Cloning, overexpression, and structural characterization of a novel archaeal thermostable neopullulanase from Desulfurococcus mucosus DSM 2162.

The main purpose of the present study is to introduce the biochemical characteristics of the industrial valuable thermostable pullulan degrading enzyme from Desulfurococcus mucosus DSM2162. Recombinant protein was purified by a combination of thermal treatment and affinity chromatography, with a yield of 15.94% and 7.69-fold purity. Purified enzyme showed the molecular mass of 55,787 Da with optimum activity at 70 °C and a broad range of pH (5.0-9.0) with kcat of 2150 min-1 and Km of 6.55 mg.mL-1, when using starch as substrate. The enzyme activity assay on various polysaccharide substrates revealed the substrate preference of pullulan > amylopectin > ß cyclodextrin > starch > glycogen; therefore, it classified as a neopullulanase. The neopullulanase structural analysis by spectrofluorometer, FT-IR, and circular dichroism spectroscopy indicated the corporation of α-helix (47.3%) and ß-sheet (31.6%) in its secondary structure. The melting temperature and specific heat capacity calculations using differential scanning calorimetry confirmed its extreme thermal stability. Further, salt-elevated concentrations resulted in oligomeric state dominancy without any significant influence on the starch-degrading ability. The newly cloned archaeal neopullulanase was with broad activity on polysaccharide substrates, with thermal and salt stability. Thus, the Desulfurococcus mucosus DSM2162 neopullulanase can be introduced as a good candidate to be used in carbohydrate industry.

Functional compartmentalization and metabolic separation in a prokaryotic cell.

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC.

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.

Structure of an Influenza A virus N9 neuraminidase with a tetrabrachion-domain stalk.

The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3 Šresolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.

Metabolic reconstruction and experimental verification of glucose utilization in Desulfurococcus amylolyticus DSM 16532.

Desulfurococcus amylolyticus DSM 16532 is an anaerobic and hyperthermophilic crenarchaeon known to grow on a variety of different carbon sources, including monosaccharides and polysaccharides. Furthermore, D. amylolyticus is one of the few archaea that are known to be able to grow on cellulose. Here, we present the metabolic reconstruction of D. amylolyticus' central carbon metabolism. Based on the published genome, the metabolic reconstruction was completed by integrating complementary information available from the KEGG, BRENDA, UniProt, NCBI, and PFAM databases, as well as from available literature. The genomic analysis of D. amylolyticus revealed genes for both the classical and the archaeal version of the Embden-Meyerhof pathway. The metabolic reconstruction highlighted gaps in carbon dioxide-fixation pathways. No complete carbon dioxide-fixation pathway such as the reductive citrate cycle or the dicarboxylate-4-hydroxybutyrate cycle could be identified. However, the metabolic reconstruction indicated that D. amylolyticus harbors all genes necessary for glucose metabolization. Closed batch experimental verification of glucose utilization by D. amylolyticus was performed in chemically defined medium. The findings from in silico analyses and from growth experiments are discussed with respect to physiological features of hyperthermophilic organisms.

Archaea S-layer nanotube from a "black smoker" in complex with cyclo-octasulfur (S8 ) rings.

Elemental sulfur exists primarily as an S80 ring and serves as terminal electron acceptor for a variety of sulfur-fermenting bacteria. Hyperthermophilic archaea from black smoker vents are an exciting research tool to advance our knowledge of sulfur respiration under extreme conditions. Here, we use a hybrid method approach to demonstrate that the proteinaceous cavities of the S-layer nanotube of the hyperthermophilic archaeon Staphylothermus marinus act as a storage reservoir for cyclo-octasulfur S8. Fully atomistic molecular dynamics (MD) simulations were performed and the method of multiconfigurational thermodynamic integration was employed to compute the absolute free energy for transferring a ring of elemental sulfur S8 from an aqueous bath into the largest hydrophobic cavity of a fragment of archaeal tetrabrachion. Comparisons with earlier MD studies of the free energy of hydration as a function of water occupancy in the same cavity of archaeal tetrabrachion show that the sulfur ring is energetically favored over water.

Multi-omics analysis provides insight to the Ignicoccus hospitalis-Nanoarchaeum equitans association.

