Regulation of Streptomyces differentiation and antibiotic production by SoxR

描述(由申请人提供)： 这项研究的广泛目标是了解大自然如何适应氧化还原感应转录因子SoxR，以满足具有不同生理和环境挑战的生物体的需求。SOXR传统上被认为是大肠杆菌和肠沙门氏菌中氧化应激反应的中介，但这一作用可能仅限于肠道细菌。在绝大多数非肠道药物中，SoxR被预测为介导对内源性产生的氧化还原活性抗生素的反应。这一点已在铜绿假单胞菌中得到证实，其中SoxR感知内源性抗生素并控制涉及其运输和加工的基因的表达。拟议的研究计划旨在研究SoxR在进化上不同的抗生素生产商天蓝色链霉菌中的功能和氧化还原敏感特性。天蓝色假单胞菌产生多种次生代谢产物，包括两种氧化还原活性的有色抗生素放线菌素和十一烷基灵芝菌素。天蓝色链霉菌中SoxR的缺失导致这些色素抗生素的加速发育和超量生产，这表明SoxR参与了控制发育和抗生素代谢基因的调控网络。在一个色素缺陷突变体中，两个SoxR靶基因的表达显著降低，这表明，就像在铜绿假单胞菌中一样，SoxR可能介导了天蓝色链霉菌对内源抗生素的反应。将进行微阵列研究，以确定在天蓝色链霉菌发育周期中受SoxR直接和间接调控的基因。这些将独立地通过实时定量聚合酶链式反应来验证，直接的SoxR-靶标将通过体外凝胶移位分析来确认。SoxR调节子的鉴定将有助于更好地理解SoxR如何调控这一抗生素产生模式中的形态和生理分化。天蓝色链霉菌SoxR的氧化还原敏感机制将在体内进行研究。[2Fe-2S]簇的重要性将通过检查簇缺陷突变体补充？soxR突变体中缺陷的能力来评估。同样的策略将被用来探索潜在的调控结构域的重要性，该结构域由一个扩展的C-末端区域(带有两个半胱氨酸残基)组成，该区域是链霉菌物种SoxRs所特有的。我们将分别检测放线菌素缺失突变体和十一烷基灵芝菌素缺失突变体中SoxR的转录活性，以确定相关的生理信号。最后，将在色素缺陷突变体中评估外源添加的氧化还原循环化学物质诱导SOxR活性的能力，这将揭示信号识别的特异性。综上所述，这些实验将进一步深入了解铁硫调节蛋白的氧化还原传感和转导机制。最后，作为一个领域的建议，这项研究将培训本科生设计和执行假设驱动的实验和数据分析。 公共卫生相关性： 链霉菌属的成员以产生临床和兽医中使用的三分之二的生物活性代谢物(包括抗生素)而闻名。这一迹象表明，SoxR在天蓝色链霉菌模型中控制细胞机械来处理和/运输抗生素，这对于优化链霉菌生产生物活性分子具有重要意义，并可能进一步为我们日益减少的有效抗生素清单带来新的候选者。

DESCRIPTION (provided by applicant): The broad goal of this research is to understand how nature has adapted the redox-sensing transcription factor SoxR to serve the needs of organisms with different physiologies and environmental challenges. SoxR has traditionally been known as the mediator of an oxidative stress response in Escherichia coli and Salmonella enterica, but this role may be restricted to enteric bacteria. In the vast majority of non-enterics, SoxR is predicted to mediate a response to endogenously-produced redox-active antibiotics. This has been confirmed in Pseudomonas aeruginosa where SoxR senses endogenous antibiotics and controls the expression of genes involved in their transport and processing. The proposed research program is designed to examine the function and redox-sensing properties of SoxR in the evolutionarily divergent antibiotic producer Streptomyces coelicolor. S. coelicolor produces several secondary metabolites including two redox-active pigmented antibiotics, actinorhodin and undecylprodigiosin. Deletion of soxR in S. coelicolor causes accelerated development and hyper-production of these pigmented antibiotics, suggesting that SoxR participates in a regulatory network that controls developmental and antibiotic-metabolizing genes. The expression of two SoxR-target genes is significantly reduced in a pigment-deficient mutant suggesting that, as in P. aeruginosa, SoxR may mediate its effects in response to endogenous antibiotics in S. coelicolor. Microarray studies will be conducted to identify genes that are directly and indirectly regulated by SoxR as S. coelicolor proceeds through its developmental cycle. These will be independently verified by quantitative real time PCR and direct SoxR-targets will be confirmed by gel shift assays in vitro. The identification of the SoxR regulon will help to forge a better understanding of how SoxR regulates morphological and physiological differentiation in this model antibiotic-producer. The mechanism of redox-sensing by S. coelicolor SoxR will be investigated in vivo. The importance of the [2Fe-2S] clusters will be assessed by examining the ability of a cluster-deficient mutant to complement the defects in the ?soxR mutant. The same strategy will be employed to probe the importance of a potential regulatory domain consisting of an extended C-terminal region (with two cysteine residues) that is unique to SoxRs from Streptomyces species. The transcriptional activity of SoxR in an actinorhodin-deficient mutant, and separately in an undecylprodigiosin-deficient mutant will be examined to identify the relevant physiological signal. Finally, the ability of exogenously added redox-cycling chemicals to elicit SoxR activity will be assessed in a pigment-deficient mutant, which will reveal specificity of signal recognition. Together, these experiments will provide further insight into redox-sensing and transduction mechanisms by iron-sulfur regulatory proteins. Finally, as an AREA proposal, the research will train undergraduate students in the design and execution of hypothesis-driven experimentation and data analysis. PUBLIC HEALTH RELEVANCE: Members of the Streptomyces genus are notable for producing two-thirds of the biologically active metabolites (including antibiotics) used in clinical and veterinary medicine. The indication that SoxR controls cellular machinery to process and/transport antibiotics in the model S. coelicolor, has implications for optimizing the production of bioactive molecules by Streptomyces, and may further bring new candidates to our dwindling list of effective antibiotics.