Regulation of Streptomyces coelicolor Development-AREA

天蓝色链霉菌发育调控-AREA

• 批准号: 7011318
• 负责人: Amy Gehring
• 金额: $ 20.23万
• 依托单位: WILLIAMS COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011318
• 关键词: DNA binding protein; DNA footprinting; Streptomyces; bacterial genetics; bacterial proteins; binding sites; cell differentiation; developmental genetics; gel mobility shift assay; gene induction /repression; genetic regulatory element; microorganism growth; polymerase chain reaction; protein protein interaction; protein purification; protein structure function; sporogenesis; stress; transcription factor

DESCRIPTION (provided by applicant): The broad objective of this application is to understand the molecular events that occur during development of the model bacterium Streptomyces coelicolor. The Streptomyces are common soil bacteria well-known for the prolific synthesis of bioactive molecules including the majority of antibiotics in current use. Synthesis of these secondary metabolites occurs as part of a larger developmental program in these multicellular bacteria, which culminates in the sporulation of filamentous aerial hyphae. The products of two gene clusters that have profound but different impacts on S. coelicolor development will be characterized in this study. The whiJ cluster contains genes required for the sporulation of aerial hyphae including one that encodes a putative transcription factor. WhiJ will be purified, its ability to bind DNA in vitro characterized, and the specific DNA sequence to which it binds identified using a PCR-based approach. Search of the completed S. coelicolor genome for WhiJ binding sites will thereby reveal the downstream sporulation genes regulated by WhiJ and thus elucidate the next set of molecular players in this pathway. While some gene products function to advance S. coelicolor development, others must restrict it when conditions are unsuitable. Excessive activity of the sigma factor U caused by mutation of its negative regulator abolishes differentiation presumably because the sigmaU regulon contains genes whose products halt development. The presumed complex between sigmaU and its negative regulator will be biochemically characterized using purified proteins. A transcriptional reporter will be used to identify stress conditions that activate sigmaU. Finally, members of the sigmaU regulon will be identified by genome analysis and characterization of a putative sigmaU promoter consensus sequence. The characterization of WhiJ, sigmaU and their targets will elucidate at least a partial sequence of the molecular events that mediate development in S. coelicolor and will enhance the currently incomplete understanding of this complex process. Relevance: The goal of the proposed research is to understand how Streptomyces bacteria grow and change. The more that is understood, the more likely it is that these useful bacteria can be rationally manipulated to increase the yield and variety of life-saving drugs that they make during their life cycle (such as antibiotics and anticancer drugs).

描述（由申请人提供）：本应用的广泛目的是了解模型细菌Coelicolor开发过程中发生的分子事件。链霉菌是常见的土壤细菌，众所周知，以多产的生物活性分子合成，包括大多数当前使用中的抗生素。这些次级代谢物的合成是这些多细胞细菌中较大发育计划的一部分，这些程序在丝状空气菌丝的孢子体中达到顶点。在这项研究中，将表征两个对Coelicolor发育产生深远但不同影响的基因簇的产物。 Whij簇包含孢子形成菌丝所需的基因，其中包括编码推定转录因子的基因。 Whij将被纯化，其在体外表征结合DNA的能力以及使用基于PCR的方法鉴定的特定DNA序列。因此，搜索完整的Coelicolor基因组对WHIJ结合位点的搜索将显示由WHIJ调节的下游孢子型基因，从而阐明了该途径中的下一组分子参与者。虽然某些基因产物的作用可以推进S. coelicolor的发育，但其他基因产物必须在条件不合适时限制它。 Sigma因子U的过度活性是由于其负调节剂突变引起的，因为Sigmau调节量包含其产物停止发育的基因，因此废除了分化。 Sigmau及其阴性调节剂之间的假定复合物将使用纯化的蛋白质进行生化表征。转录报告基因将用于识别激活Sigmau的应力条件。最后，将通过基因组分析和推定的Sigmau启动子共识序列的表征来鉴定Sigmau调节子的成员。 WHIJ，Sigmau及其目标的表征将阐明至少介导岩链球葡萄球菌发展的分子事件的部分序列，并将增强对这一复杂过程的当前不完整的理解。相关性：拟议的研究的目的是了解链霉菌细菌如何生长和变化。了解的越多，就越有可能在合理地操纵这些有用的细菌，以增加它们在生命周期（例如抗生素和抗生素药物）中生命的产量和挽救生命的药物。