The Experts below are selected from a list of 909 Experts worldwide ranked by ideXlab platform
Cheryl A Kerfeld - One of the best experts on this subject based on the ideXlab platform.
-
structure of a synthetic β Carboxysome shell
Plant Physiology, 2019Co-Authors: Markus Sutter, Thomas G Laughlin, Nancy B Sloan, Daniel Serwas, Karen M Davies, Cheryl A KerfeldAbstract:Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the Carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional Carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified β-Carboxysome shell. The structure reveals the sidedness and the specific interactions between the Carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the Carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the Carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.
-
β Carboxysome bioinformatics identification and evolution of new bacterial microcompartment protein gene classes and core locus constraints
Journal of Experimental Botany, 2017Co-Authors: Manuel Sommer, Matthew R Melnicki, Cheryl A KerfeldAbstract:: Carboxysomes are bacterial microcompartments (BMCs) that enhance CO2 fixation in all cyanobacteria. Structurally, Carboxysome shell proteins are classified according to the type of oligomer formed: hexameric (BMC-H), trimeric (BMC-T) and pentameric (BMC-P) proteins. To understand the forces driving the evolution of the Carboxysome shell, we conducted a bioinformatic study of genes encoding β-Carboxysome shell proteins, taking advantage of the recent large increase in sequenced cyanobacterial genomes. In addition to the four well-established BMC-H (CcmK1-4) classes, our analysis reveals two new CcmK classes, which we name CcmK5 and CcmK6. CcmK5 is phylogenetically closest to CcmK3 and CcmK4, and the ccmK5 gene is found only in genomes lacking ccmK3 and ccmk4 genes. ccmK6 is found predominantly in heterocyst-forming cyanobacteria. The gene encoding the BMC-T homolog CcmO is associated with the main Carboxysome locus (MCL) in only 60% of all species. We find five evolutionary origins of separation of ccmO from the MCL. Transcriptome analysis demonstrates that satellite ccmO genes, in contrast to MCL-associated ccmO genes, are never co-regulated with other MCL genes. The dispersal of Carboxysome shell genes across the genome allows for distinct regulation of their expression, perhaps in response to changes in environmental conditions.
-
production and characterization of synthetic Carboxysome shells with incorporated luminal proteins
Plant Physiology, 2016Co-Authors: Fei Cai, Susan L Bernstein, Steven C Wilson, Cheryl A KerfeldAbstract:Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial Carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The Carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering Carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces Carboxysome shells. Protein domains native to the Carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing Carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic Carboxysomes for plant engineering.
-
Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ
Scientific Reports, 2015Co-Authors: Markus Sutter, Evan W. Roberts, Raul C. Gonzalez, Cassandra Bates, Salma Dawoud, Kimberly Landry, Gordon C Cannon, Sabine Heinhorst, Cheryl A KerfeldAbstract:Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO_2 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 α-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 α-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.
-
engineering bacterial microcompartment shells chimeric shell proteins and chimeric Carboxysome shells
ACS Synthetic Biology, 2015Co-Authors: Markus Sutter, James N Kinney, Susan L Bernstein, Cheryl A KerfeldAbstract:Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorganic carbon fixation (Carboxysomes) or organic carbon utilization (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. We undertook both approaches to altering shell properties using the Carboxysome as a model system. There are two types of Carboxysomes, α and β. In both, the predominant shell protein(s) contain a single copy of th...
Benedict M. Long - One of the best experts on this subject based on the ideXlab platform.
-
rubisco condensate formation by ccmm in beta Carboxysome biogenesis
Nature, 2019Co-Authors: H Wang, Benedict M. Long, X Yan, H Aigner, Andreas Bracher, N D Nguyen, W Y Hee, G D Price, F U Hartl, Manajit HayerhartlAbstract:Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency1. The α- and β-Carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-a complex of eight large (RbcL) and eight small (RbcS) subunits-and carbonic anhydrase2-4. As HCO3- can diffuse through the proteinaceous Carboxysome shell but CO2 cannot5, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco6. The shell also prevents access to reducing agents, generating an oxidizing environment7-9. The formation of β-Carboxysomes involves the aggregation of Rubisco by the protein CcmM10, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain11. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS2-4. Here we have reconstituted the Rubisco-CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for Carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes12,13. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of Carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC114. Understanding Carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants15-19.
