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模式细菌天蓝色链霉菌发育过程中发生的分子事件。链霉菌属是常见的土壤细菌，以大量合成生物活性分子而闻名，包括目前使用的大多数抗生素。这些次级代谢产物的合成作为这些多细胞细菌中更大的发育程序的一部分而发生，其在丝状气生菌丝的孢子形成中达到高潮。两个基因簇的产物对S.本研究将描述腔棘鱼发育的特征。whiJ簇包含气生菌丝孢子形成所需的基因，包括编码推定的转录因子的基因。WhiJ将被纯化，其在体外结合DNA的能力被表征，并且使用基于PCR的方法鉴定其结合的特异性DNA序列。搜索已完成的S。因此，对腔棘鱼基因组中WhiJ结合位点的分析将揭示受WhiJ调控的下游孢子形成基因，从而阐明该途径中的下一组分子参与者。而某些基因产物具有促进S.其他人必须在条件不适合时限制它。由负调节因子突变引起的σ因子U的过度活性破坏了分化，这可能是因为σ因子U调节子含有其产物阻止发育的基因。将使用纯化的蛋白质对sigmaU及其负调节因子之间的假定复合物进行生化表征。转录报告基因将用于鉴定激活sigmaU的胁迫条件。最后，sigmaU调节子的成员将通过基因组分析和推定的sigmaU启动子共有序列的表征来鉴定。WhiJ、sigmaU及其靶点的表征将阐明介导S. coelicolor，并将加强目前对这一复杂过程的不完全理解。相关性：拟议研究的目标是了解链霉菌细菌如何生长和变化。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生产的救命药物（如抗生素和抗癌药物）的产量和种类。

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).