Relationship of Antibiotic Production and Development in Streptomyces coelicolor

描述(由申请人提供)：这项建议的广泛目标是阐明模式菌天蓝色链霉菌抗生素生产和形态发育的复杂、相互关联的过程之间的一些分子联系。链霉菌是生物活性分子的一种极其宝贵的来源，包括临床使用的大多数抗生素。这些菌丝体细菌通常合成抗生素和其他次生代谢产物，同时伴随着更广泛的发育事件，导致在菌落表面产生气生菌丝体，其丝状细胞最终形成孢子。这项研究的目的涉及两种特殊类型的蛋白质，这两种蛋白质既影响天蓝色链霉菌的抗生素生产和发育。首先，磷酸铁氨酰转移酶是催化许多抗生素生物合成酶活性所需的翻译后修饰的酶。天蓝色链霉菌中SCO6673 PPTase基因的突变会导致产生钙依赖抗生素的能力丧失，但反过来也会导致潜在的抗癌分子十一烷基灵芝红素的过度生产。在这个突变体中，产孢量也延迟了。为了了解这些不同表型效应的基础，将使用实时荧光聚合酶链式反应来评估与野生型相比，PPTase突变体中已知的抗生素调节和发育基因的表达是否存在差异。这些数据将为后续的定向突变策略提供信息，以使天蓝色葡萄球菌能够最大限度地生产十一烷基灵芝菌素。第二个目的是了解一种可能的应激反应ECF sigma因子？u的活性如何阻止天蓝色链霉菌中聚酮类抗生素的产生和气生菌丝的形成。基因芯片分析和蛋白质组学研究相结合，将野生型天蓝色链球菌与具有高活性、未受调节的？U活性的RSUA突变体进行比较，将建立？U依赖基因的完整调控以及那些受？U活性间接影响的基因。观察到的RSUA突变体胞外蛋白水解酶和磷酸戊糖途径酶活性的升高与其抗生素生产和发育缺陷的相关性将通过相关基因的突变或过度表达和对产生的菌株的分析来测试。突变分析也将被用来测试可能的膜蛋白SCO4110参与调节？U的活性。更好地了解次生代谢和发育是如何相互影响的，最终可能会为链霉菌的有效药物生产提供普遍的策略。 与公共卫生相关：拟议的研究的目标是了解链霉菌如何作为其整个生命周期的一部分来制造有用的化合物，如抗生素。我们越好地了解这些有益细菌是如何生长的，我们就越有可能利用这种生长来生产抗击感染和癌症的救命药物。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to elucidate some of the molecular connections between the complex, interrelated processes of antibiotic production and morphological development in the model bacterium Streptomyces coelicolor. The streptomycetes are an incredibly valuable source of bioactive molecules, including the majority of antibiotics clinically used. These mycelial bacteria typically synthesize antibiotics and other secondary metabolites concurrently with broader developmental events that result in the generation of an aerial mycelium on the colony surface whose filamentous cells ultimately sporulate. The aims of this study concern two particular types of proteins that influence both antibiotic production and development in S. coelicolor. First, the phosphopantetheinyl transferase are enzymes that catalyze a posttranslational modification required for the activity of many antibiotic biosynthetic enzymes. Mutation of the SCO6673 PPTase gene in S. coelicolor results in loss of the ability to produce calcium dependent-antibiotic but also conversely hyperproduction of the potential anti-cancer molecule undecylprodigiosin. Sporulation is also delayed in this mutant. In order to understand the basis for these diverse phenotypic effects, real-time PCR will be used to assess any differences in expression of known antibiotic regulatory and developmental genes in the PPTase mutant as compared to the wild type. This data will inform a subsequent targeted mutational strategy to engineer S. coelicolor for maximum undecylprodigiosin production. The second aim focuses on understanding how activity of a likely stress response ECF sigma factor, ?U, can block both polyketide antibiotic production and aerial mycelium formation in S. coelicolor. A combination of microarray analysis and proteomic studies comparing wild type S. coelicolor to a rsuA mutant with high, unregulated ?U activity will establish the complete regulon of ?U-dependent genes as well as those indirectly affected by ?U activity. The relevance of the observed elevation of both extracellular protease and pentose phosphate pathway enzyme activity in the rsuA mutant to its antibiotic production and developmental defects will be tested by mutation or overexpression of the relevant genes and analysis of the resulting strains. Mutational analysis will also be used to test the involvement of the likely membrane protein SCO4110 in the regulation of ?U activity. A better understanding of how secondary metabolism and development are mutually influenced may ultimately suggest generalizable strategies for effective pharmaceutical production in the streptomycetes. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to understand how Streptomyces bacteria make useful compounds, such as antibiotics, as part of their overall life cycle. The better we understand how these beneficial bacteria grow, the more likely it is that we can engineer that growth to produce life-saving drugs for fighting infections and cancers.