-
rubisco condensate formation by ccmm in beta Carboxysome biogenesis
Nature, 2018Co-Authors: H Wang, Benedict M. Long, X Yan, H Aigner, Andreas Bracher, N D Nguyen, W Y Hee, F U Hartl, Graeme Price, Manajit HayerhartlAbstract:Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency1. The α- and β-Carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)—a complex of eight large (RbcL) and eight small (RbcS) subunits—and carbonic anhydrase2–4. As HCO3− can diffuse through the proteinaceous Carboxysome shell but CO2 cannot5, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco6. The shell also prevents access to reducing agents, generating an oxidizing environment7–9. The formation of β-Carboxysomes involves the aggregation of Rubisco by the protein CcmM10, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain11. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS2–4. Here we have reconstituted the Rubisco–CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for Carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid–liquid phase separation in eukaryotes12,13. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of Carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC114. Understanding Carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants15–19. The structure of a Rubisco–CcmM complex sheds light on the formation of Carboxysomes in cyanobacteria.
-
Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts.
Nature communications, 2018Co-Authors: Benedict M. Long, Wei Yih Hee, Robert E. Sharwood, Benjamin D. Rae, Sarah Kaines, Yi-leen Lim, Nghiem D. Nguyen, Baxter Massey, Soumi Bala, Susanne Von CaemmererAbstract:A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called Carboxysomes. To date, chloroplastic expression of Carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified Carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-Carboxysome structural proteins. This minimal gene set produces Carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-Carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-Carboxysomes in chloroplasts.
-
Carboxysome encapsulation of the co 2 fixing enzyme rubisco in tobacco chloroplasts
Nature Communications, 2018Co-Authors: Benedict M. Long, Wei Yih Hee, Robert E. Sharwood, Benjamin D. Rae, Sarah Kaines, Yi-leen Lim, Nghiem D. Nguyen, Baxter Massey, Soumi Bala, Susanne Von CaemmererAbstract:A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called Carboxysomes. To date, chloroplastic expression of Carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified Carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-Carboxysome structural proteins. This minimal gene set produces Carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-Carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-Carboxysomes in chloroplasts.
-
Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Nature Publishing Group, 2018Co-Authors: Benedict M. Long, Wei Yih Hee, Robert E. Sharwood, Benjamin D. Rae, Sarah Kaines, Yi-leen Lim, Nghiem D. Nguyen, Baxter Massey, Soumi Bala, Susanne Von CaemmererAbstract:Previous efforts to assemble Rubisco within a cyanobacterial Carboxysome-derived protein shell in plant chloroplasts to concentrate CO2 have been unsuccessful. Here, Long et al. produce Carboxysomes in tobacco chloroplasts that encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2
Markus Sutter - One of the best experts on this subject based on the ideXlab platform.
-
structure of a synthetic β Carboxysome shell
Plant Physiology, 2019Co-Authors: Markus Sutter, Thomas G Laughlin, Nancy B Sloan, Daniel Serwas, Karen M Davies, Cheryl A KerfeldAbstract:Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the Carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional Carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified β-Carboxysome shell. The structure reveals the sidedness and the specific interactions between the Carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the Carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the Carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.
-
heterohexamers formed by ccmk3 and ccmk4 increase the complexity of beta Carboxysome shells
Plant Physiology, 2019Co-Authors: Manuel Sommer, Markus Sutter, Sayan Gupta, Henning Kirst, Aiko Turmo, Sigal Lechnoyossef, Rodney L BurtonAbstract:Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The Carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of β-Carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other Carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in Carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that Carboxysome shell permeability may be dynamically attenuated by “capping” facet-embedded hexamers with a second hexamer. Because β-Carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.
-
Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ
Scientific Reports, 2015Co-Authors: Markus Sutter, Evan W. Roberts, Raul C. Gonzalez, Cassandra Bates, Salma Dawoud, Kimberly Landry, Gordon C Cannon, Sabine Heinhorst, Cheryl A KerfeldAbstract:Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO_2 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 α-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 α-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.
-
engineering bacterial microcompartment shells chimeric shell proteins and chimeric Carboxysome shells
ACS Synthetic Biology, 2015Co-Authors: Markus Sutter, James N Kinney, Susan L Bernstein, Cheryl A KerfeldAbstract:Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorganic carbon fixation (Carboxysomes) or organic carbon utilization (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. We undertook both approaches to altering shell properties using the Carboxysome as a model system. There are two types of Carboxysomes, α and β. In both, the predominant shell protein(s) contain a single copy of th...
