EAGER: Tracking marine diazotrophy with isotope-labeling proteomics

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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.