Collaborative Research: Structural, Functional, and Ecological Characterization of the Prochlorococcus Carboxysome, the Ocean's Primary Molecular Module for Carbon Fixation

原氯球菌对全球碳循环和海洋食物网具有深远的重要性，因为原氯球菌在数量上主导着营养稀少的海洋，并在某些区域对海洋初级生产力的贡献估计为50%或更多。原氯球菌在一种叫做羧体的特殊细胞器中进行二氧化碳固定的第一步。羧基体是完全由蛋白质组成的自组装代谢模块。尽管电子显微镜已经很好地证明了羧基体的多面体形状，但只有少数非光合作用的模式生物知道它们的蛋白质组成。关于羧体结构与其在提高其包裹的二氧化碳固定酶(Rubisco)催化效率方面的作用之间的关系的结构研究直到最近才开始。我们将重点介绍两株原氯球菌模式菌株MIT9313和MED4，它们代表了重要的生态型，具有明显的生理特性和生态分布。我们的初步数据表明，MIT9313和MED4在其羧体中进化出了关键的结构和组成差异，这些差异有望影响这些菌株的羧体功能，从而影响其固碳能力。为了对这些原氯球菌菌株的碳代谢中所起的作用有一个全面的了解，我们结合生化、生物物理和遗传学的方法来分析羧体成分之间的相互作用，并研究单个蛋白质和整个碳体的结构和功能之间的关系。这包括从两个原氯球菌菌株中提纯羧体，鉴定其特定的蛋白质组成，以及使用重组蛋白来确定单个羧体基因产物的结构和测试其功能。此外，这些数据将通过在不同环境条件下的体内基因和蛋白质表达研究以及对参与公海固碳的基因的含量和表达的元基因组调查相结合的方式放在生理和生态环境中。更广泛的影响我们的研究将促进我们对原氯球菌浓缩和固定海洋中二氧化碳的机制的基本理解。此外，这项研究将有助于我们了解羧基体对水柱中自养细菌优化固碳的贡献。这一多学科研究项目将受益于私人投资机构在微生物生态和生理学、结构生物学、生物化学、分子生物学和生物信息学方面的互补专业知识。我们希望我们的研究能够揭示羧体结构在优化开放海洋二氧化碳固定中的作用的新见解。这可能导致优化或设计其他专门的细菌细胞器，以增强二氧化碳的固定。本科生将参与分析海洋调查中的DNA序列和表达数据。该项目将为三个合作机构的研究生和本科生，包括妇女和代表性不足的少数群体成员提供跨学科培训和联网机会。

The cyanobacterium Prochlorococcus is profoundly important to the global carbon cycle and the ocean's food web, since Prochlorococcus numerically dominates the oligotrophic oceans and contributes an estimated 50% or more to marine primary production in certain regions. Prochlorococcus carries out the first step of carbon dioxide fixation in a specialized organelle called the carboxysome. Carboxysomes are self-assembling metabolic modules, composed entirely of protein. Although the polyhedral shape of carboxysomes has been well documented by electron microscopy, their protein composition is known for only a few non-photosynthetic model organisms. Structural studies that address the relationship between carboxysome architecture, which appears to differ among high and low-light adapted Prochlorococcus strains, and its role in enhancing the catalytic efficiency of the carbon dioxide-fixing enzyme (RubisCO) that it encapsulates, have only recently been initiated. We will focus on two Prochlorococcus model strains, MIT9313 and MED4, which represent important ecotypes that have distinct physiological characteristics and ecological distributions. Our preliminary data suggest that MIT9313 and MED4 have evolved key structural and compositional differences in their carboxysomes, and these differences are expected to impact carboxysome function and thus, the carbon fixation capabilities of these strains. In order to achieve an integrative understanding of the role played by carboxysomes in carbon metabolism in these Prochlorococcus strains, we are combining biochemical, biophysical and genetic approaches to analyze interactions between carboxysome components and to examine the relationship between structure and function of individual proteins and of the entire carboxysome. This includes purification of carboxysomes from both Prochlorococcus strains, characterization of their specific protein composition, and the use of recombinant proteins to determine the structures and test the functions of individual carboxysome gene products. Moreover, these data will be placed in physiological and ecological contexts via a combination of in vivo gene and protein expression studies under different environmental conditions and metagenomic surveying of content and expression of genes involved in carbon fixation in the open ocean. Broader Impacts Our research will advance our fundamental understanding of the mechanisms by which Prochlorococcus concentrates and fixes carbon dioxide in the oceans. Furthermore, this study will contribute to our knowledge of the contribution of carboxysomes to optimized carbon fixation by autotrophic bacteria in the water column. This multi-disciplinary research project will benefit from the complementary expertise of the PIs in microbial ecology and physiology, structural biology, biochemistry, molecular biology and bioinformatics. We expect our study to reveal novel insights into the role of carboxysome architecture in optimizing carbon dioxide fixation in the open ocean. This could lead to optimization of or design of other specialized bacterial organelles to enhance carbon dioxide fixation. Undergraduate students will be involved in analyzing DNA sequence and expression data from an ocean survey. The project will provide interdisciplinary training and networking opportunities for graduate and undergraduate students including women and members of under-represented minorities at the three cooperating institutions.