-
Two new high-resolution crystal structures of Carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species
Photosynthesis Research, 2013Co-Authors: Markus Sutter, Steven C Wilson, Samuel Deutsch, Cheryl A KerfeldAbstract:Cyanobacteria have evolved a unique carbon fixation organelle known as the Carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO_2 concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins.
Gordon C Cannon - One of the best experts on this subject based on the ideXlab platform.
-
Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ
Scientific Reports, 2015Co-Authors: Markus Sutter, Evan W. Roberts, Raul C. Gonzalez, Cassandra Bates, Salma Dawoud, Kimberly Landry, Gordon C Cannon, Sabine Heinhorst, Cheryl A KerfeldAbstract:Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO_2 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 α-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 α-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.
-
advances in understanding Carboxysome assembly in prochlorococcus and synechococcus implicate csos2 as a critical component
Life, 2015Co-Authors: Fei Cai, Susan L Bernstein, Gordon C Cannon, Sabine Heinhorst, Cheryl A Kerfeld, Zhicheng Dou, Ryan L Leverenz, Eric B Williams, Jessup M ShivelyAbstract:The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the Carboxysome, a self-assembling proteinaceous organelle. Two types of Carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-Carboxysome, the assembly process of the α-Carboxysome is enigmatic. Moreover, an absolutely conserved α-Carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-Carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-Carboxysome. Analogies between the pathway for β-Carboxysome biogenesis and our model for α-Carboxysome assembly are discussed.
-
isolation and characterization of the prochlorococcus Carboxysome reveal the presence of the novel shell protein csos1d
Journal of Bacteriology, 2012Co-Authors: Evan W. Roberts, Gordon C Cannon, Cheryl A Kerfeld, Fei Cai, Sabine HeinhorstAbstract:Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as Carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial Carboxysomes has been the inability to obtain homogeneous Carboxysome preparations. This study describes the first successful purification and characterization of Carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 Carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for Carboxysome composition, structure, and function. Our study indicates that Carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters.
-
the Carboxysome shell is permeable to protons
Journal of Bacteriology, 2010Co-Authors: Balaraj B Menon, Jessup M Shively, Sabine Heinhorst, Gordon C CannonAbstract:Bacterial microcompartments (BMCs) are polyhedral organelles found in an increasingly wide variety of bacterial species. These structures, typified by Carboxysomes of cyanobacteria and many chemoautotrophs, function to compartmentalize important reaction sequences of metabolic pathways. Unlike their eukaryotic counterparts, which are surrounded by lipid bilayer membranes, these microbial organelles are bounded by a thin protein shell that is assembled from multiple copies of a few different polypeptides. The main shell proteins form hexamers whose edges interact to create the thin sheets that form the facets of the polyhedral BMCs. Each hexamer contains a central pore hypothesized to mediate flux of metabolites into and out of the organelle. Because several distinctly different metabolic processes are found in the various BMCs studied to date, it has been proposed that a common advantage to packaging these pathways within shell-bound compartments is to optimize the concentration of volatile metabolites in the BMC by maintaining an interior pH that is lower than that of the cytoplasm. We have tested this idea by recombinantly fusing a pH-sensitive green fluorescent protein (GFP) to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the major enzyme component inside the Carboxysome. Our results suggest that the carboxysomal pH is similar to that of its external environment and that the protein shell does not constitute a proton barrier. The explanation for the sundry BMC functions must therefore be sought in the characteristics of the pores that traverse their shells.
-
organization structure and assembly of α Carboxysomes determined by electron cryotomography of intact cells
Journal of Molecular Biology, 2010Co-Authors: Cristina V Iancu, Gordon C Cannon, Sabine Heinhorst, Zhicheng Dou, Dylan M Morris, Grant J JensenAbstract:Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined Carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that Carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby Carboxysomes. Small granular bodies were also seen within Carboxysomes. These observations suggest a functional relationship between Carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed Carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial Carboxysome was ever observed per cell. Based on these observations, we propose a model for Carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.
Murray R Badger - One of the best experts on this subject based on the ideXlab platform.
