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[This corrects the article DOI: 10.3389/fmicb.2022.872708.].

Autotrophic bacteria are able to fix CO2 in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO2 fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO2-concentrating mechanisms (CCMs). CCMs consist of CO2 and HCO3- transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophic Bacteria that use the Calvin-Benson-Basham cycle. Members of the genus Thiomicrospira lack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO2 fixation by carboxysomes from members of Thiomicrospira and potentially other Bacteria. Carboxysome enrichments from Thiomicrospira pelophila and Thiomicrospira aerophila were found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted in T. pelophila, cells could no longer grow under low-CO2 conditions, and CA activity was no longer detectable in their carboxysomes. When T. pelophila ιCA was expressed in a strain of Escherichia coli lacking native CA activity, this strain recovered an ability to grow under low CO2 conditions, and CA activity was present in crude cell extracts prepared from this strain. [ABSTRACT FROM AUTHOR]

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco—the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s−1 median versus <7 s−1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature. Synopsis: Form I rubisco, known for its slow kinetics in plants and algae, exhibits a great unexplored diversity in autotrophic bacteria. This article represents the first large-scale survey of bacterial form I rubisco kinetics and reveals unifying features of fast carboxylating rubiscos. Over 100 homologs were systematically screened, spanning the wide genetic diversity of form I rubisco enzymes across a variety of ecological niches and metabolic profiles. Phototrophy and carboxysome association are correlated with fast-carboxylating rubiscos. α-cyanobacteria emerges as the bacterial clade expressing the fastest form I rubiscos on Earth. Bacterial rubisco enzymes associated with carboxysomes have the fastest CO2-fixing rates. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: The efficacy of the cyanobacterial CO2‐concentrating mechanism (CCM) relies on its ability to concentrate CO2 around the Rubisco enzyme, which is natively encapsulated within a carboxysome. As our understanding of carboxysome biogenesis and functionality grows, there is optimism that this microcompartment and the cyanobacterial CCM as a whole, can contribute to addressing global food security. [ABSTRACT FROM AUTHOR]

ABSTRACT Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes.IMPORTANCECarboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications.

Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a "do-it yourself" analysis for the public we have devised a webserver, BMC Caller ( https://bmc-caller.prl.msu.edu ), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism's metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria, primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3 - in the cytoplasm via active transport, HCO3 - enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3 - to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: a-carboxysomes and ß-carboxysomes, which arose independently from ancestral microcompartments. The a-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and a-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some a-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysomerelated genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts. [ABSTRACT FROM AUTHOR]

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803. [ABSTRACT FROM AUTHOR]

The Calvin-Benson cycle (CBC) evolved over 2 billion years ago but has been subject to massive selection due to falling atmospheric carbon dioxide, rising atmospheric oxygen and changing nutrient and water availability. In addition, large groups of organisms have evolved carbon-concentrating mechanisms (CCMs) that operate upstream of the CBC. Most previous studies of CBC diversity focused on Rubisco kinetics and regulation. Quantitative metabolite profiling provides a top-down strategy to uncover inter-species diversity in CBC operation. CBC profiles were recently published for twenty species including terrestrial C 3 species, terrestrial C 4 species that operate a biochemical CCM, and cyanobacteria and green algae that operate different types of biophysical CCM. Distinctive profiles were found for species with different modes of photosynthesis, revealing that evolution of the various CCMs was accompanied by co-evolution of the CBC. Diversity was also found between species that share the same mode of photosynthesis, reflecting lineage-dependent diversity of the CBC. Connectivity analysis uncovers constraints due to pathway and thermodynamic topology, and reveals that cross-species diversity in the CBC is driven by changes in the balance between regulated enzymes and in the balance between the CBC and the light reactions or end-product synthesis. • Calvin-Benson cycle metabolites were profiled in 20 species. • Calvin-Benson cycle has co-evolved with CO 2 -concentrating mechanisms. • Substantial diversity between species with the same photosynthesis mode. [ABSTRACT FROM AUTHOR]

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO2-concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation. [ABSTRACT FROM AUTHOR]

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria, primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3 - in the cytoplasm via active transport, HCO3 - enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3 - to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and β-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts.

