EAGER: High-throughput, culture-independent technique identifying cyanobacteria infections to improve understanding of carbon biogeochemical cycling

EAGER：识别蓝藻感染的高通量、独立于培养的技术，以提高对碳生物地球化学循环的理解

• 批准号: 1820652
• 负责人: Sarah Preheim
• 金额: $ 29.75万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1820652&HistoricalAwards=false
• 关键词: EAGER; throughput; culture; independent; technique

Viruses in the ocean are 10 times more abundant than bacteria and kill 10-66% of bacterial cells daily. Viral infections of bacteria, such as of photosynthetic cyanobacteria that form the base of the food web, impact the flow of energy and carbon within the marine ecosystem. Thus, viral infections dramatically alter important biogeochemical and ecological factors in the ocean, such as how much carbon dioxide is respired or how many fish the ocean ecosystem can sustain. Despite their importance in the ocean ecosystem, researchers do not know the answer to the most basic question of viral biology for most environmental viruses: which bacteria do different viruses infect? Identifying these infections could help researchers understand more about how viruses shape the ecosystem through infections of keystone microbial species, infections of microbes with unique characteristics, or infection patterns that promote microbial community stability. This project is to develop a cost-effective method to substantially increase the number of infections identified within natural microbial communities. The researchers are applying this novel method to determine viral infections in cyanobacteria in the Chesapeake Bay and compare the results to standard approaches to determine viral infections. This technique can be widely used to transform our understanding of how viruses impact many ecosystems, since it is cost-effective, does not need specialized equipment and can be adapted to target different viral populations. Additionally, this project provides research opportunities for undergraduate and high school students, including underrepresented minority students and women. To develop a high-throughput, culture-independent technique to identify infections in the environment, the researchers are optimizing a previously developed method, emulsion paired concatenation-isolation PCR (epicPCR). Adapting epicPCR for viral-host associations will identify interactions by isolating actively infected single cells within a microdroplet to retain the physical proximity of the host and viral DNA during DNA extraction. Next, fusion PCR is done within the microdroplet to allow host rRNA genes to fuse to viral marker genes, such as g20 or ribonucleotide reductase, retained within the same bead. Only rRNA genes successfully fused to viral markers are amplified. Finally, high-throughput sequencing is done on the resulting fusion products. This approach is cultivation-independent, screens a larger fraction of diversity within the sample than traditional approaches, requires little additional equipment compared to microfluidic approaches, and can be scaled up to hundreds of samples because the amount of sequencing required to deeply sample a single environment is low compared to shotgun metagenomic sequencing. Although this technique will be limited to viral marker genes and suffers from the biases of PCR, it still offers great potential to investigate viral-host interactions across a large number of environments. The method is being applied to determine how viral infections influence cynaobacterial blooms in the Chesapeake Bay. The researchers will also compare the results of epicPCR to culture-based, single-cell, and bioinformatics based methods of host-virus associations to identify biases, limitations and caveats of various approaches.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

海洋中的病毒比细菌多10倍，每天杀死10-66%的细菌细胞。细菌的病毒感染，如构成食物网基础的光合蓝藻，影响海洋生态系统内能量和碳的流动。因此，病毒感染极大地改变了海洋中重要的生物地球化学和生态因素，例如呼吸多少二氧化碳或海洋生态系统可以维持多少鱼类。尽管它们在海洋生态系统中很重要，但研究人员不知道大多数环境病毒最基本的病毒生物学问题的答案：不同的病毒感染哪些细菌？识别这些感染可以帮助研究人员更多地了解病毒如何通过感染关键微生物物种、感染具有独特特征的微生物或感染模式来塑造生态系统，从而促进微生物群落的稳定性。该项目旨在开发一种具有成本效益的方法，以大幅增加在天然微生物群落中确定的感染数量。研究人员正在应用这种新方法来确定切萨皮克湾蓝藻中的病毒感染情况，并将结果与确定病毒感染的标准方法进行比较。这项技术可以广泛用于改变我们对病毒如何影响许多生态系统的理解，因为它具有成本效益，不需要专门的设备，并且可以针对不同的病毒种群进行调整。此外，该项目为本科生和高中生提供研究机会，包括代表性不足的少数民族学生和妇女。为了开发一种高通量、不依赖培养的技术来识别环境中的感染，研究人员正在优化先前开发的一种方法——乳状液配对串联分离PCR （epicPCR）。将外链pcr应用于病毒-宿主关联将通过在微滴中分离活跃感染的单细胞来识别相互作用，从而在DNA提取过程中保持宿主和病毒DNA的物理接近。接下来，在微滴内进行融合PCR，使宿主rRNA基因与病毒标记基因融合，如20或核糖核苷酸还原酶，保留在同一个头内。只有成功融合到病毒标记物的rRNA基因才会被扩增。最后，对得到的融合产物进行高通量测序。该方法与培养无关，与传统方法相比，可以在样品中筛选更大比例的多样性，与微流体方法相比，需要的额外设备很少，并且可以扩展到数百个样品，因为与霰弹枪宏基因组测序相比，对单个环境进行深度采样所需的测序量较低。虽然这项技术将仅限于病毒标记基因，并受到PCR的偏见，但它仍然为研究病毒与宿主在大量环境中的相互作用提供了巨大的潜力。该方法被应用于确定病毒感染如何影响切萨皮克湾的绿杆菌繁殖。研究人员还将把epicPCR的结果与基于培养、单细胞和基于生物信息学的宿主-病毒关联方法进行比较，以确定各种方法的偏差、局限性和警告。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。