MIP: Denitrification: Not Just for Prokaryotes Anymore?

脱氮过程，发生在缺氧（即，缺氧）或微氧环境中，被定义为硝酸盐或亚硝酸盐转化为气态产物如氮气和/或一氧化二氮。这一过程被认为是从海洋中去除生物可利用氮的主要机制，因此在长期尺度上对海洋的“肥力”起着重要作用。反硝化作用的遗传学和生物化学已经在细菌和真菌以及两种真核真菌中进行了研究。现在，根据最近的一项研究，某些底栖有孔虫原生生物能够完全脱氮。这是一个重要的发现，这表明在真核生物领域的反硝化能力扩大，虽然有孔虫反硝化的机制尚未确定。这个项目的主要目标是研究某些有孔虫的代谢能力和微生物协会，使这些有孔虫物种能够繁殖。将分析三种有孔虫物种：一种是已知的螯合硝酸盐还原叶绿体;一种是新的（即，未描述），但已知具有与其细胞质空泡相关的细菌内共生体;不具有任何内共生体的细菌。本项目将测试以下假设：1）有孔虫细胞质中的液泡储存硝酸盐，其可以由内共生体或有孔虫宿主呼吸，以及（2）反硝化作用由新型有孔虫内共生体辅助。本研究的主要目的是：（1）利用序列分析和荧光原位杂交（FISH）技术鉴定与新有孔虫相关的微生物内共生体，（2）利用mRNA-FISH技术鉴定和定位有孔虫中与反硝化作用相关的基因产物，（3）利用热氯化钒法研究细胞内硝酸盐的积累，（4）利用荧光原位杂交（FISH）技术研究与新有孔虫相关的微生物内共生体。（4）利用高压冷冻、冷冻置换、高压电子显微镜（HVEM）和二次离子质谱（西姆斯）测定有孔虫液泡的元素组成（C、N、S）;（5）有孔虫反硝化速率的测定，这将有助于更好地了解微生物对底栖缺氧和微生物的影响。有毒海洋生态系统以及全球养分循环。该项目还将增加对原生生物-原核生物共生体功能的理解，并可能有助于评估它们对真核生物生态和进化的潜在影响。该项目有各种教育组成部分，从中学到大学，并为中学教师和普通公众提供外联机会。

The process of denitrification, which occurs in anoxic (i.e., lacking oxygen) or micro-oxic environments, is defined as the conversion of nitrate or nitrite to gaseous products such as nitrogen and/or nitrous oxide. This process is believed to be the primary mechanism for removing bioavailable nitrogen from the sea and thus plays an important role in the "fertility" of the ocean on long time scales. The genetics and biochemistry of denitrification has been studied in Bacteria and Archaea, as well as two species of Eukaryotic fungi. Now, according to a recent study, certain benthic foraminiferan protists are capable of complete denitrification. This is an important finding, which suggests an expanded capability for denitrification in the Eukaryotic domain, although the mechanisms of foraminiferal denitrification have not yet been identified. The broad objective of this project is to examine the metabolic capabilities and microbial associations in certain foraminifera that allow these foraminiferal species to denitrify. Three foraminiferal species will be analyzed: one that is known to sequester nitrate-reducing chloroplasts; one that is novel (i.e., undescribed) but is known to have bacterial endsymbionts associated with its cytoplasmic vacuoles; one that does not possess any endosymbionts. This project will test the following hypotheses: 1) vacuoles in the foraminiferal cytoplasm store nitrate, which can be respired by the endobionts or the foraminiferal host, and (2) denitrification is aided by endosymbionts within the novel foraminifer. The following specific aims will be addressed: (1) identification of the microbial endosymbionts associated with the novel foraminifer using sequence analyses and fluorescent in situ hybridization (FISH); (2) identification and localization of gene products associated with denitrification in foraminifera using mRNA-FISH; (3) survey for intracellular nitrate accumulation using hot vanadium chloride assays; (4) determination of the elemental composition (C, N, S) of foraminiferal vacuoles using High Pressure Freezing, Freeze Substitution, and correlative High Voltage Electron Microscopy (HVEM) and Secondary Ion Mass Spectrometry (SIMS); (5) measurement of rates of foraminiferal denitrification.Results will enable better understanding of microbial affects on benthic anoxic and micro-oxic oceanic ecosystems, as well as global nutrient cycling. The project will also increase understanding of the functioning of protist-prokaryote symbioses and may help to assess their potential impact on eukaryotic ecology and evolution. This project has varied educational components, ranging from middle school to undergraduate studies, and outreach opportunities for secondary school teachers and the lay-public.