Uncovering the roles of the scaffolding protein CsoS2 in mediating the assembly and shape of the α-carboxysome shell.

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

Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli.

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMCs were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stoichiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome-scale modeling and 13C-fluxomics. Our results reveal that contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing to significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli.IMPORTANCEThe properties of bacterial microcompartments make them an ideal tool for building orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modeling, and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in Escherichia coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.

Conserved and repetitive motifs in an intrinsically disordered protein drive ⍺-carboxysome assembly.

All cyanobacteria and some chemoautotrophic bacteria fix CO2 into sugars using specialized proteinaceous compartments called carboxysomes. Carboxysomes enclose the enzymes Rubisco and carbonic anhydrase inside a layer of shell proteins to increase the CO2 concentration for efficient carbon fixation by Rubisco. In the ⍺-carboxysome lineage, a disordered and highly repetitive protein named CsoS2 is essential for carboxysome formation and function. Without it, the bacteria require high CO2 to grow. How does a protein predicted to be lacking structure serve as the architectural scaffold for such a vital cellular compartment? In this study, we identify key residues present in the repeats of CsoS2, VTG and Y, which are necessary for building functional ⍺-carboxysomes in vivo. These highly conserved and repetitive residues contribute to the multivalent binding interaction and phase separation behavior between CsoS2 and shell proteins. We also demonstrate 3-component reconstitution of CsoS2, Rubisco, and shell proteins into spherical condensates and show the utility of reconstitution as a biochemical tool to study carboxysome biogenesis. The precise self-assembly of thousands of proteins is crucial for carboxysome formation, and understanding this process could enable their use in alternative biological hosts or industrial processes as effective tools to fix carbon.

Corrigendum: Atypical carboxysome loci: JEEPs or junk?

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

Nanoengineering Carboxysome Shells for Protein Cages with Programmable Cargo Targeting.

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

Potential utility of bacterial protein nanoreactor for sustainable in-situ biocatalysis in wide range of bioprocess conditions.

Bacterial microcompartments (MCPs) are proteinaceous organelles that natively encapsulates the enzymes, substrates, and cofactors within a protein shell. They optimize the reaction rates by enriching the substrate in the vicinity of enzymes to increase the yields of the product and mitigate the outward diffusion of the toxic or volatile intermediates. The shell protein subunits of MCP shell are selectively permeable and have specialized pores for the selective inward diffusion of substrates and products release. Given their attributes, MCPs have been recently explored as potential candidates as subcellular nano-bioreactor for the enhanced production of industrially important molecules by exercising pathway encapsulation. In the current study, MCPs have been shown to sustain enzyme activity for extended periods, emphasizing their durability against a range of physical challenges such as temperature, pH and organic solvents. The significance of an intact shell in conferring maximum protection is highlighted by analyzing the differences in enzyme activities inside the intact and broken shell. Moreover, a minimal synthetic shell was designed with recruitment of a heterologous enzyme cargo to demonstrate the improved durability of the enzyme. The encapsulated enzyme was shown to be more stable than its free counterpart under the aforementioned conditions. Bacterial MCP-mediated encapsulation can serve as a potential strategy to shield the enzymes used under extreme conditions by maintaining the internal microenvironment and enhancing their cycle life, thereby opening new means for stabilizing, and reutilizing the enzymes in several bioprocess industries.

A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable protein shell, but the mechanisms that allow selective transport across the shell are not fully understood. Here we examine transport across the shell of the choline utilization (Cut) MCP of Escherichia coli 536, which has not been studied before. The shell of the Cut MCP is unusual in consisting of one pentameric and four hexameric bacterial microcompartment (BMC) domain proteins. It lacks trimeric shell proteins, which are thought to be required for the transport of larger substrates and enzymatic cofactors. In addition, its four hexameric BMC domain proteins are very similar in amino acid sequence. This raises questions about how the Cut MCP mediates the selective transport of the substrate, products and cofactors of choline metabolism. In this report, site-directed mutagenesis is used to modify the central pores (the main transport channels) of all four Cut BMC hexamers to assess their transport roles. Our findings indicate that a single shell protein, CmcB, plays the major role in choline transport across the shell of the Cut MCP and that the electrostatic properties of the CmcB pore also impact choline transport. The implications of these findings with regard to the higher-order structure of MCPs are discussed.

Modulation of Hybrid GRM2-type Bacterial Microcompartment Shells through BMC-H Shell Protein Fusion and Incorporation of Non-native BMC-T Shell Proteins.

