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PubMed1994

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.

Amino Acids, Essential↗

PubMed2000

Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs.

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90 degrees C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H(2), acetate, and CO(2) as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.

Archaea↗

PubMed1996

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.

Amino Acid Sequence↗

PubMed1989

Phylogenetic conservation of antigenic determinants in archaebacterial elongation factors (Tu proteins).

By using affinity chromatography methods, we have purified elongation factor Tu (EF-Tu) proteins from a host of archaebacteria covering all known divisions in the archaebacterial tree except halophiles, and from such distantly related eubacteria as Thermotoga maritima and Escherichia coli. Polyclonal antibodies were raised against the Tu proteins of Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum, and Thermotoga and used to probe the immunochemical relatedness of elongation factors both within and across kingdom boundaries. A selection of the results, presented here, indicates that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the functionally analogous proteins of eubacteria and eukaryotes, with only one possible exception concerning the recognition of eukaryotic (EF-1 alpha) factors by Thermococcus EF-Tu antibodies, and (ii) within the archaebacteria there appears to be a correlation between EF-Tu immunochemical similarities and the phylogenetic relatedness of the organisms inferred from other (sequence) criteria. On the whole, immunochemical similarity data argue against the proposal that the archaebacterial taxon should be split and redistributed between two superkingdoms.

Antigens, Bacterial↗

PubMed2006

[Cloning, expression and biochemical characterization of a novel diacetylchitobiose deacetylase from the hyperthermophilic archaeon Pyrococcus horikoshii].

Chitin is the second most abundant organic compound in nature and the degradation of this biomass is an important process in the recycling of nutrients in the environments. Several biodegradation pathway of chitin have been classified in eukaryotes and bacteria, and a unique chitin degradation pathway was proposed according to recent studies on hyperthermophilic archaeon Thermococcus kodakaraensis. In the genome of Pyrococcus horikoshii, several ORFs show high homology to the chitin-degrading related genes from T. kodakararaensis, therefore P. horikoshii is likely to have the same chitin degrading pathway as that of T. kodakaraensis. In order to further characterize the novel chitin degrading pathway in thermophilic archaea, a diacetylchitobiose deacetylase from P. horikoshii (Dacph) was studied in the present study. Dacph belongs to the LmbE-like protein family and the amino sequence is not related to the other deacetylases studied before (except that in T. kodakararaensis). The gene (Dacph, PH0499) from the hyperthermophilic archaeon P. horikoshii was amplified by polymerase chain reaction, cloned into expression vector pET15b, and expressed in E. coli BL21-codonPlus (DE3)-RIL. A soluble fraction of Dacph (31.6kDa) was obtained as shown by SDS-PAGE. TLC analysis showed that Dacph is able to deacetylate one acetyl group of GlcNAc2 and GlcNAc. By the concerted reaction with the Exo-beta-D-Glucosaminid-ase (BglAph), it is also able to convert GlcNAc2 into GlcN. It is concluded that PH0499 is a diacetylchit-obiose deacetylase. By reaction together with Exo-beta-D-Glucosaminidase in P. horikoshii, Dacph probably plays a key role in the new chitin degradation pathway in hyperthermophilic archaea (the genera Thermococcus and Pyrococcus).

Archaeal Proteins↗

PubMed2002

Chemoenzymatic syntheses of linear and branched hemithiomaltodextrins as potential inhibitors for starch-debranching enzymes.

