Collaborative Research: Prochlorococcus and its contribution to new production in the Sargasso Sea

海洋原绿球藻（Prochlorococcus marinus）在亚热带和热带海洋中普遍存在，在某些地区可贡献高达82%的初级生产力。与大多数其他浮游植物相比，培养的原绿球藻分离物不能吸收NO3-。然而，Lomas团队已经使用流式细胞术和稳定同位素示踪剂证明了马尾藻海原绿球藻对NO3-的直接同化。为了支持这些发现，Martiny和他的同事已经证明，生活在混合层中的原绿球藻细胞携带NO2-和NO3-同化基因，这些基因在野外种群中是功能性的和表达的。综合结果表明，未培养的原绿球藻谱系能够同化NO3-，并有助于在许多海洋地区产生新的产物，但总体意义尚不清楚。该项目的首要假设是，细胞特异性NO3-同化速率是环境营养浓度和细胞代谢潜力（即编码NO2-和NO3-同化的基因的存在）的函数。本项目的具体研究问题是：1)NO3-是否是原绿球藻在数量上的重要营养来源，是否对原绿球藻的新产量有贡献？2)氮底物（NH4+、尿素、NO2-和no3 -）的季节和垂直变化对原绿球藻基因组含量和氧化氮吸收速率有何影响？为了回答这些问题，pi将结合使用高灵敏度的营养测量，Lomas开发的流式细胞分析方法来量化特定分类群中的氮同化，以及宏基因组学和qPCR方法来确定与原绿球藻相关的亚硝酸盐（nirA）和硝酸盐还原酶（narB）基因的发生。利用这些工具，他们将量化原绿球藻在三个完整的季节周期内的NO3-同化和NO3-同化基因的分布。此外，这些直接测量将通过操纵中观实验（互惠移植和营养添加实验）来增强，以明确测试其假设的各个方面。PIs希望实现对NO3-同化速率的直接（氮物种浓度的变化）和间接控制（原绿球藻的基因组适应）的机制理解。该项目最令人兴奋的成果之一将是更全面地了解原绿球藻在野外组合中的营养生态学。pi选择在马尾藻海进行这项研究，因为该地点提供了丰富的必要支持数据和后勤基础设施，而且他们已经证明原绿球藻能够在该地区同化硝酸盐。更广泛的影响：本项目将在许多层面上产生更广泛的影响。首先，原绿球藻在no2 -和NO3-同化作用下具有以前未被认识到的广泛生产力，这对理解原绿球藻的生物地理学及其在海洋碳氮循环中的作用具有重要意义。其次，它将举例说明基因组进化，细胞生理学和环境变量如何相互作用，以塑造细菌在海洋中的生物地球化学作用。pi将以一种易于纳入生物地球化学和生态系统模型的方式展示其结果，包括那些与响应气候驱动变率的区域和全球生物过程变化有关的模型。该项目将为研究生（UCI一个）和本科生提供研究机会。该项目还将建立一个与国际挂钩的教育推广方案，为加利福尼亚和百慕大的高中教师和学生提供海洋微生物生态学及其与全球海洋过程和生物地球化学关系的实践经验。

The cyanobacterium Prochlorococcus marinus is ubiquitous in the oligotrophic subtropical and tropical oceans and can contribute up to 82% of the primary productivity in certain regions. In contrast to most other phytoplankton, cultured Prochlorococcus isolates cannot assimilate NO3-. However, Lomas' group has used flow cytometry and stable isotope tracers to demonstrate direct NO3- assimilation by Prochlorococcus in the Sargasso Sea. In support of these findings, Martiny and colleagues have shown that Prochlorococcus cells residing in the mixed layer carry genes for NO2- and NO3- assimilation, and that these genes are functional and expressed in field populations. The combined results suggest that uncultured lineages of Prochlorococcus are capable of NO3- assimilation and can contribute to new production in many oceanic regions - but the overall significance is yet unknown. The overarching hypothesis of this project is that cell-specific NO3- assimilation rate is a function of both the ambient nutrient concentrations and the metabolic potential of the cell (i.e. presence of genes encoding for NO2- and NO3- assimilation). The specific research questions of this project are: 1) Is NO3- a quantitatively important nutrient source for Prochlorococcus and does Prochlorococcus contribute to new production? 2) What is the influence of seasonal and vertical variation in nitrogen substrates (NH4+, urea, NO2-, andNO3-) on the genome content of Prochlorococcus and oxidized nitrogen uptake rates? To answer these questions, PIs will use the combination of high-sensitivity nutrient measurements, a flow cytometric assay developed by Lomas to quantify nitrogen assimilation in specific taxonomic groups, and metagenomics and a qPCR assay to determine the occurrence of nitrite (nirA) and nitrate reductase (narB) genes associated with Prochlorococcus. Using these tools, they will quantify NO3- assimilation and the distribution of NO3- assimilation genes in Prochlorococcus through three full seasonal cycles and over the entire euphotic zone. In addition, these direct measurements will be augmented by manipulative mesocosm experiments (reciprocal transplant and nutrient addition experiments) to explicitly test aspects of their hypotheses. The PIs hope to achieve a mechanistic understanding of direct (variations in the concentration of nitrogen species) and indirect controls (genomic adaptation in Prochlorococcus) on NO3- assimilation rates. One of the most exciting outcomes from this project will be a more complete understanding of the nutritional ecology of Prochlorococcus in field assemblages. The PIs have selected to conduct this study in the Sargasso Sea, because of the wealth of necessary supporting data and logistical infrastructure that this site provides, and because they have already shown that Prochlorococcus is capable of nitrate assimilation in this region. Broader Impacts: This project will have broader impacts on many levels. First, the potential for previously unrecognized widespread productivity by Prochlorococcus fueled byNO2- and NO3- assimilation has significant implications for the understanding of the biogeography of Prochlorococcus and its role in oceanic carbon and nitrogen cycles. Secondly, it will exemplify how genome evolution, cell physiology, and environmental variables interact to shape the biogeochemical role of bacteria in the ocean. The PIs will present their results in a manner that can be easily incorporated in biogeochemical and ecosystem models, including those related to changes in regional and global biological processes in response to climate-driven variability. This project will provide research opportunities for graduate (one at UCI) and undergraduate students. This project will also establish an internationally linked education outreach program to provide high school teachers and students in California and Bermuda with hands-on experience in marine microbial ecology and its relationship to global ocean processes and biogeochemistry.