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO2-concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

Summary: The carboxysome plays an essential role in the carbon concentration mechanism in cyanobacteria. Although significant progress has been made in the structural analysis of the carboxysome, little is still known about its biosynthesis.We identified slr1911, a gene encoding a protein of unknown function in cyanobacterium Synechocystis sp. Strain PCC 6803 (Syn6803), which we termed ccmS by screening a low CO2‐sensitive mutant. CcmS interacts with CcmK1 and CcmM. The former is a shell protein of the β‐carboxysome and the latter is a crucial component of the β‐carboxysome, which is responsible for aggregating RuBisCO and recruiting shell proteins.The deletion of ccmS lowers the accumulation and assembly of CcmK1, resulting in aberrant carboxysomes, suppressed photosynthetic capacities, and leads to a slow growth phenotype, especially under CO2‐limited conditions.These observations suggest that CcmS stabilizes the assembly of the β‐carboxysome shell and likely connects the carboxysome core with the shell. Our results provide a molecular view of the role played by CcmS in the formation of the β‐carboxysome and its function in Syn6803. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The intracellular accumulation of inorganic carbon (Ci) by microalgae and cyanobacteria under ambient atmospheric CO2 levels was first documented in the 80s of the 20th Century. Hence, a third variety of the CO2-concentrating mechanism (CCM), acting in aquatic photoautotrophs with the C3 photosynthetic pathway, was revealed in addition to the then-known schemes of CCM, functioning in CAM and C4 higher plants. Despite the low affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of microalgae and cyanobacteria for the CO2 substrate and low CO2/O2 specificity, CCM allows them to perform efficient CO2 fixation in the reductive pentose phosphate (RPP) cycle. CCM is based on the coordinated operation of strategically located carbonic anhydrases and CO2/HCO3− uptake systems. This cooperation enables the intracellular accumulation of HCO3−, which is then employed to generate a high concentration of CO2 molecules in the vicinity of Rubisco's active centers compensating up for the shortcomings of enzyme features. CCM functions as an add-on to the RPP cycle while also acting as an important regulatory link in the interaction of dark and light reactions of photosynthesis. This review summarizes recent advances in the study of CCM molecular and cellular organization in microalgae and cyanobacteria, as well as the fundamental principles of its functioning and regulation. [ABSTRACT FROM AUTHOR]

Abstract Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a “do-it yourself” analysis for the public we have devised a webserver, BMC Caller ( https://bmc-caller.prl.msu.edu ), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism’s metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

Cyanobacteria mostly rely on the active uptake of hydrated CO2 (i.e., bicarbonate ions) from the surrounding media to fuel their inorganic carbon assimilation. The dehydration of bicarbonate in close vicinity of RuBisCO is achieved through the activity of carboxysomal carbonic anhydrase (CA) enzymes. Simultaneously, many cyanobacterial genomes encode extracellular α- and β-class CAs (EcaA, EcaB) whose exact physiological role remains largely unknown. To date, the CahB1 enzyme of Sodalinema gerasimenkoae (formerly Microcoleus/Coleofasciculus chthonoplastes) remains the sole described active extracellular β-CA in cyanobacteria, but its molecular features strongly suggest it to be a carboxysomal rather than a secreted protein. Upon expression of CahB1 in Synechocystis sp. PCC6803, we found that its expression complemented the loss of endogenous CcaA. Moreover, CahB1 was found to localize to a carboxysome-harboring and CA-active cell fraction. Our data suggest that CahB1 retains all crucial properties of a cellular carboxysomal CA and that the secretion mechanism and/or the machinations of the Sodalinema gerasimenkoae carboxysome are different from those of Synechocystis. [ABSTRACT FROM AUTHOR]

Aquatic autotrophs that fix carbon using ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) frequently expend metabolic energy to pump inorganic carbon towards the enzyme's active site. A central requirement of this strategy is the formation of highly concentrated Rubisco condensates (or Rubiscondensates) known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes, respectively. Recent data indicate that these condensates form by the mechanism of liquid–liquid phase separation. This mechanism requires networks of weak multivalent interactions typically mediated by intrinsically disordered scaffold proteins. Here we comparatively review recent rapid developments that detail the determinants and precise interactions that underlie diverse Rubisco condensates. The burgeoning field of biomolecular condensates has few examples where liquid–liquid phase separation can be linked to clear phenotypic outcomes. When present, Rubisco condensates are essential for photosynthesis and growth, and they are thus emerging as powerful and tractable models to investigate the structure–function relationship of phase separation in biology. [ABSTRACT FROM AUTHOR]

