Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography

Abstract

Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…The major challenges have been the poor specificity of immunoblots and mass spectrometry, given the homology of carboxysome proteins and the lack of effective purification of intact carboxysomes from host cells, as well as the heterogeneity of carboxysome structures (Long et al, 2005). The previous model of carboxysome protein stoichiometry was based on the total amount of proteins in cell lysates (Long et al, 2011) and does not directly reflect the stoichiometry of carboxysome proteins in the organelle, given the possible free-standing carboxysome components in the cytosol (Dai et al, 2018). We have recently reported the isolation of b-carboxysomes from Syn7942 and the structural and mechanical exploration of the organelles (Faulkner et al, 2017).…”

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…The major challenges have been the poor specificity of immunoblot and mass spectrometry, given the homology of carboxysome proteins and the lack of effective purification of intact carboxysomes from host cells, as well as the heterogeneity of carboxysome structures (Long et al, 2005). The previous model of carboxysome protein stoichiometry was based on the total amount of proteins in cell lysates (Long et al, 2011) and does not directly reflect the stoichiometry of carboxysome proteins in the organelle, given the possible free-standing carboxysome components in the cytosol (Dai et al, 2018). We have recently reported the isolation of β-carboxysomes from Syn7942 and the structural and mechanical exploration of the organelles (Faulkner et al, 2017).…”

A Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene

“…Cyanobacteria, the oldest and largest phylum of prokaryotes that perform the plant-like photosynthesis (Schirrmeister et al, 2015), are regarded as the ancestors of the plant chloroplast (Ponce-Toledo et al, 2017) and the purveyors of the oxygen that shaped our biosphere (Hamilton et al, 2016). Contemporary cyanobacteria still capture a vast quantity of solar energy to assimilate huge amounts of CO 2 (Dai et al, 2018) and nitrogen (nitrate, ammonium or urea) (Singh et al, 2016;Veaudor et al, 2019) to produce an enormous biomass that sustain most life forms on our planet. In colonizing most waters (fresh, brackish and marine) and soils (even deserts) biotopes, cyanobacteria have developed as widely diverse organisms.…”