Thermophilic Bacterium Thermus Aquaticus Research Articles

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10−5 bp−1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded β-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76°C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the β1–β2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674s−1. The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.

The Effect of Deleting a Putative Salt Bridge on the Properties of the Thermostable Subtilisin-Like Proteinase, Aqualysin I

Aqualysin I, is a subtilisin-like serine proteinase, from the thermophilic bacterium Thermus aquaticus. It is predicted that the enzyme contains a salt bridge, D17-R259, connecting the N- and C-terminal regions of the enzyme. Previously we reported on the stabilizing effect of the incorporation of a salt bridge at a corresponding site in VPR, a related cold adapted enzyme from a marine Vibrio sp. Here we describe the effect of the reverse change, i.e. the elimination of the salt bridge on the thermal stability and kinetic properties of aqualysin I. Deletion of the putative salt bridge in the D17N mutant of the enzyme destabilized the enzyme by 8-9 °C in terms of T₅₀%, determined by thermal inactivation and over 4 °C in T(m), as measured from melting curves of the inhibited enzyme. The mutation, however, had no significant effect on the kinetic parameters of the enzyme under standard assay conditions.

METHODS: An alternate method for purification of recombinantTaqDNA polymerase using an aqueous two-phase system

Abstract A simple, cost-efficient method for purification of E. coli-derived recombinant Taq DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus is described. Recombinant Taq DNA polymerase was purified using an aqueous two-phase extraction method following a primary treatment of heat denaturation. The extraction method selectively enriched Taq DNA polymerase in the lower salt phase with complete recovery and high specific activity. The purified Taq DNA polymerase protein displayed nearly eightfold purification with ~117 % activity recovery with an insignificant amount of host DNA. This process was found to be rapid and scalable in comparison to the conventional ammonium sulfate precipitation method.

Characterization of a rolling-circle replication plasmid from Thermus aquaticus NTU103

The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames ( orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication.

Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.

A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli

The gene ( malM) encoding for 4-α-glucanotransferase (4αGTase) from the hyperthermophilic bacterium Aquifex aeolicus was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and its behavior towards various substrates was examined. The enzyme was hyperthermostable, exhibiting maximal activity at 90 °C and it retained 70% of its original activity after incubation at 90 °C for 30 min. A low affinity was observed for the enzyme towards maltose ( K m=71 mM) and the k cat/ K m value for maltotriose was approximately 166 times greater than that observed for maltose but the values did not change significantly with larger maltooligosaccharides. The A. aeolicus 4αGTase produced cycloamylose with a minimum degree of polymerization (DP) of 16, whereas the cycloamylose produced by the amylomaltase from the thermophilic bacterium Thermus aquaticus, which is also a Type II 4αGTase, produced a cycloamylose with a minimum DP of 22. These findings indicate that the A. aeolicus 4αGTase differs from the T. aquaticus amylomaltase and the glucanotransferases produced by other hyperthermophilic organisms.

Cloning, Overexpression, and Characterization of Peroxiredoxin and NADH Peroxiredoxin Reductase from Thermus aquaticus

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.

Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.

Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA. Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits). Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA. rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.

Aspartate aminotranferase Thermus aquaticus YT-1. stability from the thermophilic bacterium Functional characterisation and stability

Abstract: Aspartate aminotranferase from the thermophilic bacterium Thermus aquaticus has been purified to homogeneity. The enzyme is pyridoxal-5-phosphate dependent and is composed of two subunits having an Mr 44000 each. T.aquaticus AspAT shows a pi of 4.5 and is a thermophilic enzyme exhibiting its maximum activity at 80°C in the pH range 7-8. Itis also resistant against denaturation to hea(and several chemical agents.

Purification and Characterization of a Thermostable Trehalose Synthase from Thermus aquaticus.

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80-82% at 30-40°C. The activity was inhibited by Cu(2+) , Hg(2+), Zn(2+), and Tris.

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.

Multi-enzyme complexes in thermophilic organisms: isolation and characterization of the pyruvate dehydrogenase complex from Thermus aquaticus AT62

Summary: The pyruvate dehydrogenase complex from the thermophilic bacterium Thermus aquaticus was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from Bacillus subtilis, with four bands: the α-chain (M r 39600) and β-chain (M r 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetytransferase component (M r 58500) and the dihydrolipoamide dehydrogenase component (M r 53900). Antibodies against the purified T. aquaticus pyruvate dehydrogenase complex cross-reacted with the enzyme complex from B. subtilis and, to a minor extent, with that from bovine heart. No cross-reactivity could be observed with the enzyme complex from Escherichia coli. The T. aquaticus enzyme complex had a temperature maximum at 72°C. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from T. aquaticus by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.

Electron image analysis of ribosomal subunits from Thermus aquaticus

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.

Studies on immobilization of the thermophilic bacterium Thermus aquaticus YT-1 by entrapment in various matrices

Immobilization of the thermophilic bacterium Thermus aquaticus YT-1 has been studied using various entrapment techniques. Alginate, κ-carrageenan, agar, agarose and polyacrylamide were tested as supports during operation at 65°C at which the cells are known to produce protease when grown free in solution. Alginate showed toxic effects and no viability was observed after entrapment in Ca alginate or even after exposure of free living cells to sodium alginate. Polyacrylamide was observed to be the best support. Protease activity was closely related to the appearance of free cells in the medium.

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.

We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.

Purification and properties of the RNA polymerase of an extremely thermophilic bacterium Thermus aquaticus T2

The RNA polymerase of the extremely thermophilic bacterium Thermus aquaticus T2 has been purified to homogeneity. The apparent molecular weights of the subunits of the enzyme are: 165 000, 130 000, 92 000 and 44 000. The in vitro temperature optimum of enzyme activity is around 65°C. The enzyme has a preference towards the homologous template and is strongly inhibited by KCl. Rifampicin inhibits the enzyme only to 50% even at very high concentrations. Heparin inhibits it completely, but only at higher molar excess than in the case of the Escherichia coli enzyme. The enzyme can form heparin-resistant complexes at elevated temperatures on the homologous template, but not on E. coli DNA.

Occurrence of phosphenolpyruvate carboxylase in the extremely thermophilic bacterium Thermus aquaticus.

In the extreme thermophile Thermus aquaticus, phosphoenolpyruvate carboxylase catalyzes carbon dioxide fixation on the C3 metabolite phosphoenolpyruvate, producing oxaloacetate. In a moderately thermophilic Bacillus species this function is fulfilled by pyruvate carboyxlase. Like several of its mesophilic counterparts, the Thermus enzyme exhibits a requirement for acetyl coenzyme A.