Benedict M. Long

Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate.

ABSTRACT Cell quotas of microcystin (QMCYST; femtomoles of MCYST per cell), protein, and chlorophyll a(Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship betweenQMCYST and specific growth rate (μ), from which we propose a generalized model that enablesQMCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts QMCYST from μ, μmax(maximum specific growth rate), QMCYSTmax(maximum cell quota), and QMCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict aQMCYSTmax of 0.129 fmol cell−1 at μmax and a QMCYSTmin of 0.050 fmol cell−1 at μ = 0. Net MCYST production rate (RMCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell−1 day−1at μmax. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chla ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.

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

In cyanobacteria, the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. Cyanobacteria with Form IB Rubisco produce β-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 β-carboxysome structure. In an effort to identify such interactions within the carboxysomes of the β-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 L8S8 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 CO2-requiring ccmM deletion mutant (ΔccmM) can achieve nearly normal growth rates at ambient CO2 after complementation with both wild type and chimeric (His6-tagged) forms of CcmM. Although a significant amount of independent L8S8 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.

Functions, Compositions, and Evolution of the Two Types of Carboxysomes: Polyhedral Microcompartments That Facilitate CO2 Fixation in Cyanobacteria and Some Proteobacteria

SUMMARY Cyanobacteria are the globally dominant photoautotrophic lineage. Their success is dependent on a set of adaptations collectively termed the CO2-concentrating mechanism (CCM). The purpose of the CCM is to support effective CO2 fixation by enhancing the chemical conditions in the vicinity of the primary CO2-fixing enzyme, d-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to promote the carboxylase reaction and suppress the oxygenase reaction. In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within carboxysomes, which are examples of a group of proteinaceous bodies called bacterial microcompartments. Carboxysomes encapsulate the CO2-fixing enzyme within the selectively permeable protein shell and simultaneously encapsulate a carbonic anhydrase enzyme for CO2 supply from a cytoplasmic bicarbonate pool. These bodies appear to have arisen twice and undergone a process of convergent evolution. While the gross structures of all known carboxysomes are ostensibly very similar, with shared gross features such as a selectively permeable shell layer, each type of carboxysome encapsulates a phyletically distinct form of RubisCO enzyme. Furthermore, the specific proteins forming structures such as the protein shell or the inner RubisCO matrix are not identical between carboxysome types. Each type has evolutionarily distinct forms of the same proteins, as well as proteins that are entirely unrelated to one another. In light of recent developments in the study of carboxysome structure and function, we present this review to summarize the knowledge of the structure and function of both types of carboxysome. We also endeavor to cast light on differing evolutionary trajectories which may have led to the differences observed in extant carboxysomes.

Functional Cyanobacterial β-Carboxysomes Have an Absolute Requirement for Both Long and Short Forms of the CcmM Protein

Carboxysomes are an essential part of the cyanobacterial CO2-concentrating mechanism, consisting of a protein shell and an interior of Rubisco. The β-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 γ-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, ΔccmM. Expression of wild-type ccmM in ΔccmM 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.

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

Cyanobacterial CO2-fixation is supported by a CO2-concentrating mechanism which improves photosynthesis by saturating the primary carboxylating enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), with its preferred substrate CO2. The site of CO2-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-CO2-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 CO2 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.

Cyanobacterial carboxysomes: microcompartments that facilitate CO2 fixation.

Carboxysomes are extraordinarily efficient proteinaceous microcompartments that encapsulate the primary CO2-fixing enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) in cyanobacteria and some proteobacteria. These microbodies form part of a CO2-concentrating mechanism (CCM), operating together with active CO2 and HCO3- uptake transporters which accumulate HCO3- in the cytoplasm of the cell. Cyanobacteria (also known as blue-green algae) are highly productive on a global scale, especially those species from open-ocean niches, which collectively contribute nearly 30% of global net primary fixation. This productivity would not be possible without a CCM which is dependent on carboxysomes. Two evolutionarily distinct forms of carboxysome are evident that encapsulate proteobacterial RuBisCO form-1A or higher-plant RuBisCO form- 1B, respectively. Based partly on RuBisCO phylogeny, the two carboxysome types are known either as α-carboxysomes, found in predominantly oceanic cyanobacteria (α-cyanobacteria) and some proteobacteria, or as β-carboxysomes, found mainly in freshwater/estuarine cyanobacteria (β-cyanobacteria). Both carboxysome types are believed to have evolved in parallel as a consequence of fluctuating atmospheric CO2 levels and evolutionary pressure acting via the poor enzymatic kinetics of RuBisCO. The three-dimensional structures and protein components of each carboxysome type reflect distinct evolutionarily strategies to the same major functions: subcellular compartmentalization and RuBisCO encapsulation, oxygen exclusion, and CO2 concentration and fixation.

Over-expression of the β-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 CO2-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% CO2-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.

Comparing the in Vivo Function of α-Carboxysomes and β-Carboxysomes in Two Model Cyanobacteria

Despite evolutionary and structural differences between carboxysomes, Rubisco kinetics and in vivo performance are similar. 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 β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-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 β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3−) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-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.

Leaf-level photosynthetic capacity in lowland Amazonian and high-elevation Andean tropical moist forests of Peru.

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.

Cyanobacterial CO2-concentrating mechanism components: function and prospects for plant metabolic engineering.

Global population growth is projected to outpace plant-breeding improvements in major crop yields within decades. To ensure future food security, multiple creative efforts seek to overcome limitations to crop yield. Perhaps the greatest limitation to increased crop yield is photosynthetic inefficiency, particularly in C3 crop plants. Recently, great strides have been made toward crop improvement by researchers seeking to introduce the cyanobacterial CO2-concentrating mechanism (CCM) into plant chloroplasts. This strategy recognises the C3 chloroplast as lacking a CCM, and being a primordial cyanobacterium at its essence. Hence the collection of solute transporters, enzymes, and physical structures that make cyanobacterial CO2-fixation so efficient are viewed as a natural source of genetic material for C3 chloroplast improvement. Also we highlight recent outstanding research aimed toward the goal of introducing a cyanobacterial CCM into C3 chloroplasts and consider future research directions.