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110, has three ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isoenzymes, CbbM, CbbLS-1, and CbbLS-2, which differ in CO 2 /O 2 specificity factor values. Expressions of CbbM and CbbLS-1 are regulated differently by transcriptional regulators of the LysR family, CbbRm and CbbR1, respectively. CbbLS-2 has the highest specificity and is induced under low CO 2 conditions, but the regulator for the cbbL2S2 genes encoding CbbLS-2 remains unidentified. In this study, the cbbR2 gene encoding the third CbbR-type regulator was identified in the downstream region of the cbbL2S2 and carboxysome gene cluster via transposon mutagenesis. CO 2 depletion induced the cbbR2 gene. The cbbR2 knockout mutant could not grow under low CO 2 conditions and did not produce CbbLS-2. Recombinant CbbR2 protein was bound to the promoter region of the cbbL2S2 genes. These results indicate that CbbR2 is the specific regulator for CbbLS-2 expression. [ABSTRACT FROM AUTHOR]

Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer (QconCAT)-based quantitative mass spectrometry, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the central enzyme for converting atmospheric CO 2 into organic molecules, playing a crucial role in the global carbon cycle. In cyanobacteria and some chemoautotrophs, Rubisco complexes, along with carbonic anhydrase, are enclosed within specific proteinaceous microcompartments, known as carboxysomes. The polyhedral carboxysome shell ensures a dense packaging of Rubisco and creates a high-CO 2 internal environment to facilitate the fixation of CO 2 . Rubisco and carboxysomes have been popular targets for bioengineering, with the intent of enhancing plant photosynthesis, crop yields, and biofuel production. However, efficient generation of Form 1B Rubisco and cyanobacterial β-carboxysomes in heterologous systems remains challenging. Here, we developed genetic systems to efficiently engineer functional cyanobacterial Form 1B Rubisco in E. coli, by incorporating Rubisco assembly factor Raf1 and modulating the RbcL/S stoichiometry. We further accomplished effective reconstitution of catalytically active β-carboxysomes in E. coli with cognate Form 1B Rubisco by fine-tuning the expression levels of individual β-carboxysome components. In addition, we investigated the encapsulation mechanism of Rubisco into carboxysomes via constructing hybrid carboxysomes; this was achieved by creating a chimeric encapsulation peptide incorporating SSLDs that permits the encapsulation of Form 1B Rubisco into α-carboxysome shells. Our study provides insights into the assembly mechanisms of plant-like Form 1B Rubisco and its encapsulation principles in both β-carboxysomes and hybrid carboxysomes, and highlights the inherent modularity of carboxysome structures. The findings lay the framework for rational design and repurposing of CO 2 -fixing modules in bioengineering applications, e.g. crop engineering, biocatalyst production, and molecule delivery., (Copyright © 2024. Published by Elsevier Inc.)

Carboxysomes are a family of bacterial microcompartments (BMCs), present in all cyanobacteria and some proteobacteria, which encapsulate the primary CO 2 -fixing enzyme, Rubisco, within a virus-like polyhedral protein shell. Carboxysomes provide significantly elevated levels of CO 2 around Rubisco to maximize carboxylation and reduce wasteful photorespiration, thus functioning as the central CO 2 -fixation organelles of bacterial CO 2 -concentration mechanisms. Their intriguing architectural features allow carboxysomes to make a vast contribution to carbon assimilation on a global scale. In this review, we discuss recent research progress that provides new insights into the mechanisms of how carboxysomes are assembled and functionally maintained in bacteria and recent advances in synthetic biology to repurpose the metabolic module in diverse applications. CO 2 -concentrating mechanisms (CCMs) provide a means for accumulating CO 2 around Rubisco to overcome the inherent limitations of Rubisco and enhance CO 2 fixation. Carboxysomes are proteinaceous organelles in cyanobacteria and some proteobacteria which serve as the central CO 2 -fixing factory of CCMs. Carboxysomes sequester the cellular Rubisco and carbonic anhydrase from the cytoplasm, using a selectively permeable shell that structurally resembles virus capsids. Great efforts have been made recently to advance our understanding of the molecular mechanisms underlying carboxysome structure, assembly, biogenesis, and physiology. Advances in fundamental knowledge about carboxysome assembly and function has stimulated rational design and engineering of the protein organelles for improving CO 2 fixation and new functions. [ABSTRACT FROM AUTHOR]

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria , primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3− in the cytoplasm via active transport, HCO3− enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3− to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and β-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts. [ABSTRACT FROM AUTHOR]

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria, primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3− in the cytoplasm via active transport, HCO3− enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3− to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and β-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts.

