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Dieter Soll - One of the best experts on this subject based on the ideXlab platform.
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crystal structure and assembly of the functional Nanoarchaeum equitans trna splicing endonuclease
Nucleic Acids Research, 2009Co-Authors: Michelle Mitchell, Lennart Randau, Song Xue, Rachel Erdman, Dieter SollAbstract:The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (alphabeta)(2) family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 A containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 A. The functional enzyme resembles previously known alpha(2) and alpha(4) endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.
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life without rnase p
Nature, 2008Co-Authors: Lennart Randau, Imke Schroder, Dieter SollAbstract:Transfer RNAs are first synthesized as longer forms which are then reprocessed. RNase P is the enzyme that clips off excess RNA to give the mature 5' end, and it is found universally. Except, that is, in Nanoarchaeum equitans. Randau et al. have now uncovered the secret of this novel microbe's 'life without RNaseP'. Rearrangements in the promoters of this archaeon's tRNA genes generate ready-to-go 'leaderless' tRNAs. Transfer RNAs are synthesized as longer forms that need to be processed, and ribonuclease P (RNase P) is the ribonuclease that clips off excess RNA to give the mature 5' end. It is found universally, except in Nanoarchaeum equitans, and this paper uncovers how rearrangements in the promoters of this archaeon's tRNA genes seem to have facilitated the generation of 'leaderless' tRNAs that no longer required RNase P processing. The universality of ribonuclease P (RNase P), the ribonucleoprotein essential for transfer RNA (tRNA) 5′ maturation1,2, is challenged in the archaeon Nanoarchaeum equitans. Neither extensive computational analysis of the genome nor biochemical tests in cell extracts revealed the existence of this enzyme. Here we show that the conserved placement of its tRNA gene promoters allows the synthesis of leaderless tRNAs, whose presence was verified by the observation of 5′ triphosphorylated mature tRNA species. Initiation of tRNA gene transcription requires a purine, which coincides with the finding that tRNAs with a cytosine in position 1 display unusually extended 5′ termini with an extra purine residue. These tRNAs were shown to be substrates for their cognate aminoacyl-tRNA synthetases. These findings demonstrate how nature can cope with the loss of the universal and supposedly ancient RNase P through genomic rearrangement at tRNA genes under the pressure of genome condensation.
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protein trans splicing and characterization of a split family b type dna polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans
Journal of Molecular Biology, 2006Co-Authors: Jeong Jin Choi, Dieter Soll, Ki Hoon Nam, Bokkee Min, Sangjin Kim, Suktae KwonAbstract:Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.
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the heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge helix bulge motifs of joined trna halves
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Lennart Randau, Mircea Podar, Katherine Calvin, Michelle Hall, Jing Yuan, Dieter SollAbstract:Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate.
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the complete set of trna species in Nanoarchaeum equitans
FEBS Letters, 2005Co-Authors: Lennart Randau, Michael Pearson, Dieter SollAbstract:The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requiring the assembly of seperate 5′ and 3′ tRNA halves [Randau, L., Munch, R., Hohn, M.J., Jahn, D. and Soll, D. (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves. Nature 433, 537–541]. Biochemical evidence was missing for one of the computationally-predicted, joined tRNAs designated as tRNATrp. Our RT-PCR and sequencing results identify this tRNA as tRNALys (CUU) joined at the alternative position between bases 30 and 31. We show that the intron-containing tRNATrp was misidentified in the initial Nanoarchaeum equitans genome annotation [E. Waters et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100, 12984–12988]. Along with a previously unidentified joined tRNAGln (UUG), Nanoarchaeum equitans exhibits 44 tRNAs and is enabled to read all 61 sense codons. Features unique to this set of tRNA molecules are discussed.
