Diverse Bacterial Microcompartment Organelles

SUMMARY

Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.     

The protein shells of bacterial microcompartment organelles

Details are emerging on the structure and function of a remarkable class of capsid-like protein assemblies that serve as simple metabolic organelles in many bacteria. These bacterial microcompartments consist of a few thousand shell proteins, which encapsulate two or more sequentially acting enzymes in order to enhance or sequester certain metabolic pathways, particularly those involving toxic or volatile intermediates. Genomic data indicate that bacterial microcompartment shell proteins are present in a wide range of bacterial species, where they encapsulate varied reactions. Crystal structures of numerous shell proteins from distinct types of microcompartments have provided keys for understanding how the shells are assembled and how they conduct molecular transport into and out of microcompartments. The structural data emphasize a high level of mechanistic sophistication in the protein shell, and point the way for further studies on this fascinating but poorly appreciated class of subcellular structures.     

Encapsulins: molecular biology of the shell

Compartmentalization is both a fundamental principle of cellular organization and an emerging theme in prokaryotic biology. Work in the past few decades has shown that protein-based organelles called microcompartments enhance the function of encapsulated cargo proteins. More recently, the repertoire of known prokaryotic organelles has expanded beyond microcompartments to include a new class of smaller proteinaceous compartments, termed nanocompartments (also known as encapsulins). Nanocompartments are icosahedral capsids that are smaller and less complex than microcompartments. Encapsulins are formed by a single species of shell protein that self-assembles and typically encapsulates only one type of cargo protein. Significant progress has been made in understanding the structure of nanocompartment shells and the loading of cargo to the interior. Recent analysis has also demonstrated the prevalence of encapsulin genes throughout prokaryotic genomes and documented a large diversity of cargo proteins with a variety of novel functions, suggesting that nanocompartments play an important role in many microbes. Here we review the current understanding of encapsulin structure and function and highlight exciting open questions of physiological significance.     

Bacterial microcompartments: their properties and paradoxes

Many bacteria conditionally express proteinaceous organelles referred to here as microcompartments (Fig. 1). These microcompartments are thought to be involved in a least seven different metabolic processes and the number is growing. Microcompartments are very large and structurally sophisticated. They are usually about 100-150 nm in cross section and consist of 10,000-20,000 polypeptides of 10-20 types. Their unifying feature is a solid shell constructed from proteins having bacterial microcompartment (BMC) domains. In the examples that have been studied, the microcompartment shell encases sequentially acting metabolic enzymes that catalyze a reaction sequence having a toxic or volatile intermediate product. It is thought that the shell of the microcompartment confines such intermediates, thereby enhancing metabolic efficiency and/or protecting cytoplasmic components. Mechanistically, however, this creates a paradox. How do microcompartments allow enzyme substrates, products and cofactors to pass while confining metabolic intermediates in the absence of a selectively permeable membrane? We suggest that the answer to this paradox may have broad implications with respect to our understanding of the fundamental properties of biological protein sheets including microcompartment shells, S-layers and viral capsids.     

Two new high-resolution crystal structures of carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species

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.     

Two‐dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three‐dimensional crystals

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two‐dimensional (2D) layers within the context of three‐dimensional (3D) crystals. Here, we show that well‐ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His‐tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.     

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.     

Using comparative genomics to uncover new kinds of protein‐based metabolic organelles in bacteria

Bacterial microcompartment (MCP) organelles are cytosolic, polyhedral structures consisting of a thin protein shell and a series of encapsulated, sequentially acting enzymes. To date, different microcompartments carrying out three distinct types of metabolic processes have been characterized experimentally in various bacteria. In the present work, we use comparative genomics to explore the existence of yet uncharacterized microcompartments encapsulating a broader set of metabolic pathways. A clustering approach was used to group together enzymes that show a strong tendency to be encoded in chromosomal proximity to each other while also being near genes for microcompartment shell proteins. The results uncover new types of putative microcompartments, including one that appears to encapsulate B₁₂‐independent, glycyl radical‐based degradation of 1,2‐propanediol, and another potentially involved in amino alcohol metabolism in mycobacteria. Preliminary experiments show that an unusual shell protein encoded within the glycyl radical‐based microcompartment binds an iron‐sulfur cluster, hinting at complex mechanisms in this uncharacterized system. In addition, an examination of the computed microcompartment clusters suggests the existence of specific functional variations within certain types of MCPs, including the alpha carboxysome and the glycyl radical‐based microcompartment. The findings lead to a deeper understanding of bacterial microcompartments and the pathways they sequester.     

An allosteric model for control of pore opening by substrate binding in the EutL microcompartment shell protein

The ethanolamine utilization (Eut) microcompartment is a protein-based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of this elaborate protein complex is composed from a few thousand copies of BMC-domain shell proteins, which form a semi-permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear. Previous work has revealed two conformations of the EutL shell protein, providing substantial evidence for a gated pore that might allow the passage of large cofactors. Here we report structural and biophysical evidence to show that ethanolamine, the substrate of the Eut microcompartment, acts as a negative allosteric regulator of EutL pore opening. Specifically, a series of X-ray crystal structures of EutL from Clostridium perfringens, along with equilibrium binding studies, reveal that ethanolamine binds to EutL at a site that exists in the closed-pore conformation and which is incompatible with opening of the large pore for cofactor transport. The allosteric mechanism we propose is consistent with the cofactor requirements of the Eut microcompartment, leading to a new model for EutL function. Furthermore, our results suggest the possibility of redox modulation of the allosteric mechanism, opening potentially new lines of investigation.     

Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39 ± 2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.     

Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport

Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by analysis of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.     

Variety of size and form of GRM2 bacterial microcompartment particles

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi‐permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non‐native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo‐EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi‐layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.     

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat‐shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data.     

Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2

Salmonella enterica forms polyhedral organelles during coenzyme B(12)-dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B(12)-dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B(12)-dependent 1,2-PD degradation by S. enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B(12)-dependent diol dehydratase. Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB'CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB'JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO(2) fixation), suggesting that the S. enterica organelles and carboxysomes have a related multiprotein shell. In addition, S. enterica organelles contained four enzymes: B(12)-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the S. enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S. enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.     

Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging from global carbon fixation to enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid‐based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best‐studied MCPs highlighting atomic‐level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications of an unusual compartment designed by nature to optimize metabolic processes within a cellular context.     

The Pentameric Vertex Proteins Are Necessary for the Icosahedral Carboxysome Shell to Function as a CO2 Leakage Barrier

Background

Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO₂ fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria.

Methodology/Principal Findings

We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected.

Conclusions/Significance

In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO₂ and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome.     

Protein-based organelles in bacteria: carboxysomes and related microcompartments

Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置:胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和用途 。     

Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO₂ in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.     

Biochemical and structural insights into bacterial organelle form and biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.