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

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes., Importance: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO 2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications., Competing Interests: The authors declare no conflict of interest.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO 2 -fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire β-carboxysome. This study and recent findings on the β-carboxysome structure provide insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications., Competing Interests: Competing Interests The authors declare no conflict of interest.

All cyanobacteria and some chemoautotrophic bacteria fix CO 2 into sugars using specialized proteinaceous compartments called carboxysomes. Carboxysomes enclose the enzymes Rubisco and carbonic anhydrase inside a layer of shell proteins to increase the CO 2 concentration for efficient carbon fixation by Rubisco. In the ⍺-carboxysome lineage, a disordered and highly repetitive protein named CsoS2 is essential for carboxysome formation and function. Without it, the bacteria require high CO 2 to grow. How does a protein predicted to be lacking structure serve as the architectural scaffold for such a vital cellular compartment? In this study, we identify key residues present in the repeats of CsoS2, VTG and Y, which are necessary for building functional ⍺-carboxysomes in vivo. These highly conserved and repetitive residues contribute to the multivalent binding interaction and phase separation behavior between CsoS2 and shell proteins. We also demonstrate 3-component reconstitution of CsoS2, Rubisco, and shell proteins into spherical condensates and show the utility of reconstitution as a biochemical tool to study carboxysome biogenesis. The precise self-assembly of thousands of proteins is crucial for carboxysome formation, and understanding this process could enable their use in alternative biological hosts or industrial processes as effective tools to fix carbon., Competing Interests: Conflict of interest D. F. S. is a co-founder and scientific advisory board member of Scribe Therapeutics. All other authors declare that thye have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This document is a corrigendum for an article titled "Atypical carboxysome loci: JEEPs or junk?" published in Frontiers in Microbiology. The corrigendum addresses an error in the acknowledgments section of the original article, where the names of the students who contributed to the manuscript were incorrectly listed. The corrected names of the students are provided in the corrigendum. The authors apologize for the error and state that it does not affect the scientific conclusions of the article. [Extracted from the article]

Cyanobacteria mostly rely on the active uptake of hydrated CO2 (i.e., bicarbonate ions) from the surrounding media to fuel their inorganic carbon assimilation. The dehydration of bicarbonate in close vicinity of RuBisCO is achieved through the activity of carboxysomal carbonic anhydrase (CA) enzymes. Simultaneously, many cyanobacterial genomes encode extracellular α- and β-class CAs (EcaA, EcaB) whose exact physiological role remains largely unknown. To date, the CahB1 enzyme of Sodalinema gerasimenkoae (formerly Microcoleus/Coleofasciculus chthonoplastes) remains the sole described active extracellular β-CA in cyanobacteria, but its molecular features strongly suggest it to be a carboxysomal rather than a secreted protein. Upon expression of CahB1 in Synechocystis sp. PCC6803, we found that its expression complemented the loss of endogenous CcaA. Moreover, CahB1 was found to localize to a carboxysome-harboring and CA-active cell fraction. Our data suggest that CahB1 retains all crucial properties of a cellular carboxysomal CA and that the secretion mechanism and/or the machinations of the Sodalinema gerasimenkoae carboxysome are different from those of Synechocystis.

The introduction of the carboxysome-based CO 2 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., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Protein nanocages have emerged as promising candidates for enzyme immobilization and cargo delivery in biotechnology and nanotechnology. Carboxysomes are natural proteinaceous organelles in cyanobacteria and proteobacteria and have exhibited great potential in creating versatile nanocages for a wide range of applications given their intrinsic characteristics of self-assembly, cargo encapsulation, permeability, and modularity. However, how to program intact carboxysome shells with specific docking sites for tunable and efficient cargo loading is a key question in the rational design and engineering of carboxysome-based nanostructures. Here, we generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and Coiled-coil protein coupling systems. The systematic analysis demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. Our study provides insights into the encapsulation principles of the α-carboxysome and establishes a solid foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, thereby enabling applications in catalysis, delivery, and medicine.

