A novel ADP-forming succinyl-CoA synthetase in Thermococcus kodakaraensis structurally related to the archaeal nucleoside diphosphate-forming acetyl-CoA synthetases

We have identified and characterized a structurally novel succinyl-CoA synthetase (SCS) from the hyperthermophilic Archaea Thermococcus kodakaraensis. The presence of an SCS completes the metabolic pathway from glutamate to succinate in Thermococcales, which had not been clarified because of the absence of classical SCS homologs on their genomes. The SCS from T. kodakaraensis (SCS(Tk)) is a heteromeric enzyme (alpha(2)beta(2)) encoded by TK1880 (alpha-subunit) and TK0943 (beta-subunit). Although both SCS(Tk) and classical SCSs harbor the five domains present in enzymes of the acyl-CoA synthetase (nucleoside diphosphate-forming) superfamily, the domain order and distribution among subunits in SCS(Tk) (alpha-subunit, domains 1-2-5; beta-subunit, domains 3-4) are distinct from those of classical SCSs (alpha-subunit, domains 1-2; beta-subunit, domains 3-4-5) and instead resemble the acetyl-CoA synthetases from Pyrococcus furiosus (ACSs I(Pf) and II(Pf)). Comparison of the four Thermococcales genomes revealed that each strain harbors five alpha- and two beta-subunit homologs. Sequence similarity suggests that the beta-subunit of SCS(Tk) is also a component of the presumed ACS II from T. kodakaraensis (ACS II(Tk)). We coexpressed the alpha/beta-genes of SCS(Tk) (TK1880/TK0943) and of ACS II(Tk) (TK0139/TK0943). ACS II(Tk) recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS II(Pf), whereas SCS(Tk) displays a distinct and relatively strict substrate specificity for several acids, including succinate. This indicates that the alpha-subunits are responsible for the distinct substrate specificities of SCS(Tk) and ACS II(Tk).     

Agmatine is essential for the cell growth of Thermococcus kodakaraensis

TK0149 (designated as Tk-PdaD) of a hyperthermophilic archaeon, Thermococcus kodakaraensis, was annotated as pyruvoyl-dependent arginine decarboxylase, which catalyzes agmatine formation by the decarboxylation of arginine as the first step of polyamine biosynthesis. In order to investigate its physiological roles, Tk-PdaD was purified as a recombinant form, and its substrate dependency was examined using the candidate compounds arginine, ornithine and lysine. Tk-PdaD, expressed in Escherichia coli, was cleaved into alpha and beta subunits, as other pyruvoyl-dependent enzymes, and the resulting subunits formed an (alphabeta)(6) complex. The Tk-PdaD complex catalyzed the decarboxylation of arginine but not that of ornithine and lysine. A gene disruptant lacking Tk-pdaD was constructed, showing that it grew only in the medium in the presence of agmatine but not in the absence of agmatine. The obtained results indicate that Tk-pdaD encodes a pyruvoyl-dependent arginine decarboxylase and that agmatine is essential for the cell growth of T. kodakaraensis.     

Formation of dAMP-glycerol and dAMP-Tris derivatives by Thermococcus kodakaraensis DNA primase

In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.     

Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis

Archaeal swimming motility is driven by archaella: rotary motors attached to long extracellular filaments. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar basal body in vivo in Thermococcus kodakaraensis KOD1. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplasmic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frustum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associates with chemosensory arrays and ribosome‐excluding material and may function as a polar organizing center for the coccoid cells.     

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 DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.     

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.     

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.     

Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl(2) method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.     

Novel archaeal alanine:glyoxylate aminotransferase from Thermococcus litoralis

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.     

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.     

Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (k(ass)) of 5.2×103 M?1 s?1 at 40°C and 3.1×10? M?1 s?1 at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k(ass) values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3-7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.