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

尽管稀有且大部分不溶性，但微量金属是复杂酶（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（低于1000米）的生物地球化学循环。例如，金属酶几乎参与了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，但暗海洋中痕量金属分布与生物氮循环过程之间的关系很少被探索，这可能是由于与他们的研究相关的技术挑战。自主水下航行器（AUV） Clio的可用性克服了这一挑战，该采样平台能够通过大体积过滤微生物颗粒材料来收集高分辨率垂直剖面样本，用于生化和微生物测量。因此，该研究项目计划进行跨学科的化学、生物学和工程学的努力，以验证以下假设：某些化学反应，如亚硝酸盐氧化，可能会受到中上层金属可用性的限制，而亚硝酸盐氧化细菌对微量金属的需求可能会在低氧条件下增加。本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Clio生物地球化学AUV作为一种社区资源。此外，元蛋白质组学数据将被存入最近启动的海洋蛋白质门户网站，以便海洋学家和生物界的金属学家检查蛋白质和金属酶在海洋中的分布。所有三所院校的本科生都将得到该项目的支持，并努力招收少数民族学生。拟议的研究还将与早期社区建设工作的目标协同作用，以潜在的全球规模的微生物生物地球化学项目为模型，模仿GEOTRACES项目的成功，暂时称为“生物地理景观：变化中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随环境中氧和微量金属的梯度呈空间动态变化；(2)热带太平洋东部中远洋微生物蛋白质亚硝酸盐氧化还原酶无法完成生物合成，从而限制了亚硝酸盐氧化；(3)亚硝酸盐氧化细菌在低氧条件下增加了对金属酶的需求。影响溶解性和颗粒性金属在氧最小带内的分布。描述中远洋生物地球化学特征的挑战之一是无法对其进行有效取样。作为采样平台，我们将使用新型生物地球化学AUV Clio，通过在东热带太平洋的研究考察中对微生物颗粒材料进行大体积过滤，实现高分辨率垂直剖面样品的生化和微生物测量。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Clio收集的剖面样品的元蛋白质组学结果来探索。假设2将通过利用clio收集的孵育样品的15NO2-氧化率进行孵育实验来验证。假设3将通过基于qPCR（定量聚合酶链反应）的亚硝酸盐氧化细菌丰度（NOB）来划分靶向亚硝酸盐氧化还原酶蛋白拷贝数，以确定细胞拷贝数是否随氧气分布而变化，并通过对NOB培养物的金属蛋白质组学分析来验证。再矿化过程中微量金属的限制，而不仅仅是初级生产的证明，将改变我们对金属在生物地球化学循环中的作用的理解，并为解释海洋中溶解和颗粒微量金属分布的剖面数据提供新的方法。氧气可能在控制微量金属方面发挥着以前未被重视的作用，这不仅是因为金属的物理化学性质，还因为生物需求的变化，这一想法将提高我们在面对海洋氧气含量下降时预测微量金属分布的能力。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。

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.