-
functions compositions and evolution of the two types of Carboxysomes polyhedral microcompartments that facilitate co2 fixation in cyanobacteria and some proteobacteria
Microbiology and Molecular Biology Reviews, 2013Co-Authors: Benedict M. Long, Murray R Badger, Graeme PriceAbstract:SUMMARY Cyanobacteria are the globally dominant photoautotrophic lineage. Their success is dependent on a set of adaptations collectively termed the CO2-concentrating mechanism (CCM). The purpose of the CCM is to support effective CO2 fixation by enhancing the chemical conditions in the vicinity of the primary CO2-fixing enzyme, d-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to promote the carboxylase reaction and suppress the oxygenase reaction. In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within Carboxysomes, which are examples of a group of proteinaceous bodies called bacterial microcompartments. Carboxysomes encapsulate the CO2-fixing enzyme within the selectively permeable protein shell and simultaneously encapsulate a carbonic anhydrase enzyme for CO2 supply from a cytoplasmic bicarbonate pool. These bodies appear to have arisen twice and undergone a process of convergent evolution. While the gross structures of all known Carboxysomes are ostensibly very similar, with shared gross features such as a selectively permeable shell layer, each type of Carboxysome encapsulates a phyletically distinct form of RubisCO enzyme. Furthermore, the specific proteins forming structures such as the protein shell or the inner RubisCO matrix are not identical between Carboxysome types. Each type has evolutionarily distinct forms of the same proteins, as well as proteins that are entirely unrelated to one another. In light of recent developments in the study of Carboxysome structure and function, we present this review to summarize the knowledge of the structure and function of both types of Carboxysome. We also endeavor to cast light on differing evolutionary trajectories which may have led to the differences observed in extant Carboxysomes.
-
structural determinants of the outer shell of β Carboxysomes in synechococcus elongatus pcc 7942 roles for ccmk2 k3 k4 ccmo and ccml
PLOS ONE, 2012Co-Authors: Benjamin D. Rae, Murray R Badger, Benedict M. Long, Dean G PriceAbstract:Cyanobacterial CO2-fixation is supported by a CO2-concentrating mechanism which improves photosynthesis by saturating the primary carboxylating enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), with its preferred substrate CO2. The site of CO2-concentration is a protein bound micro-compartment called the Carboxysome which contains most, if not all, of the cellular RuBisCO. The shell of β-type Carboxysomes is thought to be composed of two functional layers, with the inner layer involved in RuBisCO scaffolding and bicarbonate dehydration, and the outer layer in selective permeability to dissolved solutes. Here, four genes (ccmK2-4, ccmO), whose products were predicted to function in the outer shell layer of β-Carboxysomes from Synechococcus elongatus PCC 7942, were investigated by analysis of defined genetic mutants. Deletion of the ccmK2 and ccmO genes resulted in severe high-CO2-requiring mutants with aberrant Carboxysomes, whilst deletion of ccmK3 or ccmK4 resulted in cells with wild-type physiology and normal ultrastructure. However, a tandem deletion of ccmK3-4 resulted in cells with wild-type Carboxysome structure, but physiologically deficient at low CO2 conditions. These results revealed the minimum structural determinants of the outer shell of β-Carboxysomes from this strain: CcmK2, CcmO and CcmL. An accessory set of proteins was required to refine the function of the pre-existing shell: CcmK3 and CcmK4. These data suggested a model for the facet structure of β-Carboxysomes with CcmL forming the vertices, CcmK2 forming the bulk facet, and CcmO, a “zipper protein,” interfacing the edges of Carboxysome facets.
