Types Of Carboxysomes Research Articles

Towards engineering a hybrid carboxysome

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 β-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 β-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.

A taxonomy of bacterial microcompartment loci constructed by a novel scoring method.

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications.

Engineering bacterial microcompartment shells: chimeric shell proteins and chimeric carboxysome shells.

Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorganic carbon fixation (carboxysomes) or organic carbon utilization (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. We undertook both approaches to altering shell properties using the carboxysome as a model system. There are two types of carboxysomes, α and β. In both, the predominant shell protein(s) contain a single copy of the BMC domain (pfam00936), but they are significantly different in primary structure. Indeed, phylogenetic analysis shows that the two types of carboxysome shell proteins are more similar to their counterparts in metabolosomes than to each other. We solved high resolution crystal structures of the major shell proteins, CsoS1 and CcmK2, and the presumed minor shell protein CcmK4, representing both types of cyanobacterial carboxysomes and then tested the interchangeability. The in vivo study presented here confirms that both engineering pores to mimic those of other shell proteins and the construction of chimeric shells is feasible.

Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria.

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.

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.

Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants

Cyanobacteria have evolved a significant environmental adaptation, known as a CO(2)-concentrating-mechanism (CCM), that vastly improves photosynthetic performance and survival under limiting CO(2) concentrations. The CCM functions to transport and accumulate inorganic carbon actively (Ci; HCO(3)(-), and CO(2)) within the cell where the Ci pool is utilized to provide elevated CO(2) concentrations around the primary CO(2)-fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). In cyanobacteria, Rubisco is encapsulated in unique micro-compartments known as carboxysomes. Cyanobacteria can possess up to five distinct transport systems for Ci uptake. Through database analysis of some 33 complete genomic DNA sequences for cyanobacteria it is evident that considerable diversity exists in the composition of transporters employed, although in many species this diversity is yet to be confirmed by comparative phenomics. In addition, two types of carboxysomes are known within the cyanobacteria that have apparently arisen by parallel evolution, and considerable progress has been made towards understanding the proteins responsible for carboxysome assembly and function. Progress has also been made towards identifying the primary signal for the induction of the subset of CCM genes known as CO(2)-responsive genes, and transcriptional regulators CcmR and CmpR have been shown to regulate these genes. Finally, some prospects for introducing cyanobacterial CCM components into higher plants are considered, with the objective of engineering plants that make more efficient use of water and nitrogen.

Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942

Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase–oxygenase (Rubisco). Sequencing of cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes. Cyanobacteria containing form 1A Rubisco possess α-carboxysomes, while those with form 1B Rubisco possess β-carboxysomes. Given the central importance of carboxysomes in the CO2-concentrating mechanism of cyanobacteria, understanding the nature and composition of these structures is of considerable importance. In an effort to develop techniques for the characterization of the structure of β-carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the Percoll–Mg2+ carboxysome enrichment technique using the freshwater cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorption–ionization – time of flight mass spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of 38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.Key words: CO2-concentrating mechanism, cyanobacteria, carboxysomes, proteomics.

Evolution and diversity of CO2 concentrating mechanisms in cyanobacteria.

Cyanobacteria have developed an effective photosynthetic CO2 concentrating mechanism (CCM) for improving the efficiency of carboxylation by a relatively inefficient Rubisco. The development of this CCM was presumably in response to the decline in atmospheric CO2 levels and rising O2, both of which were triggered by the development of oxygenic photosynthesis by cyanobacteria themselves. In the past few years there has been a rapid expansion in our understanding of the mechanism and genes responsible for the CCM. In addition, there has been a recent expansion in the availability of complete cyanobacterial genomes, thus increasing our potential to examine questions regarding both the evolution and diversity of components of the CCM across cyanobacteria. This paper considers various CCM and photosynthesis gene components across eight cyanobacteria where significant genomic information is available. Significant conclusions from our analysis of the distribution of various genes indicated the following. Firstly, cyanobacteria have developed with two types of carboxysomes, and this is correlated with the form of Rubisco present. We have coined the terms α-cyanobacteria to refer to cyanobacteria containing Form 1A Rubisco and α-carboxysomes, and β-cyanobacteria having Form 1B Rubisco and β-carboxysomes. Secondly, there are two NDH-1 CO2 uptake systems distributed variably, withProchlorococcus marinus species appearing to lack this CO2 uptake system. There are at least two HCO3- transport systems distributed variably, with some α-cyanobacteria having an absence of systems identified in β-cyanobacteria. Finally, there are multiple forms of carbonic anhydrases (CAs), but with only β-carboxysomal CA having a clearly shown role at present. The α-cyanobacteria appear to lack a clearly identifiable carboxysomal CA. A pathway for the evolution of cyanobacterial CCMs is proposed. The acquisition of carboxysomes triggered by the rapid decline of atmospheric CO2 in the Phanerozoic is argued to be the initial step. This would then be followed by the development of NDH-1 CO2-uptake systems, followed by the development of low-and high-affinity HCO3- transporters. An intriguing question is, were carboxysomes developed first in cyanobacteria, or did they originate by the lateral transfer of pre-existing proteobacterial bacterial microcompartment genes? The potentially late evolution of the CCM genes in cyanobacteria argues for a polyphyletic and separate evolution of CCMs in cyanobacteria, algae, and higher plants.

The response of Synechococcus PCC 7942 (Cyanophyta) to changes in CO 2supply in relation to the acclimation of the CO 2-concentrating mechanism. II. carboxysomes

Distinct types of carboxysomes were distinguished in Synechococcus PCC 7942: electron-clear, electron-intermediate, carboxysomes with internal electron-clear areas, typical electron-dense and bar-shaped carboxysomes. Immunogold location with antibodies against the Rubisco large subunit showed specific label in all carboxysomes. The positive correlation between electron-density, the density of immunogold label, and the percentage of labeled structures within each type support a model of carboxysome biogenesis whereby electron-clear evolve to electron-intermediate and then to electron-dense carboxysomes by the progressive sequestering of Rubisco molecules. Cells responded to limitation in CO 2 supply by increasing carboxysome frequency and the proportion of typical electron-dense carboxysomes, the extent of the response depending on the degree of limitation. The time course of carboxysome expression during transfers between different conditions of CO 2 supply indicated that, under our experimental conditions, there were different levels of response, depending on the degree of limitation. The first level occured at atmospheric levels of CO 2 and involved changes in the affinity of the CCM and in carboxysome, which occurred simultaneously. More severe limitation of CO 2 supply affected carboxysomes exclusively, without further improvement in the affinity of the CCM.