Features and implications of nitrogen assimilation trait variability in populations of Prochlorococcus

海洋细菌原绿球藻是亚热带开阔海洋食物网的中心部分，是地球上最大的生物群落之一。像陆地上的植物一样，原绿球藻和其他浮游植物能够进行光合作用，利用光将二氧化碳转化为糖和其他有机物。这种物质是海里所有生物的食物。与陆生植物类似，原绿球藻需要额外的营养或“肥料”来生长和光合作用。在这些营养物质中，在大部分阳光普照的海洋中，氮通常是稀缺的。因此，了解原绿球藻获取和利用氮的方式对了解海洋中氮和碳循环是如何耦合的具有重要意义。并非所有原绿球藻细胞都具有利用所有无机氮来源的遗传能力，即硝酸盐、亚硝酸盐和铵。有些可以用全部三种，有些用最后两种，有些只用铵。可以利用硝酸盐的细胞必须依次将其转化为亚硝酸盐，然后再转化为铵，然后才能制造蛋白质的基本构件。该项目的起源是由于观察到一些原绿球藻细胞在硝酸盐生长过程中向海水中释放亚硝酸盐。该项目研究了这些细胞系的生理特征，并询问释放亚硝酸盐的细胞是否可以支持其他细胞的生长，而不是不能使用硝酸盐，实际上创造了一个交叉喂养的情况，可以使系统更健壮。了解不同方式获取氮（海洋中的关键货币）的细胞共存背后的驱动因素，将为海洋生态系统中氮的流动提供重要的见解。该项目还揭示了微生物之间相互作用的结构，并为更广泛的科学界（例如，研究与人类健康和疾病或农业相关的各种微生物组的科学界）提供了一个关于微生物如何形成有益伙伴关系的新视角。该项目为本科生提供沉浸式实验室研究体验，他们设计和执行与项目总体目标直接相关的实验。该项目进一步支持研究人员与公众就与浮游植物、光合作用和这些海洋生物提供的生态系统服务有关的主题进行接触。在本项目关注的原绿球藻（Prochlorococcus）适应弱光的LLI分支中，几乎所有细胞都具有硝酸盐同化途径的下游一半（用于亚硝酸盐的同化）。然而，只有一小部分LLI细胞具有完整的硝酸盐同化途径。在硝酸盐作为唯一氮源的生长过程中，LLI细胞观察到硝酸盐的不完全同化还原，并伴有亚硝酸盐的释放。此外，生长在硝酸盐上的细胞释放的亚硝酸盐可以支持原绿球藻的生长，而原绿球藻只能使用亚硝酸盐而不能使用氧化程度更高的硝酸盐。总体而言，在一组密切相关的原绿球藻中，存在与亚硝酸盐的产生和消耗相关的基因型和表型多样性，亚硝酸盐是氮循环的中心中间体。研究人员建议进一步发展原绿球藻作为一个模型系统，以探索亚硝酸盐在群体中的循环，并为性状变异和互补功能的选择如何促进微生物群体的稳健性和/或弹性提供新的见解。总体假设是，不同功能类型的原绿球藻在种群水平上的聚集是通过相互作用出现的，这种相互作用部分是由亚硝酸盐的交叉摄食介导的。为了解决这个广泛的假设，研究人员将重点放在以下目标上：1)通过亚硝酸盐还原酶的体外生化表征和细胞在光和温度胁迫下的转录谱分析，评估不完全同化硝酸盐还原和亚硝酸盐释放的生理基础；2)检测原绿球藻菌株在不同环境梯度（如光、温度和温度）下的亚硝酸盐产生和消耗速率；3)在不同的环境条件和扰动下，确定实验室和野外种群中氮同化基因型的频率和活性。目标1和2的结果有助于限制与不完全硝酸盐还原和亚硝酸盐释放（氮限制细胞的宝贵商品）相关的权衡，以促进氮同化基因型之间的伙伴关系如何构建的建模和解释。这些见解有助于指导目标3中的实验，其目的是检查受控的实验室共培养和现场种群，以便产生关于原绿球藻种群的涌现特征的定量数据，其中相互作用是由亚硝酸盐交叉喂养介导的。这些数据被用来更好地理解由共同公共利益介导的相互作用如何产生种群的涌现特性，包括对扰动的恢复能力和更高的种群范围内的氮同化效率。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。

The marine bacterium, Prochlorococcus, is a central part of the food web in the subtropical open ocean, one of the largest biomes on the planet. Like plants on land, Prochlorococcus and other phytoplankton are capable of photosynthesis, harnessing light to convert carbon dioxide into sugars and other organic matter. This matter feeds all the life in the sea. Akin to terrestrial plants, Prochlorococcus requires additional nutrients or “fertilizer” to grow and photosynthesize. Among these nutrients, nitrogen is often scarce across much of the sunlit ocean. Thus, understanding the means through which nitrogen is obtained and used by Prochlorococcus has important consequences for understanding how the nitrogen and carbon cycles are coupled in the ocean. Not all Prochlorococcus cells have the genetic capacity to use all sources of inorganic nitrogen, i.e. nitrate, nitrite, and ammonium. Some can use all three, some the last two, and some only ammonium. Cells that can use nitrate must sequentially transform it to nitrite and then to ammonium before they can make the building blocks for proteins. The genesis of this project derives from the observation that some Prochlorococcus cells release nitrite into the seawater during growth on nitrate. This project examines this feature of the physiology of these cell lines, and asks whether cells that release nitrite can support the growth of other cells than cannot use nitrate, in effect creating a cross-feeding situation that could make the system more robust. Understanding the drivers behind the coexistence of cells with different ways of obtaining nitrogen, a key currency in the ocean, will provide important insights on the flow of nitrogen in marine ecosystems. This project also sheds light on the structure of interactions between microbes and provide the broader scientific community (for instance, those studying diverse microbiomes