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

光合作用微生物为海洋中几乎所有其他生命提供食物。它们的新陈代谢产生被称为代谢物的有机分子，可以从细胞中泄漏出来，被故意排泄到海水中，或者在细胞死亡期间被释放。一旦在细胞外，这些代谢物是微生物之间特定相互作用的基础，并决定群落结构和活动。然而，目前对海洋中代谢物的了解受到历史上缺乏测量它们的能力的限制。这里提出的工作将使用最近开发的分析方法来扩展当前关于代谢物结构、浓度和产生率的知识。这些方法已经发现，1933年首次在龙虾中发现的取代吡啶--高丙氨酸--是北太平洋微生物群落中最丰富的可检测代谢物。虽然Homarine被认为是捕食者的威慑剂、渗透保护剂、甲基供体和抗生物污染剂，但对其在微生物群落动态中的作用缺乏研究。这项拟议的工作将阐明高盐碱在海洋微生物群落中的作用。这项工作将创建一个开放源码的代谢物数据库，为更广泛的代谢组学领域服务，代谢组学是环境、工程和医学中一个日益增长的领域。这项合作还将促进研究生和博士后研究员的职业生涯，以及来自以本科生为主的机构(PUI)中代表不足的群体的早期职业教授。来自这两个学院的本科生将为项目的开发和实施、在R.V.Carson号上的当地巡航、实验室工作和成果传播做出贡献。这项研究将被整合到普吉特海湾大学200级遗传学课程本科生的基于课程的研究体验中。这项拟议的工作将进行实地研究和实验室实验，以验证代谢物是流经微生物群落的碳和氮的数量显著形式这一假设。代谢物的特性、数量和生产率也将被确定。对于Homarine，将确定负责其转化的酶和有机体。具体的拟议活动将1)量化浮游植物培养物和海洋表层水群落中的氮代谢物库及其净生产量(颗粒和溶解的)；2)分离高盐碱消耗异养菌，并使用突变技术、转录组分和稳定同位素辅助代谢组学来注释基因和表征与高盐碱降解有关的生化反应；3)在浮游植物培养物、对高盐碱敏感的异养细菌和自然群落中进行稳定同位素标记的高盐碱的孵育，以定量评估高盐碱对生长的影响，通过代谢途径追踪高盐碱，并确定高苯丙氨酸的吸收动力学；4)通过挖掘现有环境中的高血糖分解代谢基因，确定高血糖消费者和在环境中使用高血糖的生化途径。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议的开源出版物以及向科学界和公众发表的演示文稿来传播。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.