Collaborative Research: An Integrated Approach to Understanding the Function of the Potent Hepatotoxin Microcystin in the Growth & Ecology of Microcystis

合作研究：了解强效肝毒素微囊藻毒素在生长中功能的综合方法

• 批准号: 1451528
• 负责人: Steven Wilhelm
• 金额: $ 71.94万
• 依托单位: University of Tennessee Knoxville
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1451528&HistoricalAwards=false
• 关键词: Collaborative; Research; Integrated; Approach; Understanding

Blooms of toxic photosynthetic bacteria (cyanobacteria) are occurring globally with expanding frequency, duration and intensity in lakes, reservoirs and river systems. Most recently blooms of the toxic cyanobacterium Microcystis shut down the water supply of the city of Toledo, OH for a weekend in August of 2014. While the scientific community has developed a solid understanding of the factors that contribute to the blooms of Microcystis, previous research has not explained why cells make the hepato- (liver) toxin microcystin. As a potent inhibitor of a key class of enzymes - protein phosphatases - microcystin might play important roles inside Microcystis cells, and once released, inside the cells of other (target) organisms. This project will use advanced tools in molecular biology (RNA sequencing), microbial genetics, the quantification of small metabolites (metabolomics) and enzyme analyses to understand how the presence of microcystin shapes the activity of both the cells that make the compound and the community of microorganisms around them. Experiments in the laboratory will be complemented by field surveys of bloom events across naturally occurring toxin gradients - areas of historically high and low concentrations of toxin during the summer bloom season. State-of-the-art statistical analyses combined with these advanced scientific approaches will transform the understanding of why these cyanobacteria make this toxic compound. Understanding of the biological functions of the microcystin, will lead to better stewardship of a valuable natural resource: potable water. The total research effort will train students, including those from underrepresented groups, and broadly disseminate information to the public, systems managers and the scientific community. A significant component will feed into state-associated, in-class 4H training that will expose as many as 200,000 students to cyanobacteria as a model system to examine complex biochemical questions.The goal of this project is to develop a deeper understanding of the biochemical role of microcystins, a potent protein phosphatase inhibitor, within cells and communities, and address both ecological and evolutionary questions concerning the maintenance of this and other expensive biosynthetic pathways for non-ribosomally encoded secondary metabolites within a (sub)population of cells. To determine how microcystin shapes cellular biochemistry and physiology, controlled lab experiments with Microcystis isolates that make microcystin, engineered strains where the biosynthetic gene has been knocked out, and wild-type Microcystis cells that lack the biosynthetic pathway will be conducted. Other cyanobacterial pairs (Planktothrix and Anabaena spp.) that make or do not make the toxin, engineered bacteria that produce this compound and a set of microorganisms isolated from Lake Erie that co-occur with Microcystis and may be influenced by toxin will also be tested. Experiments in the presence and absence of exogenous toxin will be conducted with both producers and non-toxin producers. State-of-the-art techniques in metabolic (LC-MS and LC-MS/MS metabolomics and lipidomics), transcriptional (Illumina mRNA-sequencing), enzymatic (4:3:3-regulated processes) and physiological analyses (e.g., cellular growth rates, primary production, and photosynthetic efficiency) for these defined lab strains will be employed to develop "fingerprints" of cellular function and elucidate how microcystin shapes these biochemical pathways and the physiological ecology of these cells. Lab experiments will be complemented by field surveys of bloom events across naturally occurring and well documented toxin gradients. Relationships will be identified using univariate and multivariate techniques. This novel integration of sequencing, small molecule chemistry, physiological and enzymatic approaches will permit the mapping of the physiological biochemistry of cells and identify both isolated as well as synergistic effects: indeed this work may transform the study of secondary metabolites in complex microbial systems and provide insights into microbial evolutionary ecology.

有毒光合细菌（蓝细菌）的花朵在全球发生，湖泊，水库和河流系统的频率，持续时间和强度不断扩大。最近，有毒的蓝细胞小杆菌的大花朵关闭了2014年8月的俄亥俄州托莱多市的供水。虽然科学界已经对有助于微囊炎的繁殖的因素有着深入的了解，但先前的研究并未阐明细胞为何使Hepato-（liver- liver-（liver-liver）toxin toxin MicrocyStins促进细胞。作为关键类酶（蛋白质磷酸酶）的有效抑制剂 - 微囊藻蛋白可能在微囊细胞内起重要作用，并且一旦释放，在其他（靶）生物体的细胞内释放。该项目将在分子生物学（RNA测序），微生物遗传学，小型代谢产物（代谢组学）和酶分析中使用先进的工具，以了解微囊蛋白的存在如何塑造它们使周围微生物的化合物和细胞的活性。实验室中的实验将通过自然发生的毒素梯度的Bloom事件进行现场调查，这是夏季盛开季节在历史上高和低浓度毒素的区域。最先进的统计分析与这些先进的科学方法相结合，将改变人们对这些蓝细菌为何使这种有毒化合物的理解。了解微囊蛋白的生物学功能将导致更好地管理宝贵的自然资源：饮用水。总的研究工作将培训学生，包括来自代表性不足的群体的学生，并向公众，系统经理和科学界广泛传播信息。 A significant component will feed into state-associated, in-class 4H training that will expose as many as 200,000 students to cyanobacteria as a model system to examine complex biochemical questions.The goal of this project is to develop a deeper understanding of the biochemical role of microcystins, a potent protein phosphatase inhibitor, within cells and communities, and address both ecological and evolutionary questions concerning the maintenance of this and other expensive （亚）细胞群体中非核糖体编码的二级代谢产物的生物合成途径。为了确定微型细胞素如何形成细胞生物化学和生理学，用微囊菌分离株进行了受控的实验室实验，这些实验是使生物合成基因被敲除出的微囊蛋白，工程性菌株，并且野生型微囊细胞缺乏生物合成途径。其他蓝细菌对（Planktothrix和Anabaena spp。）产生或不产生产生这种化合物的毒素，工程细菌，并从伊利湖分离出的一组微生物，这些微生物与微囊藻共同发生，并且可能受到毒素的影响。在存在和不存在外源性毒素的情况下，将与生产者和非毒素生产者进行实验。代谢（LC-MS和LC-MS/MS代谢组学和脂科学）的最先进技术，转录（Illumina mRNA-sequesting），酶促（4：3：3：3：3：3：3受调节的过程）和生理分析（例如，细胞生长率，一级生产和照相效率）的特定效率（例如，生产率），以创建这些特定效果，并创建了对这些定义的效果，并效应了范围的效果。细胞功能并阐明微囊藻毒素如何塑造这些生化途径和这些细胞的生理生态学。实验实验将与天然发生且有充分记录的毒素梯度的Bloom事件进行现场调查相辅相成。将使用单变量和多元技术来确定关系。测序，小分子化学，生理和酶促方法的这种新型整合将允许映射细胞的生理生物化学，并确定孤立的和协同作用：确实，这项工作可能会改变复杂微生物系统中次生代谢物的研究，并将洞察力用于微生物进化生态学。