Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ.

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.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

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.

Kinetics and control of self-assembly of ABH1 hydrophobin from the edible white button mushroom.

Hydrophobins are small fungal proteins that self-assemble at hydrophobic/hydrophilic interfaces to form stable, amyloid membranes that are resistant to denaturation. Their remarkable surface activity has driven intense research for their potential utility in biomedical and cosmetic applications. In this research, the self-assembly characteristics of the Class I hydrophobin ABH1 from Agaricus bisporus , the edible white button mushroom, were evaluated as a function of solution and interface properties, in an attempt to gain greater mechanistic understanding.

Isolation and characterization of the Prochlorococcus carboxysome reveal the presence of the novel shell protein CsoS1D.

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.

The carboxysome shell is permeable to protons.

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.

Bacterial microcompartments.

Bacterial microcompartments (BMCs) are organelles composed entirely of protein. They promote specific metabolic processes by encapsulating and colocalizing enzymes with their substrates and cofactors, by protecting vulnerable enzymes in a defined microenvironment, and by sequestering toxic or volatile intermediates. Prototypes of the BMCs are the carboxysomes of autotrophic bacteria.

Organization, structure, and assembly of alpha-carboxysomes determined by electron cryotomography of intact cells.

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.

The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier.

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.

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation.

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO(2) concentrations by employing a CO(2)-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO(2)-fixing enzyme, RubisCO. The efficiency of CO(2) fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO(2).

Identification and structural analysis of a novel carboxysome shell protein with implications for metabolite transport.

Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans.

Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species.

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.

Protein-based organelles in bacteria: carboxysomes and related microcompartments.

Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO.

Atomic-level models of the bacterial carboxysome shell.

The carboxysome is a bacterial microcompartment that functions as a simple organelle by sequestering enzymes involved in carbon fixation. The carboxysome shell is roughly 800 to 1400 angstroms in diameter and is assembled from several thousand protein subunits. Previous studies have revealed the three-dimensional structures of hexameric carboxysome shell proteins, which self-assemble into molecular layers that most likely constitute the facets of the polyhedral shell.

CO2 fixation kinetics of Halothiobacillus neapolitanus mutant carboxysomes lacking carbonic anhydrase suggest the shell acts as a diffusional barrier for CO2.

The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO(2), to permit its efficient fixation onto ribulose 1,5-bisphosphate.

Transcript analysis of the Halothiobacillus neapolitanus cso operon.

Carboxysomes are polyhedral microcompartments that sequester the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase in many autotrophic bacteria. Their protein constituents are encoded by a set of tightly clustered genes that are thought to form an operon (the cso operon). This study is the first to systematically address transcriptional regulation of carboxysome protein expression.

Structural analysis of CsoS1A and the protein shell of the Halothiobacillus neapolitanus carboxysome.

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.

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus.

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes.

Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune.

Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques.

The structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two.

CsoSCA (formerly CsoS3) is a bacterial carbonic anhydrase localized in the shell of a cellular microcompartment called the carboxysome, where it converts HCO(3)(-) to CO(2) for use in carbon fixation by ribulose-bisphosphate carboxylase/oxygenase (RuBisCO). CsoSCA lacks significant sequence similarity to any of the four known classes of carbonic anhydrase (alpha, beta, gamma, or delta), and so it was initially classified as belonging to a new class, epsilon. The crystal structure of CsoSCA from Halothiobacillus neapolitanus reveals that it is actually a representative member of a new subclass of beta-carbonic anhydrases, distinguished by a lack of active site pairing.

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell.

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth.

Experimental evidence for multiple assembled states of Sc3 from Schizophyllum commune.

The hydrophobin Sc3 from the fungus Schizophyllum commune assembles from the aqueous phase into ordered structures with substantially different characteristics depending upon experimental conditions. Under the first condition, a vortexing procedure widely reported in the literature, interfacial assembly yields highly ordered, stacked beta-sheets. We have also observed a previously unreported assembly of Sc3 under a second condition, which occurs in a time-dependent manner from quiescent solution.

Organization of carboxysome genes in the thiobacilli.

The order of genes in the carboxysome gene clusters of four thiobacilli was examined and the possibility of the cluster forming an operon evaluated. Furthermore, carboxysome peptide homologs were compared with respect to similarities in primary sequence, and the unique structural features of the shell protein CsoS2 were described.

The DNA-compacting protein DCP68 from soybean chloroplasts is ferredoxin:sulfite reductase and co-localizes with the organellar nucleoid.

The multiple copies of the chloroplast genome (plastome) are condensed and organized into nucleoids by a set of proteins. One of these, the DNA-binding protein DCP68 from soybean, has previously been shown to compact DNA and to inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is the siroheme protein sulfite reductase, a ferredoxin-dependent enzyme that participates in sulfur assimilation for cysteine and methionine biosynthesis.