Collaborative Research: Underexplored connections between nitrogen and trace metal cycling in oxygen minimum zones mediated by metalloenzyme inventories

虽然微量金属稀少且基本上不溶，但它们是复杂酶（加速生化反应的蛋白质分子）的关键成分，这些酶参与了黑暗海洋（1000米以下）的地球化学循环。 例如，金属酶几乎参与氮循环中的每一个反应。然而，尽管痕量金属和氮循环之间存在直接联系，但在黑暗海洋中痕量金属分布和生物氮循环过程之间的关系很少被探索，可能是由于与其研究相关的技术挑战。 自主式水下航行器克利奥是一个采样平台，能够收集高分辨率垂直剖面样本，通过大量过滤微生物颗粒材料进行生物化学和微生物测量，该平台的可用性克服了这一挑战。 因此，该研究项目计划跨学科的化学，生物学和工程学的努力，以测试的假设，某些化学反应，如亚硝酸盐氧化，可能会受到上层中层金属的可用性和微量金属的亚硝酸盐氧化细菌的需求可能会增加低氧条件下。 这项研究的更广泛影响包括通过开发和测试其高分辨率和自适应采样能力，继续开发和应用克利奥生物地球化学自动潜航器作为社区资源。此外，元蛋白质组数据将存入最近推出的海洋蛋白质门户网站，让海洋学家和生物界的金属研究海洋中的蛋白质和金属酶的分布。该项目将为所有三所机构的本科生提供支持，并努力招收少数族裔学生。拟议的研究还将与早期社区建设努力的目标协同作用，以GEOTRACES计划的成功为蓝本，建立一个潜在的全球规模微生物生物地球化学计划，暂时称为“生物地球景观：不断变化的星球上的海洋代谢和营养循环”。拟议的研究项目将测试以下三个假设：（1）中远洋微生物金属酶的分布在空间上是动态的，以响应氧和微量金属的环境梯度，（二）热带太平洋东部的亚硝酸盐氧化可能受到上层中层中的铁可利用性的限制，这是由于不能完成微生物蛋白质亚硝酸盐氧化还原酶的生物合成，和（3）亚硝酸盐氧化细菌在低氧下增加它们的金属酶需求，影响溶解和颗粒金属在氧最小区域内的分布。描述中层海洋生物地球化学特征的挑战之一是无法对其进行有效采样。作为采样平台，我们将使用新型的生物地球化学AUV克利奥，它可以通过对热带东太平洋研究考察中的微生物颗粒物质进行大体积过滤，获得高分辨率的垂直剖面样品，用于生物化学和微生物测量。将安排具体的研究活动来检验这些假设。 假设1将通过比较水文、微生物分布、溶解和颗粒金属数据以及与克利奥收集的剖面样本的元蛋白质组学结果来探索。假设2将通过孵育实验进行检验，使用Clio收集的孵育样品的15 NO2-氧化率。假设3将通过将靶向亚硝酸盐氧化还原酶蛋白拷贝除以基于qPCR（定量聚合酶链反应）的亚硝酸盐氧化细菌丰度（NOB）以确定细胞拷贝数是否随氧分布而变化，并通过NOB培养物的金属蛋白质组学分析进行检验。证明微量金属限制的矿化过程，而不仅仅是初级生产，将改变我们的理解的作用，金属在海洋地球化学循环，并提供新的方法来解释截面数据的溶解和颗粒微量金属分布在海洋中。氧在控制痕量金属方面可能发挥了以前未被充分认识的作用，这不仅是由于金属的物理化学性质，而且还由于生物需求的变化，该奖项反映了NSF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进行评估，被认为值得支持的搜索.

Though scarce and largely insoluble, trace metals are key components of sophisticated enzymes (protein molecules that speed up biochemical reactions) involved in biogeochemical cycles in the dark ocean (below 1000m). For example, metalloenzymes are involved in nearly every reaction in the nitrogen cycle. Yet, despite direct connections between trace metal and nitrogen cycles, the relationship between trace metal distributions and biological nitrogen cycling processes in the dark ocean have rarely been explored, likely due to the technical challenges associated with their study. Availability of the autonomous underwater vehicle (AUV) Clio, a sampling platform capable of collecting high-resolution vertical profile samples for biochemical and microbial measurements by large volume filtration of microbial particulate material, has overcome this challenge. Thus, this research project plans an interdisciplinary chemistry, biology, and engineering effort to test the hypothesis that certain chemical reactions, such as nitrite oxidation, could become limited by metal availability within the upper mesopelagic and that trace metal demands for nitrite-oxidizing bacteria may be increased under low oxygen conditions. Broader impacts of this study include the continued development and application of the Clio Biogeochemical AUV as a community resource by developing and testing its high-resolution and adaptive sampling capabilities. In addition, metaproteomic data will be deposited into the recently launched Ocean Protein Portal to allow oceanographers and the metals in biology community to examine the distribution of proteins and metalloenzymes in the ocean. Undergraduate students will be supported by this project at all three institutions, with an effort to recruit minority students. The proposed research will also be synergistic with the goals of early community-building efforts for a potential global scale microbial biogeochemistry program modeled after the success of the GEOTRACES program, provisionally called "Biogeoscapes: Ocean metabolism and nutrient cycles on a changing planet".The proposed research project will test the following three hypotheses: (1) the microbial metalloenzyme distribution of the mesopelagic is spatially dynamic in response to environmental gradients in oxygen and trace metals, (2) nitrite oxidation in the Eastern Tropical Pacific Ocean can be limited by iron availability in the upper mesopelagic through an inability to complete biosynthesis of the microbial protein nitrite oxidoreductase, and (3) nitrite-oxidizing bacteria increase their metalloenzyme requirements at low oxygen, impacting the distribution of both dissolved and particulate metals within oxygen minimum zones. One of the challenges to characterizing the biogeochemistry of the mesopelagic ocean is an inability to effectively sample it. As a sampling platform, we will use the novel biogeochemical AUV Clio that enables high-resolution vertical profile samples for biochemical and microbial measurements by large volume filtration of microbial particulate material on a research expedition in the Eastern Tropical Pacific Ocean. Specific research activities will be orchestrated to test the hypotheses. Hypothesis 1 will be explored by comparison of hydrographic, microbial distributions, dissolved and particulate metal data, and metaproteomic results with profile samples collected by Clio. Hypothesis 2 will be tested by incubation experiments using 15NO2- oxidation rates on Clio-collected incubation samples. Hypothesis 3 will be tested by dividing targeted nitrite oxidoreductase protein copies by qPCR (quantitative polymerase chain reaction)-based nitrite oxidizing bacteria abundance (NOB) to determine if cellular copy number varies with oxygen distributions, and by metalloproteomic analyses of NOB cultures. The demonstration of trace metal limitation of remineralization processes, not just primary production, would transform our understanding of the role of metals in biogeochemical cycling and provide new ways with which to interpret sectional data of dissolved and particulate trace metal distributions in the ocean. The idea that oxygen may play a previously underappreciated role in controlling trace metals due not just to metals' physical chemistry, but also from changing biological demand, will improve our ability to predict trace metal distributions in the face of decreasing ocean oxygen content.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.