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Harald Huber - One of the best experts on this subject based on the ideXlab platform.
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Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC
Archives of Microbiology, 2020Co-Authors: Dagmar Koschnitzki, Reinhard Rachel, Reinhard Wirth, Harald Huber, Kristina Beblo-vranesevic, Ralf Moeller, Stefan Leuko, Bartos Przybyla, Petra RettbergAbstract: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 D _10-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 .
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The hyperthermophilic partners Nanoarchaeum and Ignicoccus stabilize their tRNA T-loops via different but structurally equivalent modifications
Nucleic Acids Research, 2020Co-Authors: Simon Rose, Harald Huber, Sylvie Auxilien, Jesper F. Havelund, Finn Kirpekar, Henri Grosjean, Stephen DouthwaiteAbstract:The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m(5)U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m(5)U54 is subsequently thiolated to form m(5)s(2)U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m(1)s(4)Psi. The methyl and thiol groups in m(1)s(4)Psi and m(5)s(2)U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m(1)A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.
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Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.
Journal of bacteriology, 2019Co-Authors: Lydia Kreuter, Andrea Weinfurtner, Alexander Ziegler, Julia Weigl, Jan Hoffmann, Nina Morgner, Volker Müller, Harald HuberAbstract:In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized and structurally compared to the respective enzymes of the other Ignicoccus species to shed light on energy conservation in this unique group of Archaea. The crenarchaeal genus Ignicoccus comprises three described species: I. hospitalis and I. islandicus from hot marine sediments near Iceland and I. pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all Archaea due to the unusual cell envelope consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly for being the only known host for the potentially parasitic archaeon Nanoarchaeum equitans . I. hospitalis grows chemolithoautotrophically, and its sole energy yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electro-chemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes for nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A 1 A O complex seems to be at pH 9. Interestingly, the soluble A 1 subcomplexes of the different Ignicoccus species exhibit significant differences in their apparent molecular masses in native electrophoresis, although their behavior in gel filtration and mass spectrometry chromatography is very similar. IMPORTANCE The Crenarchaeota represent one of the major phyla within the domain of the Archaea. This study describes the successful purification of a crenarchaeal ATP synthase. So far, all information about A-type ATP synthases stem from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now, points towards significant differences in their stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among the Crenarchaeota, since it is the only known host of N. equitans . The energy metabolism in this system is still poorly comprehended and our results can help understand the unique relationship between these two microbes.
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Bypassing rRNA methylation by RsmA/Dim1during ribosome maturation in the hyperthermophilic archaeon Nanoarchaeum equitans.
Nucleic acids research, 2017Co-Authors: Kenneth H. Seistrup, Harald Huber, Simon Rose, Ulf Birkedal, Henrik Nielsen, Stephen DouthwaiteAbstract:In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate here that the I. hospitalis host possesses the homolog Igni_1059, which dimethylates the N6-positions of two invariant adenosines within helix 45 of 16S rRNA in a manner identical to other RsmA/Dim1 enzymes. However, Igni_1059 is not transferred from I. hospitalis to N. equitans across their fused cell membrane structures and the corresponding nucleotides in N. equitans 16S rRNA remain unmethylated. An alternative mechanism for ribosomal subunit maturation in N. equitans is suggested by sRNA interactions that span the redundant RsmA/Dim1 site to introduce 2΄-O-ribose methylations within helices 44 and 45 of the rRNA.
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In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.
The FEBS journal, 2016Co-Authors: Kristian Parey, Reinhard Rachel, Harald Huber, Alistair J. Fielding, Matthias Sörgel, Christine Ziegler, Chitra RajendranAbstract: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 towards 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 A 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 homologues 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. This article is protected by copyright. All rights reserved.
Reinhard Rachel - One of the best experts on this subject based on the ideXlab platform.
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Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC
Archives of Microbiology, 2020Co-Authors: Dagmar Koschnitzki, Reinhard Rachel, Reinhard Wirth, Harald Huber, Kristina Beblo-vranesevic, Ralf Moeller, Stefan Leuko, Bartos Przybyla, Petra RettbergAbstract: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 D _10-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 .
