CAREER: Activation of the Vibrio fischeri Luminescence Operon via Quorum Sensing

来自费氏弧菌的StevensLuxR是一种新的细菌转录激活因子家族中最受了解的成员。这些激活剂参与细胞密度依赖性的群体感应现象和对自生成的酰基高丝氨酸内酯环境信号的反应。本研究项目的目标是确定在群体感应过程中luxI启动子转录激活的分子机制。主要的重点将是进一步确定LuxR和RNA聚合酶（RNAP）在这一过程中的作用，使用遗传和生化方法的结合。将纯化活性全长LuxR并对其体外活性进行表征。RNAP的特定区域对luxI启动子的转录激活至关重要。LuxR上对luxI启动子转录激活至关重要的残基将进一步定义。鉴定LuxR和RNAP之间蛋白相互作用位点的基因间抑制子将被分离出来。最后，LuxR中定义域间相互作用的基因内抑制子也将被分离。从这些研究中获得的知识将与越来越多的细菌系统相关，这些细菌系统已被发现使用群体感应和反应调节方案。将群体感应等新兴研究领域的最新发现和理论纳入教学课程，对培养下一代微生物学家至关重要。因此，还制定了一项教育计划，其主要目标是使本科生和研究生接触到分子微生物学的当前研究领域，特别强调微生物生理学和遗传学。这将需要在系一级协调讨论课程，然后设计和实施适当的新讲座和实验课程。为了帮助优化学生在这些课程和其他课程中的学习体验质量，讲师将继续获得学习理论、教学设计、评估和教育技术领域的知识。这些努力的结果是，现代分子微生物学将成为本系课程的一个标准部分，用于教育和培训学生未来在微生物学和相关学科的专业生涯。细菌细胞能够作为多细胞单位通过所谓的群体感应过程进行细胞间通信和协调行为的想法是微生物学领域中一个相对较新的但重要的概念。细菌细胞能够通过被称为转录调节因子的蛋白质分子的活性在基因表达水平上控制这些过程。LuxR是转录调控因子家族中最容易理解的成员，其成员都具有相似的功能。LuxR家族的转录调控因子控制一系列靶基因的表达，这些基因参与了许多具有科学、医学、农业和经济重要性的细菌过程。其中一些，包括LuxR，存在于与植物、动物或人类宿主形成有益的共生关系或有害的致病关系的细菌中。对LuxR转录调控家族的精确分子和生化功能的详细了解可能有助于识别干扰这些关联的化合物。本研究的重点是进一步定义LuxR的一些属性。

StevensLuxR from Vibrio fischeri is the best understood member of a new family of bacterial activators of transcription. These activators are involved in the cell density-dependent phenomenon of quorum sensing and response to self-generated acyl homoserine lactone environmental signals. The goal of this research project is to determine the molecular mechanism of transcriptional activation employed at the luxI promoter during quorum sensing. The primary focus will be to further define the roles of LuxR and RNA polymerase (RNAP) in this process using a combination of genetic and biochemical approaches. Active full-length LuxR will be purified and its activity characterized in vitro. Specific regions of RNAP that are essential for transcriptional activation at the luxI promoter will be identified. The residues on LuxR that are essential for transcriptional activation at the luxI promoter will be further defined. Intergenic suppressors that identify protein-protein interaction sites between LuxR and RNAP will be isolated. Finally, intragenic suppressors within LuxR that define interdomain interactions will also be isolated. The knowledge gained from these studies will be relevant to the growing number of bacterial systems that have been found to use a quorum sensing and response regulatory scheme. The incorporation into the instructional curriculum of the latest findings and theories coming out of emerging research fields like quorum sensing, is essential to the training of the next generation of microbiologists. Thus, an educational plan has also been developed with the primary goal of exposing undergraduate and graduate students to the current field of research in molecular microbiology, with a particular emphasis on microbial physiology and genetics. This will necessitate a coordinated discussion of curriculum at the departmental level followed by the design and implementation of appropriate new lecture and laboratory courses. To help optimize the quality of the students' learning experiences in these and other courses, the instructor will continue to gain knowledge in the areas of learning theory, instructional design, assessment and educational technologies. As a result of these efforts, modern molecular microbiology will become a standard part of the departmental curriculum used to educate and train students for their future professional careers in microbiology and related disciplines. The idea that bacterial cells are capable of intercellular communication and coordinated behavior as multicellular units through a process known as quorum sensing is a relatively new, but important concept in the field of microbiology. Bacterial cells are able to control these processes at the level of gene expression through the activity of protein molecules know as transcriptional regulators. LuxR is the name given to the best understood member of a family of transcriptional regulators whose members all have similar functions. This LuxR family of transcriptional regulators controls the expression of a range of target genes involved in a number of bacterial processes of scientific, medical, agricultural and economic importance. Several of them, including LuxR, are found in bacteria that enter either helpful symbiotic, or harmful pathogenic associations with plant, animal or human hosts. A detailed knowledge of the precise molecular and biochemical functions of the LuxR family of transcriptional regulators may be useful for identifying compounds that interfere with these associations. This research focuses on further defining some of these properties for LuxR.