BACKGROUND: Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS: We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS: Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS: This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE: Our study applies statistical and visualization techniques to a mixed-source omics dataset to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.

Biochemical characterization of TyrA enzymes from Ignicoccus hospitalis and Haemophilus influenzae: A comparative study of the bifunctional and monofunctional dehydrogenase forms.

Biosynthesis of l-tyrosine (l-Tyr) is directed by the interplay of two enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which is then converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD). This work reports the first characterization of the independently expressed PD domain of bifunctional CM-PD from the crenarchaeon Ignicoccus hospitalis and the first functional studies of both full-length CM-PD and the PD domain from the bacterium Haemophilus influenzae. All proteins were hexa-histidine tagged, expressed in Escherichia coli and purified. Expression and purification of I. hospitalis CM-PD generated a degradation product identified as a PD fragment lacking the protein's first 80 residues, Δ80CM-PD. A comparable stable PD domain could also be generated by limited tryptic digestion of this bifunctional enzyme. Thus, Δ80CM-PD constructs were prepared in both organisms. CM-PD and Δ80CM-PD from both organisms were dimeric and displayed the predicted enzymatic activities and thermal stabilities in accord with their hyperthermophilic and mesophilic origins. In contrast with H. influenzae PD activity which was NAD+-specific and displayed >75% inhibition with 50µM l-Tyr, I. hospitalis PD demonstrated dual cofactor specificity with a preference for NADP+ and an insensitivity to l-Tyr. These properties are consistent with a model of the I. hospitalis PD domain based on the previously reported structure of the H. influenzae homolog. Our results highlight the similarities and differences between the archaeal and bacterial TyrA proteins and reveal that the PD activity of both prokaryotes can be successfully mapped to a functionally independent unit.

In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis lives in symbiosis with Nanoarchaeum equitans providing essential cell components and nutrients to its symbiont. Ignicoccus hospitalis shows an intriguing morphology that points toward an evolutionary role in driving compartmentalization. Therefore, the bioenergetics of this archaeal host-symbiont system remains a pressing question. To date, the only electron acceptor described for I. hospitalis is elemental sulfur, but the organism comprises genes that encode for enzymes involved in nitrogen metabolism, e.g., one nitrate reductase and two octaheme cytochrome c, Igni_0955 (IhOCC) and Igni_1359. Herein, we detail functional and structural studies of the highly abundant IhOCC, including an X-ray crystal structure at 1.7 Å resolution, the first three-dimensional structure of an archaeal OCC. The trimeric IhOCC is membrane associated and exhibits significant structural and functional differences to previously characterized homologs within the hydroxylamine oxidoreductases (HAOs) and octaheme cytochrome c nitrite reductases (ONRs). The positions and spatial arrangement of the eight hemes are highly conserved, but the axial ligands of the individual hemes 3, 6 and 7 and the protein environment of the active site show significant differences. Most notably, the active site heme 4 lacks porphyrin-tyrosine cross-links present in the HAO family. We show that IhOCC efficiently reduces nitrite and hydroxylamine, with possible relevance to detoxification or energy conservation. DATABASE: Structural data are available in the Protein Data Bank under the accession number 4QO5.

Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics.

Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome these limitations is to unambiguously identify the key structural players involved in catalysis. Here, we report the E117A I-DmoI mutant crystal structure at 2.2 Šresolution that, together with the wt and Q42A/K120M constructs, is combined with computational approaches to shed light on protein cleavage activity. The cleavage mechanism was related both to key structural effects, such as the position of water molecules and ions participating in the cleavage reaction, and to dynamical effects related to protein behavior. In particular, we found that the protein perturbation pattern significantly changes between cleaved and noncleaved DNA strands when the ions and water molecules are correctly positioned for the nucleophilic attack that initiates the cleavage reaction, in line with experimental enzymatic activity. The proposed approach paves the way for an effective, general, and reliable procedure to analyze the enzymatic activity of endonucleases from a very limited data set, i.e., structure and dynamics.

The Iho670 fibers of Ignicoccus hospitalis are anchored in the cell by a spherical structure located beneath the inner membrane.

