MIP: Denitrification: Not Just for Prokaryotes Anymore?

反硝化过程发生在缺氧（即缺氧）或微氧环境中，定义为硝酸盐或亚硝酸盐转化为气态产物，如氮和/或氧化亚氮。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。对细菌和古细菌以及两种真核真菌的反硝化作用进行了遗传和生物化学研究。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反硝化。这是一个重要的发现，虽然有孔虫反硝化的机制尚未确定，但这表明在真核生物领域中反硝化的能力得到了扩展。该项目的主要目标是研究某些有孔虫的代谢能力和微生物关联，这些有孔虫允许这些有孔虫物种进行反硝化。将分析三种有孔虫：一种是已知的吸收硝酸盐还原叶绿体的；一种新的（即未描述的）但已知与其细胞质液泡相关的细菌内共生体；不具有任何内共生体的生物。该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，这些硝酸盐可以被内生生物或有孔虫宿主呼吸；(2)新型有孔虫体内的内生共生体有助于反硝化。以下具体目标将被解决：(1)利用序列分析和荧光原位杂交（FISH）鉴定与新型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反硝化相关基因产物；(3)热氯化钒法测定细胞内硝酸盐积累；(4)利用高压冷冻、冷冻取代和相关的高压电子显微镜（HVEM）和次级离子质谱（SIMS）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。结果将有助于更好地了解微生物对底栖缺氧和微氧海洋生态系统的影响，以及全球营养循环。该项目还将增加对原生生物-原核生物共生功能的了解，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。这个项目有不同的教育组成部分，从中学到本科学习，并为中学教师和普通公众提供推广机会。

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.