Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly

Bacterial microcompartments are organelles composed of a protein shell that surrounds functionally related proteins. Bioinformatic analysis of sequenced genomes indicates that homologs to shell protein genes are widespread among bacteria and suggests that the shell proteins are capable of encapsulating diverse enzymes. The carboxysome is a bacterial microcompartment that enhances CO(2) fixation in cyanobacteria and some chemoautotrophs by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase in the microcompartment shell. Here, we report the in vitro and in vivo characterization of CcmN, a protein of previously unknown function that is absolutely conserved in β-carboxysomal gene clusters. We show that CcmN localizes to the carboxysome and is essential for carboxysome biogenesis. CcmN has two functionally distinct regions separated by a poorly conserved linker. The N-terminal portion of the protein is important for interaction with CcmM and, by extension, ribulose-1,5-bisphosphate carboxylase/oxygenase and the carbonic anhydrase CcaA, whereas the C-terminal peptide is essential for interaction with the carboxysome shell. Deletion of the peptide abolishes carboxysome formation, indicating that its interaction with the shell is an essential step in microcompartment formation. Peptides with similar length and sequence properties to those in CcmN can be bioinformatically detected in a large number of diverse proteins proposed to be encapsulated in functionally distinct microcompartments, suggesting that this peptide and its interaction with its cognate shell proteins are common features of microcompartment assembly.     

Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA

In cyanobacteria, the key enzyme for photosynthetic CO(2) fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. Cyanobacteria with Form IB Rubisco produce beta-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 beta-carboxysome structure. In an effort to identify such interactions within the carboxysomes of the beta-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 L(8)S(8) 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 CO(2)-requiring ccmM deletion mutant (DeltaccmM) can achieve nearly normal growth rates at ambient CO(2) after complementation with both wild type and chimeric (His(6)-tagged) forms of CcmM. Although a significant amount of independent L(8)S(8) Rubisco is confined to the center of the carboxysome, we speculate that the CcmM-CcaA-Rubisco complex forms an important assembly coordination within the carboxysome shell. A speculative carboxysome structural model is presented.     

Complex structure reveals CcmM and CcmN form a heterotrimeric adaptor in β‐carboxysome

Carboxysome is an icosahedral self‐assembled microcompartment that sequesters RuBisCO and carbonic anhydrases within a selectively permeable protein shell. The scaffolding proteins, CcmM, and CcmN were proposed to act as adaptors that crosslink the enzymatic core to shell facets. However, the details of interaction pattern remain unknown. Here we obtained a stable heterotrimeric complex of CcmM γ‐carbonic anhydrase domain (termed CcmM^NT) and CcmN, with a 1:2 stoichiometry, which interacts with the shell proteins CcmO and CcmL in vitro. The 2.9 Å crystal structure of this heterotrimer revealed an asymmetric bundle composed of one CcmM^NT and two CcmN subunits, all of which adopt a triangular left‐handed β‐helical barrel structure. The central CcmN subunit packs against CcmM^NT and another CcmN subunit via a wall‐to‐edge or wall‐to‐wall pattern, respectively. Together with previous findings, we propose CcmM^NT‐CcmN functions as an adaptor to facilitate the recruitment of shell proteins and the assembly of intact β‐carboxysome.     

Over-expression of the ��-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content

Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.     

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.     

Structural Determinants of the Outer Shell of β-Carboxysomes in Synechococcus elongatus PCC 7942: Roles for CcmK2, K3-K4, CcmO, and CcmL

Cyanobacterial CO(2)-fixation is supported by a CO(2)-concentrating mechanism which improves photosynthesis by saturating the primary carboxylating enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), with its preferred substrate CO(2). The site of CO(2)-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-CO(2)-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 CO(2) 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.     

