Halothiobacillus diazotrophicus sp. nov., a chemolithoautotrophic sulphur-oxidizing and nitrogen-fixing bacterium isolated from freshwater.

A sulphur-oxidizing and nitrogen-fixing bacterium, designated strain LS2T, was isolated from freshwater collected from the Pearl River in Guangzhou, PR China. The strain was an obligate chemolithoautotroph, utilizing reduced sulphur compounds (sulphide, sulphite, elemental sulphur, thiosulphate and tetrathionate) as energy sources and electron donors. Diazotrophic growth of strain LS2T was observed at 15-40 °C, pH 5-9, with a NaCl concentration range of 0-0.68 mol l-1 and with oxygen content higher than 21 %. The major cellular fatty acids were summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The DNA G+C content of the complete genome sequence was 60.7 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain LS2T formed a lineage within the family Halothiobacillaceae, showing gene sequence identity of 96.8 % with its closest relative Halothiobacillus neapolitanus c2. The genome of strain LS2T contains multiple genes encoding sulphur-oxidizing enzymes that catalyse the oxidation of reduced sulphur compounds and an nif complex encoding enzymes for nitrogen fixation. In addition, the genome contains genes encoding cbb3-type cytochrome c oxidase, aa3-type cytochrome c oxidase, bd-type quinol oxidase and cytochrome o oxidase, which enable the survival strain LS2T under oxic and microaerophilic conditions. On the basis of phenotypic, genotypic and phylogenetic data, strain LS2T is considered to represent a novel species of the genus Halothiobacillus, for which the name Halothiobacillus diazotrophicus sp. nov. is proposed. The type strain is LS2T (=GDMCC 1.4095T=JCM 39442T).

Structure and assembly of cargo Rubisco in two native α-carboxysomes.

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.

Decoding the Absolute Stoichiometric Composition and Structural Plasticity of α-Carboxysomes.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO2-fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications. IMPORTANCE A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco. The self-assembling organelle is generated by hundreds of proteins and plays important roles in converting carbon dioxide to sugar, a process known as carbon fixation. In this study, we uncovered the exact stoichiometry of all building components and the structural plasticity of the functional α-carboxysome, using newly developed quantitative mass spectrometry together with biochemistry, electron microscopy, and enzymatic assay. The study advances our understanding of the architecture and modularity of natural carboxysomes. The knowledge learned from natural carboxysomes will suggest feasible ways to produce functional carboxysomes in other hosts, such as crop plants, with the overwhelming goal of boosting cell metabolism and crop yields.

The McdAB system positions α-carboxysomes in proteobacteria.

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 ß-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 ß-cyanobacteria, which possess ß-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 ß-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 ß-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 CO2 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.

Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production.

Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation.

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

DABs are inorganic carbon pumps found throughout prokaryotic phyla.

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.

Response of cbb gene transcription levels of four typical sulfur-oxidizing bacteria to the CO2 concentration and its effect on their carbon fixation efficiency during sulfur oxidation.

The variability in carbon fixation capability of four sulfur-oxidizing bacteria (Thiobacillus thioparus DSM 505, Halothiobacillus neapolitanus DSM 15147, Starkeya novella DSM 506, and Thiomonas intermedia DSM 18155) during sulfur oxidation was studied at low and high concentrations of CO2. The mechanism underlying the variability in carbon fixation was clarified by analyzing the transcription of the cbb gene, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. DSM 15147 and DSM 505 fixed carbon more efficiently during sulfur oxidation than DSM 506 and DSM 18155 at 0.5% and 10% CO2, which was mainly because their cbb gene transcription levels were much higher than those of DSM 506 and DSM 18155. A high CO2 concentration significantly stimulated the carbon fixation efficiency of DSM 505 by greatly increasing the cbb gene transcription efficiency. Moreover, the influence of the CO2 concentration on the carbon fixation efficiency of the four strains differed greatly during sulfur oxidation.

A data set from flash X-ray imaging of carboxysomes.

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.

