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大部分不溶，但它们是参与黑暗海洋(1000米以下)生物地球化学循环的复杂酶(加快生化反应的蛋白质分子)的关键成分。例如，金属酶几乎参与了氮循环中的每一种反应。然而，尽管痕量金属和氮循环之间有直接联系，但很少有人探索黑暗海洋中痕量金属分布和生物氮循环过程之间的关系，这可能是因为与它们的研究相关的技术挑战。自主水下航行器(AUV)CLIO是一个采样平台，能够通过大量过滤微生物颗粒材料收集用于生化和微生物测量的高分辨率垂直剖面样本，该平台的问世克服了这一挑战。因此，这项研究项目计划进行化学、生物学和工程学的交叉努力，以检验以下假设：某些化学反应，如亚硝酸盐氧化，可能会受到中上层海洋中金属有效性的限制，以及在低氧条件下，亚硝酸盐氧化细菌对痕量金属的需求可能会增加。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，继续开发和应用克里奥生物地球化学AUV作为社区资源。此外，元蛋白质组数据将被存入最近启动的海洋蛋白质门户网站，使海洋学家和生物界的金属能够检查海洋中蛋白质和金属酶的分布。这三所院校的本科生都将得到这一项目的支持，努力招收少数民族学生。这项拟议的研究还将与潜在的全球规模微生物生物地球化学计划的早期社区建设努力的目标相协调，该计划仿照GEOTRACES计划的成功，暂时被称为“生物地理景观：变化星球上的海洋新陈代谢和营养循环”。拟议的研究项目将检验以下三个假设：(1)中大洋微生物金属酶的分布是对氧和痕量金属环境梯度的响应；(2)东热带太平洋的亚硝酸盐氧化可能受到中上层海洋中铁的有效性的限制，因为无法完成微生物亚硝酸盐氧化还原酶的生物合成。(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，影响了溶解金属和颗粒金属在最低含氧量区域内的分布。描述中层海洋的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，我们将使用新型生物地球化学AUV CLIO，它能够通过对东热带太平洋的研究考察中的微生物颗粒材料进行大量过滤，获得高分辨率的垂直剖面样本，用于生化和微生物测量。具体的研究活动将被安排来检验这些假说。假说1将通过将水文、微生物分布、溶解和颗粒金属数据以及变蛋白组学结果与CLIO收集的剖面样本进行比较来进行探索。假设2将通过在收集的克里奥培养样本上使用15NO2-氧化率的培养实验来验证。假设3将通过将目标亚硝酸氧化还原酶蛋白拷贝与基于qPCR(定量聚合酶链式反应)的亚硝酸盐氧化细菌丰度(NOB)分开来检验，以确定细胞拷贝数是否随氧分布而变化，并通过NOB培养的金属蛋白质组分析来检验。对再矿化过程的痕量金属限制的论证，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痕量金属分布的剖面数据提供新的方法。由于金属的物理化学以及生物需求的变化，氧气在控制痕量金属方面可能扮演着以前被低估的角色，这一想法将提高我们在海洋氧含量下降的情况下预测痕量金属分布的能力。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.