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

有毒光合作用细菌(蓝藻)的水华正在全球范围内发生，在湖泊、水库和河流系统中的频率、持续时间和强度都在不断扩大。最近一次爆发的有毒蓝藻微囊藻在2014年8月的一个周末切断了俄亥俄州托莱多市的供水。虽然科学界已经对导致微囊藻大量繁殖的因素有了坚实的理解，但之前的研究还没有解释为什么细胞会产生肝(肝)毒素微囊藻毒素。作为一类关键的酶-蛋白磷酸酶的有效抑制剂，微囊藻毒素可能在微囊藻细胞内发挥重要作用，一旦释放，可能会在其他(靶标)生物的细胞内发挥作用。该项目将使用分子生物学(RNA测序)、微生物遗传学、小分子代谢物的量化(代谢组学)和酶分析方面的先进工具，以了解微囊藻毒素的存在如何塑造形成化合物的细胞和周围微生物群落的活动。实验室中的实验将通过对自然产生的毒素梯度--夏季花季毒素浓度历史最高和最低的地区--的水华事件进行实地调查来补充。最先进的统计分析与这些先进的科学方法相结合，将改变对这些蓝藻为什么会产生这种有毒化合物的理解。了解微囊藻毒素的生物学功能，将有助于更好地管理一种宝贵的自然资源：饮用水。整个研究工作将培训学生，包括那些来自代表性不足的群体的学生，并向公众、系统管理人员和科学界广泛传播信息。一个重要的组成部分将进入国家相关的课堂4H培训，这将使多达200,000名学生接触蓝藻作为一个模型系统，以检查复杂的生化问题。该项目的目标是加深对微囊藻毒素的生化作用的理解，微囊藻毒素是一种有效的蛋白质磷酸酶抑制剂，在细胞和群落中，并解决与维持这一途径和其他昂贵的生物合成途径有关的生态学和进化学问题，在(亚)细胞种群中非核糖体编码的次生代谢物的生物合成途径。为了确定微囊藻毒素如何塑造细胞生化和生理学，将对制造微囊藻毒素的微囊藻分离株、生物合成基因已被敲除的工程菌株以及缺乏生物合成途径的野生型微囊藻细胞进行对照实验室实验。其他蓝藻对(Planktothrix和Anabaena spp.)制造或不制造毒素的细菌、产生这种化合物的工程菌，以及从伊利湖分离出来的一组微生物也将接受测试，这些微生物与微囊藻共存，可能受到毒素的影响。在存在和不存在外源毒素的情况下，将对生产者和非毒素生产者进行实验。在代谢(LC-MS和LC-MS/MS代谢组学和脂类组学)、转录(Illumina mRNA测序)、酶(4：3：3调节过程)和生理分析(例如细胞生长速度、初级生产和光合作用效率)方面的最先进技术将被用于开发细胞功能的“指纹”，并阐明微囊藻毒素如何塑造这些细胞的生化途径和生理生态。实验室实验将得到对自然发生的和有充分记录的毒素梯度上的水华事件的实地调查的补充。将使用单变量和多变量技术来确定关系。这种将测序、小分子化学、生理和酶方法相结合的新方法将允许绘制细胞的生理生化图，并识别单独和协同作用：事实上，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，并为微生物进化生态学提供见解。

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.