Bacterial microcompartments (BMCs) are organelle-like structures in bacteria that facilitate a wide range of enzymatic reactions. The microcompartment shell contains an encapsulated enzymatic core and, in contrast to phospholipid-based eukaryotic organelle membranes, has a pseudoicosahedral shape composed of BMC-H, BMC-T, and BMC-P proteins with conserved structures. This semipermeable microcompartment shell delineates the enzymatic core assemblies and the intermediates from the rest of the cell. It is also thought to function as a barrier against toxic intermediates as well as to increase the reaction rate. These properties of BMCs have made them intriguing candidates for biotechnological applications, for which it is important to explore the potential scope of the BMC shell modulation possibilities. In this work, we explore two BMC shell modulation mechanisms: first, confirming the incorporation of three trimeric BMC-T shell proteins and two truncated BMC-T shell proteins into Klebsiella pneumoniae GRM2-type BMC protein shells containing no representatives of this group, and second, producing BMC particles from double- and triple-fused hexameric BMC-H shell proteins. These results reveal the potential for "mix and match" synthetic BMC shell formation to ensure shell properties specifically suited to the encapsulated cargo and show for the first time the involvement of an essentially dimeric pseudohexameric shell protein in BMC shell formation.

Kinetic Growth of Multicomponent Microcompartment Shells.

An important goal of systems and synthetic biology is to produce high value chemical species in large quantities. Microcompartments, which are protein nanoshells encapsulating catalytic enzyme cargo, could potentially function as tunable nanobioreactors inside and outside cells to generate these high value species. Modifying the morphology of microcompartments through genetic engineering of shell proteins is one viable strategy to tune cofactor and metabolite access to encapsulated enzymes. However, this is a difficult task without understanding how changing interactions between the many different types of shell proteins and enzymes affect microcompartment assembly and shape. Here, we use multiscale molecular dynamics and experimental data to describe assembly pathways available to microcompartments composed of multiple types of shell proteins with varied interactions. As the average interaction between the enzyme cargo and the multiple types of shell proteins is weakened, the shell assembly pathway transitions from (i) nucleating on the enzyme cargo to (ii) nucleating in the bulk and then binding the cargo as it grows to (iii) an empty shell. Atomistic simulations and experiments using the 1,2-propanediol utilization microcompartment system demonstrate that shell protein interactions are highly varied and consistent with our multicomponent, coarse-grained model. Furthermore, our results suggest that intrinsic bending angles control the size of these microcompartments. Overall, our simulations and experiments provide guidance to control microcomparmtent size and assembly by modulating the interactions between shell proteins.

Anaerobic purinolytic enzymes enable dietary purine clearance by engineered gut bacteria.

Uric acid, the end product of purine degradation, causes hyperuricemia and gout, afflicting hundreds of millions of people. The debilitating effects of gout are exacerbated by dietary purine intake, and thus a potential therapeutic strategy is to enhance purine degradation in the gut microbiome. Aerobic purine degradation involves oxidative dearomatization of uric acid catalyzed by the O2-dependent uricase. The enzymes involved in purine degradation in strictly anaerobic bacteria remain unknown. Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

Computational and biochemical methods to measure the activity of carboxysomes and protein organelles in vivo.

Cyanobacteria are photosynthetic microorganisms that play important ecological roles as major contributors to global nutrient cycles. Cyanobacteria are highly efficient in carrying out oxygenic photosynthesis because they possess carboxysomes, a class of bacterial microcompartments (BMC) in which a polyhedral protein shell encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase and functions as the key component of the cyanobacterial CO2-concentrating mechanism (CCM). Elevated CO2 levels within the carboxysome shell as a result of carbonic anhydrase activity increase the efficiency of RuBisCO. Yet, there remain many questions regarding the flux or exclusion of metabolites across the shell and how the activity of BMCs varies over time. These questions have been difficult to address using traditional ensemble techniques due to the heterogeneity of BMCs extracted from their native hosts or with heterologous expression. In this chapter, we describe a method to film and extract quantitative information about carboxysome activity using molecular biology and live cell, timelapse microscopy. In our method, the production of carboxysomes is first controlled by deleting the native genes required for carboxysome assembly and then re-introducing them under the control of an inducible promoter. This system enables carboxysomes to be tracked through multiple generations of cells and provides a way to quantify the total biomass accumulation attributed to a single carboxysome. While the method presented here was developed specifically for carboxysomes, it could be modified to track and quantify the activity of bacterial microcompartments in general.

Phase Separation of Rubisco by the Folded SSUL Domains of CcmM in Beta-Carboxysome Biogenesis.

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO3- ions into the carboxysome, where they are converted to CO2 by CA. Thus, the carboxysome functions as a CO2-concentrating mechanism (CCM), enhancing the efficiency of Rubisco in CO2 fixation. In ß-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the ß-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions.

