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Cyanobacteria uniquely contain a primitive water-soluble carotenoprotein, the orange carotenoid protein (OCP). Nearly all extant cyanobacterial genomes contain genes for the OCP or its homologs, implying an evolutionary constraint for cyanobacteria to conserve its function. Genes encoding the OCP and its two constituent structural domains, the N-terminal domain, helical carotenoid proteins (HCPs), and its C-terminal domain, are found in the most basal lineages of extant cyanobacteria. These three carotenoproteins exemplify the importance of the protein for carotenoid properties, including protein dynamics, in response to environmental changes in facilitating a photoresponse and energy quenching. Here, we review new structural insights for these carotenoproteins and situate the role of the protein in what is currently understood about their functions.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

Photosynthetic organisms harvest light using pigment-protein complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show in Synechocystis sp. PCC 6803 that the recently discovered PBS linker protein ApcG (sll1873) interacts specifically with PSII through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG causes reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with PSII is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with the thylakoid membrane, suggesting a regulatory role mediated by phosphorylation of ApcG. Low-temperature fluorescence measurements showed decreased PSI fluorescence in apcG deletion and complementation strains. The PSI fluorescence was the lowest in the phospho-mimicking complementation strain, while the pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and imply that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria that encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semipermeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We used molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed the overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

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

The orange carotenoid protein (OCP) functions as a sensor of the ambient light intensity and as a quencher of bilin excitons when it binds to the core of the cyanobacterial phycobilisome. We show herein that the photoactivation mechanism that converts the resting, orange-colored state, OCPO, to the active red-colored state, OCPR, requires a sequence of two reactions, each requiring absorption of a single photon by an intrinsic ketocarotenoid chromophore. Global analysis of absorption spectra recorded during continuous illumination of OCPO preparations from Synechocystis sp. PCC 6803 detects the reversible formation of a metastable intermediate, OCPI, in which the ketocarotenoid canthaxanthin exhibits an absorption spectrum with a partial red shift and a broadened vibronic structure compared to that of the OCPO state. While the dark recovery from OCPR to OCPI is a first-order, unimolecular reaction, the subsequent conversion of OCPI to the resting OCPO state is bimolecular, involving association of two OCPO monomers to form the dark-stable OCPO dimer aggregate. These results indicate that photodissociation of the OCPO dimer to form the monomeric OCPO intermediate is the first step in the photoactivation mechanism. Formation of the OCPO monomer from the dimer increases the mean value and broadens the distribution of the solvent-accessible surface area of the canthaxanthin chromophore measured in molecular dynamics trajectories at 300 K. The second step in the photoactivation mechanism is initiated by absorption of a second photon, by canthaxanthin in the OCPO monomer, which obtains the fully red-shifted and broadened absorption spectrum detected in the OCPR product state owing to displacement of the C-terminal domain and the translocation of canthaxanthin more than 12 Å into the N-terminal domain. Both steps in the photoactivation reaction of OCP are likely to involve changes in the structure of the C-terminal domain elicited by excited-state conformational motions of the ketocarotenoid.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures.

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.

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3-6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

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.

Bacterial microcompartments (BMCs) are self-assembling prokaryotic organelles which encapsulate enzymes within a polyhedral protein shell. The shells are comprised of only two structural modules, distinct domains that form pentagonal and hexagonal building blocks, which occupy the vertices and facets, respectively. As all BMC loci encode at least one hexamer-forming and one pentamer-forming protein, the evolutionary history of BMCs can be interrogated from the perspective of their shells. Here, we discuss how structures of intact shells and detailed phylogenies of their building blocks from a recent phylogenomic survey distinguish families of these domains and reveal clade-specific structural features. These features suggest distinct functional roles that recur across diverse BMCs. For example, it is clear that carboxysomes independently arose twice from metabolosomes, yet the principles of shell assembly are remarkably conserved.

Bacterial microcompartments (BMCs) are organelles that segregate segments of metabolic pathways which are incompatible with surrounding metabolism. BMCs consist of a selectively permeable shell, composed of three types of structurally conserved proteins, together with sequestered enzymes that vary among functionally distinct BMCs. Genes encoding shell proteins are typically clustered with those for the encapsulated enzymes. Here, we report that the number of identifiable BMC loci has increased twenty-fold since the last comprehensive census of 2014, and the number of distinct BMC types has doubled. The new BMC types expand the range of compartmentalized catalysis and suggest that there is more BMC biochemistry yet to be discovered. Our comprehensive catalog of BMCs provides a framework for their identification, correlation with bacterial niche adaptation, experimental characterization, and development of BMC-based nanoarchitectures for biomedical and bioengineering applications.

Bacterial microcompartments (BMCs) are protein-based organelles that expand the metabolic potential of many bacteria by sequestering segments of enzymatic pathways in a selectively permeable protein shell. Sixty-eight different types/subtypes of BMCs have been bioinformatically identified based on the encapsulated enzymes and shell proteins encoded in genomic loci. BMCs are found across bacterial phyla. The organisms that contain them, rather than strictly correlating with specific lineages, tend to reflect the metabolic landscape of the environmental niches they occupy. From our recent comprehensive bioinformatic survey of BMCs found in genome sequence data, we find many in members of the human microbiome. Here we survey the distribution of BMCs in the different biotopes of the human body. Given their amenability to be horizontally transferred and bioengineered they hold promise as metabolic modules that could be used to probiotically alter microbiomes or treat dysbiosis.

