Collaborative Research: Resolving the production and fate of nitrogenous metabolites in the surface ocean

光合微生物为海洋中几乎所有其他生命提供食物。它们的新陈代谢会产生被称为代谢物的有机分子，这些分子可以从细胞中泄漏出来，被故意排泄到海水中，或者在细胞死亡时被释放出来。一旦离开细胞，这些代谢物就成为微生物之间特定相互作用的基础，并决定了群落结构和活性。然而，由于历史上缺乏测量海洋代谢物的能力，目前对海洋代谢物的了解受到限制。这里提出的工作将扩大现有的知识代谢物的结构，浓度和生产速率使用最近开发的分析方法。这些方法已经导致人们发现，1933年首次在龙虾中发现的取代吡啶，是北太平洋微生物群落中可检测到的最丰富的代谢物。虽然苦参碱被认为是一种捕食者威慑剂、渗透保护剂、甲基供体和抗菌剂，但对其在微生物群落动态中的作用的研究尚缺乏。这项工作将阐明海碱在海洋微生物群落中的作用。这项工作将创建一个开源的代谢物数据库，服务于更广泛的代谢物组学领域，这是一个在环境、工程和医学科学中不断发展的领域。这项合作还将促进一名研究生和一名博士后研究员的职业生涯，以及一名来自主要本科院校（PUI）代表性不足群体的早期职业教授。两所大学的本科生将参与项目的开发和实施、在R.V. Carson上的当地巡游、实验室工作和结果的传播。这项研究将被整合到普吉特海湾大学（PUI, University of Puget Sound） 200级遗传学课程的本科生课程研究经验中。拟议的工作将进行实地研究和实验室实验，以验证代谢物是在微生物群落中流动的碳和氮的定量重要形式的假设。代谢产物的特性、数量和生产速率也将被确定。对于天甘素，将确定负责其转化的酶和生物体。具体的拟议活动将：1)量化浮游植物培养物和海洋地表水群落中的氮代谢物池及其净产量（颗粒和溶解）；2)分离消耗苦参碱的异养菌，利用诱变技术、转录组学和稳定同位素辅助代谢组学对苦参碱降解相关的基因进行注释和生化反应表征；3)在浮游植物培养物、对苦参碱敏感的异养细菌培养物和自然群落中进行苦参碱稳定同位素标记的培养，定量评价苦参碱对生长的影响，跟踪苦参碱的代谢途径，确定苦参碱的摄取动力学；4)通过挖掘现有的苦参碱分解代谢基因的环境亚转录组，确定苦参碱的消费者和在环境中使用苦参碱的生化途径。这些方法的结合将更好地了解含氮代谢物在海洋微生物生态系统中的流动。这项工作的结果将通过同行评审的开源出版物以及向科学界和公众发表的报告进行传播。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。

Photosynthetic microbes provide food for nearly all other life in the ocean. Their metabolism produces organic molecules called metabolites that can leak out of cells, be intentionally excreted into seawater, or be released during cell death. Once outside the cell, these metabolites are the basis for specific interactions among microbes and determine community structure and activity. Yet, current understanding of metabolites in the ocean is limited by a historical lack of ability to measure them. The work proposed here will expand current knowledge of metabolite structures, concentrations, and production rates using recently developed analytical methods. These methods have already led to the discovery that homarine, a substituted pyridine first found in lobster in 1933, is the most abundant detectable metabolite in microbial communities of the North Pacific Ocean. While homarine is known as a predator deterrent, osmoprotectant, methyl donor, and antibiofouling agent, studies of its role in microbial community dynamics are lacking. The work proposed will clarify the role of homarine in the ocean’s microbial communities. This work will create an open-source metabolite database that will serve the broader field of metabolomics, a growing area in environmental, engineering, and medical sciences. This collaboration will also promote the careers of a graduate student and a postdoctoral researcher as well as an early career professor from an underrepresented group at a primarily undergraduate institution (PUI). Undergraduates from both institutions will contribute to project development and implementation, local cruises on the R.V. Carson, lab work, and dissemination of results. This research will be integrated into a curriculum-based research experience for undergraduates in a 200-level genetics course at the PUI, University of Puget Sound. The proposed work will carry out field studies and laboratory experiments to test the hypothesis that metabolites are quantitatively significant forms of carbon and nitrogen flowing through microbial communities. The identity, quantity, and production rates of metabolites will also be determined. For homarine, the enzymes and organisms responsible for its transformations will be determined. Specific proposed activities will 1) Quantify nitrogenous metabolite pools and their net production rates (particulate and dissolved) in phytoplankton cultures and in marine surface water communities; 2) Isolate homarine consuming heterotrophic bacteria and use mutagenesis techniques, transcriptomics, and stable isotope assisted metabolomics to annotate genes and characterize the biochemical reactions involved in the degradation of homarine; 3) Carry out incubations of stable isotope labeled homarine in phytoplankton cultures, heterotrophic bacterial cultures sensitive to homarine, and natural communities to quantitatively evaluate the effect of homarine on growth, track homarine through metabolic pathways, and determine the kinetics of homarine uptake; 4) Identify homarine consumers and biochemical pathways for homarine use in the environment by mining existing environmental metatranscriptomes for homarine catabolism genes. The combination of these approaches will provide better understanding of the flow of nitrogen containing metabolites through marine microbial ecosystems. Results from this work will be disseminated through peer reviewed open-source publications as well as presentations to the scientific community and the general public.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.