Extremophilic nitrite-oxidizing Chloroflexi from Yellowstone hot springs.

Nitrifying microorganisms occur across a wide temperature range from 4 to 84 °C and previous studies in geothermal systems revealed their activity under extreme conditions. Archaea were detected to be responsible for the first step of nitrification, but it is still a challenging issue to clarify the identity of heat-tolerant nitrite oxidizers. In a long-term cultivation approach, we inoculated mineral media containing ammonium and nitrite as substrates with biofilms and sediments of two hot springs in Yellowstone National Park (USA). The nitrifying consortia obtained at 70 °C consisted mostly of novel Chloroflexi as revealed by metagenomic sequencing. Among these, two deep-branching novel Chloroflexi were identified as putative nitrite-oxidizing bacteria (NOB) by the presence of nitrite oxidoreductase encoding genes in their genomes. Stoichiometric oxidation of nitrite to nitrate occurred under lithoautotrophic conditions, but was stimulated by organic matter. Both NOB candidates survived long periods of starvation and the more abundant one formed miniaturized cells and was heat resistant. This detection of novel thermophilic NOB exemplifies our still incomplete knowledge of nitrification, and indicates that nitrite oxidation might be an ancient and wide-spread form of energy conservation.

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 ß, 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 ß-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 ß-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

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. 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.

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. 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.

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. 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.

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. 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.

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. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO(2)-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 CO(2) into and out of the carboxysome interior.

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. Quantification of transcript levels derived from the cso operon of Halothiobacillus neapolitanus, the sulfur oxidizer that has emerged as the model organism for carboxysome structural and functional studies, indicated that all cso genes are transcribed, albeit at different levels. Combined with comparative genomic evidence, this study supports the premise that the cso gene cluster constitutes an operon. Characterization of transcript 5'- and 3'-ends and examination of likely regulatory sequences and secondary structure elements within the operon suggested potential strategies by which the vastly different levels of individual carboxysome proteins in the microcompartment could have arisen.

Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography.

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.

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.

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. 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.

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.

Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270.

Acidithiobacillus ferrooxidans ATCC 23270 possesses two copies of form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The nucleotide sequence identity between the two large and two small subunit peptides was 75% and 58%, respectively. It is proposed that the two copies resulted from lateral gene transfer.

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.

Rhodobacter capsulatus genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbLS) and neighbouring genes were acquired by a horizontal gene transfer.

Analysis of the nucleotide sequence of the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes (cbbL and cbbS) of the non-sulfur purple bacterium Rhodobacter capsulatus indicated that the deduced amino acid sequence of the large subunit was not closely homologous to the large subunit from related organisms. Indeed, phylogenetic analysis suggested that the large subunit protein (CbbL) more closely resembled the enzyme from alpha/beta/gamma purple bacteria and cyanobacteria and is within a 'green-like' radiation of the RubisCO phylogenetic tree, well separated from CbbL of the related organism Rhodobacter sphaeroides. A cbbQ gene was discovered downstream of cbbS in Rh. capsulatus, a gene arrangement which also appears to be limited to certain organisms containing a 'green-like' RubisCO. Upstream, and divergently transcribed from cbbLSQ, is a gene (cbbRI) that encodes a LysR-type transcriptional activator. Phylogenetic analysis of the deduced amino acid sequence of CbbRI also suggests that this protein is quite distinct from the Rh. sphaeroides CbbR protein, and is even distinct from the previously described CbbRII protein, the gene of which is upstream and divergently transcribed from the cbbII operon of Rh. capsulatus. Interestingly, Rh. capsulatus CbbRI is more closely related to CbbR from bacteria whose RubisCO falls within the 'green-like' radiation of the CbbL tree. These studies suggest that the cbbRI-cbbL-cbbS-cbbQ genes were acquired by Rh. capsulatus via horizontal gene transfer from a bacterial species containing a 'green-like' RubisCO.