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In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.
The FEBS journal, 2016Co-Authors: Kristian Parey, Reinhard Rachel, Harald Huber, Alistair J. Fielding, Matthias Sörgel, Christine Ziegler, Chitra RajendranAbstract: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 towards 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 A 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 homologues 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. This article is protected by copyright. All rights reserved.
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Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain.
Proceedings of the National Academy of Sciences, 2016Co-Authors: Tatjana Braun, Matthijn R. J. Vos, Nir Kalisman, Nicholas E. Sherman, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder, Edward H. EgelmanAbstract:The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-A resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.
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Cytochromes c in Archaea: distribution, maturation, cell architecture, and the special case of Ignicoccus hospitalis
Frontiers in microbiology, 2015Co-Authors: Arnulf Kletzin, Reinhard Rachel, Thomas Heimerl, Jennifer Flechsler, Laura Van Niftrik, Andreas KlinglAbstract:Cytochromes c (Cytc) are widespread electron transfer proteins and important enzymes in the global nitrogen and sulfur cycles. The distribution of Cytc in more than 300 archaeal proteomes deduced from sequence was analyzed with computational methods including pattern and similarity searches, secondary and tertiary structure prediction. Two hundred and fifty-eight predicted Cytc (with single, double, or multiple heme c attachment sites) were found in some but not all species of the Desulfurococcales, Thermoproteales, Archaeoglobales, Methanosarcinales, Halobacteriales, and in two single-cell genome sequences of the Thermoplasmatales, all of them Cren- or Euryarchaeota. Other archaeal phyla including the Thaumarchaeota are so far free of these proteins. The archaeal Cytc sequences were bundled into 54 clusters of mutual similarity, some of which were specific for Archaea while others had homologs in the Bacteria. The cytochrome c maturation system I (CCM) was the only one found. The highest number and variability of Cytc were present in those species with known or predicted metal oxidation and/or reduction capabilities. Paradoxical findings were made in the haloarchaea: several Cytc had been purified biochemically but corresponding proteins were not found in the proteomes. The results are discussed with emphasis on cell morphologies and envelopes and especially for double-membraned Archaea-like Ignicoccus hospitalis. A comparison is made with compartmentalized bacteria such as the Planctomycetes of the Anammox group with a focus on the putative localization and roles of the Cytc and other electron transport proteins.
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Life on the edge: functional genomic response of Ignicoccus hospitalis to the presence of Nanoarchaeum equitans
The ISME Journal, 2015Co-Authors: Richard J. Giannone, Reinhard Rachel, Harald Huber, Thomas Heimerl, Robert L. Hettich, Louie L. Wurch, Stanton Martin, Zamin Yang, Mircea PodarAbstract:The marine hyperthermophilic crenarchaeon Ignicoccus hospitalis supports the propagation on its surface of Nanoarchaeum equitans , an evolutionarily enigmatic archaeon that resembles highly derived parasitic and symbiotic bacteria. The cellular and molecular mechanisms that enable this interarchaea relationship and the intimate physiologic consequences to I. hospitalis are unknown. Here, we used concerted proteomic and transcriptomic analyses to probe into the functional genomic response of I. hospitalis as N. equitans multiplies on its surface. The expression of over 97% of the genes was detected at mRNA level and over 80% of the predicted proteins were identified and their relative abundance measured by proteomics. These indicate that little, if any, of the host genomic information is silenced during growth in the laboratory. The primary response to N. equitans was at the membrane level, with increases in relative abundance of most protein complexes involved in energy generation as well as that of several transporters and proteins involved in cellular membrane stabilization. Similar upregulation was observed for genes and proteins involved in key metabolic steps controlling nitrogen and carbon metabolism, although the overall biosynthetic pathways were marginally impacted. Proliferation of N. equitans resulted, however, in selective downregulation of genes coding for transcription factors and replication and cell cycle control proteins as I. hospitalis shifted its physiology from its own cellular growth to that of its ectosymbiont/parasite. The combination of these multiomic approaches provided an unprecedented level of detail regarding the dynamics of this interspecies interaction, which is especially pertinent as these organisms are not genetically tractable.
Ulrike Jahn - One of the best experts on this subject based on the ideXlab platform.
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Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins
Archives of Microbiology, 2008Co-Authors: Tillmann Burghardt, Ulrike Jahn, Carolin Meyer, Sonja Gürster, Frank Siedler, Manfred Saller, Daniel Müller, Eduard Hochmuth, Rainer Deutzmann, Patrick BabingerAbstract:Ignicoccus hospitalis , a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans . Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans , as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.
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aspekte der zellbiologie des archaeellen wirt parasit systems Ignicoccus hospitalis und nanoarchaeum equitans zentrale stoffwechselwege lipide histone
2008Co-Authors: Ulrike JahnAbstract:Biosynthesewege in I. hospitalis wurden durch eine Kombination aus drei voneinander unabhangigen Methoden (enzymatische Analysen, Genomanalysen und 13C-Langzeit-Markierungen von Aminosauren) untersucht. Dabei konnte gezeigt werden, dass sich I. hospitalis eines bisher unbekannten CO2-Fixierungswegs bedient. Der erste CO2-Fixierungsschritt ist offensichtlich die Carboxylierung von Acetyl-CoA zu Pyruvat, welches zu PEP umgewandelt wird. Den zweiten CO2-Fixierungsschritt stellt die Carboxylierung von PEP zu Oxalacetat dar. Oxalacetat mundet in einen unvollstandigen Citrat-Zyklus, dem die 2-Oxoglutarat:Akzeptor-Oxidoreduktase fehlt. Bisher ungeklart ist die Regeneration/Synthese des primaren CO2-Akzeptors Acetyl-CoA. Ob der unvollstandige Citrat-Zyklus an der Regeneration des Acetats beteilig ist, oder nur anabolen Zwecken dient, ist Gegenstand der aktuellen Forschung. Des Weiteren wurde gezeigt, dass I. hospitalis uber eine re-spezifische Citrat-Synthase verfugt. Die Gluconeogenese scheint in I. hospitalis uber einen ruckwarts gerichteten (modifizierten) Embden-Meyerhoff-Weg stattzufinden. Die Biosynthese von Pentosephosphaten erfolgt wahrscheinlich uber eine Deformylierung von Hexosephosphat, uber den Ribulose-Monophosphat-Weg. I. hospitalis fehlen vermutlich alle glycolytischen Wege zur Gewinnung von Energie oder Zellbausteinen aus Zuckern. Die Aminosaure-Biosynthesewege verlaufen in I. hospitalis fur die meisten Aminosauren uber die bekannten, konventionellen Wege. Aromatische Aminosauren werden uber den konventionellen Shikimat-Weg und nicht uber den kurzlich beschriebenen Aspartat-Semialdehyd-Weg synthetisiert. Zur Synthese von Lysin wird offensichtlich der 2-Aminoadipatweg genutzt, der bisher nur bei primitiven Eukaryota, Bacteria der Gattung Thermus und dem Crenarchaeum Thermoproteus neutrophilus entdeckt wurde. Die Synthese von Isoleucin erfolgt den hier gezeigten Ergebnissen nach uber den Citramalat-Weg, der bisher ausschlieslich in Bacteria der Gattung Leptospira, methanogenen Archaea und Thermoproteus neutrophilus gefunden wurde. Die Membranen von I. hospitalis und N. equitans wurden mittels GC-MS und LC-MS auf ihre Lipidzusammensetzung hin untersucht. Dabei wurden sowohl die Kohlenwasserstoffketten und die Zucker der Lipide (GC-MS), als auch die intakten polaren Lipide (LC-MS) bestimmt. Die Membranen wiesen eine einfache Lipidzusammensetzung aus Glycerin-Diphytan-Diether- und Glycerin-Dibiphytan-Glycerin-Tetraetherlipiden auf, die glycosyliert (mit Mannose oder Glucoseresten) und phosphoryliert sein konnten. Vergleichende Lipidanalysen zeigten, dass N. equitans seine Lipide offensichtlich vollstandig von seinem Wirt I. hospitalis ubernimmt. Auf welche Weise dieser Lipidtransfer stattfindet, ist ungeklart. Spezifische Biomarker wurden fur keinen der beiden Organismen detektiert. I. hospitalis-Zellen, die bei der minimalen, optimalen und maximalen Wachstumstemperatur gezuchtet wurden, zeigten