Structural Insight into the Mechanisms of Transport across the Salmonella enterica Pdu Microcompartment Shell

Bacterial microcompartments are a functionally diverse group of proteinaceous organelles that confine specific reaction pathways in the cell within a thin protein-based shell. The propanediol utilizing (Pdu) microcompartment contains the reactions for metabolizing 1,2-propanediol in certain enteric bacteria, including Salmonella. The Pdu shell is assembled from a few thousand protein subunits of several different types. Here we report the crystal structures of two key shell proteins, PduA and PduT. The crystal structures offer insights into the mechanisms of Pdu microcompartment assembly and molecular transport across the shell. PduA forms a symmetric homohexamer whose central pore appears tailored for facilitating transport of the 1,2-propanediol substrate. PduT is a novel, tandem domain shell protein that assembles as a pseudohexameric homotrimer. Its structure reveals an unexpected site for binding an [Fe-S] cluster at the center of the PduT pore. The location of a metal redox cofactor in the pore of a shell protein suggests a novel mechanism for either transferring redox equivalents across the shell or for regenerating luminal [Fe-S] clusters.     

Exploring Bacterial Organelle Interactomes: A Model of the Protein-Protein Interaction Network in the Pdu Microcompartment

Bacterial microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a “universal hub” for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding β-barrel of the hexameric PduU shell protein. Preliminary experiments involving a bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a bacterial microcompartment provides fresh insight into the organization of the Pdu MCP.     

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella

Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effects of each mutation on 1,2-PD degradation and MCP structure were determined by electron microscopy and growth studies. Deletion of the pduBB', pduJ, or pduN gene severely impaired MCP formation, and the observed defects were consistent with roles as facet, edge, or vertex protein, respectively. Metabolite measurements showed that pduA, pduBB', pduJ, or pduN deletion mutants accumulated propionaldehyde to toxic levels during 1,2-PD catabolism, indicating that the integrity of the shell was disrupted. Deletion of the pduK, pduT, or pduU gene did not substantially affect MCP structure or propionaldehyde accumulation, suggesting they are nonessential to MCP formation. However, the pduU or pduT deletion mutants grew more slowly than the wild type on 1,2-PD at saturating B(12), indicating that they are needed for maximal activity of the 1,2-PD degradative enzymes encased within the MCP shell. Considering recent crystallography studies, this suggests that PduT and PduU may mediate the transport of enzyme substrates/cofactors across the MCP shell. Interestingly, a pduK deletion caused MCP aggregation, suggesting a role in the spatial organization of MCP within the cytoplasm or perhaps in segregation at cell division.     

Alanine Scanning Mutagenesis Identifies an Asparagine–Arginine–Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment

Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic, and structural analysis of 20 mutants allowed us to determine that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.     

The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.     

The PduM protein is a structural component of the microcompartments involved in coenzyme B(12)-dependent 1,2-propanediol degradation by Salmonella enterica

Diverse bacteria use proteinaceous microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of metabolic enzymes encased within a protein shell that provides a defined environment. In Salmonella enterica, a MCP is involved in B(12)-dependent 1,2-propanediol utilization (Pdu MCP). In this report, we show that the protein PduM is required for the assembly and function of the Pdu MCP. The results of tandem mass spectrometry and Western blot analyses show that PduM is a component of the Pdu MCP. Electron microscopy shows that a pduM deletion mutant forms MCPs with abnormal morphology. Growth tests and metabolite measurements establish that a pduM deletion mutant is unable to form functional MCPs. PduM is unrelated in sequence to proteins of known function and hence may represent a new class of MCP structural proteins. We also report a modified protocol for the purification of Pdu MCP from Salmonella which allows isolation of milligram amounts of MCPs in about 4 h. We believe that this protocol can be extended or modified for the purification of MCPs from diverse bacteria.     

The effects of time,temperature, and pH on the stability of PDU bacterial microcompartments

Bacterial microcompartments (MCPs) are subcellular organelles that are composed of a protein shell and encapsulated metabolic enzymes. It has been suggested that MCPs can be engineered to encapsulate protein cargo for use as in vivo nanobioreactors or carriers for drug delivery. Understanding the stability of the MCP shell is critical for such applications. Here, we investigate the integrity of the propanediol utilization (Pdu) MCP shell of Salmonella enterica over time, in buffers with various pH, and at elevated temperatures. The results show that MCPs are remarkably stable. When stored at 4°C or at room temperature, Pdu MCPs retain their structure for several days, both in vivo and in vitro. Furthermore, Pdu MCPs can tolerate temperatures up to 60°C without apparent structural degradation. MCPs are, however, sensitive to pH and require conditions between pH 6 and pH 10. In nonoptimal conditions, MCPs form aggregates. However, within the aggregated protein mass, MCPs often retain their polyhedral outlines. These results show that MCPs are highly robust, making them suitable for a wide range of applications.     

