RESUMEN
Synthetically reconstructed carboxysomes form the basis of CO2-concentrating mechanisms (CCMs) that could enhance the photosynthetic efficiency of crops and improve yield. Recently, Chen et al. revealed another step toward the reconstruction of bacterial carboxysomes in plants, reporting the formation of almost-complete carboxysomes in the chloroplast of Nicotiana tabacum.
Asunto(s)
Cianobacterias , Dióxido de Carbono , Ribulosa-Bifosfato Carboxilasa , Orgánulos , CloroplastosRESUMEN
The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.
Asunto(s)
Dióxido de Carbono , Cloroplastos , Productos Agrícolas , Ribulosa-Bifosfato Carboxilasa , Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Fotosíntesis/fisiología , Cianobacterias/metabolismo , Cianobacterias/fisiología , Cianobacterias/genética , Plantas Modificadas GenéticamenteRESUMEN
The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacterial CO2-concentrating mechanism, could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.
RESUMEN
Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3-, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3- accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3- accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.
Asunto(s)
Ciclo del Carbono/genética , Dióxido de Carbono/metabolismo , Fotosíntesis/genética , Ribulosa-Bifosfato Carboxilasa/química , Synechococcus/genética , Carbono/química , Carbono/metabolismo , Dióxido de Carbono/química , Anhidrasas Carbónicas , Orgánulos/metabolismo , Proteobacteria/química , Proteobacteria/metabolismo , Protones , Ribulosa-Bifosfato Carboxilasa/metabolismo , Synechococcus/química , Synechococcus/metabolismoRESUMEN
Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the ß-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta, work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in ß-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.
Asunto(s)
Anhidrasas Carbónicas , Cianobacterias , Ribulosa-Bifosfato Carboxilasa/metabolismo , Orgánulos/metabolismo , Cloroplastos/metabolismo , Cianobacterias/metabolismo , Anhidrasas Carbónicas/metabolismo , Dióxido de Carbono/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.
Asunto(s)
Nitrógeno , Agua , AustraliaRESUMEN
We examined whether variations in photosynthetic capacity are linked to variations in the environment and/or associated leaf traits for tropical moist forests (TMFs) in the Andes/western Amazon regions of Peru. We compared photosynthetic capacity (maximal rate of carboxylation of Rubisco (Vcmax ), and the maximum rate of electron transport (Jmax )), leaf mass, nitrogen (N) and phosphorus (P) per unit leaf area (Ma , Na and Pa , respectively), and chlorophyll from 210 species at 18 field sites along a 3300-m elevation gradient. Western blots were used to quantify the abundance of the CO2 -fixing enzyme Rubisco. Area- and N-based rates of photosynthetic capacity at 25°C were higher in upland than lowland TMFs, underpinned by greater investment of N in photosynthesis in high-elevation trees. Soil [P] and leaf Pa were key explanatory factors for models of area-based Vcmax and Jmax but did not account for variations in photosynthetic N-use efficiency. At any given Na and Pa , the fraction of N allocated to photosynthesis was higher in upland than lowland species. For a small subset of lowland TMF trees examined, a substantial fraction of Rubisco was inactive. These results highlight the importance of soil- and leaf-P in defining the photosynthetic capacity of TMFs, with variations in N allocation and Rubisco activation state further influencing photosynthetic rates and N-use efficiency of these critically important forests.
Asunto(s)
Altitud , Bosques , Humedad , Fotosíntesis/fisiología , Hojas de la Planta/fisiología , Clima Tropical , Dióxido de Carbono/metabolismo , Pruebas de Enzimas , Cinética , Modelos Biológicos , Nitrógeno/metabolismo , Perú , Hojas de la Planta/anatomía & histología , Hojas de la Planta/química , Ribulosa-Bifosfato Carboxilasa/metabolismo , Especificidad de la Especie , TemperaturaRESUMEN
Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.
Asunto(s)
Cianobacterias/genética , Embryophyta/genética , Fotosíntesis , Plantas Modificadas Genéticamente/genética , Biofisica , Dióxido de Carbono/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Understanding of the extent of acclimation of light-saturated net photosynthesis (An ) to temperature (T), and associated underlying mechanisms, remains limited. This is a key knowledge gap given the importance of thermal acclimation for plant functioning, both under current and future higher temperatures, limiting the accuracy and realism of Earth system model (ESM) predictions. Given this, we analysed and modelled T-dependent changes in photosynthetic capacity in 10 wet-forest tree species: six from temperate forests and four from tropical forests. Temperate and tropical species were each acclimated to three daytime growth temperatures (Tgrowth ): temperate - 15, 20 and 25 °C; tropical - 25, 30 and 35 °C. CO2 response curves of An were used to model maximal rates of RuBP (ribulose-1,5-bisphosphate) carboxylation (Vcmax ) and electron transport (Jmax ) at each treatment's respective Tgrowth and at a common measurement T (25 °C). SDS-PAGE gels were used to determine abundance of the CO2 -fixing enzyme, Rubisco. Leaf chlorophyll, nitrogen (N) and mass per unit leaf area (LMA) were also determined. For all species and Tgrowth , An at current atmospheric CO2 partial pressure was Rubisco-limited. Across all species, LMA decreased with increasing Tgrowth . Similarly, area-based rates of Vcmax at a measurement T of 25 °C (Vcmax25 ) linearly declined with increasing Tgrowth , linked to a concomitant decline in total leaf protein per unit leaf area and Rubisco as a percentage of leaf N. The decline in Rubisco constrained Vcmax and An for leaves developed at higher Tgrowth and resulted in poor predictions of photosynthesis by currently widely used models that do not account for Tgrowth -mediated changes in Rubisco abundance that underpin the thermal acclimation response of photosynthesis in wet-forest tree species. A new model is proposed that accounts for the effect of Tgrowth -mediated declines in Vcmax25 on An , complementing current photosynthetic thermal acclimation models that do not account for T sensitivity of Vcmax25 .