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO 2 concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO 2 fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO 2 . This creates a high CO 2 concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO 2 hydration. This method allows us to determine the kinetic parameters k cat , K M and k cat /K M for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO 2 and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well., (Copyright © 2024. Published by Elsevier Inc.)

Carboxysomes are proteinaceous bacterial microcompartments that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating several enzymes, including ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural understanding of native carboxysomes is currently limited to low-resolution studies. Here, we report the characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryoelectron microscopy (cryo-EM). We have determined the structure of its RuBisCO enzyme, and obtained low-resolution maps of its icosahedral shell, and of its concentric interior organization. Using integrative modeling approaches, we have proposed a complete atomic model of an intact carboxysome, providing insight into its organization and assembly. This is critical for a better understanding of the carbon fixation mechanism and toward repurposing carboxysomes in synthetic biology for biotechnological applications. [Display omitted] • Carboxysomes are prokaryotic microcompartments that sequester photosynthetic enzymes • We used single-particle cryo-EM analysis to characterize intact carboxysome complexes • We have proposed a model of how enzymes are arranged within carboxysomes • We also have generated a structural model of its shell architecture Carboxysomes are proteinaceous microcompartments found in cyanobacteria, which facilitate carbon fixation, the first step of photosynthesis, by sequestering the corresponding enzymes. Evans et al. used cryoelectron microscopy to characterize intact carboxysomes, which allowed them to establish a structural model for the carboxysome shell and for its internal enzyme organization. [ABSTRACT FROM AUTHOR]

Aquatic autotrophs that fix carbon using ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) frequently expend metabolic energy to pump inorganic carbon towards the enzyme's active site. A central requirement of this strategy is the formation of highly concentrated Rubisco condensates (or Rubiscondensates) known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes, respectively. Recent data indicate that these condensates form by the mechanism of liquid-liquid phase separation. This mechanism requires networks of weak multivalent interactions typically mediated by intrinsically disordered scaffold proteins. Here we comparatively review recent rapid developments that detail the determinants and precise interactions that underlie diverse Rubisco condensates. The burgeoning field of biomolecular condensates has few examples where liquid-liquid phase separation can be linked to clear phenotypic outcomes. When present, Rubisco condensates are essential for photosynthesis and growth, and they are thus emerging as powerful and tractable models to investigate the structure-function relationship of phase separation in biology., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO 2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO 3 ions into the carboxysome, where they are converted to CO - by CA. Thus, the carboxysome functions as a CO 2 by CA. Thus, the carboxysome functions as a CO 2 fixation. In β-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the β-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions.2 fixation. In β-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the β-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cyanobacteria are photosynthetic microorganisms that play important ecological roles as major contributors to global nutrient cycles. Cyanobacteria are highly efficient in carrying out oxygenic photosynthesis because they possess carboxysomes, a class of bacterial microcompartments (BMC) in which a polyhedral protein shell encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase and functions as the key component of the cyanobacterial CO 2 -concentrating mechanism (CCM). Elevated CO 2 levels within the carboxysome shell as a result of carbonic anhydrase activity increase the efficiency of RuBisCO. Yet, there remain many questions regarding the flux or exclusion of metabolites across the shell and how the activity of BMCs varies over time. These questions have been difficult to address using traditional ensemble techniques due to the heterogeneity of BMCs extracted from their native hosts or with heterologous expression. In this chapter, we describe a method to film and extract quantitative information about carboxysome activity using molecular biology and live cell, timelapse microscopy. In our method, the production of carboxysomes is first controlled by deleting the native genes required for carboxysome assembly and then re-introducing them under the control of an inducible promoter. This system enables carboxysomes to be tracked through multiple generations of cells and provides a way to quantify the total biomass accumulation attributed to a single carboxysome. While the method presented here was developed specifically for carboxysomes, it could be modified to track and quantify the activity of bacterial microcompartments in general., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The availability of inorganic carbon (C i) as the source for photosynthesis is fluctuating in aquatic environments. Despite the involvement of transcriptional regulators CmpR and NdhR in regulating genes encoding C i transporters at limiting CO 2 , the C i -sensing mechanism is largely unknown among cyanobacteria. Here we report that a cAMP-dependent transcription factor SyCRP1 mediates C i response in Synechocystis. The mutant ∆ sycrp1 exhibited a slow-growth phenotype and reduced maximum rate of bicarbonate-dependent photosynthetic electron transport (V max) compared to wild-type at the scarcity of CO 2. The number of carboxysomes was decreased significantly in the ∆ sycrp1 at low CO 2 consistent with its reduced V max. The DNA microarray analysis revealed the upregulation of genes encoding C i transporters in ∆sycrp1. The membrane-localized SyCRP1 was released into the cytosol in wild-type cells shifted from low to high CO 2 or upon cAMP treatment. Soluble His-tagged SyCRP1 was shown to target DNA-binding sites upstream of the C i -regulated genes sbtA and ccmK 3. In addition, cAMP enhanced the binding of SyCRP1 to its target sites. Our data collectively suggest that the C i is sensed through the second messenger cAMP releasing membrane-bound SyCRP1 into cytoplasm under sufficient CO 2 conditions. Hence, SyCRP1 is a possible regulator of carbon concentrating mechanism, and such a regulation might be mediated via sensing C i levels through cAMP in Synechocystis. • It has been known that cAMP receptor proteins (CRP) play a crucial role in regulation of gene expression in bacteria. • Transcription regulators of CCM genes were decribed previously, however C i -sensing mechanism in cyanobacteria is unclear. • A membrane associated Synechocystis CRP (SyCRP1), releases to cytosol when bound to CAMP to regulate CCM. • Thus, SyCRP1 mediates C i response through the signalling molecule cAMP in Synechocytis. [ABSTRACT FROM AUTHOR]

