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

蓝藻原绿球藻对全球碳循环和海洋食物网非常重要，因为原绿球藻在低营养海洋中占主导地位，在某些地区对海洋初级生产的贡献估计达到50%或更多。原绿球藻在称为羧基体的特殊细胞器中进行二氧化碳固定的第一步。羧基小体是一种完全由蛋白质组成的自组装代谢模块。虽然电子显微镜已经很好地记录了羧体的多面体形状，但它们的蛋白质组成仅在少数非光合模式生物中已知。最近才开始进行结构研究，以解决羧基体结构之间的关系，这种结构在高光和弱光适应原绿球藻菌株中似乎有所不同，以及它在提高其封装的二氧化碳固定酶（RubisCO）的催化效率方面的作用。我们将重点研究两个原绿球藻模型菌株MIT9313和MED4，这两个菌株代表了具有不同生理特征和生态分布的重要生态型。我们的初步数据表明，MIT9313和MED4在其羧基体上进化出了关键的结构和组成差异，这些差异预计会影响这些菌株的羧基体功能，从而影响这些菌株的固碳能力。为了全面了解这些原绿球藻菌株中羧基体在碳代谢中的作用，我们结合生物化学、生物物理和遗传学的方法分析了羧基体组分之间的相互作用，并研究了单个蛋白质和整个羧基体的结构和功能之间的关系。这包括从两种原绿球藻菌株中纯化羧基体，表征其特定蛋白质组成，以及使用重组蛋白确定单个羧基体基因产物的结构和测试功能。此外，这些数据将通过不同环境条件下的体内基因和蛋白质表达研究以及开放海洋中参与碳固定的基因含量和表达的宏基因组研究相结合，置于生理和生态背景下。更广泛的影响我们的研究将促进我们对原绿球藻在海洋中集中和固定二氧化碳的机制的基本理解。此外，该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。这个多学科研究项目将受益于pi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羧基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，以加强二氧化碳固定。本科生将参与分析来自海洋调查的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民族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.