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.     

PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.     

Structural Insights into Higher Order Assembly and Function of the Bacterial Microcompartment Protein PduA

Bacterial microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing microcompartment is assembled from seven different types of shell proteins, one of which is PduA. It is one of the more abundant components of the shell and intriguingly can form nanotubule-like structures when expressed on its own in the cytoplasm of Escherichia coli. We propose a model that accounts for the size and appearance of these PduA structures and underpin our model using a combinatorial approach. Making strategic mutations at Lys-26, Val-51, and Arg-79, we targeted residues predicted to be important for PduA assembly. We present the effect of the amino acid residue substitution on the phenotype of the PduA higher order assemblies (transmission electron microscopy) and the crystal structure of the K26D mutant with one glycerol molecule bound to the central pore. Our results support the view that the hexamer-hexamer interactions seen in PduA crystals persist in the cytoplasmic structures and reveal the profound influence of the two key amino acids, Lys-26 and Arg-79, on tiling, not only in the crystal lattice but also in the bacterial cytoplasm. Understanding and controlling PduA assemblies is valuable in order to inform manipulation for synthetic biology and biotechnological applications.     

Structure of the PduU shell protein from the Pdu microcompartment of Salmonella

The Pdu microcompartment is a proteinaceous, subcellular structure that serves as an organelle for the metabolism of 1,2-propanediol in Salmonella enterica. It encapsulates several related enzymes within a shell composed of a few thousand protein subunits. Recent structural studies on the carboxysome, a related microcompartment involved in CO(2) fixation, have concluded that the major shell proteins from that microcompartment form hexamers that pack into layers comprising the facets of the shell. Here we report the crystal structure of PduU, a protein from the Pdu microcompartment, representing the first structure of a shell protein from a noncarboxysome microcompartment. Though PduU is a hexamer like other characterized shell proteins, it has undergone a circular permutation leading to dramatic differences in the hexamer pore. In view of the hypothesis that microcompartment metabolites diffuse across the outer shell through these pores, the unique structure of PduU suggests the possibility of a special functional role.     

Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins

Bacterial microcompartments (MCPs) are large proteinaceous structures comprised of a roughly icosahedral shell and a series of encapsulated enzymes. MCPs carrying out three different metabolic functions have been characterized in some detail, while gene expression and bioinformatics studies have implicated other types, including one believed to perform g lycyl r adical‐based metabolism of 1,2‐p ropanediol (Grp). Here we report the crystal structure of a protein (GrpN), which is presumed to be part of the shell of a Grp‐type MCP in Rhodospirillum rubrum F11. GrpN is homologous to a family of proteins (EutN/PduN/CcmL/CsoS4) whose members have been implicated in forming the vertices of MCP shells. Consistent with that notion, the crystal structure of GrpN revealed a pentameric assembly. That observation revived an outstanding question about the oligomeric state of this protein family: pentameric forms (for CcmL and CsoS4A) and a hexameric form (for EutN) had both been observed in previous crystal structures. To clarify these confounding observations, we revisited the case of EutN. We developed a molecular biology‐based method for accurately determining the number of subunits in homo‐oligomeric proteins, and found unequivocally that EutN is a pentamer in solution. Based on these convergent findings, we propose the name bacterial microcompartment vertex for this special family of MCP shell proteins.     

Choline transporter‐like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell‐to‐cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell‐to‐cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map‐based cloning of one mutant revealed a point mutation in the choline transporter‐like 1 (CHER1) protein, changing glycine²⁴⁷ into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD‐associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level.     