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. Whereas a typical beta-carbonic anhydrase maintains a pair of active sites organized within a two-fold symmetric homodimer or pair of fused, homologous domains, the two domains in CsoSCA have diverged to the point that only one domain in the pair retains a viable active site. We suggest that this defunct and somewhat diminished domain has evolved a new function, specific to its carboxysomal environment. Despite the level of sequence divergence that separates CsoSCA from the other two subclasses of beta-carbonic anhydrases, there is a remarkable level of structural similarity among active site regions, which suggests a common catalytic mechanism for the interconversion of HCO(3)(-) and CO(2). Crystal packing analysis suggests that CsoSCA exists within the carboxysome shell either as a homodimer or as extended filaments.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO(2) uptake systems and two HCO(3)(-) transporters. We transformed a mutant impaired in CO(2) uptake and in cmpA-D encoding a HCO(3)(-)transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO(3)(-) and grow in low CO(2) at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO(2), requires Na(+), and plays the major role in HCO(3)(-) uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na(+)/K(+)-translocating protein) abolished the ability of cells to grow at [Na(+)] higher than 100 mm and severely depressed the activity of the SbtA-mediated HCO(3)(-) transport. We propose that the SbtA-mediated HCO(3)(-) transport is driven by DeltamuNa(+) across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na(+)-dependent HCO(3)(-) transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO(2) supply is limited, or in Prochlorococcus strains that do not possess CO(2) uptake systems or Cmp-dependent HCO(3)(-) transport.     

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.     

羧酶体结构及其CO_2浓缩机制研究进展

羧酶体(Carboxysome)是高效的固碳微体,在CO2浓缩机制(CO2-concentrating mechanism,CCM)中发挥重要作用。在蓝藻及某些化能自养菌中,羧酶体作为类细胞器包裹1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)和碳酸酐酶(Carbonic anhydrase,CA),它与无机碳转运蛋白共同在胞质中积累HCO3–,通过增加RubisCO周围的CO2浓度来提高固碳效率。随着羧酶体结构和功能的阐明,异源表达羧酶体已成功实现,并且已鉴定出编码羧酶体壳蛋白及内部组分的基因。首先简要介绍羧酶体的发现和种类,然后系统分析其结构及在CCM机制中的作用,并对其在代谢工程上的广阔应用前景进行了展望。     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

In situ characterization of carbonic anhydrase activity in isolated rat lungs

Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.     

A comparison of the kinetic properties of native bovine muscle carbonic anhydrase and an activated derivative with modified thiol groups

Steady-state and equilibrium kinetic properties of native bovine carbonic anhydrase III (carbonate hydrolyase, EC 4.2.1.1) and a derivative modified with methyl methanethiosulfonate were investigated. The modified enzyme has a markedly increased CO2 hydration activity compared to the native form with a 3-times higher value of kcat and a 6-10-times higher value of kcat/Km. Qualitatively, the activated enzyme shows the same kinetic behavior as native isoenzyme III. This is reflected in similar pH dependences of the kinetic parameters for CO2 hydration, similar solvent hydrogen isotope effects on these parameters, similar deviations from Michaelis-Menten kinetics for the HCO3- dehydration reaction, and similar behavior of the kinetics of CO2/HCO3- exchange at chemical equilibrium as measured by a 13C-NMR magnetization transfer technique. It is concluded that the conversion of -SH groups to -S-S-CH3 moieties does not change the catalytic mechanism, but leads to an increased rate of CO2/HCO3- interconversion as well as to an increased rate of proton transfer between the active site and the reaction medium.     

The effects of exogenous extracellular carbonic anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering capacity

The buffering capacity (beta) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3- within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma beta (-3.9+/-0.3 mmol 1(-1) pH unit(-1)), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) elevated plasma beta (-12.1+/-1.1 mmol 1(-1) pH unit(-1)). An injection of 5 mg kg(-1) carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (-0.23+/-0.05 Torr) or high (-0.20+/-0.05 Torr) plasma beta; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3- dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3- to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl-/HCO3- exchange governs HCO3- accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl-/HCO3- exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3- dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma beta was elevated in the present study was insufficient to drive a substantially increased component of HCO3- dehydration through the plasma.     

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. 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.     