Asunto(s)
Aclimatación , Bosques , Fotosíntesis , Dióxido de Carbono , Hojas de la Planta , Ribulosa-Bifosfato Carboxilasa , ÁrbolesRESUMEN
The carbon dioxide (CO2)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO2 concentrations at the site of fixation, providing an advantage in low-CO2 environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain ß-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and ß-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and ß-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3-) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and ß-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external Ci and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and Ci pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic Ci, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm Ci. This finding has bearing on the viability of transplanting functional carboxysomes into the C3 chloroplast.
Asunto(s)
Dióxido de Carbono/metabolismo , Cianobacterias/metabolismo , Orgánulos/metabolismo , Bicarbonatos/metabolismo , Carbono/farmacología , Cianobacterias/efectos de los fármacos , Cianobacterias/efectos de la radiación , Cianobacterias/ultraestructura , Ácidos Glicéricos/metabolismo , Cinética , Luz , Espectrometría de Masas , Orgánulos/efectos de los fármacos , Orgánulos/efectos de la radiación , Fotosíntesis/efectos de los fármacos , Fotosíntesis/efectos de la radiación , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosafosfatos/metabolismo , Synechococcus/efectos de los fármacos , Synechococcus/metabolismo , Synechococcus/efectos de la radiación , Synechococcus/ultraestructuraRESUMEN
In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia fabaâ L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.
Asunto(s)
Fotosíntesis , Células Vegetales/fisiología , Temperatura , Vicia faba/metabolismo , Respiración de la Célula , Proteínas Mitocondriales/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Vicia faba/citología , Vicia faba/crecimiento & desarrolloRESUMEN
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 ß-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 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) 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.
Asunto(s)
Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Nostoc/enzimología , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
In this issue of Structure, Kong et al. utilized cryoelectron tomography to closely examine Rubisco packaging within ß-carboxysomes. They observed unique Rubisco packaging arrangements that may have important implications for carboxysome structural integrity.
Asunto(s)
Microscopía por Crioelectrón , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Tomografía con Microscopio Electrónico , Modelos MolecularesRESUMEN
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.
Asunto(s)
Anhidrasas Carbónicas , Cianobacterias , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/genética , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/química , Cianobacterias/metabolismo , Cianobacterias/genética , Cianobacterias/enzimología , Regulación Alostérica , Filogenia , Ribulosafosfatos/metabolismo , Modelos Moleculares , Multimerización de Proteína , Dióxido de Carbono/metabolismo , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
The carboxysome is a versatile paradigm of prokaryotic organelles and is a proteinaceous self-assembling microcompartment that plays essential roles in carbon fixation in all cyanobacteria and some chemoautotrophs. The carboxysome encapsulates the central CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), using a polyhedral protein shell that is selectively permeable to specific metabolites in favor of Rubisco carboxylation. There is tremendous interest in repurposing carboxysomes to boost carbon fixation in heterologous organisms. Here, we develop the design and engineering of α-carboxysomes by coexpressing the Rubisco activase components CbbQ and CbbO with α-carboxysomes in Escherichia coli. Our results show that CbbQ and CbbO could assemble into the reconstituted α-carboxysome as intrinsic components. Incorporation of both CbbQ and CbbO within the carboxysome promotes activation of Rubisco and enhances the CO2-fixation activities of recombinant carboxysomes. We also show that the structural composition of these carboxysomes could be modified in different expression systems, representing the plasticity of the carboxysome architecture. In translational terms, our study informs strategies for engineering and modulating carboxysomes in diverse biotechnological applications.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa , Activador de Tejido Plasminógeno , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciclo del Carbono , Dióxido de Carbono/metabolismo , Orgánulos/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Carboxysomes are an essential part of the cyanobacterial CO2-concentrating mechanism, consisting of a protein shell and an interior of Rubisco. The beta-carboxysome shell protein CcmM forms two peptides via a proposed internal ribosomal entry site (IRES) within the ccmM transcript in Synechococcus PCC7942. The abundant short form (35 kD, M35) consists of Rubisco small subunit-like repeats and binds Rubisco. The lower abundance long form (58 kD, M58) also contains a gamma-carbonic anhydrase-like domain, which binds the carboxysomal carbonic anhydrase, CcaA. We examined whether these CcmM forms arise via an IRES or by other means. Mutations of a putative internal start codon (GTG) and Shine-Dalgarno sequence within ccmM, along with a gene coding for M35 alone, were examined in the high-CO2-requiring (HCR) carboxysomeless mutant, DeltaccmM. Expression of wild-type ccmM in DeltaccmM restored the wild-type phenotype, while mutation of putative start and Shine-Dalgarno sequences led to as much as 20-fold reduction in M35 content with no recovery from HCR phenotype. These cells also contained small electron-dense structures. Cells producing little or no M58, but sufficient M35, were found to contain large electron-dense structures, no CcaA, and had a HCR phenotype. Large subcellular aggregates can therefore form in the absence of M58, suggesting a role for M35 in internal carboxysome Rubisco packing. The results confirm that M35 is independently translated via an IRES within ccmM. Importantly, the data reveal that functional carboxysomes require both M35 and M58 in sufficient quantities and with a minimum stoichiometry of close to 1:1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Synechococcus/metabolismo , Codón Iniciador , Synechococcus/genéticaRESUMEN
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 ß-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the ß-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 ß-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the ß-carboxysome. A modified ß-carboxysome packing model is presented.