Increasing efficiency is an important driving force behind cellular organization and often achieved through compartmentalization. Long recognized as a core principle of eukaryotic cell organization, its widespread occurrence in prokaryotes has only recently come to light. Despite the early discovery of a few microcompartments, such as gas vesicles and carboxysomes, the vast majority of these structures in prokaryotes are less than 100-nm in diameter-too small for conventional light microscopy and electron microscopic thin sectioning. Consequently, these smaller nanocompartments have been discovered serendipitously and then through bioinformatics shown to be broadly distributed. Their small uniform size, robust self-assembly, high stability, excellent biocompatibility, and large cargo capacity make them excellent candidates for biotechnology applications. This review will highlight our current knowledge of nanocompartments and the prospects for applications, as well as open questions and challenges that need to be addressed to fully understand these important structures. [ABSTRACT FROM AUTHOR]

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO 2 -fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications. IMPORTANCE A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco. The self-assembling organelle is generated by hundreds of proteins and plays important roles in converting carbon dioxide to sugar, a process known as carbon fixation. In this study, we uncovered the exact stoichiometry of all building components and the structural plasticity of the functional α-carboxysome, using newly developed quantitative mass spectrometry together with biochemistry, electron microscopy, and enzymatic assay. The study advances our understanding of the architecture and modularity of natural carboxysomes. The knowledge learned from natural carboxysomes will suggest feasible ways to produce functional carboxysomes in other hosts, such as crop plants, with the overwhelming goal of boosting cell metabolism and crop yields.

The availability of inorganic carbon (C i ) as the source for photosynthesis is fluctuating in aquatic environments. Despite the involvement of transcriptional regulators CmpR and NdhR in regulating genes encoding C i transporters at limiting CO 2 , the C i -sensing mechanism is largely unknown among cyanobacteria. Here we report that a cAMP-dependent transcription factor SyCRP1 mediates C i response in Synechocystis. The mutant ∆sycrp1 exhibited a slow-growth phenotype and reduced maximum rate of bicarbonate-dependent photosynthetic electron transport (V max ) compared to wild-type at the scarcity of CO 2 . The number of carboxysomes was decreased significantly in the ∆sycrp1 at low CO 2 consistent with its reduced V max . The DNA microarray analysis revealed the upregulation of genes encoding C i transporters in ∆sycrp1. The membrane-localized SyCRP1 was released into the cytosol in wild-type cells shifted from low to high CO 2 or upon cAMP treatment. Soluble His-tagged SyCRP1 was shown to target DNA-binding sites upstream of the C i -regulated genes sbtA and ccmK3. In addition, cAMP enhanced the binding of SyCRP1 to its target sites. Our data collectively suggest that the C i is sensed through the second messenger cAMP releasing membrane-bound SyCRP1 into cytoplasm under sufficient CO 2 conditions. Hence, SyCRP1 is a possible regulator of carbon concentrating mechanism, and such a regulation might be mediated via sensing C i levels through cAMP in Synechocystis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 CO 2 -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 CO 2 -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.