Regulation of Streptomyces differentiation and antibiotic production by SoxR

SoxR 对链霉菌分化和抗生素生产的调节

• 批准号: 7881169
• 负责人: Monica Chander
• 金额: $ 20.28万
• 依托单位: BRYN MAWR COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881169
• 关键词: Animal Model; Antibiotics; Bacteria; Binding; Biochemical; C-terminal; Chemicals; Clinical; Complement; Cysteine; DNA Binding; Data Analyses; Defect; Development; Developmental Gene; Electrophoretic Mobility Shift Assay; Enteral; Enterobacteriaceae; Enzymes; Escherichia coli; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Histidine; In Vitro; Iron; Mediating; Mediator of activation protein; Metabolism; Mixed Function Oxygenases; Modeling; Monitor; Nature; Organism; Oxidation-Reduction; Oxidative Stress; Phenazines; Physiological; Physiology; Pigments; Play; Production; Promoter Regions; Property; Proteins; Pseudomonas aeruginosa; Regulation; Regulon; Research; Research Proposals; Role; Salmonella enterica; Signal Transduction; Soil; Solid; Source; Specificity; Streptomyces; Streptomyces coelicolor; Students; Sulfur; Supervision; Time; Training; Transport Process; Veterinary Medicine; actinorhodin; base; biological adaptation to stress; cofactor; design; forging; genetic regulatory protein; in vivo; insight; member; mutant; programs; public health relevance; research study; response; transcription factor

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.

描述（由申请人提供）： 这项研究的广泛目标是了解自然界如何适应氧化还原传感转录因子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沙门氏菌中氧化应激反应的介体，但这种作用可能仅限于肠道细菌。在绝大多数非肠道药物中，预计SoxR介导对内源性产生的氧化还原活性抗生素的反应。这已经在铜绿假单胞菌中得到证实，其中SoxR感测内源性抗生素并控制参与其运输和加工的基因的表达。拟议的研究计划旨在研究SoxR在进化趋异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。S.腔棘鱼产生几种次级代谢物，包括两种氧化还原活性色素抗生素，放线菌紫素和十一烷基灵菌红素。S. coelicolor导致这些色素抗生素的加速发育和过度生产，这表明SoxR参与了控制发育和代谢基因的调控网络。两个SoxR靶基因的表达在色素缺陷型突变体中显著降低，这表明与铜绿假单胞菌一样，SoxR可能介导其对S.天蓝色。将进行微阵列研究以鉴定直接和间接受SoxR调控的基因，如S。腔棘鱼在其发育周期中进行。这些将通过定量真实的时间PCR进行独立验证，直接SoxR靶点将通过体外凝胶迁移试验进行确认。SoxR调节子的鉴定将有助于更好地理解SoxR如何调节该模型中的植物生产者的形态和生理分化。对S.将在体内研究腔棘鱼SoxR。[2Fe-2S]集群的重要性将通过检查集群缺陷突变体的能力，以补充在？soxR突变体。相同的策略将被用来探测一个潜在的调节结构域的重要性，该结构域由一个扩展的C-末端区域（具有两个半胱氨酸残基）组成，该区域是链霉菌属物种的SoxRs所特有的。将检查放线菌红素缺陷突变体中SoxR的转录活性，并分别检查十一烷基灵菌红素缺陷突变体中SoxR的转录活性，以鉴定相关的生理信号。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引发SoxR活性的能力，这将揭示信号识别的特异性。总之，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，作为一个区域的建议，本研究将培养本科生的假设驱动的实验和数据分析的设计和执行。 公共卫生相关性： 链霉菌属的成员以生产临床和兽医学中使用的三分之二的生物活性代谢物（包括抗生素）而闻名。SoxR在模型S中控制细胞机器以加工和/转运抗生素的指示。coelicolor，具有优化链霉菌生产生物活性分子的意义，并可能进一步为我们不断减少的有效抗生素名单带来新的候选人。