EAGER: Tracking marine diazotrophy with isotope-labeling proteomics

在全球大部分海洋中，固氮作用——将大气中丰富的氮气转化为生命可用的化学形式——是生物活动的重要营养来源。特别是，生命需要氮来制造蛋白质，而蛋白质是为所有细胞提供能量的基本生化机器。海洋中的固氮是由各种微生物进行的，海洋学家正在努力揭示其惊人的多样性和生态学。该项目使用一种新的质谱技术直接跟踪氮气通过固氮进入蛋白质的氮流。这些测量揭示了哪些固氮生物在海洋的特定部分是活跃的，它们用它们固定的氮制造了什么蛋白质，以及这些氮最终是如何流向整个海洋生物群落的。这些信息将更好地为海洋微生物生态系统及其基本生物地球化学如何响应海洋变化条件的模型和预测提供信息。该项目聘请了一名本科生研究员，从科学领域代表性不足的群体中招募，从事实地和实验室工作。重氮营养化（生物固氮）是海洋中最大的固定氮输入，由多种多样的海洋微生物进行，但我们对海洋重氮营养化的全部多样性的固氮活性进行量化的能力有限，也无法确定它们所固定的氮最终如何流向更广泛的微生物群落。由于固定氮的主要生物合成目的是生产蛋白质，蛋白质组学将为我们理解海洋氮循环，特别是重氮营养体的分子生理和生态作用做出重大贡献。本项目提出了一种新颖的15n跟踪蛋白质组学方法，以解决海洋重氮营养盐生态学中的三个突出问题：1)整个群落的N2固定活性如何在不同重氮营养盐分类群中分配？重氮营养体及其共生伙伴如何利用它们固定的氮进行生物合成？3)有多少重氮营养来源的氮被重新分配到特定的非重氮营养类群？研究人员正在夏威夷海洋时间序列（HOT）站ALOHA进行15n跟踪蛋白质组学实验，分析从15n标记的二氮到重氮营养体和更广泛的微生物群落蛋白质中的氮结合情况。这是第一次使用蛋白质组学来跟踪重氮营养15N2的掺入，代表了一种新的，分子水平的方法来研究海洋固氮，包括整个微生物群落的高分类分辨率。15n跟踪蛋白质组学数据通过细胞蛋白质组学中的关键生物学账户，以及海洋重氮营养体的生态生理和生物地球化学作用，为氮货币的流动提供了独特的见解。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。

Throughout much of the global ocean, nitrogen fixation – the conversion of abundant atmospheric nitrogen gas into chemical forms usable by life – is a crucial source of nutrients for biological activity. In particular, life needs nitrogen to make proteins, the essential biochemical machines that power all cells. Nitrogen fixation in the oceans is carried out by a variety of microorganisms, whose surprising diversity and ecology oceanographers are working to uncover. This project uses a novel mass spectrometry technique to directly track the flow of nitrogen from nitrogen gas, via nitrogen fixation, into proteins. These measurements reveal which nitrogen-fixing organisms are active in a given part of the ocean, what proteins they make with the nitrogen they fix, and how that nitrogen ultimately flows to feed the entire marine biological community. This information will better inform models and predictions of how marine microbial ecosystems, and the essential biogeochemistry they perform, responds to changing conditions in the ocean. This project engages an undergraduate student researcher, recruited from groups underrepresented in science, in both the field and laboratory work.Diazotrophy (biological N2 fixation) is the largest input of fixed nitrogen to the ocean and is carried out by a wide diversity of marine microbes, but we have limited ability to quantify the N2-fixation activity of the full diversity of marine diazotrophs, or to resolve how the N they fix ultimately flows to supply the broader microbial community. Since the primary biosynthetic fate of fixed N2 is protein production, proteomics is poised to make substantial contributions to our understanding of the marine nitrogen cycle, and to the molecular physiology and ecological roles of diazotrophs in particular. This project brings a novel 15N-tracking proteomics methodology to bear on three outstanding questions in marine diazotroph ecology:1) How is whole-community N2 fixation activity apportioned among different diazotroph taxa?2) How do diazotrophs and their symbiotic partners make biosynthetic use of the N they fix?3) How much diazotroph-derived N is redistributed to particular non-diazotroph taxa?The investigators are conducting 15N2-tracking proteomics experiments at the Hawaii Ocean Time-series (HOT) Station ALOHA, assaying N incorporation from 15N-labeled dinitrogen into proteins of both diazotrophs and the broader microbial community. This is the first use of proteomics to track diazotrophic 15N2 incorporation, representing a novel, molecular-level approach for investigating marine nitrogen fixation that encompasses the entire microbial community with high taxonomic resolution. 15N-tracking proteomics data provides unique insight into the flow of nitrogen currency through its key biological accounts in cellular proteomes, and into the ecophysiology and biogeochemical roles of marine diazotrophs.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.