Bacterial Microcompartments (BMCs) are used by diverse bacteria to compartmentalize enzymatic reactions, functioning analogously to the organelles of eukaryotes. The bounding membrane and encapsulated components are composed entirely of protein, which makes them ideal targets for modification by genetic engineering. In contrast to viruses, in which generally only one protein forms the capsid, the shells of BMCs consist of a variety of shell proteins, each a potential unit of selection. Despite their differences in permeability, the shell proteins are surprisingly interchangeable. Recent developments have shown that they are also highly amenable to engineered modifications which poise them for a variety of biotechnological applications. Given their modular structure, with a module defined as a semi-autonomous functional unit, BMCs can be considered apps for programming metabolism that can be de-bugged by adaptive evolution.

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.

In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is photoactivated under excess light conditions and binds to the light-harvesting antenna, triggering the dissipation of captured light energy. In low light, the OCP relaxes to the native state, a process that is accelerated in the presence of fluorescence recovery protein (FRP). Despite the importance of the OCP in photoprotection, the precise mechanism of photoactivation by this protein is not well-understood. Using time-resolved X-ray-mediated in situ hydroxyl radical labeling, we probed real-time solvent accessibility (SA) changes at key OCP residues during photoactivation and relaxation. We observed a biphasic photoactivation process in which carotenoid migration preceded domain dissociation. We also observed a multiphasic relaxation process, with collapsed domain association preceding the final conformational rearrangement of the carotenoid. Using steady-state hydroxyl radical labeling, we identified sites of interaction between the FRP and OCP. In combination, the findings in this study provide molecular-level insights into the factors driving structural changes during OCP-mediated photoprotection in cyanobacteria, and furnish a basis for understanding the physiological relevance of the FRP-mediated relaxation process.

An increasing number of microbes are being identified that organize catabolic pathways within self-assembling proteinaceous structures known as bacterial microcompartments (BMCs). Most BMCs are characterized by their singular substrate specificity and commonly employ B12-dependent radical mechanisms. In contrast, a less-well-known BMC type utilizes the B12-independent radical chemistry of glycyl radical enzymes (GREs). Unlike B12-dependent enzymes, GREs require an activating enzyme (AE) as well as an external source of electrons to generate an adenosyl radical and form their catalytic glycyl radical. Organisms encoding these glycyl radical enzyme-associated microcompartments (GRMs) confront the challenge of coordinating the activation and maintenance of their GREs with the assembly of a multienzyme core that is encapsulated in a protein shell. The GRMs appear to enlist redox proteins to either generate reductants internally or facilitate the transfer of electrons from the cytosol across the shell. Despite this relative complexity, GRMs are one of the most widespread types of BMC, with distinct subtypes to catabolize different substrates. Moreover, they are encoded by many prominent gut-associated and pathogenic bacteria. In this review, we will focus on the diversity, function, and physiological importance of GRMs, with particular attention given to their associated and enigmatic redox proteins.

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of β-carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that carboxysome shell permeability may be dynamically attenuated by "capping" facet-embedded hexamers with a second hexamer. Because β-carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.

Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which solution scattering data are collected using X-ray exposures below rotational diffusion times, resulting in angularly anisotropic X-ray snapshots that provide several orders of magnitude more information than traditional solution scattering data. Such experiments can be performed using the ultrashort X-ray pulses provided by a free-electron laser source, allowing one to collect a large number of diffraction patterns in a relatively short time. Here, we describe a test data set for FXS, obtained at the Linac Coherent Light Source, consisting of close to 100 000 multi-particle diffraction patterns originating from approximately 50 to 200 Paramecium Bursaria Chlorella virus particles per snapshot. In addition to the raw data, a selection of high-quality pre-processed diffraction patterns and a reference SAXS profile are provided.

Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.

Bacterial microcompartments (BMCs) are self-assembling organelles that consist of an enzymatic core that is encapsulated by a selectively permeable protein shell. The potential to form BMCs is widespread and found across the kingdom Bacteria. BMCs have crucial roles in carbon dioxide fixation in autotrophs and the catabolism of organic substrates in heterotrophs. They contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. Although BMCs were first visualized more than 60 years ago, it is mainly in the past decade that progress has been made in understanding their metabolic diversity and the structural basis of their assembly and function. This progress has not only heightened our understanding of their role in microbial metabolism but is also beginning to enable their use in a variety of applications in synthetic biology. In this Review, we focus on recent insights into the structure, assembly, diversity and function of BMCs.

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B12-dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B12-independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

Using a phylogenomic approach, we have identified and subclassified a new family of carotenoid-binding proteins. These proteins have sequence homology to the N-terminal domain (NTD) of the Orange Carotenoid Protein (OCP), and are referred to as Helical Carotenoid Proteins (HCPs). These proteins comprise at least nine distinct clades and are found in diverse organisms, frequently as multiple paralogs representing the distinct clades. These seem to be out-paralogs maintained from ancient duplications associated with subfunctionalization. All of the HCPs share conservation of the residues for carotenoid binding, and we confirm that carotenoid binding is a fundamental property of HCPs. We solved two crystal structures of the Nostoc sp. PCC 7120 HCP1 protein, each binding a different carotenoid, suggesting that the proteins flexibly bind a range of carotenoids. Based on a comprehensive phylogenetic analysis, we propose that one of the HCP subtypes is likely the evolutionary ancestor of the NTD of the OCP, which arose following a domain fusion event. However, we predict that the majority of HCPs have functions distinct from the NTD of the OCP. Our results demonstrate that the HCPs are a new family of functionally diverse carotenoid-binding proteins found among ecophysiologically diverse cyanobacteria.

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]

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.

Cyanobacteria have evolved a unique carbon fixation organelle known as the carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO2 concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins.