Effects of hydrostatic pressure and temperature on physiological traits of Thermococcus guaymasensis and Thermococcus aggregans growing on starch

The effects of temperature and hydrostatic pressure on growth of two novel Thermococcus species, T. guaymasensis and T. aggregans, were investigated. These archaea, isolated from the Guaymas Basin hydrothermal vent site at 2000 meters depth, are able to grow on starch in sulfur-depleted medium producing significant amounts of amylases and pullulanases. At 85 degrees C, T. guaymasensis exhibited a barophilic response at 20 and 35 MPa but inhibition of growth occurred at 50 MPa; at 50 MPa, cell replication was repressed, the mean cell size increased, and production of starch-hydrolysing enzymes was significantly stimulated. Barophily was also expressed by T. guaymasensis under 20 MPa at sub-optimal temperature (70 C) but morphological alterations of cells were observed earlier (35 MPa). No barophily was exhibited by T. aggregans at 85 degrees C. In this case, cell replication was repressed at 20 MPa and remarkable inhibition of growth occurred at 50 MPa. Only when T. aggregans was cultivated at 75 degrees C, a significant barophilic response was exhibited at 20 MPa, as shown by the rate of replication and metabolism. These results show that Thermococcus species, although isolated from the same ecosystem, differ with regard to the effects of pressure and temperature on cell physiology. The metabolic responses and their significance for potential biotechnological applications are also discussed.     

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir

Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799-2,287 m; temperature, 60 degrees-84 degrees C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%-98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40 degrees and 88 degrees C, with the optimum at 78 degrees C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%-2.0%. The doubling time at optimal growth conditions was about 43 min. The G+C content of the DNA was 38.4 mol%. The DNA-DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.     

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.     

Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus spp.

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, α-glutamate, di-myo-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated α-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.     

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.     

Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum.

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.     

Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.     

Growth Physiology of the Hyperthermophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Defined Medium, Characterization of an Exopolysaccharide, and Evidence of Biofilm Formation

Nutritional characteristics of the hyperthermophilic archaeon Thermococcus litoralis have been investigated with emphasis on the development of a sulfur-free, defined growth medium, analysis of an exocellular polysaccharide, and formation of a biofilm. An artificial-seawater-based medium, containing 16 amino acids, adenine, uracil, vitamins, and trace elements, allowed T. litoralis to attain growth rates and cell densities similar to those found with complex media. Four amino acids (alanine, asparagine, glutamine, and glutamate) were not included due to their lack of effect on growth rates and cell yields. In this medium, cultures reached densities of 10(sup8) cells per ml, with doubling times of 55 min (without maltose) or 43 min (with maltose). Neither the addition of elemental sulfur nor the presence of H(inf2) significantly affected cell growth. A sparingly soluble exopolysaccharide was produced by T. litoralis grown in either defined or complex media. Analysis of the acid-hydrolyzed exopolysaccharide yielded mannose as the only monosaccharidic constituent. This exopolysaccharide is apparently involved in the formation of a biofilm on polycarbonate filters and glass slides, which is inhabited by high levels of T. litoralis. Biofilm formation by hyperthermophilic microorganisms in geothermal environments has not been examined to any extent, but further work in this area may provide information related to the interactions among high-temperature organisms.     

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.     

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.     

Characteristics of the ionic composition and ultrastructure of Bordetella pertussis in controlled regulation of the mineral element content in a growth medium

The content of trace elements necessary for the normal growth of bacteria was found to have no effect on the intracellular concentration of Ca2+ and Al3+. The content of Cu2+, Fe3+, Mn2+, Mg2+ was considerably reduced. The addition of Mg2+ at different concentrations to this culture medium stimulated the capacity of cells for accumulating not only Mg2+, but also some other ions. Their maximum intracellular concentration was observed when the concentration of Mg2+ in the culture medium was 41 mM. The growth of microbial cells in the standard culture medium containing Mg2+ at a concentration of 4 mM was accompanied by the increased consumption of elements actively participating in redox reactions (Cu2+, Fe3+, Mn2+). Shifts in the ionic composition of microbial cells were manifested by the morphological features of B. pertussis, linked with the increased synthesis of crystalloid structures. The influence of Mn2+, Al3+, Zn2+ at different concentrations on the ionic composition and morphology of B. pertussis was studied.     

Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)     

New high fidelity polymerases from Thermococcus species

Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability.     

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.     

Complete genome sequence of the hyperthermophilic, piezophilic, heterotrophic, and carboxydotrophic archaeon Thermococcus barophilus MP

Thermococcus barophilus is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m. T. barophilus is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of T. barophilus. The genome data reveal a close proximity with Thermococcus sibiricus, another Thermococcus isolated from the deep biosphere and a possible connection to life in the depths.     

Effect of carbon and nitrogen sources on growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon Thermococcus litoralis and bacterium Thermotoga maritima

Batch and continuous cultures were used to compare specific physiological features of the hyperthermophilic archaeon, Thermococcus litoralis (T(opt) of 85 degrees to 88 degrees C), to another fermentative hyperthermophile that reduces S degrees facultatively, that is, the bacterium Thermotoga maritima (T(opt) of 80 degrees to 85 degrees C). Under nutritionally optimal conditions, these two hyperthermophiles had similar growth yields on maltose and similar cell formula weights based on elemental analysis: CH(1.7)O(0. 7)N(0.2)S(0.006) for T. litoralis and CH(1.6)O(0.6)N(0.2)S(0.005) for T. maritima. However, they differed with respect to nitrogen source, fermentation product patterns, and propensity to form exopolysaccharides (EPS). T. litoralis could be cultured in the absence or presence of maltose on an amino acid-containing defined medium in which amino acids served as the sole nitrogen source. T. maritima, on the other hand, did not utilize amino acids as carbon, energy, or nitrogen sources, and could be grown in a similar defined medium only when supplemented with maltose and ammonium chloride. Not only was T. litoralis unable to utilize NH(4)Cl as a nitrogen source, its growth was inhibited at certain levels. At 1 g/L ( approximately 20 mM) NH(4)Cl, the maximum growth yield (Y(x/s(max))) for T. litoralis was reduced to 13 g cells dry weight (CDW)/mol glucose from 40 g CDW/mol glucose in media lacking NH(4)Cl. Alanine production increased with increasing NH(4)Cl concentrations and was most pronounced if growth on NH(4)Cl was carried out in an 80% H(2) atmosphere. In T. maritima cultures, which would not grow in an 80% H(2) atmosphere, alanine and EPS were produced at much lower levels, which did not change with NH(4)Cl concentration. EPS production rose sharply at high dilution rates for both organisms, such that maltose utilization plots were biphasic. Wall growth effects were also noted, because cultures failed to wash out at dilution rates significantly above maximum growth rates determined from batch growth experiments. This study illustrates the importance of effective cultivation methods for addressing physiological issues related to the growth of hyperthermophilic heterotrophs.     

Dependence of the superficial layers of Spirillum putridiconchylium on Ca2+ or Sr2+.

Chelating agents disrupted the superficial layers on Spirillum putridiconchylium and adsorption of cationized ferritin indicated that both upper and lower surfaces of superficial layer fragments, as well as the outer membrane surface, possessed areas which were negatively charged. Growth of the bacterium in 1% casamino acids (vitamin free) resulted in cells which were devoid of the superficial layers, and negative staining of these cells revealed in amorphous precipitate together with a vesicular outer membrane component extruding from their surfaces into the medium. Addition of either 1 mM Ca2+ or 1 mM Sr2+ to the growth medium produced the typical regularly structured cell surface, whereas addition of equal concentrations of Li+, Na+, K+, Mg2+, Ba2+, Mn2+, Fe3+, or three polyamines produced the structureless surface.     

Purification and characterization of a new DNA polymerase modulator from the hyperthermophilic archaeon Thermococcus fumicolans

During purification of the native alpha-like DNA polymerase from the hyperthermophilic euryarchaeote Thermococcus fumicolans, two activity peaks were detected after cation-exchange chromatography. One of the peaks (Ppol) was identified as the T. fumicolans DNA polymerase and the second peak (Pf) was shown to contain a factor which increased the DNA polymerase activity over 70-fold when tested with activated calf thymus DNA as substrate. The factor also stimulated nucleotide incorporation when using primed lambda DNA as substrate (approximately 8-fold), while inducing a very large decrease in the turnover rate of the enzyme. The factor, therefore, maximizes the ability of the DNA polymerase to synthesize small fragments, which is compatible with DNA repair or lagging strand DNA replication.