Jessup M. Shively

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-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.

A Novel Evolutionary Lineage of Carbonic Anhydrase (ε 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. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

Microcompartments in prokaryotes: carboxysomes and related polyhedra.

All cyanobacteria and many chemoautotrophs contain polyhedral inclusion bodies that are bound by a unilamellar protein shell (15, 63). Isolation and enzymatic analysis of the bodies from Halothiobacillus neapolitanus (previously Thiobacillus neapolitanus) revealed that they are filled with ribulose 1,5bisphosphate carboxylase/oxygenase (RuBisCO); therefore they were given the name “carboxysomes” (59). Subsequent studies of both cyanobacteria and chemoautotrophic bacteria have led to the well-accepted conclusion that the “organelles” or microcompartments function to enhance the catalytic properties of the RuBisCO they contain, although the mechanism of this catalytic enhancement is unclear (51, 65). Localization and characterization of the genes encoding carboxysome components has underscored the apparent common function of these bodies in carboxysome-containing autotrophic bacteria. More surprising is the finding that a number of heterotrophic prokaryotes harbor genes homologous to those for carboxysome shell proteins (9, 32, 62). Under proper growth conditions, these bacteria produce polyhedral inclusion bodies that are morphologically similar to carboxysomes, although the cells expressing these bodies contain no RuBisCO and do not fix CO 2 via the Calvin-Benson-Bassham cycle as a major part of their carbon metabolism. This review evaluates the evidence that relates carboxysome structure to function in the carbon metabolism of autotrophic prokaryotes and examines similarities to newly discovered particles found in heterotrophs. The possibility is explored that microcompartmentalization of key metabolic enzymes by carboxysomes and their relatives is a more widely utilized regulatory mechanism in prokaryotes than was previously envisioned.

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, CO2, to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO2-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO2 into and out of the carboxysome interior.

Relations between D-Ribulose-1,5-Bis-Phosphate Carboxylase, Carboxysomes and Co2 Fixing Capacity in the Obligate Chemolithotroph Thiobacillus-Neapolitanus Grown under Different Limitations in the Chemostat

An adaptation of the d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity to changing CO2 concentrations in the growth medium in the chemostat was observed in the obligate chemolithotroph Thiobacillus neapolitanus. RuBPCase activity has been separated in a soluble and particulate fraction. The activity of the particulate fraction appeared to be associated with the carboxysomes.The total activity of RuBPCase of CO2 limited cultures was about 5-fold higher than the activity of thiosulphate limited cultures grown in the presence of 5% CO2 whilst the particulate activity and the soluble activity were about 8- and 1.5-fold higher, respectively. The fluctuation of the total and particulate RuBPCase activity correlated with the changes in volume density of carboxysomes in the cell.An inverse correlation between maximal CO2 fixing capacity by whole cells and the volume density of carboxysomes was observed. The change in ratio of soluble RuBPCase activity to particulate RuBPCase activity paralleled the change in maximal CO2 fixation by whole cells during the different growth conditions.

Characterization of a homogenous preparation of carboxysomes from Thiobacillus neapolitanus

A homogenous preparation of carboxysomes was isolated from Thiobacillus neapolitanus by means of density gradient centrifugation and preparative electrophoresis through agarose. Analysis of the carboxysomes by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of 12–15 polypeptides. Approximately 62% of the total protein was found to consist of the large and small subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO). Two polypeptides were found to be components of the carboxysome shell. Ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and fructose-1,6-bisphosphatase could not be detected in the carboxysome. Purified carboxysomal and cytoplasmic RubisCO were shown to be similar if not identical by several criteria including specific activity, carboxylase/oxygenase activity, and electrophoretic mobility.

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 α-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.

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. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.

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 CO2 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 CO2 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.

Carboxysome genomics: a status report

Carboxysomes, microcompartments that enhance the fixation of carbon dioxide by Rubisco, are found in several chemoautotrophs and in all cyanobacteria thus far examined. The genes for Rubisco large (cbbL) and small (cbbS) subunits (cbb for Calvin-Benson-Bassham), along with the genes (csoS) for the carboxysome shell peptides, are organized in a putative operon in Halothiobacillus neapolitanus in the following order: cbbL,cbbS, csoS2, csoS3, orfA, orfB, csoS1C, csoS1A, and csoS1B. DNA sequencing has revealed essentially the same operon in three other thiobacilli, Acidithiobacillus ferrooxidans, Thiomonas intermedia, and Thiobacillus denitrificans. The carboxysome genes are also clustered inSynechococcus sp. and Synechocystis sp., although in some cases certain genes lie outside the cluster. The genes, labelled ccm for CO2 concentrating mechanism, exist in Synechococcus PCC7942 in the order ccmK, ccmL, ccmM, ccmN, and ccmO, and are located upstream of the Rubisco genes. ccmO is absent, and multiple copies of ccmK exist in some species. The ccmK/ccmO and ccmL genes are homologues of csoS1CAB andorfAB, respectively. The ccmM and ccmN genes have no apparent counterpart in the thiobacilli. More recently, the genome sequence of four additional cyanobacteria has become available. The carboxysome genes in Nostoc punctiforme are clustered like, and are similar to, the genes of the earlier mentioned cyanobacteria. However, the three marine organisms Prochlorococcus marinus MIT9313, P. marinus MED4, and Synechococcus WH8102, possess an operon nearly identical to that found in thiobacilli. Furthermore, the genes exhibit surprising sequence identity to the carboxysome genes of the thiobacilli.