Formate supplementation can increase nickel recovery by Halothiobacillus halophilus.

Acidophilic thiobacilli are traditional biotechnological agents for metal recovery from sulfide ores. Major industrial strains belong to autotrophic bacteria which are used without any organic supplements to stimulate the process. The autotrophic strain Acidithiobacillus ferrooxidans ATCC 21834 is known to use formate as a source of energy under special laboratory conditions. We showed the presence of formate dehydrogenase in the type strain of another autotrophic species Halothiobacillus halophilus representing another genus of thiobacilli. This finding prompted studies of bioleaching stimulation by formate. Canadian sulfide nickel ore was chosen for model investigation as leached substrate and the moderate acidophilic strain H. halophilus DSM 6132 was used as the leaching agent. In bench-scale bioleaching experiments, inoculation of the ore with H. halophilus supplemented with 0.3 % formate increased the recovery of nickel 70-fold as compared with formate-free inoculation (1008.0 vs. 13.8 mg Ni/L per 34 days). Bacteria H. halophilus belong to moderate acidophilic microorganisms; thus, the results were obtained with initial pH 7.4 and final pH 5.4. The mechanism of formate stimulation is under discussion.

Isolation and characterization of an obligately chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy source.

Molecular and microbiological analysis of a laboratory bioreactor biomass oxidizing thiocyanate at autotrophic conditions and at 1 M NaCl showed a domination of a single chemolithoautotrophic sulfur-oxidizing bacterium (SOB) capable of using thiocyanate as an energy source. The bacterium was isolated in pure cultures and identified as a member of the Halothiobacillus halophilus/hydrothermalis clade. This clade includes moderately halophilic chemolithoautotrophic SOB from marine and hypersaline habitats for which the ability to utilize thiocyanate as an electron donor has not been previously demonstrated. Halothiobacillus sp. strain SCN-R1 grew with thiocyanate as the sole energy and nitrogen source oxidizing it to sulfate and ammonium via the cyanate pathway. The pH range for thiocyanate oxidation was within a neutral region between 7 and 8 and the range of salinity was from 0.2 to 1.5 M NaCl, with an optimum at 0.5 M. Despite the close phylogenetic relatedness, none of the tested type strains and other isolates from the H. halophilus/hydrothermalis group exhibited thiocyanate-oxidizing capacity.

Modularity of a carbon-fixing protein organelle.

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

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.

Sulphide oxidation to elemental sulphur in a membrane bioreactor: performance and characterization of the selected microbial sulphur-oxidizing community.

In leather tanning industrial areas sulphide management represents a major problem. However, biological sulphide oxidation to sulphur represents a convenient solution to this problem. Elemental sulphur is easy to separate and the process is highly efficient in terms of energy consumption and effluent quality. As the oxidation process is performed by specialized bacteria, selection of an appropriate microbial community is fundamental for obtaining a good yield. Sulphur oxidizing bacteria (SOB) represent a wide-ranging and highly diversified group of microorganisms with the capability of oxidizing reduced sulphur compounds. Therefore, it is useful to select new microbes that are able to perform this process efficiently. For this purpose, an experimental membrane bioreactor for sulphide oxidation was set up, and the selected microbial community was characterized by constructing 16S rRNA gene libraries and subsequent screening of clones. Fluorescence in situ hybridization (FISH) was then used to assess the relative abundance of different bacterial groups. Sulphide oxidation to elemental sulphur proceeded in an efficient (up to 79% conversion) and stable way in the bioreactor. Both analysis of clone libraries and FISH experiments revealed that the dominant operational taxonomic unit (OTU) in the bioreactor was constituted by Gammaproteobacteria belonging to the Halothiobacillaceae family. FISH performed with the specifically designed probe tios_434 demonstrated that this OTU constituted 90.6+/-1.3% of the bacterial community. Smaller fractions were represented by bacteria belonging to the classes Betaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Clostridia, Mollicutes, Sphingobacteria, Bacteroidetes and Chlorobia. Phylogenetic analysis revealed that clone sequences from the dominant OTU formed a stable clade (here called the TIOS44 cluster), within the Halothiobacillaceae family, with sequences from many organisms that have not yet been validly described. The data indicated that bacteria belonging to the TIOS44 cluster were responsible for the oxidation process.