-
functional cyanobacterial β Carboxysomes have an absolute requirement for both long and short forms of the ccmm protein
Plant Physiology, 2010Co-Authors: Benedict M. Long, Murray R Badger, L Tucker, Graeme PriceAbstract:Carboxysomes are an essential part of the cyanobacterial CO2-concentrating mechanism, consisting of a protein shell and an interior of Rubisco. The β-Carboxysome shell protein CcmM forms two peptides via a proposed internal ribosomal entry site (IRES) within the ccmM transcript in Synechococcus PCC7942. The abundant short form (35 kD, M35) consists of Rubisco small subunit-like repeats and binds Rubisco. The lower abundance long form (58 kD, M58) also contains a γ-carbonic anhydrase-like domain, which binds the carboxysomal carbonic anhydrase, CcaA. We examined whether these CcmM forms arise via an IRES or by other means. Mutations of a putative internal start codon (GTG) and Shine-Dalgarno sequence within ccmM, along with a gene coding for M35 alone, were examined in the high-CO2-requiring (HCR) Carboxysomeless mutant, ΔccmM. Expression of wild-type ccmM in ΔccmM restored the wild-type phenotype, while mutation of putative start and Shine-Dalgarno sequences led to as much as 20-fold reduction in M35 content with no recovery from HCR phenotype. These cells also contained small electron-dense structures. Cells producing little or no M58, but sufficient M35, were found to contain large electron-dense structures, no CcaA, and had a HCR phenotype. Large subcellular aggregates can therefore form in the absence of M58, suggesting a role for M35 in internal Carboxysome Rubisco packing. The results confirm that M35 is independently translated via an IRES within ccmM. Importantly, the data reveal that functional Carboxysomes require both M35 and M58 in sufficient quantities and with a minimum stoichiometry of close to 1:1.
-
advances in understanding the cyanobacterial co2 concentrating mechanism ccm functional components ci transporters diversity genetic regulation and prospects for engineering into plants
Journal of Experimental Botany, 2008Co-Authors: Dean G Price, Murray R Badger, Fiona J Woodger, Benedict M. LongAbstract:: Cyanobacteria have evolved a significant environmental adaptation, known as a CO(2)-concentrating-mechanism (CCM), that vastly improves photosynthetic performance and survival under limiting CO(2) concentrations. The CCM functions to transport and accumulate inorganic carbon actively (Ci; HCO(3)(-), and CO(2)) within the cell where the Ci pool is utilized to provide elevated CO(2) concentrations around the primary CO(2)-fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). In cyanobacteria, Rubisco is encapsulated in unique micro-compartments known as Carboxysomes. Cyanobacteria can possess up to five distinct transport systems for Ci uptake. Through database analysis of some 33 complete genomic DNA sequences for cyanobacteria it is evident that considerable diversity exists in the composition of transporters employed, although in many species this diversity is yet to be confirmed by comparative phenomics. In addition, two types of Carboxysomes are known within the cyanobacteria that have apparently arisen by parallel evolution, and considerable progress has been made towards understanding the proteins responsible for Carboxysome assembly and function. Progress has also been made towards identifying the primary signal for the induction of the subset of CCM genes known as CO(2)-responsive genes, and transcriptional regulators CcmR and CmpR have been shown to regulate these genes. Finally, some prospects for introducing cyanobacterial CCM components into higher plants are considered, with the objective of engineering plants that make more efficient use of water and nitrogen.
-
analysis of Carboxysomes from synechococcus pcc7942 reveals multiple rubisco complexes with carboxysomal proteins ccmm and ccaa
Journal of Biological Chemistry, 2007Co-Authors: Benedict M. Long, Murray R Badger, Spencer M Whitney, Dean G PriceAbstract:In cyanobacteria, the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called Carboxysomes. Cyanobacteria with Form IB Rubisco produce-Carboxysomes whose putative shell proteins are encoded by the ccm-type genes. To date, very little is known of the protein-protein interactions that form the basis of -Carboxysome structure. In an effort to identify such interactions within the Carboxysomes of the -cyanobacterium Synechococcus sp. PCC7942, we have used polyhistidine-tagging approaches to identify at least three carboxysomal subcomplexes that contain active Rubisco. In addition to the expected L8S8 Rubisco, which is the major component of Carboxysomes, we have identified two Rubisco complexes containing the putative shell protein CcmM, one of which also contains the carboxysomal carbonic anhydrase, CcaA. The complex containing CcaA consists of Rubisco and the full-length 58-kDa form of CcmM (M58), whereas the other is made up of Rubisco and a short 35-kDa form of CcmM (M35), which is probably translated independently of M58 via an internal ribosomal entry site within the ccmM gene. We also show that the high CO2-requiring ccmM deletion mutant (ccmM) can achieve nearly normal growth rates at ambient CO2 after complementation with both wild type and chimeric (His6-tagged) forms of CcmM. Although a significant amount of independent L8S8 Rubisco is confined to the center of the Carboxysome, we speculate that the CcmMCcaA-Rubisco complex forms an important assembly coordination within the Carboxysome shell. A speculative Carboxysome structural model is presented.