The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene

Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. In this study, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2‐propanediol (1,2‐PD) utilization (Pdu) MCP of Salmonella. Prior studies showed that PduA mediates the transport of 1,2‐PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2‐PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2‐PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein‐protein interactions essential to the function of the MCP shell.     

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.     

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 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.     

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.     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Diverse Bacterial Microcompartment Organelles

SUMMARY

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

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.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Ocean Warming,More than Acidification,Reduces Shell Strength in a Commercial Shellfish Species during Food Limitation

Ocean surface pH levels are predicted to fall by 0.3–0.4 pH units by the end of the century and are likely to coincide with an increase in sea surface temperature of 2–4°C. The combined effect of ocean acidification and warming on the functional properties of bivalve shells is largely unknown and of growing concern as the shell provides protection from mechanical and environmental challenges. We examined the effects of near-future pH (ambient pH –0.4 pH units) and warming (ambient temperature +4°C) on the shells of the commercially important bivalve, Mytilus edulis when fed for a limited period (4–6 h day⁻¹). After six months exposure, warming, but not acidification, significantly reduced shell strength determined as reductions in the maximum load endured by the shells. However, acidification resulted in a reduction in shell flex before failure. Reductions in shell strength with warming could not be explained by alterations in morphology, or shell composition but were accompanied by reductions in shell surface area, and by a fall in whole-body condition index. It appears that warming has an indirect effect on shell strength by re-allocating energy from shell formation to support temperature-related increases in maintenance costs, especially as food supply was limited and the mussels were probably relying on internal energy reserves. The maintenance of shell strength despite seawater acidification suggests that biomineralisation processes are unaffected by the associated changes in CaCO₃ saturation levels. We conclude that under near-future climate change conditions, ocean warming will pose a greater risk to shell integrity in M. edulis than ocean acidification when food availability is limited.     

Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B₁₂ (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B₁₂ and hydroxocobalamin. Assimilation of these compounds requires reduction of their central cobalt atom from Co³⁺ to Co²⁺ to Co⁺, followed by adenosylation to AdoCbl. In this work, the His₆-tagged PduS cobalamin reductase from S. enterica was produced at high levels in Escherichia coli, purified, and characterized. The anaerobically purified enzyme reduced cob(III)alamin to cob(II)alamin at a rate of 42.3 ± 3.2 μmol min⁻¹ mg⁻¹, and it reduced cob(II)alamin to cob(I)alamin at a rate of 54.5 ± 4.2 nmol min⁻¹ mg⁻¹ protein. The apparent K_m values of PduS-His₆ were 10.1 ± 0.7 μM for NADH and 67.5 ± 8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent K_m values for cob(II)alamin reduction were 27.5 ± 2.4 μM with NADH as the substrate and 72.4 ± 9.5 μM with cob(II)alamin as the substrate. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) indicated that each monomer of PduS contained one molecule of noncovalently bound flavin mononucleotide (FMN). Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD, supporting a role in cobalamin reduction in vivo. Further studies demonstrated that the PduS protein is a component of the Pdu microcompartments (MCPs) used for 1,2-PD degradation and that it interacts with the PduO adenosyltransferase, which catalyzes the terminal step of AdoCbl synthesis. These studies further characterize PduS, an unusual MCP-associated cobalamin reductase, and, in conjunction with prior results, indicate that the Pdu MCP encapsulates a complete cobalamin assimilation system.Coenzyme B₁₂ (adenosylcobalamin, AdoCbl) is an indispensable cofactor for a variety of enzymes that are widely distributed among microbes and higher animals (2, 55). Organisms obtain AdoCbl by de novo synthesis or by assimilation of complex precursors, such as vitamin B₁₂ (cyanocobalamin, CN-Cbl) and hydroxocobalamin (OH-Cbl), which can be enzymatically converted to AdoCbl. De novo synthesis occurs only in prokaryotes, but the assimilation of complex precursors is more widespread, taking place in many microbes and in higher animals (56). A model for the assimilation of CN-Cbl and OH-Cbl to AdoCbl, based on work done in a number of laboratories, is shown in Fig. ​Fig.1.1. CN-Cbl is first reductively decyanated to cob(II)alamin (22, 30, 68). Next, cob(II)alamin is reduced to cob(I)alamin, and ATP:cob(I)alamin adenosyltransferase (ATR) transfers a 5′ deoxyadenosyl group from ATP to cob(I)alamin to form AdoCbl (10, 11, 28, 29, 35, 63, 64, 72). Studies indicate that prior to reduction cob(II)alamin binds the ATR and undergoes a transition to the 4-coordinate base-off conformer (41, 48, 59, 61, 62). Transition to the 4-coordinate state raises the midpoint potential of the cob(II)alamin/cob(I)alamin couple by about 250 mV, facilitating reduction (60). OH-Cbl assimilation occurs by a similar pathway except that the first step is reduction of OH-Cbl to cob(II)alamin by cobalamin reductase or by the reducing environment of the cell (19, 69).Open in a separate windowFIG. 1.Cobalamin assimilation and recycling pathway. Many organisms are able to take up CN-Cbl and OH-Cbl and convert them to the active coenzyme form, AdoCbl. This process involves reduction of the central cobalt atom of the corrin ring followed by addition of a 5′ deoxyadenosyl (Ado) group via a carbon-cobalt bond. The Ado group is unstable in vivo, and AdoCbl breaks down to form OH-Cbl. Consequently, cobalamin recycling is required for AdoCbl-dependent processes, and recycling uses the same pathway that functions in the assimilation of cobalamin from the environment. PPPi, triphosphate.The pathway used for the assimilation of OH-Cbl and CN-Cbl is also used for intracellular cobalamin recycling. During catalysis the adenosyl group of AdoCbl is periodically lost due to by-reactions and is usually replaced by a hydroxyl group, resulting in the formation of an inactive OH-Cbl enzyme complex (66). Cobalamin recycling begins with a reactivase that converts the inactive OH-Cbl-enzyme complex to OH-Cbl and apoenzyme (43, 44). Next, the process described in Fig. ​Fig.11 converts OH-Cbl to AdoCbl, which spontaneously associates with apoenzyme to form active holoenzyme (43, 44, 66). In the organisms that have been studied, cobalamin recycling is essential, and genetic defects in this process block AdoCbl-dependent metabolism (3, 16, 29).Salmonella enterica degrades 1,2-propanediol (1,2-PD) via an AdoCbl-dependent pathway (27). 1,2-PD is a major product of the anaerobic degradation of common plant sugars rhamnose and fucose and is thought to be an important carbon and energy source in natural environments (38, 46). Twenty-four genes for 1,2-PD utilization (pdu) are found in a contiguous cluster (pocR, pduF, and pduABB′CDEGHJKLMNOPQSTUVWX) (7, 27). This locus encodes enzymes for the degradation of 1,2-PD and cobalamin recycling, as well as proteins for the formation of a bacterial microcompartment (MCP) (7). Bacterial MCPs are simple proteinaceous organelles used by diverse bacteria to optimize metabolic pathways that have toxic or volatile intermediates (6, 13, 14, 71). They are polyhedral in shape, 100 to 150 nm in cross section (about the size of a large virus), and consist of a protein shell that encapsulates sequentially acting metabolic enzymes. Sequence analyses indicate that MCPs are produced by 20 to 25% of all bacteria and function in seven or more different metabolic processes (14). The function of the Pdu MCP is to confine the propionaldehyde formed in the first step of 1,2-PD degradation in order to mitigate its toxicity and prevent DNA damage (7, 23, 24, 51). Prior studies indicate that 1,2-PD traverses the protein shell and enters the lumen of the Pdu MCP, where it is converted to propionaldehyde and then to propionyl-coenzyme A (CoA) by AdoCbl-dependent diol dehydratase (DDH; PduCDE) and propionaldehyde dehydrogenase (PduP) (8, 33). Propionyl-CoA then exits the MCP into the cytoplasm, where it is converted to 1-propanol or propionate or enters central metabolism via the methylcitrate pathway (25, 47). The shell of the Pdu MCP is thought to limit the diffusion of propionaldehyde in order to protect cytoplasmic components from toxicity. The Pdu MCP was purified, and 14 major polypeptide components were identified (PduABB′CDEGHJKOPTU), all of which are encoded by the pdu locus (23). PduABB′JKTU are confirmed or putative shell proteins (23, 24, 51). PduCDE and PduP catalyze the first 2 steps of 1,2-PD degradation as described above (7, 8, 23, 33). The PduO and PduGH enzymes are used for cobalamin recycling. PduO is an adenosyltransferase (29), and PduGH is a homolog of the Klebsiella DDH reactivase, which mediates the removal of OH-Cbl from an inactive OH-Cbl-DDH complex (43, 44). However, a reductase which is also required for cobalamin recycling was not previously identified as a component of the Pdu MCP (23). This raises the question of how cobalamin is recycled for the AdoCbl-dependent DDH that resides within the Pdu MCP.Prior studies indicated that the PduS enzyme (which is encoded by the pdu locus) is a cobalamin reductase (52). Very recently PduS was purified from S. enterica and shown to be a flavoprotein that can mediate the reduction of 4-coordinate cob(II)alamin bound to ATR but was not further characterized (40). In this study, PduS from S. enterica is purified and more extensively characterized, including identification of its cofactor requirements and kinetic properties. In addition, we show that PduS is a component of the Pdu MCP. This finding in conjunction with prior work indicates that, in addition to 1,2-PD degradative enzymes, the Pdu MCP encapsulates a complete cobalamin recycling system.     

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.     

Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.     

The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa,Caused by the Fungus Moniliophthora perniciosa

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.     

Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium,and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P < 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P < 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.     

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from Bacillus licheniformis, N-acetyl-d-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to N-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.