Lennart Randau - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH Open Access RNA processing in the minimal organism
2013Co-Authors: Nanoarchaeum Equitans, Lennart RandauAbstract:Background: The minimal genome of the tiny, hyperthermophilic archaeon Nanoarchaeum equitans contains several fragmented genes and revealed unusual RNA processing pathways. These include the maturation of tRNA molecules via the trans-splicing of tRNA halves and genomic rearrangements to compensate for the absence of RNase P. Results: Here, the RNA processing events in the N. equitans cell are analyzed using RNA-Seq deep sequencing methodology. All tRNA half precursor and tRNA termini were determined and support the tRNA trans-splicing model. The processing of CRISPR RNAs from two CRISPR clusters was verified. Twenty-seven C/D box small RNAs (sRNAs) and a H/ACA box sRNA were identified. The C/D box sRNAs were found to flank split genes, to form dicistronic tRNA-sRNA precursors and to be encoded within the tRNAMet intron. Conclusions: The presented data provide an overview of the production and usage of small RNAs in a cell that has to survive with a highly reduced genome. N. equitans lost many essential metabolic pathways but maintains highly active CRISPR/Cas and rRNA modification systems that appear to play an important role in genome fragmentation. Backgroun
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RNA processing in the minimal organism Nanoarchaeum equitans
Genome Biology, 2012Co-Authors: Lennart RandauAbstract:Background The minimal genome of the tiny, hyperthermophilic archaeon Nanoarchaeum equitans contains several fragmented genes and revealed unusual RNA processing pathways. These include the maturation of tRNA molecules via the trans-splicing of tRNA halves and genomic rearrangements to compensate for the absence of RNase P.
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crystal structure and assembly of the functional Nanoarchaeum equitans trna splicing endonuclease
Nucleic Acids Research, 2009Co-Authors: Michelle Mitchell, Lennart Randau, Song Xue, Rachel Erdman, Dieter SollAbstract:The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (alphabeta)(2) family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 A containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 A. The functional enzyme resembles previously known alpha(2) and alpha(4) endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.
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life without rnase p
Nature, 2008Co-Authors: Lennart Randau, Imke Schroder, Dieter SollAbstract:Transfer RNAs are first synthesized as longer forms which are then reprocessed. RNase P is the enzyme that clips off excess RNA to give the mature 5' end, and it is found universally. Except, that is, in Nanoarchaeum equitans. Randau et al. have now uncovered the secret of this novel microbe's 'life without RNaseP'. Rearrangements in the promoters of this archaeon's tRNA genes generate ready-to-go 'leaderless' tRNAs. Transfer RNAs are synthesized as longer forms that need to be processed, and ribonuclease P (RNase P) is the ribonuclease that clips off excess RNA to give the mature 5' end. It is found universally, except in Nanoarchaeum equitans, and this paper uncovers how rearrangements in the promoters of this archaeon's tRNA genes seem to have facilitated the generation of 'leaderless' tRNAs that no longer required RNase P processing. The universality of ribonuclease P (RNase P), the ribonucleoprotein essential for transfer RNA (tRNA) 5′ maturation1,2, is challenged in the archaeon Nanoarchaeum equitans. Neither extensive computational analysis of the genome nor biochemical tests in cell extracts revealed the existence of this enzyme. Here we show that the conserved placement of its tRNA gene promoters allows the synthesis of leaderless tRNAs, whose presence was verified by the observation of 5′ triphosphorylated mature tRNA species. Initiation of tRNA gene transcription requires a purine, which coincides with the finding that tRNAs with a cytosine in position 1 display unusually extended 5′ termini with an extra purine residue. These tRNAs were shown to be substrates for their cognate aminoacyl-tRNA synthetases. These findings demonstrate how nature can cope with the loss of the universal and supposedly ancient RNase P through genomic rearrangement at tRNA genes under the pressure of genome condensation.
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the heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge helix bulge motifs of joined trna halves
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Lennart Randau, Mircea Podar, Katherine Calvin, Michelle Hall, Jing Yuan, Dieter SollAbstract:Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate.
Wolfgang Eisenreich - One of the best experts on this subject based on the ideXlab platform.
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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: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang EisenreichAbstract: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: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang EisenreichAbstract: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, Martin Gallenberger, Wolfgang Eisenreich, Walter Paper, Benjamin Junglas, Reinhard RachelAbstract: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.
