Adaptation of Streptomyces coelicolor to endogenous antibiotics mediated by a red

描述（由申请人提供）： 这项更新R15提案的目标是进一步深入了解氧化还原传感的作用， 在产芽孢杆菌天蓝色链霉菌（Streptomyces coelicolor）中的转录因子SoxR。链霉菌属的成员产生大多数已知的抗生素，但允许这些生物体适应内源性抗生素的机制尚不清楚。我们有证据表明，SoxR可能在这种适应中发挥作用。In S.在腔棘鱼中，SoxR调节基因的表达响应于氧化还原活性抗生素放线菌紫素的产生。SoxR调控基因编码ABC转运蛋白和几个推定的蛋白质与同源性的approxic-tailoring酶。与亲本菌株相比，δsoxR突变体过量产生放线菌紫素，这表明SoxR调节通常减少这种氧化还原活性代谢物产生的机制，从而使这种生物体避免自毒。我们建议，SoxR调节蛋白质修改放线菌紫素生物合成途径中的有毒中间体，然后将它们从细胞中排出。因此，我们期望发现野生型和δsoxR细胞产生的代谢物的差异，我们将使用高压液相色谱（HPLC）和质谱（MS）分析的组合来确定。为了鉴定作为SoxR调节酶底物的途径中间体，我们将在野生型和δsoxR背景中创建放线菌紫素生物合成途径的阻断（通过突变），以迫使特定中间体的积累。如果这些中间体中的任何一种通过SoxR依赖性机制代谢，则与含有SoxR的细胞相比，它们在SoxR缺陷细胞中的水平将更高，并且这将再次通过HPLC-MS分析检测。将通过定量实时PCR监测SoxR-调节子在Act生物合成突变体中的表达，以确定SoxR是否感测Act的氧化还原活性前体，从而诱导解毒基因伴随或先于毒性分子的产生。为了更好地了解SoxR介导其功能的机制，将删除单个SoxR调节基因（直接执行SoxR的解毒功能），并对突变体进行遗传和生物化学表征。此外，我们将构建一个菌株，这是缺乏在两个SoR和一个假定的放线菌紫素出口（不是SoxR调节子的一部分），并评估由此产生的突变体的健身。总之，这些研究将增强我们对SoxR如何帮助抗生素生产商适应有毒内源性代谢物的理解。最后，作为一个区域的建议，本研究将培养学生在假设驱动的实验和数据分析的设计和执行。

DESCRIPTION (provided by applicant): The goal of this renewal R15 proposal is to gain further insight into the role of the redox-sensing transcription factor SoxR in the antibiotic-producing bacterium Streptomyces coelicolor. Members of the Streptomyces genus produce the majority of known antibiotics, but the mechanisms that allow these organisms to adapt to endogenous antibiotics are not well understood. We have evidence that SoxR may play a role in this adaptation. In S. coelicolor, SoxR-regulated genes are expressed in response to the production of the redox-active antibiotic, actinorhodin. SoxR-regulated genes encode an ABC-transporter and several putative proteins with homology to antibiotic-tailoring enzymes. A δsoxR mutant overproduces actinorhodin compared to the parental strain, suggesting that SoxR regulates mechanisms that normally reduce production of this redox-active metabolite thereby allowing this organism to avoid self-toxicity. We propose that SoxR-regulated proteins modify toxic intermediates in the actinorhodin biosynthetic pathway and then expel them from the cell. As such, we expect to find differences in the metabolites produced by wild type and δsoxR cells, which we will determine using a combination of high pressure liquid chromatography (HPLC) and mass spectroscopy (MS) analyses. In order to identify the pathway intermediates that serve as substrates for SoxR-regulated enzymes, we will create blocks in the actinorhodin biosynthetic pathway (by mutations) in the wild type and δsoxR backgrounds, to force the accumulation of specific intermediates. If any of these intermediates are metabolized by SoxR-dependent mechanisms, their levels will be higher in SoxR-deficient cells compared to SoxR-containing cells, and this wil again be detected via HPLC-MS analysis. The expression of the SoxR- regulon in the act biosynthetic mutants will be monitored by quantitative real-time PCR to determine if SoxR senses a redox-active precursor of Act, and thus induces detoxification genes to accompany or precede the production of toxic molecules. To obtain a better understanding of the mechanism via which SoxR mediates its function, individual SoxR-regulated genes (which directly execute SoxR's proposed detoxification function) will be deleted, and the mutants characterized genetically and biochemically. In addition, we will construct a strain that is deficient in both SoR and a putative actinorhodin exporter (not part of the SoxR regulon) and assess the fitness of the resulting mutant. Together, these studies will enhance our understanding of how SoxR helps antibiotic producers adapt to toxic endogenous metabolites. Finally, as an AREA proposal, the research will train students in the design and execution of hypothesis-driven experimentation and data analysis.