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.     

Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B₁₂-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics.     

Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.     

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.     

The Structure of CcmP,a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome,Forms a Subcompartment Within a Microcompartment

The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO₂ fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all β-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a β-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-containing shell protein that may weakly bind 3-phosphoglycerate, the product of CO₂ fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.     

The Carboxysome Shell Is Permeable to Protons

Bacterial microcompartments (BMCs) are polyhedral organelles found in an increasingly wide variety of bacterial species. These structures, typified by carboxysomes of cyanobacteria and many chemoautotrophs, function to compartmentalize important reaction sequences of metabolic pathways. Unlike their eukaryotic counterparts, which are surrounded by lipid bilayer membranes, these microbial organelles are bounded by a thin protein shell that is assembled from multiple copies of a few different polypeptides. The main shell proteins form hexamers whose edges interact to create the thin sheets that form the facets of the polyhedral BMCs. Each hexamer contains a central pore hypothesized to mediate flux of metabolites into and out of the organelle. Because several distinctly different metabolic processes are found in the various BMCs studied to date, it has been proposed that a common advantage to packaging these pathways within shell-bound compartments is to optimize the concentration of volatile metabolites in the BMC by maintaining an interior pH that is lower than that of the cytoplasm. We have tested this idea by recombinantly fusing a pH-sensitive green fluorescent protein (GFP) to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the major enzyme component inside the carboxysome. Our results suggest that the carboxysomal pH is similar to that of its external environment and that the protein shell does not constitute a proton barrier. The explanation for the sundry BMC functions must therefore be sought in the characteristics of the pores that traverse their shells.Clearly, the subcellular organization of bacteria is much more complex than was once assumed (reviewed in references 17 and 26), and many bacteria are able to compartmentalize metabolic processes into distinct organelles. Among these, the bacterial microcompartments (BMCs) have garnered attention because the genetic potential to form these structures, which consist entirely of protein, is widespread among the bacteria (1). BMCs have been credited with enhancing the activity of the enzyme(s) they contain by providing a unique environment with optimized substrate concentrations or pH, facilitating metabolite channeling, or protecting the cell by sequestering toxic intermediates (1). Based on comparative genomic and biochemical analyses, the interiors of BMCs in various bacteria are populated by different complements of enzymes, suggesting that, collectively, these organelles play a role in a multitude of metabolic pathways. The bounding shells of all BMCs, on the other hand, appear to be built from multimeric assemblies of proteins that belong to the same two families (pfam 00936 and pfam 03319) (7, 11, 12, 29, 30, 32). Despite some structural differences between individual members of the two main shell protein types, the central pores in the pentamers and hexamers formed by these proteins have been implicated in mediating metabolite traffic across the BMC shell and, in some cases, may actively regulate transfer of substrates and products across the shell through a gating mechanism (12, 30). The variations in pore sizes and surface properties between individual BMC shell proteins likely reflect differences in interactions with the metabolites that pass through them and beg the question about functional differences among BMCs and between BMC protein shells and the lipid bilayer-based membranes of eukaryotic organelles.The carboxysome, the first BMC to be discovered (25), is found in all cyanobacteria and in many nonphotosynthetic chemoautotrophs, exemplified by the aerobic sulfur bacterium Halothiobacillus neapolitanus and its relatives. Its interior is filled with ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) (25), the enzyme that is crucial for carbon assimilation by these bacteria because it catalyzes the fixation of inorganic carbon onto the organic acceptor molecule, ribulose-1,5-bisphosphate. The enzyme is a relatively inefficient catalyst; it has a high K_m for its substrate, CO₂, a low turnover number, and is also able to fix O₂ through a competing, unproductive reaction. The carboxysome compensates for these shortcomings by providing a microcompartment that enhances the catalytic efficiency of RubisCO. Cytosolic bicarbonate is thought to enter the carboxysome freely through the many pores in the shell, but it cannot be used by RubisCO. The action of the carboxysomal carbonic anhydrase that is cosequestered with RubisCO rapidly converts HCO₃⁻ to the RubisCO substrate, CO₂ (5, 19, 28). The carboxysome shell retards diffusion of CO₂ out of the organelle (5) and thereby contributes to the generation of an elevated steady-state concentration of CO₂ in the vicinity of the RubisCO active site, a condition that favors the carboxylation reaction. In addition, the carboxysomal shell may reduce the concentration of the competing RubisCO substrate O₂ by excluding it from the microcompartment interior (3, 14). Quantitative modeling of CO₂ fixation in cyanobacteria supports the role of the shell as a barrier for CO₂ diffusion out of the carboxysome (21). Measurements of CO₂ hydration rates in purified intact and disrupted carboxysomes suggest that access of CO₂ to the interior of the organelle is likewise limited by the shell (5).The shells of other BMCs have also been proposed to influence the flux of metabolites into and out of the microcompartment interior. The Pdu BMC of Salmonella enterica, which participates in the B₁₂-dependent degradation of propanediol, prevents the toxic intermediate propionaldehyde from diffusing into the cytoplasm (6, 24). The Eut BMC, also found in S. enterica, is thought to encapsulate several enzymes of the ethanolamine utilization pathway, in which acetaldehyde is a central intermediate. The shell of the Eut BMC is also postulated to prevent loss of a crucial metabolite, in this case acetaldehyde, from the interior by diffusion (22). Acetaldehyde, which like propionaldehyde is volatile, is thought to be captured within the Eut BMC not to protect cellular structures from damage but to prevent escape of this important intermediate from the cell (20). In an effort to identify a common mechanism for BMC function, Penrod and Roth (20) made the intriguing suggestion that all BMCs may constitute compartments with an interior pH that is lower than that of the surrounding cytoplasm. Such an environment would promote the conversion of aldehydes in the Pdu and Eut BMCs to less volatile acetals, which are less likely to escape the downstream pathway enzymes localized in the BMC interior. In the carboxysomes of autotrophic bacteria, a lower pH would presumably favor higher concentrations of CO₂ by shifting the equilibrium from HCO₃⁻ toward CO₂.This unifying model of BMC function necessitates the assumption that the BMC shell resists passage of protons out of the compartment to maintain a pH gradient. Although it is not known whether a thin protein layer can impede proton diffusion, one needs only to look at the mechanism of proton discrimination described for the pores of aquaporins (2, 31) to imagine that the multiple pores in the BMC shell might likewise fulfill the structural requirements of a proton permeability barrier.An extended discussion of this model at a recent symposium on BMCs (at the 109th General Meeting of the American Society for Microbiology in 2009) prompted us to design a study that directly determined if the BMC shell is able to maintain a lumen pH that is different from that of its surrounding medium.     

Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO(2). In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme B(12) are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.     

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.     

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.     

Comparative analysis of carboxysome shell proteins

Carboxysomes are metabolic modules for CO(2) fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome's icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO(2) fixation in other organisms or creating novel biological nanostructures.     

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle

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.     

Role of carboxysomes in cyanobacterial CO2 assimilation: CO2 concentrating mechanisms and metabolon implications

Many carbon-fixing organisms have evolved CO₂ concentrating mechanisms (CCMs) to enhance the delivery of CO₂ to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O₂. These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called ‘carboxysome’ in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO₂ around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO₂. The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin–Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO₂ fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.     

A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3- dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the beta-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3- dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal gamma-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3- but was unable to catalyze HCO3- dehydration, suggesting that it may potentially regulate BDC activity.     

Development of liver regenerative therapy using glycoside-modified bone marrow cells

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.     

Isolation and characterization of the Prochlorococcus carboxysome reveal the presence of the novel shell protein CsoS1D

Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters.     

Enzyme variation in the brook lamprey,Lampetra planeri (Bloch), a member of the vertebrate group Agnatha

A natural population of the brook lamprey, Lampetra planeri, was assayed for electrophoretically detectable variation at 30 enzyme loci. The mean heterozygosity per locus of this primitive vertebrate, a member of the Agnatha, was found to be 0.076 ± 0.031, a value similar to those recorded for other vertebrates. The high chromosome numbers recorded for this and related species have been attributed to polyploidy, but our studies do not indicate the existence of large numbers of duplicated loci. Indeed, several enzymes that are encoded by duplicate loci in other vertebrate species appear to be encoded by single loci in the lamprey. It is suggested that studies on the biology and taxonomy of lampreys will benefit greatly from an electrophoretic approach.     

