Determining the Role of csoS2 in Carboxysome Assembly and Regulation

PROJECT SUMMARY/ABSTRACT Bacteria Microcompartments (BMCs) are pseudo-organelles comprised of a highly organized, semi-permeable protein shell which self-assembles around components of specific metabolic pathways. BMCs offer bacteria an advantage to survive in harsh conditions, to organize the cytosol by separating reactive components from each other, or to increase efficiency of enzymatic pathways by tuning local concentrations. Genetic models suggest that the compact organization of the BMC loci is ideally suited for lateral transfer to other bacteria, increasing the potential for greater prevalence of this feature in the future. Understanding the mechanisms of BMC assembly, organization, and stability are important to developing new approaches involving human-bacteria interactions and symbiosis. The model BMC is a key component in the CO2 concentrating mechanism (CCM) found in cyanobacteria called the carboxysome (CB). CBs sequester Rubisco with carbonic anhydrase and CO2 increasing carbon fixing efficiency. Rubisco is the most abundant enzyme on earth and the protein responsible for carbon fixation in plants, bacteria, and algae. Rubisco has low selectivity for its substrate CO2 and has a slow enzymatic rate, resulting in an inefficient metabolic pathway. By sequestering Rubisco in CBs along with additional co-factors, cyanobacteria and many chemoautotrophs have enhanced carbon fixation. We propose studying shell assembly and cargo organization in vivo and in vitro using cryogenic electron tomography (cryoET) and live cell imaging using total internal reflection fluorescence microscopy (TIRFM) to understand internal and external factors that affect BMC stability, permeability, and function. Understanding how this model BMC organizes during assembly and throughout its life cycle will be critical for developing potential bioreactors with custom cargo for pharmaceutical applications or increasing carbon fixation in photosynthetic organizations for improved crop yields and decreasing atmospheric carbon.