Isolation and characterization of thermostable DNA polymerase of the hyperthermophilic archaeum <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> Sh1AM

Overall, 30 strains of hyperthermophilic archaea, representing seven species of the genera Thermococcus, Desulfurococcus, Thermoproteus, and Acidilobus, were tested for the presence of thermostable DNA polymerases. Thermostabilities of the polymerases varied distinctly among the strains within one species. Polymerases of five strains retained 60–100% activity upon incubation of the preparations at 95°C for 120 min. A new DNA polymerase was isolated from the strain Thermococcus litoralis Sh1AM, possessing the enzyme with the most promising properties, and characterized. Molecular weight of the enzyme is 90–100% kDa. The purified DNA polymerase preserved 50% of the initial activity upon incubation at 95°C for 120 min. The polymerase isolated displayed an associated 3–5 exonuclease activity. The error rate when extending a DNA strand was at least twofold lower compared with Taq polymerase. The main physicochemical and enzymatic properties of the new polymerase are similar to the known DNA polymerases of family B.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 40–47.Original Russian Text Copyright © 2005 by Slobodkina, Chernykh, Lopatin, Ilina, Bannikova, Ankenbauer, Eldarov, Varlamov, Bonch-Osmolovskaya.     

Isolation and characteristics of new thermostable DNA ligase from archaea of the genus Thermococcus

The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl2, pH 7.0-10.5, and temperature 70 degrees C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80 degrees C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.     

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.     

Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter. The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C. The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.     

A DNA polymerase from a thermoacidophilic archaebacterium: evolutionary and technological interests

The archaebacteria constitute a group of prokaryotes with an intermediate phylogenetic position between eukaryotes and eubacteria. The study of their DNA polymerases may provide valuable information about putative evolutionary relationships between prokaryotic and eukaryotic DNA polymerases. As a first step towards this goal, we have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. This enzyme is a monomeric protein of 100 kDa which can catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template. The activity is optimal at 70 degrees C and the enzyme is thermostable up to 80 degrees C; however, it can still polymerize up to 200 nucleotides at 100 degrees C. These remarkable thermophilic properties and thermostability permit examination of the mechanism of DNA synthesis under conditions of decreased stability of the DNA helix. Furthermore, these properties make S. acidocaldarius DNA polymerase a very efficient enzyme to be used in DNA amplification by the recently developed polymerase chain reaction method (PCR) as well as in the Sanger DNA sequencing technique.     

Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1

Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.     

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.     

DNA-dependent RNA polymerase from an extremely thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum

Extremely thermophilic bacterium Calderobacterium hydrogenophilum contains DNA-dependent RNA polymerase with unusual properties. Purified enzyme is thermoresistant (40 min at 100 degrees C) and exhibits similar subunit composition as eubacterial RNA polymerases (e.g. Escherichia coli). However, the enzyme is not susceptible to antibiotics which inhibit eubacterial RNA polymerases (rifampicin and streptolydigin). The activity of the enzyme is inhibited by actinomycin D, daunomycin and heparin.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase

Processivity of T7 DNA polymerase relies on the coupling of its cofactor Escherichia coli thioredoxin (Trx) to gene 5 protein (gp5) at 1:1 stoichiometry. We designed a coexpression system for gp5 and Trx that allows in vivo reconstitution of subunits into a functional enzyme. The properties of this enzyme were compared with the activity of commercial T7 DNA polymerase. Examination of purified enzymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the thioredoxin subunit of the two enzymes did not comigrate. To our surprise, we identified a mutation (Phe102 to Ser) in the Trx component from the commercial T7 DNA polymerase (gp5/TrxS102) that was not in the enzyme from the coexpression system (wild type gp5/Trx). A comparison of polymerase activity of the T7 DNA polymerases shows that both enzymes possessed similar specific activity but they were different in their residual activity at 37 degrees C. The half-life of gp5/TrxS102 was 7 min at 37 degrees C and 12 min for gp5/Trx. gp5/TrxS102 polymerase activity was reduced by fourfold with 3'-5' exonuclease activity as the prominent activity detected after 10 min of heat inactivation at 37 degrees C. Supplementation of reaction mixtures containing gp5/TrxS102 with exogenous nonmutant thioredoxin restored the enzyme activity levels. Pulse proteolysis was used to demonstrate that TrxS102 unfolded at lower urea concentrations than wild type thioredoxin. Thus, Ser substitution at position 102 affected the structural stability of thioredoxin resulting in a reduced binding affinity for gp5 and loss of processivity.     

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.     

Cloning and characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum

In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom Crenarchaeota, Pyrodictium occultum and Aeropyrum pernix, and several members of the kingdom Euryarchaeota. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3'-5' exonuclease motifs were also found. The gene was expressed in Escherichia coli, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from P. islandicum, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations and N-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90 degrees C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3'-5' exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify lambda DNA fragments of up to 1,500 bp.     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

Characterization of a dUTPase from the Hyperthermophilic Archaeon <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80°C and pH 8.0, and it was highly thermostable with a half-life (t _1/2) of 170 min at 95°C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl₂, KCl, (NH₄)₂SO₄, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.     

Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.

DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.     

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. Received: September 17, 1999 / Accepted: March 21, 2000     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.