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Insights into the Autotrophic CO2 Fixation Pathway of the Archaeon Ignicoccus hospitalis: Comprehensive Analysis of the Central Carbon Metabolism
Journal of bacteriology, 2007Co-Authors: Ulrike Jahn, Wolfgang Eisenreich, Michael HüglerAbstract:Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo 13C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO2 fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO2 acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the α-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.
Mircea Podar - One of the best experts on this subject based on the ideXlab platform.
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Multi-omics analysis provides insight to the Ignicoccus hospitalis-Nanoarchaeum equitans association.
Biochimica et biophysica acta. General subjects, 2017Co-Authors: Rachel A. Rawle, Mircea Podar, Richard J. Giannone, Robert L. Hettich, Louie L. Wurch, Timothy Hamerly, Brian P. Tripet, Valérie Copié, Brian BothnerAbstract:Abstract 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.
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Proteomic characterization of cellular and molecular processes that Life 2013, 3 117 enable the Nanoarchaeum. equitansIgnicoccus. hospitalis relationship
2016Co-Authors: Richard J. Giannone, Thomas Heimerl, Martin Keller, Tatiana V. Karpinets, Robert L. Hettich, Mircea PodarAbstract: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 energeti
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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, 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.
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Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth
2015Co-Authors: Richard J. Giannone, Mircea Podar, Louie L. Wurch, Robert L. HettichAbstract:The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitans proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins
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insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from obsidian pool yellowstone national park
Biology Direct, 2013Co-Authors: Mircea Podar, Kira S Makarova, David E. Graham, Yuri I Wolf, Eugene V Koonin, Annalouise ReysenbachAbstract:Background: A single cultured marine organism, Nanoarchaeum equitans, represents the Nanoarchaeota branch of symbiotic Archaea, with a highly reduced genome and unusual features such as multiple split genes. Results: The first terrestrial hyperthermophilic member of the Nanoarchaeota was collected from Obsidian Pool, a thermal feature in Yellowstone National Park, separated by single cell isolation, and sequenced together with its putative host, a Sulfolobales archaeon. Both the new Nanoarchaeota (Nst1) and N. equitans lack most biosynthetic capabilities, and phylogenetic analysis of ribosomal RNA and protein sequences indicates that the two form a deepbranching archaeal lineage. However, the Nst1 genome is more than 20% larger, and encodes a complete gluconeogenesis pathway as well as the full complement of archaeal flagellum proteins. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. These findings imply that, rather than representing ancestral characters, the extremely compact genomes and multiple split genes of Nanoarchaeota are derived characters associated with their symbiotic or parasitic lifestyle. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales. Conclusions: Comparison of the N. equitans and Nst1 genomes suggests that the marine and terrestrial lineages of Nanoarchaeota share a common ancestor that was already a symbiont of another archaeon. The two distinct Nanoarchaeota-host genomic data sets offer novel insights into the evolution of archaeal symbiosis and parasitism, enabling further studies of the cellular and molecular mechanisms of these relationships.
Ulrike Jahn - One of the best experts on this subject based on the ideXlab platform.
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DOI 10.1007/s00203-008-0399-xORIGINAL PAPER Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins
2016Co-Authors: Tillmann Burghardt, Ulrike Jahn, Carolin Meyer, Sonja Gürster, Frank Siedler, Manfred Saller, Daniel Müller, Eduard Hochmuth, Rainer Deutzmann, Patrick BabingerAbstract:chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the Wrst among Archaea. Membranes are of fundamental importance for the interaction of I. hospi-talis and N. equitans, as they harbour the proteins neces-sary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospi-talis 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 extracel-lular. The quantitatively dominating proteins in the cyto-plasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells ar
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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: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang EisenreichAbstract: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: Martin Gallenberger, Ivan A. Berg, Ulrike Jahn, Eva Eylert, Daniel Kockelkorn, Wolfgang EisenreichAbstract: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.
