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1.  The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon Thermoproteus tenax 
PLoS ONE  2013;8(4):e61354.
The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins.
PMCID: PMC3634074  PMID: 23626675
2.  Characterization of the CRISPR/Cas Subtype I-A System of the Hyperthermophilic Crenarchaeon Thermoproteus tenax 
Journal of Bacteriology  2012;194(10):2491-2500.
CRISPR (clustered regularly interspaced short palindromic repeats) elements and cas (CRISPR-associated) genes are widespread in Bacteria and Archaea. The CRISPR/Cas system operates as a defense mechanism against mobile genetic elements (i.e., viruses or plasmids). Here, we investigate seven CRISPR loci in the genome of the crenarchaeon Thermoproteus tenax that include spacers with significant similarity not only to archaeal viruses but also to T. tenax genes. The analysis of CRISPR RNA (crRNA) transcription reveals transcripts of a length between 50 and 130 nucleotides, demonstrating the processing of larger crRNA precursors. The organization of identified cas genes resembles CRISPR/Cas subtype I-A, and the core cas genes are shown to be arranged on two polycistronic transcripts: cascis (cas4, cas1/2, and csa1) and cascade (csa5, cas7, cas5a, cas3, cas3′, and cas8a2). Changes in the environmental parameters such as UV-light exposure or high ionic strength modulate cas gene transcription. Two reconstitution protocols were established for the production of two discrete multipartite Cas protein complexes that correspond to their operonic gene arrangement. These data provide insights into the specialized mechanisms of an archaeal CRISPR/Cas system and allow selective functional analyses of Cas protein complexes in the future.
PMCID: PMC3347209  PMID: 22408157
3.  Connection between Multimetal(loid) Methylation in Methanoarchaea and Central Intermediates of Methanogenesis▿† 
Applied and Environmental Microbiology  2011;77(24):8669-8675.
In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH3Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH3Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of M. mazei cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation in vivo is attributed to side reactions of CH3Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.
PMCID: PMC3233109  PMID: 22003009
4.  Production of Toxic Volatile Trimethylbismuth by the Intestinal Microbiota of Mice 
Journal of Toxicology  2011;2011:491039.
The biotransformation of metals and metalloids into their volatile methylated derivatives by microbes growing under anaerobic conditions (e.g., the mammalian intestinal microbiota) plays an important role in spreading these compounds in the environment. In this paper, we could show that the presence of an intact intestinal microbiota of mice provides the conditio sine qua non for the production of these mostly toxic derivatives. To document the indispensible role of the intestinal microbiota in methylating metals and metalloids to volatile derivatives under in vivo conditions, we compared the methylation capability of conventionally raised (CONV) and germ-free (GF) B6-mice fed with chow containing colloidal bismuth subcitrate (CBS) as the starting material for the formation of volatile methylated metal(loid)s. Permethylated volatile trimethylbismuth ((CH3)3Bi) was only detected in the blood of the conventionally raised mice. Concomitantly, a higher bismuth concentration was found in organs such as liver, lung, testicles, and brain of the CONV mice as compared to those of GF mice (P > 0.01), strongly suggesting a correlation between the intestinal biomethylation of bismuth and its accumulation in mammalian tissues.
PMCID: PMC3191823  PMID: 22007211
5.  The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota 
PLoS ONE  2011;6(10):e24222.
Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078T) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.
PMCID: PMC3189178  PMID: 22003381
6.  Toxicity of Methylated Bismuth Compounds Produced by Intestinal Microorganisms to Bacteroides thetaiotaomicron, a Member of the Physiological Intestinal Microbiota 
Journal of Toxicology  2011;2011:608349.
