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PubMed2005

Continuous enrichment culture and molecular monitoring to investigate the microbial diversity of thermophiles inhabiting deep-sea hydrothermal ecosystems.

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90 degrees C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and epsilon-Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.

Atlantic Ocean↗
PubMed2002

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions.

The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants [ Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf] were investigated to examine the role of both monopolar and dipolar interactions. The reduction potentials of all variants decreased as the polarity of the solvent decreased. The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies. The entropic contribution [delta( Tdelta S degrees )] to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution [delta(-delta H degrees )] was larger for the other mutants. The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states. Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term [delta( Tdelta S degrees )] and enthalpic term [delta(-delta H degrees )] with opposite signs, was observed. A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.

Bacterial Proteins↗
PubMed2003

Spectroscopic characterization of the [Fe(His)(4)(Cys)] site in 2Fe-superoxide reductase from Desulfovibrio vulgaris.

The electronic and vibrational properties of the [Fe(His)(4)(Cys)] site (Center II) responsible for catalysis of superoxide reduction in the two-iron superoxide reductase (2Fe-SOR) from Desulfovibrio vulgaris have been investigated using the combination of EPR, resonance Raman, UV/visible/near-IR absorption, CD, and VTMCD spectroscopies. Deconvolution of the spectral contributions of Center II from those of the [Fe(Cys)(4)] site (Center I) has been achieved by parallel investigations of the C13S variant, which does not contain Center I. The resonance Raman spectrum of ferric Center II has been assigned based on isotope shifts for (34)S and (15)N globally labeled proteins. As for the [Fe(His)(4)(Cys)] active site in 1Fe-SOR from Pyrococcus furiosus, the spectroscopic properties of ferric and ferrous Center II in D. vulgaris 2Fe-SOR are indicative of distorted octahedral and square-pyramidal coordination geometries, respectively. Differences in the properties of the ferric [Fe(His)(4)(Cys)] sites in 1Fe- and 2Fe-SORs are apparent in the rhombicity of the S=5/2 ground state ( E/ D=0.06 and 0.28 in 1Fe- and 2Fe-SORs, respectively), the energy of the CysS(-)(p(pi))-->Fe(3+)(d(pi)) CT transition (15150+/-150 cm(-1) and 15600+/-150 cm(-1) in 1Fe- and 2Fe-SORs, respectively) and in changes in the Fe-S stretching region of the resonance Raman spectrum indicative of a weaker Fe-S(Cys) bond in 2Fe-SORs. These differences are interpreted in terms of small structural perturbations in the Fe coordination sphere with changes in the Fe-S(Cys) bond strength resulting from differences in the peptide N-H.S(Cys) hydrogen bonding within a tetrapeptide bidentate "chelate". Observation of the characteristic intervalence charge transfer transition of a cyano-bridged [Fe(III)-NC-Fe(II)(CN)(5)] unit in the near-IR VTMCD spectra of ferricyanide-oxidized samples of both P. furiosus 1Fe-SOR and D. vulgaris 2Fe-SOR has confirmed the existence of novel ferrocyanide adducts of the ferric [Fe(His)(4)(Cys)] sites in both 1Fe- and 2Fe-SORs.

Binding Sites↗
PubMed2002

Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network.

A bacterial thermostable citrate synthase has been analyzed to investigate the structural basis of its thermostability, and to compare such features with those previously identified in archaeal citrate synthases. The gene encoding the citrate synthase from Thermus aquaticus was identified from a gene library by screening with a PCR fragment amplified from genomic DNA using a primer based on the determined N-terminal amino acid sequence and a citrate synthase consensus primer. Apart from high sequence similarities with citrate synthase sequences within the Thermus/ Deinococcus group, the analyzed enzyme has highest similarities with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The recombinant enzyme is a dimer with high specific activity. Compared to its thermoactivity (T(opt)at 80 degrees C), the thermal stability of the enzyme is high, as judged from its T(m) (101 degrees C), and from irreversible thermal inactivation assays. Molecular modeling of the structure revealed an inter-subunit ion-pair network, comparable in size to the network found in the citrate synthase from P. furiosus; these networks are discussed in relation to the high thermal stability of these bacterial and archaeal enzymes.

Amino Acid Sequence↗
PubMed2004

A simple fluorimetric method for the estimation of DNA-DNA relatedness between closely related microorganisms by thermal denaturation temperatures.

Determination of whole-genome DNA-DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA-DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA-DNA relatedness between closely related species.

Benzothiazoles↗
PubMed2006

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate.

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying beta-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of beta-glucosidase at 90 degrees C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90 degrees C with an overall DeltaG degrees of approximately 20 kcal mol(-1). The high temperatures needed to chemically denature P. furiosus beta-glucosidase and the large DeltaG degrees of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of beta-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.

Archaeal Proteins↗
PubMed1997

Shuttle vectors for hyperthermophilic archaea.

Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea.

Alcohol Dehydrogenase↗
PubMed1998

Sugar utilization and its control in hyperthermophiles.

Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its alpha- and beta-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine beta-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase.

Carbohydrate Metabolism↗
PubMed2000

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

Cellulase↗
PubMed2002

Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs.

Glutamate dehydrogenases (GDHs) from fresh-water and marine hyperthermophilic Archaea were compared with respect to their responses to different salt concentrations. A gene encoding GDH from the terrestrial hyperthermophilic archaeon Thermococcus waiotapuensis (Twaio) was cloned, sequenced, and expressed at a high level in Escherichia coli. The deduced amino acid sequence, which consists of 418 amino acid residues, revealed a high degree of similarity with GDHs from related marine strains such as Thermococcus litoralis (Tl) and Pyrococcus furiosus (Pfu). Recombinant Twaio GDH was purified 27-fold to homogeneity. The enzyme is hexameric with a molecular weight of 259,000. The effects of several salts (KCl, CaCl, MgSO4), temperature, and pH on enzyme activity were determined and compared in three hyperthermophilic GDHs, including T. waiotapuensis, and GDHs from two marine species, T. litoralis and P. furiosus. Kinetic studies suggested a biosynthetic role for the nicotinamide adenine dinucleotide phosphate- (NADP-) specific Twaio GDH in the cell. Interestingly, Twaio GDH revealed no salt responses, whereas the two marine GDHs showed substantial enhancement of activity as well as thermostability at increasing salt concentrations. Because electrostatic interactions between charged amino acid residues are thought to be a key feature of structural integrity and thermostability in hyperthermophilic GDHs, salt availability and its effects on marine enzymes could partially explain a higher thermal stability in marine species than in phyletically related fresh-water species.

Amino Acid Sequence↗
PubMed2004

Heterologous gene expression in Thermus thermophilus: beta-galactosidase, dibenzothiophene monooxygenase, PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase, and chloramphenicol acetyl transferase.

Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus, the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile ( bglA from Pyrococcus woesei), a cofactor-requiring enzyme ( dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme ( carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.

Bacillus subtilis↗
PubMed2005

Protein production and crystallization at SECSG -- an overview.

Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins, which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.

Animals↗
PubMed2006

Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin.

The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 degrees Celsius or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 degrees Celsius or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.

Affinity Labels↗
PubMed2006

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors.

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.

Aminopeptidases↗
PubMed2004

An archaeal histone-like protein as an efficient DNA carrier in gene transfer.

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.

Animals↗
PubMed2005

Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress.

Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.

Endoplasmic Reticulum↗
PubMed2005

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase.

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.

Alcohol Oxidoreductases↗
PubMed2006

Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.

The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.

Amino Acid Sequence↗