Stable sulfur isotope fractionation and discrimination between the sulfur atoms of thiosulfate during oxidation by Halothiobacillus neapolitanus.

Growing cultures and nongrowing suspensions of Halothiobacillus neapolitanus selectively fractionated (32)S and (34)S during the oxidation of the sulfane- and sulfonate-sulfur atoms of thiosulfate. Sulfate was enriched in (32)S, with delta(34)S reaching -6.3 per thousand relative to the precursor sulfonate-sulfur of thiosulfate, which was progressively resynthesized from the thiosulfate-sulfane-sulfur during thiosulfate metabolism. Polythionates, principally trithionate, accumulated during thiosulfate oxidation and showed progressive increase in the relative (34)S content of their sulfonate groups, with delta(34)S values up to +20 per thousand, relative to the substrate sulfur. The origins of the sulfur in the sulfate and polythionate products of oxidation were tracked by the use thiosulfate labelled with (35)S in each of its sulfur atoms, enabling determination of the flow of the sulfur atoms into the oxidation products. The results confirm that highly significant fractionation of stable sulfur isotopes can be catalyzed by thiobacilli oxidizing thiosulfate, but that differences in the (34)S/(32)S ratios of the nonequivalent constituent sulfur atoms of the thiosulfate used as substrate mean that the oxidative fate of each atom needs separate determination. The data are very significant to the understanding of bacterial sulfur-compound oxidation and highly relevant to the origins of biogenic sulfate minerals.

The effect of silica fume on biodegradation of cement paste and its capacity to immobilize strontium during exposure to microbial sulfur oxidation.

The disposal of low-level radioactive waste containing isotopes such as strontium by immobilization in cement paste has become common practice. However, the stability of cement paste in the environment may be impaired by sulfuric acid produced by sulfur-oxidizing bacteria. Since biodegradation rates in the environment of most radioactive waste burial sites are too low to be measured, determination of the degradation kinetics of cement paste is a difficult task. This study reports on the development of an accelerated biodegradation system for cement pastes in which the cement paste is exposed to a continuous culture of the sulfur-oxidizing bacterium Halothiobacillus neapolitanus. This system facilitated detection of the biodegradation processes in cement paste after as short a time as 15 days. A comparison of the durability of a cement paste blended with silica fume with that of unblended cement paste showed that the silica fume induced an increase in the leaching of Ca(+2) and Si and enhanced weight loss, indicating rapid deterioration in the structural integrity of the cement paste. The leaching of Sr(+2) from the silica fume amended cement paste was slightly reduced as compared with the non amended cement paste, indicating an increase in immobilization of strontium. Nevertheless, our findings do not support the use of silica fume as a suitable additive for immobilization of low-level radioactive waste.

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. 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.

Inhibitory effects of sulfamic acid on three thiosulfate-oxidizing chemolithotrophs.

Sulfamate is an analogue of thiosulfate, and the sodium and potassium salts of sulfamic acid inhibited the chemolithoautotrophic growth on thiosulfate of Acidithiobacillus ferrooxidans and Halothiobacillus neapolitanus. The chemo-organotrophic growth of Paracoccus versutus on sucrose was similarly inhibited by sulfamate. Thiosulfate oxidation by suspensions of H. neapolitanus was, however, unaffected by sulfamate, showing that sulfamate did not directly affect thiosulfate uptake, activation or oxidation. Inhibition of P. versutus was not relieved by cysteine and methionine, indicating that sulfate uptake and sulfur amino acid biosynthesis were not directly affected by sulfamate. Sulfamate was not degraded by any of the bacteria, and so could not serve as an alternative to thiosulfate as an energy-yielding substrate. Sulfamate is also an analogue of ammonia and might act like hydrazine by inhibiting ammonium uptake or an essential enzyme activity.

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.