The Iho670 fibers of the hyperthermophilic crenarchaeon of Ignicoccus hospitalis were shown to contain several features that indicate them as type IV pilus-like structures. The application of different visualization methods, including electron tomography and the reconstruction of a three-dimensional model, enabled a detailed description of a hitherto undescribed anchoring structure of the cell appendages. It could be identified as a spherical structure beneath the inner membrane. Furthermore, pools of the fiber protein Iho670 could be localized in the inner as well as the outer cellular membrane of I. hospitalis cells and in the tubes/vesicles in the intermembrane compartment by immunological methods.

[Capacity of hyperthermophilic Crenarchaeota for decomposition of refractory protiens (α- and ß-keratins)].

Anaerobic thermophilic archaea of the genera Thermogladius and Desulfurococcus capable of a- and P3-keratin decomposition were isolated from hot springs of Kamchatka and Kunashir Island. For two of them (strains 2355k and 3008g), the presence of high-molecular mass, cell-bound endopeptidases active against nonhydrolyzed and partially hydrolyzed proteins at high values of temperature and pH was shown. Capacity for ß-keratin decomposition was also found in collection strains (type strains of Desulfurococcus amylolyticus subsp. amylolyticus, D. mucosus subsp. mobilis, and D. fermentans).

Complete genome sequence of the hyperthermophilic cellulolytic crenarchaeon "Thermogladius cellulolyticus" 1633.

Strain 1633, a novel member of the genus Thermogladius, isolated from a freshwater hot spring, is an anaerobic hyperthermophilic crenarchaeon capable of fermenting proteinaceous and cellulose substrates. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof pathway for glucose metabolism, cytoplasmic NADP-dependent hydrogenase, and several energy-coupling membrane-bound oxidoreductases.

Filaments from Ignicoccus hospitalis show diversity of packing in proteins containing N-terminal type IV pilin helices.

Bacterial motility is driven by the rotation of flagellar filaments that supercoil. The supercoiling involves the switching of coiled-coil protofilaments between two different states. In archaea, the flagellar filaments responsible for motility are formed by proteins with distinct homology in their N-terminal portion to bacterial Type IV pilins. The bacterial pilins have a single N-terminal hydrophobic α-helix, not the coiled coil found in flagellin. We have used electron cryo-microscopy to study the adhesion filaments from the archaeon Ignicoccus hospitalis. While I. hospitalis is non-motile, these filaments make transitions between rigid stretches and curved regions and appear morphologically similar to true archaeal flagellar filaments. A resolution of ~7.5Å allows us to unambiguously build a model for the packing of these N-terminal α-helices, and this packing is different from several bacterial Type IV pili whose structure has been analyzed by electron microscopy and modeling. Our results show that the mechanism responsible for the supercoiling of bacterial flagellar filaments cannot apply to archaeal filaments.

The unusual cell biology of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis is an anaerobic, obligate chemolithoautotrophic hyperthermophile, growing by reduction of elemental sulfur using molecular hydrogen as electron donor. Together with Nanoarchaeum equitans it forms a unique, archaeal biocoenosis, in which I. hospitalis serves as host for N. equitans. Both organisms can be cultivated in a stable coculture which is mandatory for N. equitans but not for I. hospitalis. This strong dependence is affirmed by the fact that N. equitans obtains its lipids and amino acids from the host. I. hospitalis cells exhibit several unique features: they can adhere to surfaces by extracellular appendages ('fibers') which are not used for motility; they use a novel CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate pathway; and they exhibit a unique cell envelope for Archaea consisting of two membranes but lacking an S-layer. These membranes form two cell compartments, a tightly packed cytoplasm surrounded by a weakly staining intermembrane compartment (IMC) with a variable width from 20 to 1,000 nm. In this IMC, many round or elongated vesicles are found which may function as carriers of lipids or proteins out of the cytoplasm. Based on immuno-EM analyses and immuno-fluorescence experiments it was demonstrated recently that the A(1)A(O) ATP synthase, the H(2):sulfur oxidoreductase complex and the acetyl-CoA synthetase (ACS) of I. hospitalis are located in its outermost membrane. Therefore, this membrane is energized and is here renamed as "outer cellular membrane" (OCM). Among all prokaryotes possessing two membranes in their cell envelope, I. hospitalis is the first organism with an energized outermost membrane and ATP synthesis outside the cytoplasm. Since DNA and ribosomes are localized in the cytoplasm, energy conservation is separated from information processing and protein biosynthesis in I. hospitalis. This raises questions concerning the function and characterization of the two membranes, the two cell compartments and of a possible ATP transfer to N. equitans.