Monocyte chemoattractant protein-1 in obesity and type 2 diabetes. Insulin sensitivity study

Objective: Our goal was to test any association between human plasma circulating levels of monocyte chemoattractant protein‐1 (cMCP‐1) and insulin resistance and to compare monocyte chemoattractant protein‐1 (MCP‐1) adipose tissue gene expression and cMCP‐1 in relation with inflammatory markers. Research Methods and Procedures: cMCP‐1 was measured in n = 116 consecutive control male subjects to whom an insulin sensitivity (S_i) test was performed. Circulating levels of soluble CD14, soluble tumor necrosis factor receptor type 2 (sTNFR2), soluble interleukin‐6 (sIL‐6), and adiponectin also were measured. Subcutaneous adipose tissue samples were obtained from n = 107 non‐diabetic and type 2 diabetic subjects with different degrees of obesity. Real‐time polymerase chain reaction was used to measure gene expression of MCP‐1, CD68, tumor necrosis factor‐α (TNF‐α), and its receptor TNFR2. Results: In the S_i study, no independent effect of cMCP‐1 levels on insulin sensitivity was observed. In the expression study, in non‐diabetic subjects, MCP‐1 mRNA had a positive correlation with BMI (r = 0.407, p = 0.003), TNF‐α mRNA (r = 0.419, p = 0.002), and TNFR2 mRNA (r = 0.410, p = 0.003). In these subjects, cMCP‐1 was found to correlate with waist‐to‐hip ratio (r = 0.322, p = 0.048). In patients with type 2 diabetes, MCP‐1 mRNA was up‐regulated compared with non‐diabetic subjects. TNF‐α mRNA was found to independently contribute to MCP‐1 mRNA expression. In this group, CD68 mRNA was found to correlate with BMI (r = 0.455, p = 0.001). Discussion: cMCP‐1 is not associated with insulin sensitivity in apparently healthy men. TNF‐α is the inflammatory cytokine associated with MCP‐1 expression in subcutaneous adipose tissue.     

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.     

Identification of an Arabidopsis solute carrier critical for intracellular transport and inter-organ allocation of molybdate

Plants represent an important source of molybdenum in the human diet. Recently, MOT1 has been identified as a transport protein responsible for molybdate import in Arabidopsis thaliana L.; however, the function of the homologous protein MOT2 has not been resolved. Interestingly, MOT2‐GFP analysis indicated a vacuolar location of this carrier protein. By site directed mutagenesis at the N‐terminal end of MOT2, we identified a di‐leucine motif that is essential for driving the protein into the vacuolar membrane. Molybdate quantification in isolated vacuoles showed that this organelle serves as an important molybdate store in Arabidopsis cells. When grown on soil, leaves from mot2 T‐DNA mutants contained more molybdate, whereas mot2 seeds contained significantly less molybdate than corresponding wild‐type (Wt) tissues. Remarkably, MOT2 mRNA accumulates in senescing leaves and mot2 leaves from plants that had finished their life cycle had 15‐fold higher molybdate levels than Wt leaves. Reintroduction of the endogenous MOT2 gene led to a Wt molybdate phenotype. Thus, mot2 mutants exhibit impaired inter‐organ molybdate allocation. As total concentrations of the molybdenum cofactor (Moco) and its precursor MPT correlates with leaf molybdate levels, we present novel evidence for an adjustment of Moco biosynthesis in response to cellular MoO₄^2? levels. We conclude that MOT2 is important for vacuolar molybdate export, an N‐terminal di‐leucine motif is critical for correct subcellular localisation of MOT2 and activity of this carrier is required for accumulation of molybdate in Arabidopsis seeds. MOT2 is a novel element in inter‐organ translocation of an essential metal ion.     

Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus

The type 4 pill of Pseudomonas aeruginosa are important cell-associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of fiagetia-independent surface translocation known as‘twitching motility, and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non-piliated (pil) strain, PAO6609. Sequence analysis of a 3.2 kb region directly downstream of pilG revealed the presence of three genes, which have been designated pilH, pill, and pilJ. The predicted translation product of the pilH gene (13 272 Da), like PilG, exhibits significant amino acid identity with the enteric single-domain response regulator CheY. The putative Pili protein (19933 Da) is 28% identical to the FrzA protein, a CheW homologue of the gliding bacterium Myxococcus xanthus, and the PMJ protein (72 523 Da) is 26% identical to the enteric methyl-accepting chemotaxis protein (MCP) Tsr. Mutants containing insertions in pill and pilJ were severely impaired in their ability to produce pili and did not translocate across solid surfaces. The pilH mutant remained capable of pilus production and twitching motility, but displayed an altered motility pattern characterized by the presence of many doughnut-shaped swirls. Each of these pil mutants, however, produced zones that were at least as large as the parent in flagellar-mediated swarm assays. The sequence similarities between the putative pilG, H, I and J gene products and several established chemotaxis proteins, therefore, lend strong support to the hypothesis that these proteins are part of a signal-transduction network that controls P. aeruginosa pilus biosynthesis and twitching motility.