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.
Globally, the small photosynthetic marine bacteria (cyanobacteria) of the genus Prochlorococcus make significant contributions to the conversion of atmospheric carbon dioxide (CO2) to biomass. The process of CO2 fixation by Prochlorococcus and related organisms is greatly enhanced by their polyhedral protein bodies, called carboxysomes, which house the CO2 fixing enzyme within a thin protein shell. The objectives of this study were to determine protein composition and performance of Prochlorococcus carboxysomes. A large-scale culture method was established for two Prochlorococcus strains that provided the requisite amount of cellular material for the purification of carboxysomes. A purification procedure was developed that yielded the first pure carboxysome preparation from a cyanobacterium. An inventory of the Prochlorococcus marinus MED4 carboxysome proteins revealed the presence and structure of a novel shell protein that was subsequently shown to be present and fulfill an important function in carboxysomes of other bacteria as well. Importantly, this protein is not part of the gene cluster that encodes the major carboxysome proteins, prompting a search for additional, previously unknown carboxysome components. Given the large contribution of Prochlorococcus to primary production in the oceans, our study has broadened our understanding of the basic biochemical processes that govern the global carbon balance and the levels of atmospheric carbon dioxide. Through this project, a graduate student and a postdoc received training in carboxysome biochemistry. Six female undergraduate students were trained in microbiological and molecular biological techniques and experimental approaches. Two high school students (one of them African American) performed microbioloigcal research projects in the PIs’ labs.