AMP-forming acetyl coenzyme A synthetase in the outermost membrane of the hyperthermophilic crenarchaeon Ignicoccus hospitalis.

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

Proteomic characterization of cellular and molecular processes that enable the Nanoarchaeum equitans--Ignicoccus hospitalis relationship.

Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis.

Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5'-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Šresolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation.

Thermogladius shockii gen. nov., sp. nov., a hyperthermophilic crenarchaeote from Yellowstone National Park, USA.

A hyperthermophilic heterotrophic archaeon (strain WB1) was isolated from a thermal pool in the Washburn hot spring group of Yellowstone National Park, USA. WB1 is a coccus, 0.6-1.2 µm in diameter, with a tetragonal S-layer, vacuoles, and occasional stalk-like protrusions. Growth is optimal at 84°C (range 64-93°C), pH 5-6 (range 3.5-8.5), and <1 g/l NaCl (range 0-4.6 g/l NaCl). Tests of metabolic properties show the isolate to be a strict anaerobe that ferments complex organic substrates. Phylogenetic analysis of the 16S rRNA gene sequence places WB1 in a clade of previously uncultured Desulfurococcaceae and shows it to have ≤ 96% 16S rRNA sequence identity to Desulfurococcus mobilis, Staphylothermus marinus, Staphylothermus hellenicus, and Sulfophobococcus zilligii. The 16S rRNA gene contains a large insertion similar to homing endonuclease introns reported in Thermoproteus and Pyrobaculum species. Growth is unaffected by the presence of S(0) or SO(4)(2-), thereby differentiating the isolate from its closest relatives. Based on phylogenetic and physiological differences, it is proposed that isolate WB1 represents the type strain of a novel genus and species within the Desulfurococcaceae, Thermogladius shockii gen. nov., sp. nov. (RIKEN = JCM-16579, ATCC = BAA-1607, Genbank 16S rRNA gene = EU183120).

Importance of sulfate reducing bacteria in mercury methylation and demethylation in periphyton from Bolivian Amazon region.

Sulfate reducing bacteria (SRB) are important mercury methylators in sediments, but information on mercury methylators in other compartments is ambiguous. To investigate SRB involvement in methylation in Amazonian periphyton, the relationship between Hg methylation potential and SRB (Desulfobacteraceae, Desulfobulbaceae and Desulfovibrionaceae) abundance in Eichhornia crassipes and Polygonum densiflorum root associated periphyton was examined. Periphyton subsamples of each macrophyte were amended with electron donors (lactate, acetate and propionate) or inhibitors (molybdate) of sulfate reduction to create differences in SRB subgroup abundance, which was measured by quantitative real-time PCR with primers specific for the 16S rRNA gene. Mercury methylation and demethylation potentials were determined by a stable isotope tracer technique using 200HgCl and CH3(202)HgCl, respectively. Relative abundance of Desulfobacteraceae (<0.01-12.5%) and Desulfovibrionaceae (0.01-6.8%) were both highly variable among samples and subsamples, but a significant linear relationship (p<0.05) was found between Desulfobacteraceae abundance and net methylmercury formation among treatments of the same macrophyte periphyton and among all P. densiflorum samples, suggesting that Desulfobacteraceae bacteria are the most important mercury methylators among SRB families. Yet, molybdate only partially inhibited mercury methylation potentials, suggesting the involvement of other microorganisms as well. The response of net methylmercury production to the different electron donors and molybdate was highly variable (3-1104 pg g(-1) in 12 h) among samples, as was the net formation in control samples (17-164 pg g(-1) in 12 h). This demonstrates the importance of community variability and complexity of microbial interactions for the overall methylmercury production in periphyton and their response to external stimulus.