einen Abfall des Tetraetherlipid-Anteils von der optimalen zur minimalen/maximalen Wachstumstemperatur. Ein erhohter Tetraetherlipid-Anteil ist in I. hospitalis somit nicht zur Aufrechterhaltung der Membranstabilitat bei erhohten Temperaturen erforderlich. Die mutmaslichen Histongene Neq288 und Neq348 von N. equitans wurden in E. coli exprimiert. Nach Aufreinigung der rekombinanten Histone zeigten Dimerisierungs- und DNA-Kompaktierungsanalysen, dass die Gene fur funktionelle Histone kodieren. NEQ288 weist im Gegensatz zu allen anderen archaeellen Histonen einen Lysinrest in Loop 1 auf, welchem bei eukaryotischen Histonen eine wichtige Rolle bei DNA-Bindung und Kompaktierung zugeschrieben wird. In den hier durchgefuhrten Tests wies rNEQ288 tatsachlich eher eukaryotische als archaeelle Kompaktierungseigenschaften auf: rNEQ288 band an DNA, fuhrte jedoch nicht zu einer Kompaktierung. In aquimolaren Mischungen aus rNEQ348 und rNEQ288 fand hingegen eine DNA-Kompaktierung statt die starker war, als fur die gleiche Menge an rNEQ348 allein. Die genauen Ursachen sind Gegenstand weiterer Forschung.
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a dicarboxylate 4 hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic archaeum Ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Harald Huber, Ivan A. Berg, Martin Gallenberger, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang Eisenreich, Georg FuchsAbstract:Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO2 acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-14C]4-hydroxybutyrate, [1,4-13C2], [U-13C4]succinate, or [1-13C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO2 fixation mechanism.
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A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Harald Huber, Ivan A. Berg, Martin Gallenberger, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang Eisenreich, Georg FuchsAbstract:Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO2 acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-14C]4-hydroxybutyrate, [1,4-13C2], [U-13C4]succinate, or [1-13C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO2 fixation mechanism.
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Nanoarchaeum equitans and Ignicoccus hospitalis: New Insights into a Unique, Intimate Association of Two Archaea
Journal of bacteriology, 2007Co-Authors: Ulrike Jahn, Reinhard Rachel, Martin Gallenberger, Wolfgang Eisenreich, Walter Paper, Karl O Stetter, Benjamin Junglas, Harald HuberAbstract:Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.
Carolin Meyer - One of the best experts on this subject based on the ideXlab platform.
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The Iho670 Fibers of Ignicoccus hospitalis Are Anchored in the Cell by a Spherical Structure Located beneath the Inner Membrane
Journal of bacteriology, 2014Co-Authors: Carolin Meyer, Thomas Heimerl, Reinhard Wirth, Andreas Klingl, Reinhard RachelAbstract: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.
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Filaments from Ignicoccus hospitalis Show Diversity of Packing in Proteins Containing N-Terminal Type IV Pilin Helices.
Journal of molecular biology, 2012Co-Authors: Charles Goforth, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder, Carolin Meyer, Edward H. EgelmanAbstract: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 ~7.5A 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.
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AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis
Journal of bacteriology, 2012Co-Authors: Florian Mayer, Reinhard Rachel, Volker Müller, Carolin Meyer, Ulf Küper, Stefanie Daxer, Harald HuberAbstract: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.
