2的体内识别受体- - -ABC转运系统中蛋白两两的特异性相互作用，同时对.这些未知功能基因（在基因组注释中它们的功能为理论推测）进行功能和调控的研究。结.合以上结果，建立双歧杆菌AI-2体内转运的分子模型，注释一些未知功能蛋白和调控子，.从而解释双歧杆菌在肠道环境中的适应性，建立新的基因调控网络，为进一步开发以双歧.杆菌为载体的食品级表达系统奠定坚实的理论基础