Oligosaccharides embodying the S-maltosyl-6-thiomaltosyl structure have been readily synthesised by using convergent chemoenzymatic approaches. The key steps for the preparation of these molecules involved: 1) transglycosylation reactions of maltosyl fluorides onto suitable acceptors catalysed by the bacterial transglycosylase, cyclodextrin glycosyltransferase (CGTase), and 2) the SN2-type displacement of a 6-halide from acetylated acceptors by activated 1-thioglycoses. The target molecules, which were obtained in good overall yields, proved to be useful for investigating substrate binding in the active sites of several enzymes that act upon the alpha-1,6-linkage of pullulan and/or amylopectin. The compounds exhibit Ki values in the 2.5-1350 microM range with the different enzymes, and the highest affinity found by using these molecules was seen for the pullulanase from Bacillus acidopullulyticus. Both barley-malt limit dextrinase and pullulanase type II from Thermococcus hydrothermalis only recognised the longest linear thiooligosaccharide, while a branched heptasaccharide was the strongest inhibitor of pullulanase from Klebsiella planticola.

Acetylation↗

PubMed2002

Catalyzing "hot" reactions: enzymes from hyperthermophilic Archaea.

We reflect on some of our studies on the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 and its enzymes. The strain can grow at temperatures up to the boiling point and also represents one of the simplest forms of life. As expected, all enzymes displayed remarkable thermostability, and we have determined some of the basic principles that govern this feature. To our delight, many of the enzymes exhibited unique biochemical properties and novel structures not found in mesophilic proteins. Here, we focus on a few enzymes that are useful in application, and whose three-dimensional structures are characteristic of thermostable enzymes.

DNA Ligases↗

PubMed1999

In vitro heat effect on functional and conformational changes of cyclodextrin glucanotransferase from hyperthermophilic archaea.

The in vitro heat effect on protein characteristics of thermostable enzyme was examined using a cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from the hyperthermophilic archaeon Thermococcus sp. B1001 as a model protein. The recombinant form of CGTase was obtained as an inclusion body from Escherichia coli cells harboring a plasmid which carried the B1001 CGTase gene (cgtA). CGTase was solubilized by 6 M urea, refolded, purified to homogeneity, and heat treated at 80 degrees C for 20 min. Enzyme characteristics were examined compared with those of unheated CGTase. Cyclization activity was increased by in vitro heat treatment, while hydrolysis activity was decreased. The heated and unheated CGTases were analyzed for structures by circular dichroism (CD). The near- and far-UV CD spectra indicated that the structure of unheated CGTase with low cyclization activity was different from that of heated CGTase with high activity. Differential scanning calorimetry of unheated CGTase showed two absorption peaks at 87 and 106 degrees C with increasing temperature. After heat treatment, the minor peak at 87 degrees C disappeared, suggesting that heat-dependent structural conversion occurred in CGTase. These results indicate that the thermal environment plays an important role for the protein folding process of thermostable CGTase.

Calorimetry, Differential Scanning↗

PubMed2001

Two kinds of archaeal chaperonin with different temperature dependency from a hyperthermophile.

Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature.

Antibody Specificity↗

PubMed2001

Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan.

Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (alpha subunit) and Tk-TrpG (beta subunit), at 85 degrees C for 10 min. Further purification of Tk-AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk-AS complex was a heterodimer (alphabeta). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and the optimal temperature was 85 degrees C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75 degrees C, the K(m) values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 microM, respectively. The K(m) value of Mg2+ was 20.5 microM. The K(m) values of glutamine and NH4Cl were 88 microM and 5.6 mM, respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by L-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by L-tryptophan was observed, with K(i) values of 5.25 and 74 microM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by L-tryptophan in the case of Tk-AS complex are distinct from previously reported anthranilate synthases.

Amino Acid Motifs↗

PubMed2001

In vitro replication slippage by DNA polymerases from thermophilic organisms.

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.

Artifacts↗

PubMed2002

Gene conversion and the evolution of euryarchaeal chaperonins: a maximum likelihood-based method for detecting conflicting phylogenetic signals.