Disordered regions endow structural flexibility to shell proteins and function towards shell-enzyme interactions in 1,2-propanediol utilization microcompartment.

Intrinsically disordered regions in proteins have been functionally linked to the protein-protein interactions and genesis of several membraneless organelles. Depending on their residual makeup, hydrophobicity or charge distribution they may remain in extended form or may assume certain conformations upon biding to a partner protein or peptide. The present work investigates the distribution and potential roles of disordered regions in the integral proteins of 1,2-propanediol utilization microcompartments. We use bioinformatics tools to identify the probable disordered regions in the shell proteins and enzyme of the 1,2-propanediol utilization microcompartment. Using a combination of computational modelling and biochemical techniques we elucidate the role of disordered terminal regions of a major shell protein and enzyme. Our findings throw light on the importance of disordered regions in the self-assembly, providing flexibility to shell protein and mediating its interaction with a native enzyme.Communicated by Ramaswamy H. Sarma.

Encapsulins: Nanotechnology's future in a shell.

Encapsulins, virus capsid-like bacterial nanocompartments have emerged as promising tools in medicine, imaging, and material sciences. Recent work has shown that these protein-bound icosahedral 'organelles' possess distinct properties that make them exceptionally usable for nanotechnology applications. A key factor contributing to their appeal is their ability to self-assemble, coupled with their capacity to encapsulate a wide range of cargos. Their genetic manipulability, stability, biocompatibility, and nano-size further enhance their utility, offering outstanding possibilities for practical biotechnology applications. In particular, their amenability to engineering has led to their extensive modification, including the packaging of non-native cargos and the utilization of the shell surface for displaying immunogenic or targeting proteins and peptides. This inherent versatility, combined with the ease of expressing encapsulins in heterologous hosts, promises to provide broad usability. Although mostly not yet commercialized, encapsulins have started to demonstrate their vast potential for biotechnology, from drug delivery to biofuel production and the synthesis of valuable inorganic materials. In this review, we will initially discuss the structure, function and diversity of encapsulins, which form the basis for these emerging applications, before reviewing ongoing practical uses and highlighting promising applications in medicine, engineering and environmental sciences.

Delayed In Vivo Encapsulation of Enzymes Alters the Catalytic Activity of Virus-Like Particle Nanoreactors.

Encapsulation of enzymes inside protein cage structures, mimicking protein-based organelle structures found in nature, has great potential for the development of new catalytic materials with enhanced properties. In vitro and in vivo methodologies have been developed for the encapsulation of enzymes within protein cage structures of several types, particularly virus-like particles (VLPs), with the ability to retain the activity of the encapsulated enzymes. Here, we examine the in vivo encapsulation of enzymes within the bacteriophage P22 derived VLP and show that some enzymes may require a delay in encapsulation to allow proper folding and maturation before they can be encapsulated inside P22 as fully active enzymes. Using a sequential expression strategy, where enzyme cargoes are first expressed, allowed to fold, and later encapsulated by the expression of the P22 coat protein, altered enzymatic activities are obtained in comparison to enzymes encapsulated in P22 VLPs using a simultaneous coexpression strategy. The strategy and results discussed here highlight important considerations for researchers investigating the encapsulation of enzymes inside confined reaction environments via in vivo routes and provide a potential solution for those that have been unable to produce active enzymes upon encapsulation.

Getting the Payload in Place - Unravelling the Complexities of Making a Bacterial Microcompartment.

Bacterial microcompartments (BMCs) are complex macromolecular assemblies composed of any outer protein shell that encases a specific metabolic pathway cargo. Recent research is now starting to unravel some of the processes that are involved in directing the enzyme cargo to the inside of the BMC. In particular, an article in this issue of J Bacteriol by N. W. Kennedy, C. E. Mills, C. H. Abrahamson, A. Archer, et al. (J Bacteriol 204:e00576-21, 2022, https://doi.org/10.1128/jb.00576-21) highlights the role played by the shell protein PduB in coordinating this internalization process.

Atypical Carboxysome Loci: JEEPs or Junk?

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 ß-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.

Linking the Salmonella enterica 1,2-Propanediol Utilization Bacterial Microcompartment Shell to the Enzymatic Core via the Shell Protein PduB.

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here, we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is critical for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy, we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and system-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. IMPORTANCE MCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here, we provide evidence of a potential mechanism in S. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

BMC Caller: a webtool to identify and analyze bacterial microcompartment types in sequence data.

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.

The roles of diol dehydratase from pdu operon on glycerol catabolism in Klebsiella pneumoniae.

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.