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.     

Extracellular High Mobility Group Box 1 Plays a Role in the Effect of Bone Marrow Mononuclear Cell Transplantation for Heart Failure

Transplantation of unfractionated bone marrow mononuclear cells (BMCs) repairs and/or regenerates the damaged myocardium allegedly due to secretion from surviving BMCs (paracrine effect). However, donor cell survival after transplantation is known to be markedly poor. This discrepancy led us to hypothesize that dead donor BMCs might also contribute to the therapeutic benefits from BMC transplantation. High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes nucleosomes, and also acts as a multi-functional cytokine when released from damaged cells. We thus studied the role of extracellular HMGB1 in the effect of BMC transplantation for heart failure. Four weeks after coronary artery ligation in female rats, syngeneic male BMCs (or PBS only as control) were intramyocardially injected with/without anti-HMGB1 antibody or control IgG. One hour after injection, ELISA showed that circulating extracellular HMGB1 levels were elevated after BMC transplantation compared to the PBS injection. Quantitative donor cell survival assessed by PCR for male-specific sry gene at days 3 and 28 was similarly poor. Echocardiography and catheterization showed enhanced cardiac function after BMC transplantation compared to PBS injection at day 28, while this effect was abolished by antibody-neutralization of HMGB1. BMC transplantation reduced post-infarction fibrosis, improved neovascularization, and increased proliferation, while all these effects in repairing the failing myocardium were eliminated by HMGB1-inhibition. Furthermore, BMC transplantation drove the macrophage polarization towards alternatively-activated, anti-inflammatory M2 macrophages in the heart at day 3, while this was abolished by HMGB1-inhibition. Quantitative RT-PCR showed that BMC transplantation upregulated expression of an anti-inflammatory cytokine IL-10 in the heart at day 3 compared to PBS injection. In contrast, neutralizing HMGB1 by antibody-treatment suppressed this anti-inflammatory expression. These data suggest that extracellular HMGB1 contributes to the effect of BMC transplantation to recover the damaged myocardium by favorably modulating innate immunity in heart failure.     

Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome

The carboxysome is a bacterial organelle that functions to enhance the efficiency of CO₂ fixation by encapsulating the enzymes ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. The outer shell of the carboxysome is reminiscent of a viral capsid, being constructed from many copies of a few small proteins. Here we describe the structure of the shell protein CsoS1A from the chemoautotrophic bacterium Halothiobacillus neapolitanus. The CsoS1A protein forms hexameric units that pack tightly together to form a molecular layer, which is perforated by narrow pores. Sulfate ions, soaked into crystals of CsoS1A, are observed in the pores of the molecular layer, supporting the idea that the pores could be the conduit for negatively charged metabolites such as bicarbonate, which must cross the shell. The problem of diffusion across a semiporous protein shell is discussed, with the conclusion that the shell is sufficiently porous to allow adequate transport of small molecules. The molecular layer formed by CsoS1A is similar to the recently observed layers formed by cyanobacterial carboxysome shell proteins. This similarity supports the argument that the layers observed represent the natural structure of the facets of the carboxysome shell. Insights into carboxysome function are provided by comparisons of the carboxysome shell to viral capsids, and a comparison of its pores to the pores of transmembrane protein channels.     

Organization of human class I homeobox genes

We report the genomic organization of 20 human class I homeoboxes and the predicted primary sequence of the encoded homeodomains. These homeoboxes are clustered in four complex HOX loci on chromosomes 2, 7, 12, and 17. The homeoboxes of one HOX locus can be aligned to the homeoboxes of the other HOX loci so that corresponding homeodomains in all loci can share the maximal peptide sequence identity. This correspondence of individual homeoboxes in different chromosomal loci suggests the hypothesis of large-scale duplications of a single complex locus. The existence of an ancestral complex locus might have predated the divergence of vertebrates and invertebrates.