The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

Pol B, a Family B DNA Polymerase, in Thermococcus kodakarensis is Important for DNA Repair, but not DNA Replication.

Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.

Biochemical characterization and mutational studies of a thermostable uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis. The recombinant Tba UDG cleaves exclusively uracil-containing ssDNA and dsDNA at 65°C. The enzyme displays an optimal cleavage activity at 70-75°C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 11.0 with an optimal pH of 7.0-9.0. In addition, Tba UDG activity is independent on a divalent metal ion; however, both Zn2+ and Cu2+ completely inhibit the enzyme activity. Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA with the following preference: U≈U/G>U/T≈U/C>U/A. Kinetic results showed that Tba UDG cleaves uracil-containing ssDNA and dsDNA at a similar rate. The mutational studies showed that the E118A, N159A and H216A mutants completely abolish cleavage activity and retain compromised binding activity while the Y127A mutant displays similar cleavage and binding activities with the wild-type protein, suggesting that residues E118, N159 and H216 are essential for uracil removal and necessary for uracil recognition.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Genome-wide transcriptional response of the archaeon Thermococcus gammatolerans to cadmium.

Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO(4)) and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM) and a toxic (1 mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.

Indolepyruvate ferredoxin oxidoreductase: An oxygen-sensitive iron-sulfur enzyme from the hyperthermophilic archaeon Thermococcus profundus.

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T. profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1.

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.

Thermococcus thioreducens sp. nov., a novel hyperthermophilic, obligately sulfur-reducing archaeon from a deep-sea hydrothermal vent.

A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P(T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 degrees N, 33.9 degrees W). The cells of strain OGL-20P(T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within a pH range of 5.0-8.5 (optimum pH 7.0), an NaCl concentration range of 1-5 % (w/v) (optimum 3 %) and a temperature range of 55-94 degrees C (optimum 83-85 degrees C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, Fe(III) or nitrate. Proteolysis products (peptone, bacto-tryptone, Casamino acids and yeast extract) are utilized as substrates during sulfur reduction. Strain OGL-20P(T) is resistant to ampicillin, chloramphenicol, kanamycin and gentamicin, but sensitive to tetracycline and rifampicin. The G+C content of the DNA is 52.9 mol%. The 16S rRNA gene sequence analysis revealed that strain OGL-20P(T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(T) represents a new separate species within the genus Thermococcus, for which we propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(T) (=JCM 12859(T)=DSM 14981(T)=ATCC BAA-394(T)).

Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

A novel branching enzyme of the GH-57 family in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the alpha-1,4 linkage and its subsequent transfer to the alpha-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain alpha-glucans, indicating that the reaction product contained many alpha-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70 degrees C, and thermostability up to 90 degrees C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436DeltaH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436DeltaH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

Effects of dissolved sulfide, pH, and temperature on growth and survival of marine hyperthermophilic Archaea.

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.

Sulfide ameliorates metal toxicity for deep-sea hydrothermal vent archaea.

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.

Characterization of parazoanthoxanthin A binding to a series of natural and synthetic host DNA duplexes.

Parazoanthoxanthin A is a fluorescent yellow nitrogenous pigment of the group of zoanthoxanthins, which show a broad range of biological activity. These include, among others, the ability to bind to DNA. In this study we have used a variety of spectroscopic (intrinsic fluorescence emission and UV-spectroscopy) and hydrodynamic techniques (viscometry) to characterize in more detail the binding of parazoanthoxanthin A to a variety of natural and synthetic DNA duplexes in different buffer conditions. Our results reveal the following five significant features: (i) Parazoanthoxanthin A exhibits two modes of DNA binding: One binding mode exhibits properties of intercalation, while the second binding mode is predominantly electrostatic in origin. (ii) The apparent binding "site size" for parazoanthoxanthin A near physiological salt concentration (100 mM NaCl) is in the range of 7 +/- 1 base pairs for natural genomic DNA duplexes (calf thymus and salmon testes DNA) and alternating synthetic polynucleotides (poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)]). A slightly larger apparent binding site size of 9 +/- 1 bp was obtained for parazoanthoxanthin A binding to the synthetic homopolymer poly[d(A)]. poly[d(T)]. (iii) Near physiological salt concentration (100 mM NaCl) parazoanthoxanthin A binds with the same approximate binding affinity of 2-5 x 10(5) M(-1) to all DNA polymers studied. (iv) At low salt concentration, parazoanthoxanthin A preferentially binds alternating poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)] host duplexes. (v) Parazoanthoxanthin A inhibits DNA polymerase in vitro.

Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: purification and characterization of key enzymes.

Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60 degrees C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enzyme.

Isolation and characterization of Thermococcus barossii, sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent flange formation.

A new hyperthermophilic microorganism, Thermococcus barossii, was isolated from rock fragments of a hydrothermal vent flange formation, located along the East Pacific Rise of the Juan de Fuca Ridge. This organism is obligately anaerobic and grows over a temperature range of at least 60-92 degrees C in artificial seawater-based media, containing elemental sulfur, tryptone and yeast extract. The addition of a maltooligosaccharide mixture and tungsten to this medium improved growth to some extent. At the Topt for growth (82.5 degrees C), cell densities as high as 4 x 10(8) cells/ml could be obtained in 18-liter batch fermentations, with a doubling time of approximately 40 minutes, if culture access to elemental sulfur was sufficient. In continuous culture at the same temperature, comparable cell densities could be obtained but only at slower growth rates. Morphologically, T. barossii is coccoid-shaped, forming irregularly-shaped spheres; under optimal conditions, these coccoids become more regular and smaller, a characteristic of other hyperthermophilic archaea. Negatively-stained preparations showed no pili or flagella associated with the cell surface. 16S rRNA sequencing reveals that T. barossii is most similar to Thermococcus celer (99.7%). Yet, further comparisons with T. celer showed that T. barossii is a new Thermococcus species: different growth temperature optimum (82.5 degrees C vs. 88 degrees C), obligate requirement for sulfur, higher G + C content (60% vs. 56.7%) and 47.7% DNA-DNA hybridization. The nucleotide and translated amino acid sequence for the gene encoding a DNA polymerase from T. barossii was compared to sequences of related genes from other Thermacoccales. The polymerase phylogenies were congruent with those obtained from the 16S rRNA phylogenetic analyses. Based on the high degree of similarity among members of the genus Termococcus for the criteria used thus far, aspects of enzymology may be an important mechanism of differenting one species from another.