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die fibers von Ignicoccus hospitalis ultrastruktur verankerung und molekularbiologische untersuchungen
2010Co-Authors: Carolin MeyerAbstract:Im Rahmen dieser Arbeit wurden die Zellanhange von Ignicoccus hospitalis, die sogenannten Fibers, naher charakterisiert. Dabei konnte gezeigt werden, dass die Fibers Ahnlichkeiten zu bakteriellen Typ IV Pili aufweisen. Immunmarkierungsexperimente ermoglichten eine genaue Lokalisation des Hauptfiberproteins Iho670, welches sowohl in der auseren, als auch in der cytoplasmatischen Membran nachgewiesen werden konnte. Elektronenmikroskopische Untersuchungen zeigten eine Verankerung der Zellanhange unterhalb der Cytoplasmamembran an, zudem wurde ein vorlaufiges Model des Fiberankers erstellt. Polymerisationsexperimente mit rekombinaten Fiberproteinen in vitro zeigten, dass die Existenz des hydrophoben N-terminalen Bereiches von Iho670 eine untergeordnete Rolle bei der Polymerisation der Untereinheiten spielt. Eine definitive funktionelle Zuordnung der Fiber konnte nicht getroffen werden, allerdings wurden zahlreiche Hinweise gefunden, die auf eine adhasive Funktion der Fiber schliesen lassen.
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Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Ulf Küper, Reinhard Rachel, Volker Müller, Carolin Meyer, Harald HuberAbstract:ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H2:sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, Gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.
Reinhard Wirth - One of the best experts on this subject based on the ideXlab platform.
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Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC
Archives of Microbiology, 2020Co-Authors: Dagmar Koschnitzki, Reinhard Rachel, Reinhard Wirth, Harald Huber, Kristina Beblo-vranesevic, Ralf Moeller, Stefan Leuko, Bartos Przybyla, Petra RettbergAbstract: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 D _10-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 .
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Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain.
Proceedings of the National Academy of Sciences, 2016Co-Authors: Tatjana Braun, Matthijn R. J. Vos, Nir Kalisman, Nicholas E. Sherman, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder, Edward H. EgelmanAbstract:The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-A resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.
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The Iho670 Fibers of Ignicoccus hospitalis Are Anchored in the Cell by a Spherical Structure Located beneath the Inner Membrane
Journal of bacteriology, 2014Co-Authors: Carolin Meyer, Thomas Heimerl, Reinhard Wirth, Andreas Klingl, Reinhard RachelAbstract: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.
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Filaments from Ignicoccus hospitalis Show Diversity of Packing in Proteins Containing N-Terminal Type IV Pilin Helices.
Journal of molecular biology, 2012Co-Authors: Charles Goforth, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder, Carolin Meyer, Edward H. EgelmanAbstract: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 ~7.5A 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.
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survival of thermophilic and hyperthermophilic microorganisms after exposure to uv c ionizing radiation and desiccation
Archives of Microbiology, 2011Co-Authors: Kristina Beblo, Reinhard Rachel, Reinhard Wirth, Harald Huber, Thierry Douki, Gottfried Schmalz, Guenther Reitz, Petra RettbergAbstract:In this study, we investigated the ability of several (hyper-) thermophilic Archaea and phylogenetically deep-branching thermophilic Bacteria to survive high fluences of monochromatic UV-C (254 nm) and high doses of ionizing radiation, respectively. Nine out of fourteen tested microorganisms showed a surprisingly high tolerance against ionizing radiation, and two species (Aquifex pyrophilus and Ignicoccus hospitalis) were even able to survive 20 kGy. Therefore, these species had a comparable survivability after exposure to ionizing radiation such as Deinococcus radiodurans. In contrast, there was nearly no difference in survival of the tested strains after exposure to UV-C under anoxic conditions. If the cells had been dried in advance of UV-C irradiation, they were more sensitive to UV-C radiation compared with cells irradiated in liquid suspension; this effect could be reversed by the addition of protective material like sulfidic ores before irradiation. By exposure to UV-C, photoproducts were formed in the DNA of irradiated Archaea and Bacteria. The distribution of the main photoproducts was species specific, but the amount of the photoproducts was only partly dependent on the applied fluence. Overall, our results show that tolerance to radiation seems to be a common phenomenon among thermophilic and hyperthermophilic microorganisms.
