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

原绿球藻对全球碳循环和海洋食物网非常重要，因为原绿球藻在寡营养海洋中占主导地位，并在某些地区对海洋初级生产的贡献估计为50%或更多。原绿球藻在一个称为碳小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，但只有少数非光合模式生物的蛋白质组成是已知的。解决羧基结构之间的关系的结构研究，这似乎是不同的高和低光适应原绿球藻菌株，它的作用，提高催化效率的二氧化碳固定酶（RubisCO），它封装，最近才开始。我们将重点介绍两个原绿球藻模式菌株，MIT9313和MED4，这两个菌株代表了具有不同生理特性和生态分布的重要生态型。我们的初步数据表明，MIT9313和MED4在其羧基体中进化出关键的结构和组成差异，预计这些差异将影响羧基体功能，从而影响这些菌株的碳固定能力。为了实现在这些原绿球藻菌株的碳代谢中的羧基体所发挥的作用的综合理解，我们结合生物化学，生物物理学和遗传学的方法来分析羧基体组件之间的相互作用，并检查单个蛋白质和整个羧基体的结构和功能之间的关系。这包括纯化的carboxysomes从两个原绿球藻菌株，其特定的蛋白质组合物的表征，并使用重组蛋白来确定结构和测试功能的个别carboxysomes基因产物。此外，这些数据将被放置在生理和生态背景下，通过结合体内基因和蛋白质表达研究在不同的环境条件下，宏基因组调查的内容和表达的基因参与碳固定在公海。我们的研究将推进我们对原绿球藻在海洋中浓缩和固定二氧化碳的机制的基本理解。此外，这项研究将有助于我们的知识的贡献carboxysomes优化固碳自养细菌在水柱中。这项跨学科研究计划将受惠于首席研究员在微生物生态学和生理学、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碳氧体结构在优化开放海洋中二氧化碳固定中的作用。这可能导致优化或设计其他专门的细菌细胞器，以提高二氧化碳固定。本科生将参与分析海洋调查的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建立联系的机会。

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.