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins

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.A key enzyme in photosynthesis is the CO₂ fixation enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco not only fixes CO₂, resulting in carbon assimilation, but it can also fix O₂, leading to photorespiration. Suppressing the unwanted oxygenase activity of Rubisco by sequestering Rubisco with a source of CO₂ is Nature’s solution to this substrate discrimination problem. While C4 plants compartmentalize CO₂ fixation in specific cells (Hibberd et al., 2008; Parry et al., 2011), cyanobacteria have evolved a specialized organelle composed entirely of protein to encapsulate Rubisco—the carboxysome.The carboxysome is just one type of bacterial microcompartment (BMC), widespread, functionally diverse bacterial organelles (Axen et al., 2014). All BMCs consist of an enzymatic core surrounded by a selectively permeable protein shell (Kerfeld et al., 2005; Tanaka et al., 2008; Chowdhury et al., 2014; Kerfeld and Erbilgin, 2015). While the encapsulated enzymes differ among functionally distinct BMCs, they share an architecturally similar shell composed of three types of proteins: BMC-H, BMC-T, and BMC-P forming hexamers, pseudohexamers, and pentamers, respectively (Kerfeld and Erbilgin, 2015). These constitute the building blocks of a self-assembling, apparently icosahedral shell with a diameter ranging from 40 to 400 nm (Shively et al., 1973a,b, 1998; Price and Badger, 1991; Bobik et al., 1999; Iancu et al., 2007, 2010; Petit et al., 2013; Erbilgin et al., 2014). Recent studies have also shown that in the biogenesis of BMCs an encapsulation peptide (EP) (Fan and Bobik, 2011; Kinney et al., 2012; Aussignargues et al., 2015; Jakobson et al., 2015), a short (approximately 18 residues) amphipathic α-helix mediates interactions between a subset of core protein and the shell (Fan and Bobik, 2011; Choudhary et al., 2012; Kinney et al., 2012; Lawrence et al., 2014; Lin et al., 2014; Aussignargues et al., 2015). Indeed, because they are self-assembling organelles composed entirely of protein, BMCs hold great promise for diverse applications in bioengineering and development of bionanomaterials (Frank et al., 2013; Chowdhury et al., 2014; Chessher et al., 2015; Kerfeld and Erbilgin, 2015); the key features of BMCs include selective permeability, spatial colocalization of enzymes, the establishment of private cofactor pools, and the potentially beneficial effects of confinement on protein stability. For example, introducing carboxysomes into plants could provide a saltational enhancement of crop photosynthesis (Price et al., 2013; Zarzycki et al., 2013; Lin et al., 2014; McGrath and Long, 2014).The β-carboxysome, which sequesters form 1B Rubisco, has been an important model system for the study of the structural basis of carboxysome function, assembly, and engineering (Kerfeld et al., 2005; Tanaka et al., 2008; Cameron et al., 2013; Aussignargues et al., 2015; Cai et al., 2015). Beta-carboxysomes assemble from the inside out (Cameron et al., 2013; Gonzalez-Esquer et al., 2015). Two proteins that are absolutely conserved and unique to β-carboxysomes, CcmM and CcmN, play essential roles in this process: CcmM crosslinks Rubisco through its C-terminal Rubisco small subunit-like domains (SSLDs; pfam00101); CcmM and CcmN interact through their N-terminal domains; and C-terminal EP of CcmN interacts with the carboxysome shell.Here we describe a system for producing synthetic β-carboxysome shells and encapsulating nonnative cargo. We constructed a synthetic operon composed of ccmK1, ccmK2, ccmL, and ccmO, genes encoding, respectively, two BMC-H proteins, a BMC-P protein, and a BMC-T protein of the carboxysome shell of the halotolerant cyanobacterium, Halothece sp. PCC 7418 (Halo hereafter). Recombinant shells composed of all four proteins were produced and purified. We also demonstrated that the terminal α-helices of CcmK1 and CcmK2 are not, as had been proposed (Samborska and Kimber, 2012), required for the shell formation, and that the synthetic shell is a single-layered protein membrane. Cargo could be targeted to the interior of the synthetic shells using either the EP of CcmN or the N-terminal domain of CcmM; the latter observation provides new insight into the organization of the β-carboxysome. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly but also provide a platform to study carboxysome shell permeability. These results will be useful in guiding the design and optimization of carboxysomes and other BMCs for introduction into plants.     

Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic anhydrase, bicarbonate, and unstirred layers

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.