Collaborative Research: Characterizing the Constraints on Virus Infection of Cyanobacteria

“这项奖励是根据2009年美国复苏和再投资法案（公法111-5）资助的。”病毒在调节海洋生态系统的结构和功能方面发挥着重要作用。然而，我们缺乏对细菌细胞受体如何抑制病毒感染的机制理解，这对海洋微生物食物网的生态学和进化动力学具有重要意义。本项目将在海洋蓝细菌聚藻球菌（Synechococcus sp. WH7803）的外膜上鉴定三种已知的噬藻体（podo-， myo-和siphoviridae）的受体。研究人员将采用一系列经过验证的方法，使用趋化剂产生耐病毒的聚球菌群体，使用荧光病毒标签监测表面附着来缩小突变体，并使用LC-MS/MS蛋白质组学分析来表征耐药群体的外膜蛋白补充。一旦受体被确定，现有数据库的生物信息学分析将与样本档案的分子生物学查询相结合，以记录海洋蓝藻基因组中不同受体的多样性和流行程度，以及自然种群中表达的受体。实验室对趋化因子的研究将检查受体表达的调控（使用荧光病毒和qRT-PCR进行基因表达），以便开始梳理蓝藻培养群体中病毒受体表达的环境控制。所有信息将以一种适合生态竞争模型分析的方式收集，从而提供关于蓝藻面临的感染抗性成本的新见解，以及关于细胞病毒感染如何受到环境条件限制的新信息。更广泛的影响：该项目的更广泛的影响包括在EPSCoR研究所（田纳西大学）和一个研究站（凯洛格生物站）培训新一代海洋科学家，他们从事微生物学、分子生物学和蛋白质组学、海洋地球化学和古典生态学等跨学科科学。该研究将为学生提供与来自主要联邦研究机构（橡树岭国家实验室）的研究人员一起工作的机会，在那里可以使用大多数学术机构无法使用的设备。这项研究的信息将直接纳入课程作业、教育和公众宣传中，并通过与田纳西大学科学新闻项目的新型合作伙伴关系进行传播。将招募一名或多名对科学新闻感兴趣的本科生，以便为K-12学生和公众提供有关海洋微生物学和生态学的纸质和电子传播材料。pi将继续其悠久的历史，即招募代表性不足的群体并参与以方法为导向的课程。在科学上，本研究产生的信息将是全新的，扩展和改变了海洋微生物学和病毒生态学的基础，并为全球气候和海洋生物地球化学的变化如何影响海洋病毒对微生物的增殖和控制提供了新的见解。

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)." It is well established that viruses play an important role in regulating the structure and function of marine ecosystems. However, we lack a mechanistic understanding of how bacteria cell receptors constrain virus infection, which has implications for the ecology and evolutionary dynamics of marine microbial food webs. This project will identify receptors for three known groups of cyanophage (podo-, myo- and siphoviridae) on the on the outer membrane of the marine cyanobacterium Synechococcus sp. WH7803. The researchers will employ a series of proven methods to generate virus-resistant Synechococcus populations using chemostats, to narrow the mutants using fluorescent virus tags that monitor surface attachment, and to characterize the outer membrane protein compliment of the resistant populations using LC-MS/MS proteomic analyses. Once the receptors are identified, bioinformatic analyses of existing databases will be coupled with molecular biological queries of sample archives to document the diversity and prevalence of the different receptors in marine cyanobacterial genomes as well as of those expressed by natural populations. Lab studies in chemostats swill examine the regulation of receptor expression (using fluorescence viruses and qRT-PCR for gene expression) so as to begin to tease apart the environmental controls on virus receptor expression in culture populations of cyanobacteria. All information will be collected in a manner that will make it suitable for analyses by ecological competition models, thereby providing new insights on the cost-of-resistance to infection that cyanobacteria face as well as new information on how virus infection of cells may be constrained by environmental conditions.Broader Impacts: The broader impacts of this project include the training of a new generation of marine scientists in interdisciplinary sciences of microbiology, molecular biology and proteomics, marine geochemistry and classical ecology of students at an EPSCoR Institution (The University of Tennessee) and a research station (Kellogg Biological Station). The study will provide opportunities for students to work with researchers from a major federal research facility (Oak Ridge National Laboratory) where access to equipment exists that is not available at most academic institutions. Information from this study will be directly incorporated into course work, educational and public outreach, and disseminated through a novel partnership with the Science Journalism program at the University of Tennessee. One or more undergraduates interested in science journalism will be recruited in order to generate materials for paper and electronic dissemination on marine microbiology and ecology that will be appropriate for K-12 students as well as the general public. The PIs will continue their well-established history of recruitment of underrepresented groups and participation in methods-oriented courses. Scientifically the information generated by this study will be completely novel, extending and transforming the foundations of marine microbiology and virus ecology as well as providing insight into how shifts in the global climate and ocean biogeochemistry might influence microbial proliferation and control by marine viruses.