Methanoarchaea have an outstanding capability to methylate numerous metal(loid)s therefore producing toxic and highly mobile derivatives. Here, we report that the production of methylated bismuth species by the methanoarchaeum Methanobrevibacter smithii, a common member of the human intestine, impairs the growth of members of the beneficial intestinal microbiota at low concentrations. The bacterium Bacteroides thetaiotaomicron, which is of great importance for the welfare of the host due to its versatile digestive abilities and its protective function for the intestine, is highly sensitive against methylated, but not against inorganic, bismuth species. The level of methylated bismuth species produced by the methanoarchaeum M. smithii in a coculture experiment causes a reduction of the maximum cell density of B. thetaiotaomicron. This observation suggests that the production of methylated organometal(loid) species in the human intestine, caused by the activity of methanoarchaea, may affect the health of the host. The impact of the species to reduce the number of the physiological intestinal microbiota brings an additional focus on the potentially harmful role of methanoarchaea in the intestine of a higher organism.
PMCID: PMC3182067  PMID: 21966291
7.  Role of Intestinal Microbiota in Transformation of Bismuth and Other Metals and Metalloids into Volatile Methyl and Hydride Derivatives in Humans and Mice▿  
Applied and Environmental Microbiology  2008;74(10):3069-3075.
The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37°C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH3)3Bi] at rather low concentrations of bismuth (0.2 to 1 μmol kg−1 [dry weight]). An increase of bismuth up to 2 to 14 mmol kg−1 (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h−1 kg−1 (dry weight) to 2,100 pmol h−1 kg−1 (dry weight) in human feces samples and from approximately 5 pmol h−1 kg−1 (dry weight) to 120 pmol h−1 kg−1 (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH3)3Bi.
PMCID: PMC2394951  PMID: 18378667
8.  DNA Microarray Analysis of Central Carbohydrate Metabolism: Glycolytic/Gluconeogenic Carbon Switch in the Hyperthermophilic Crenarchaeum Thermoproteus tenax▿ † 
Journal of Bacteriology  2008;190(6):2231-2238.
In order to unravel the role of regulation on transcript level in central carbohydrate metabolism (CCM) of Thermoproteus tenax, a focused DNA microarray was constructed by using 85 open reading frames involved in CCM. A transcriptional analysis comparing heterotrophic growth on glucose versus autotrophic growth on CO2-H2 was performed.
PMCID: PMC2258856  PMID: 18178743
9.  CC1, a Novel Crenarchaeal DNA Binding Protein▿  
Journal of Bacteriology  2006;189(2):403-409.
The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.
PMCID: PMC1797387  PMID: 17085561
10.  Effect of Octamethylcyclotetrasiloxane on Methylation of Bismuth by Methanosarcina barkeri 
Applied and Environmental Microbiology  2006;72(10):6819-6821.
Octamethylcyclotetrasiloxane (OMCTS), a common constituent of household products, triggers the transformation of bismuth to the volatile toxic derivative trimethylbismuth by Methanosarcina barkeri, which is a representative member of the sewage sludge microflora. Comparative studies with the ionophores monensin and lasalocid, which induce effects similar to those observed for OMCTS, indicated that the stimulation of bismuth methylation is not specific for the siloxane and suggested that the stimulation observed is mainly due to facilitated membrane permeation of the metal ion.
PMCID: PMC1610270  PMID: 17021235
11.  Reconstruction of the Central Carbohydrate Metabolism of Thermoproteus tenax by Use of Genomic and Biochemical Data 
Journal of Bacteriology  2004;186(7):2179-2194.
The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.
PMCID: PMC374391  PMID: 15028704
12.  Pyruvate Kinase of the Hyperthermophilic Crenarchaeote Thermoproteus tenax: Physiological Role and Phylogenetic Aspects 
Journal of Bacteriology  2000;182(7):2001-2009.
Pyruvate kinase (PK; EC of Thermoproteus tenax was purified to homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg2+ and Mn2+. Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K+ ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.
PMCID: PMC101911  PMID: 10715009
13.  Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus 
Journal of Bacteriology  1998;180(22):5997-6004.
Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.
PMCID: PMC107676  PMID: 9811660
14.  PPi-Dependent Phosphofructokinase from Thermoproteus tenax, an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution 
Journal of Bacteriology  1998;180(8):2137-2143.
Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC, the key enzyme of glycolysis. In some organisms, the step is catalyzed by PPi-dependent PFK (PPi-PFK; EC, which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PPi-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PPi-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PPi as a cosubstrate.
PMCID: PMC107141  PMID: 9555897

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