Distinct physiological roles of the three [NiFe]-hydrogenase orthologs in the hyperthermophilic archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H₂) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S⁰) or H₂ generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H₂S- and H₂-evolving conditions. H₂S generation in PHY1 was equivalent to that of the host strain, and H₂ generation was higher in PHY1, suggesting that Hyh functions in the direction of H₂ uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H₂-evolving conditions. This fact, as well as the impaired H₂ generation activity in MHD1, indicated that Mbh is mainly responsible for H₂ evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H₂ transfer (i.e., H₂ evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H₂S-evolving conditions and exhibited a lower H₂S generation activity, indicating the involvement of Mbx in the S⁰ reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.     

Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.     

On archaeal homologs of the human RNase P proteins Pop5 and Rpp30 in the hyperthermophilic archaeon Thermococcus kodakarensis

The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.     

Genetic analyses of the functions of [NiFe]-hydrogenase maturation endopeptidases in the hyperthermophilic archaeon Thermococcus kodakarensis

The maturation of [NiFe]-hydrogenases requires a number of accessory proteins, which include hydrogenase-specific endopeptidases. The endopeptidases carry out the final cleavage reaction of the C-terminal regions of [NiFe]-hydrogenase large subunit precursors. The hyperthermophilic archaeon Thermococcus kodakarensis harbors two [NiFe]-hydrogenases, a cytoplasmic Hyh and a membrane-bound Mbh, along with two putative hydrogenase-specific endopeptidase genes. In this study, we carried out a genetic examination on the two endopeptidase genes, TK2004 and TK2066. Disruption of TK2004 resulted in a strain that could not grow under conditions requiring hydrogen evolution. The Mbh large subunit precursor (pre-MbhL) in this strain was not processed at all whereas Hyh cleavage was not affected. On the other hand, disruption of TK2066 did not affect the growth of T. kodakarensis under the conditions examined. Cleavage of the Hyh large subunit precursor (pre-HyhL) was impaired, but could be observed to some extent. In a strain lacking both TK2004 and TK2066, cleavage of pre-HyhL could not be observed. Our results indicate that pre-MbhL cleavage is carried out solely by the endopeptidase encoded by TK2004. Pre-HyhL cleavage is mainly carried out by TK2066, but TK2004 can also play a minor role in this cleavage.

     

Structural analysis of thermosome from hyperthermophilic archaeon Thermococcus

The purification and characterization of thermostable chaperonin of the thermosome family from hyperthermophilic archaeon Thermococcus profunds are described. The purified thermosome is a homooligomeric complex and an ATPase with maximal activity at 80 degrees C. The electron micrographs obtained from negatively stained as well as frozen-hydrated specimen showed an eight-fold symmetry of chaperonin. They were about 15 nm height and 16 nm in diameter with a central cavity of 5 nm. In order to understand the ATPase cycling of thermosome, we analyzed the oligomeric structure of thermosome treated with several nucleotides.     

Characterization and in vitro interaction study of a [NiFe] hydrogenase large subunit from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

The large subunit of the [NiFe] hydrogenases harbors a NiFe(CN)(2)(CO) cluster. Maturation proteins HypA, B, C, D, E, and F are required for the NiFe cluster biosynthesis. While the maturation machinery has been hitherto studied intensively, little is known about interactions between the Hyp proteins and the large subunit of the [NiFe] hydrogenase. In this study, we have purified and characterized the cytosolic [NiFe] hydrogenase large subunit HyhL from Thermococcus kodakarensis (Tk-HyhL). Tk-HyhL exists in equilibrium between monomeric and dimeric forms. In vitro interaction analyses showed that Tk-HyhL monomer forms a tight complex with Tk-HypA and weakly interacts with Tk-HypC. The expected ternary complex formation was not detected. These observations reflect a diversity in the mechanism of Ni insertion in [NiFe] hydrogenase maturation depending on the organism.     

Genome sequence of the model hyperthermophilic archaeon Thermococcus litoralis NS-C

The hyperthermophilic archaeon Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.     

Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs.     

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.     

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.     

Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

The chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, Tk-ChiA, has an interesting multidomain structure containing dual catalytic domains and triple chitin-binding domains. To determine the biochemical properties of each domain, we constructed deletion mutant genes corresponding to the individual catalytic domains and purified the recombinant proteins. A synergistic effect was observed when chitin was degraded in the presence of both catalytic domains, suggesting different cleavage specificity of these domains. Analyses of degradation products from N-acetyl-chitooligosaccharides and their chromogenic derivatives with thin layer chromatography indicated that the N-terminal catalytic domain mainly hydrolyzed the second glycosidic bond from the nonreducing end of the oligomers, whereas the C-terminal domain randomly hydrolyzed glycosidic bonds other than the first bond from the nonreducing end. Both catalytic domains formed diacetyl-chitobiose as a major end product and possessed transglycosylation activity. Further analysis of degradation products from colloidal chitin with high performance liquid chromatography showed that the N-terminal catalytic domain exclusively liberated diacetyl-chitobiose, whereas reactions with the C-terminal domain led to N-acetyl-chitooligosaccharides of various lengths. These results demonstrated that the N-terminal and C-terminal catalytic domains functioned as exo- and endochitinases, respectively. The biochemical results provide a physiological explanation for the presence of two catalytic domains with different specificity and suggest a cooperative function between the two on a single polypeptide in the degradation of chitin.     

The Fur iron regulator-like protein is cryptic in the hyperthermophilic archaeon Thermococcus kodakaraensis

Archaea, which regroup organisms with extreme living conditions, possess many predicted iron-containing proteins that may be metabolically critical; however, their need for iron remains poorly documented. In this report, iron acquisition mechanisms were investigated in the hyperthermophilic archaeon Thermococcus kodakaraensis . Thermococcus kodakaraensis requires iron for its growth and possesses many putative iron uptake systems, including several ATP-binding cassette-like transporters and two FeoAB-like receptors, showing that this organism shares similar features with bacteria. One homolog of the major bacterial iron regulator, ferric uptake regulator (Fur), with about 50% similarity to Escherichia coli Fur was also identified. Thermococcus kodakaraensis Fur was found to be able to specifically bind to a Fur-binding site consensus-like sequence of its own gene promoter. However, its expression has been hindered by a −1 frameshift mutation and the chromosomal repair of this mutation did not affect T. kodakaraensis in vivo phenotypes. Microarrays analyses helped to further characterize T. kodakaraensis iron-dependent growth and revealed no role for the Fur homolog in the global regulatory response of the cells to iron. In contrast, additional evidences indicated that the T. kodakaraensis diphtheria toxin regulator (DtxR) homolog may control the expression of the major iron acquisition effectors, while its inactivation enabled higher resistance to iron deficiency.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.     

Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1

The hydrogen (H2) production potential of the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 was evaluated at 85 degrees C. In batch cultivation using a complex medium supplemented with elemental sulfur (S0), evolution of H2S and CO2 was observed in the gas phase. When S0 was omitted and pyruvate or starch was added in the medium, the cells produced H2 at high levels instead of H2S. As the level of H2 appeared to correlate with the specific growth rate, analysis in continuous cultures was performed to develop a continuous H2 production system. In a steady-state condition at a dilution rate of 0.2 h-1, a continuous H2 production rate (per gram dry weight, gdw) of 24.9 and 14.0 mmol gdw-1 h-1 was observed in media supplemented with pyruvate and starch, respectively. In both cultivations, a high accumulation of acetate and alanine was found as metabolites. When the dilution rates were elevated in the medium with pyruvate, steady-state growth was observed up to 0.8 h-1, and a maximum H2 production rate of 59.6 mmol gdw-1 h-1 was obtained. Based on the experimental results along with data of the entire genome sequence, the metabolic pathway of the strain relating to starch and pyruvate degradation is discussed.     

Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSL(Tk)), where the hyhS(Tk) and hyhL(Tk) gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD(Tk)) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL(Tk) were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (beta, gamma, delta, and alpha), corresponding to the products of hyhBGSL(Tk), respectively. Each alphabetagammadelta unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95 degrees C for H(2) uptake and 90 degrees C for H(2) production with methyl viologen as the electron carrier. We found that NADP(+) and NADPH promoted high levels of uptake and evolution of H(2), respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.     

TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis

The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.