Regulation of Streptomyces coelicolor Development-AREA

描述（由申请人提供）：本申请的主要目的是了解在模式细菌链霉菌发育过程中发生的分子事件。链霉菌是一种常见的土壤细菌，以多产的生物活性分子合成而闻名，包括目前使用的大多数抗生素。在这些多细胞细菌中，次生代谢物的合成是一个更大的发育过程的一部分，在丝状气生菌丝的产孢过程中达到高潮。在本研究中，我们将对两个基因簇的产物进行表征，这两个基因簇对葡萄的发育有着深刻而不同的影响。whj簇包含气生菌丝产孢所需的基因，包括编码一种假定的转录因子的基因。将对whj进行纯化，鉴定其体外结合DNA的能力，并使用基于pcr的方法鉴定其结合的特定DNA序列。在完整的葡萄球菌基因组中寻找whj结合位点，将揭示受whj调控的下游产孢基因，从而阐明这一途径中的下一组分子参与者。虽然一些基因产物可以促进S. colcolcolor的发育，但当条件不合适时，其他基因产物必须限制它的发育。负调节因子突变引起的sigma因子U的过度活性会消除分化，这可能是因为sigmaU调节因子含有其产物阻碍发育的基因。假定的sigmaU及其负调节因子之间的复合物将使用纯化蛋白进行生物化学表征。转录报告器将用于识别激活sigmaU的应激条件。最后，将通过基因组分析和假定的sigmaU启动子共识序列的表征来确定sigmaU调控子的成员。whj、sigmaU及其靶点的鉴定将阐明至少部分介导南芥发育的分子事件序列，并将加强目前对这一复杂过程的不完整理解。相关性：提出的研究的目的是了解链霉菌细菌如何生长和变化。对这些了解得越多，就越有可能合理地操纵这些有用的细菌，以增加它们在生命周期中产生的救命药物的产量和种类（如抗生素和抗癌药物）。

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).