Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species.
ABSTRACT: BACKGROUND: The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO2 fixation via the Calvin-Benson-Bassham cycle. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into alpha-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition. CONCLUSIONS/SIGNIFICANCE: These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.
Project description:Carboxysomes are polyhedral bodies consisting of a proteinaceous shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). They are found in the cytoplasm of all cyanobacteria and some chemoautotrophic bacteria. Previous studies of Halothiobacillus neapolitanus and Nitrobacter agilis carboxysomes suggest that the structures are either icosahedral or dodecahedral. To determine the protein shell structure more definitively, purified H. neapolitanus carboxysomes were re-examined by cryo-electron tomography and scanning transmission electron microscopy (STEM). Due to the limited tilt angles in the electron microscope, the tomographic reconstructions are distorted. Corrections were made in the 3D orientation searching and averaging of the computationally extracted carboxysomes to minimize the missing data effects. It was found that H. neapolitanus carboxysomes vary widely in size and mass as shown by cryo-electron tomography and STEM mass measurements, respectively. We have aligned and averaged carboxysomes in several size classes from the 3D tomographic reconstruction by methods that are not model-biased. The averages reveal icosahedral symmetry of the shell, but not of the density inside it, for all the size classes.
Project description:Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.
Project description:It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO2, but requires a CO2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.
Project description:Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The ?-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The ?-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.
Project description:The carboxysome is a bacterial organelle that functions to enhance the efficiency of CO2 fixation by encapsulating the enzymes ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. The outer shell of the carboxysome is reminiscent of a viral capsid, being constructed from many copies of a few small proteins. Here we describe the structure of the shell protein CsoS1A from the chemoautotrophic bacterium Halothiobacillus neapolitanus. The CsoS1A protein forms hexameric units that pack tightly together to form a molecular layer, which is perforated by narrow pores. Sulfate ions, soaked into crystals of CsoS1A, are observed in the pores of the molecular layer, supporting the idea that the pores could be the conduit for negatively charged metabolites such as bicarbonate, which must cross the shell. The problem of diffusion across a semiporous protein shell is discussed, with the conclusion that the shell is sufficiently porous to allow adequate transport of small molecules. The molecular layer formed by CsoS1A is similar to the recently observed layers formed by cyanobacterial carboxysome shell proteins. This similarity supports the argument that the layers observed represent the natural structure of the facets of the carboxysome shell. Insights into carboxysome function are provided by comparisons of the carboxysome shell to viral capsids, and a comparison of its pores to the pores of transmembrane protein channels.
Project description:The carboxysome is a versatile paradigm of prokaryotic organelles and is a proteinaceous self-assembling microcompartment that plays essential roles in carbon fixation in all cyanobacteria and some chemoautotrophs. The carboxysome encapsulates the central CO<sub>2</sub>-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), using a polyhedral protein shell that is selectively permeable to specific metabolites in favor of Rubisco carboxylation. There is tremendous interest in repurposing carboxysomes to boost carbon fixation in heterologous organisms. Here, we develop the design and engineering of α-carboxysomes by coexpressing the Rubisco activase components CbbQ and CbbO with α-carboxysomes in <i>Escherichia coli</i>. Our results show that CbbQ and CbbO could assemble into the reconstituted α-carboxysome as intrinsic components. Incorporation of both CbbQ and CbbO within the carboxysome promotes activation of Rubisco and enhances the CO<sub>2</sub>-fixation activities of recombinant carboxysomes. We also show that the structural composition of these carboxysomes could be modified in different expression systems, representing the plasticity of the carboxysome architecture. In translational terms, our study informs strategies for engineering and modulating carboxysomes in diverse biotechnological applications.
Project description:BACKGROUND: Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO(2) fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected. CONCLUSIONS/SIGNIFICANCE: In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO(2) and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome.
Project description:Carboxysomes are protein-based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is used to equally space out β-carboxysomes across cell lengths by a two-component system (McdAB) in the model cyanobacterium Synechococcus elongatus PCC 7942. More recently, we found that McdAB systems are widespread among β-cyanobacteria, which possess β-carboxysomes, but are absent in α-cyanobacteria, which possess structurally and phyletically distinct α-carboxysomes. Cyanobacterial α-carboxysomes are thought to have arisen in proteobacteria and then horizontally transferred into cyanobacteria, which suggests that α-carboxysomes in proteobacteria may also lack the McdAB system. Here, using the model chemoautotrophic proteobacterium Halothiobacillus neapolitanus, we show that a McdAB system distinct from that of β-cyanobacteria operates to position α-carboxysomes across cell lengths. We further show that this system is widespread among α-carboxysome-containing proteobacteria and that cyanobacteria likely inherited an α-carboxysome operon from a proteobacterium lacking the mcdAB locus. These results demonstrate that McdAB is a cross-phylum two-component system necessary for positioning both α- and β-carboxysomes. The findings have further implications for understanding the positioning of other protein-based bacterial organelles involved in diverse metabolic processes. PLAIN LANGUAGE SUMMARY: Cyanobacteria are well known to fix atmospheric CO<sub>2</sub> into sugars using the enzyme Rubisco. Less appreciated are the carbon-fixing abilities of proteobacteria with diverse metabolisms. Bacterial Rubisco is housed within organelles called carboxysomes that increase enzymatic efficiency. Here we show that proteobacterial carboxysomes are distributed in the cell by two proteins, McdA and McdB. McdA on the nucleoid interacts with McdB on carboxysomes to equidistantly space carboxysomes from one another, ensuring metabolic homeostasis and a proper inheritance of carboxysomes following cell division. This study illuminates how widespread carboxysome positioning systems are among diverse bacteria. Carboxysomes significantly contribute to global carbon fixation; therefore, understanding the spatial organization mechanism shared across the bacterial world is of great interest.
Project description:Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26?nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12?min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
Project description:Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional ?-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single ?-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact ?-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial ?-carboxysomes that likely serve as the ?-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional ?-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of ?-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of ?-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.