Recombination is well known as a complicating factor in the interpretation of molecular phylogenies. Here we describe a maximum likelihood sliding window method based on a likelihood ratio test for scanning DNA sequence alignments for regions of incongruent phylogenetic signals, such as those influenced by recombination. Using this method, we identify several instances of gene conversion between paralogous chaperonin genes in euryarchaeote Archaea, many of which are not detected by two other widely used methods. In the Thermococcus/Pyrococcus lineage, where a gene duplication producing a and b paralogues predates the divergence of Thermococcus strains KS-1 and KS-8, gene conversion has homogenized portions of the a and b genes in KS-8 since the divergence of these two strains. A region near the 3' end of the a and b paralogues in the methanogen Methanobacterium thermoautotrophicum also appears to have undergone gene conversion. We apply the method to two additional test data sets, the argF gene of Neisseria and a set of actin paralogues in maize, and show that it successfully identifies all the recombinant regions that were previously detected with other methods. Our approach is relatively insensitive to the presence of divergent sequences in the alignment, making it ideal for detecting recombination between both closely and distantly related genes.

Actins↗

PubMed2005

Utilization of keratin-containing biowaste to produce biohydrogen.

A two-stage fermentation system was constructed to test and demonstrate the feasibility of biohydrogen generation from keratin-rich biowaste. We isolated a novel aerobic Bacillus strain (Bacillus licheniformis KK1) that displays outstanding keratinolytic activity. The isolated strain was employed to convert keratin-containing biowaste into a fermentation product that is rich in amino acids and peptides. The process was optimized for the second fermentation step, in which the product of keratin fermentation--supplemented with essential minerals--was metabolized by Thermococcus litoralis, an anaerobic hyperthermophilic archaeon. T. litoralis grew on the keratin hydrolysate and produced hydrogen gas as a physiological fermentation byproduct. Hyperthermophilic cells utilized the keratin hydrolysate in a similar way as their standard nutrient, i.e., bacto-peptone. The generalization of the findings to protein-rich waste treatment and production of biohydrogen is discussed and possible means of further improvements are listed.

Aerobiosis↗

PubMed2004

A simple fluorimetric method for the estimation of DNA-DNA relatedness between closely related microorganisms by thermal denaturation temperatures.

Determination of whole-genome DNA-DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA-DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA-DNA relatedness between closely related species.

Benzothiazoles↗

PubMed2001

Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex.

Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.

Amino Acid Sequence↗

PubMed2004

Molecular evolution of the ATPase subunit of three archaeal sugar ABC transporters.

The sequence of genes encoding extracellular amylopullulanase and putative maltodextrin ATP binding cassette (ABC) transporter of a hyperthermophilic archaeon, Thermococcus litoralis, was determined. The mdxK gene, which encodes an ATPase subunit of the putative maltodextrin ABC transporter, has extraordinarily high similarity with the malK gene, which encodes an ATPase subunit of trehalose/maltose ABC transporter of the same organism. DNA sequence comparison revealed that the malK gene was generated through the duplication of the mdxK gene before lateral gene transfer of the mal gene cluster from T. litoralis to Pyrococcus furiosus.

ATP-Binding Cassette Transporters↗

PubMed2004

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea.

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.

Alkyl and Aryl Transferases↗

PubMed2005

Unfolding mechanism of a hyperthermophilic protein O(6)-methylguanine-DNA methyltransferase.

Unfolding intermediates have been found only rarely in earlier studies, and how a protein unfolds is therefore poorly understood. In this paper, we show experimental evidence for multiple pathways and multiple intermediates during unfolding reaction of O(6)-methylguanine-DNA methyltransferase from hyperthermophile Thermococcus kodakaraensis (Tk-MGMT). The unfolding profiles monitored by far-UV CD and tryptophan fluorescence were both biphasic, and unfolding monitored by fluorescence was faster than that monitored by CD. GdnHCl-induced titration curves indicate that the intermediates with significant alpha-helical structure accumulate during unfolding. Dependence of kinetic phases on initial GdnHCl concentrations and cysteine reactivity of Tk-MGMT were investigated, suggesting that the heterogeneity of native conformations and parallel unfolding pathways.

Cysteine↗