related to human health and disease or agriculture) a new perspective on how microbes form beneficial partnerships. This project supports immersive laboratory-based research experiences for undergraduate students, who design and execute experiments directly related to the overall project goals. The project further supports the work of the investigators to engage with the general public on topics related to phytoplankton, photosynthesis, and the ecosystem services provided by these marine organisms.In the low-light adapted LLI clade of Prochlorococcus, the focus of this project, nearly all cells possess the downstream half of the nitrate assimilation pathway (for the assimilation of nitrite). Only a fraction of LLI cells, however, have the complete nitrate assimilation pathway. Incomplete assimilatory nitrate reduction, with concomitant nitrite release, has been observed for LLI cells during growth on nitrate as the sole nitrogen source. Further, the nitrite released by cells growing on nitrate can support the growth of Prochlorococcus that can use nitrite but not the more oxidized nitrate. Overall, within a group of closely-related Prochlorococcus, there is genotypic and phenotypic diversity related to the production and consumption of nitrite, a central intermediate in the nitrogen cycle. The investigators propose to further develop Prochlorococcus as a model system to explore nitrite cycling within populations and provide new insights on how trait variability and the selection of complementary functions facilitates robustness and/or resiliency in microbial populations. The overarching hypothesis is that the population level assembly of distinct functional types of Prochlorococcus emerges through interactions that are mediated, in part, by cross-feeding of nitrite. To address this broad hypothesis, the investigators are focusing on the following objectives: 1) assessing the physiological underpinnings of incomplete assimilatory nitrate reduction and nitrite release through in-vitro biochemical characterization of nitrite reductase enzymes and transcription profiling of cells subjected to light and temperature stress, 2) examining the nitrite production and consumption rates of Prochlorococcus strains across environmental gradients such as light, temperature, and nutrient availability in order to constrain the environmental parameters that modulate nitrite cycling, and 3) determining the frequencies and activities of nitrogen assimilation genotypes within laboratory and field populations, under varying environmental conditions and perturbations. Outcomes from objectives 1 and 2 help to constrain the tradeoffs associated with incomplete nitrate reduction and the release of nitrite (a valuable commodity to nitrogen limited cells) to facilitate modelling and interpretation of how partnerships between nitrogen assimilation genotypes are structured. These insights help to direct experiments in Objective 3, which aims to examine controlled laboratory co-cultures and field populations in order to produce quantitative data on the emergent features of Prochlorococcus populations where interactions are mediated by the cross-feeding of nitrite. These data are being used to develop an improved understanding of how interactions mediated by a common public good might give rise to emergent properties of populations, including resilience to perturbation and greater population-wide efficiency in nitrogen assimilation.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.