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Rainer Jaenicke - One of the best experts on this subject based on the ideXlab platform.
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dissection of the gene of the bifunctional pgk tim fusion protein from the Hyperthermophilic Bacterium thermotoga maritima design and characterization of the separate triosephosphate isomerase
Protein Science, 2008Co-Authors: Nicola Beaucamp, Alexandra Hofmann, Barbara Kellerer, Rainer JaenickeAbstract:Triosephosphate isomerase (TIM), from the Hyperthermophilic Bacterium Thermotoga maritima, has been shown to be covalently linked to phosphoglycerate kinase (PGK) forming a bifunctional fusion protein with TIM as the C-terminal portion of the subunits of the tetrameric protein (Schurig et al., EMBO J 14:442-451, 1995). To study the effect of the anomalous state of association on the structure, stability, and function of Thermotoga TIM, the isolated enzyme was cloned and expressed in Escherichia coli, and compared with its wild-type structure in the PGK-TIM fusion protein. After introducing a start codon at the beginning of the tpi open reading frame, the gene was expressed in E.c.BL21(DE3)/ pNBTIM. The nucleotide sequence was confirmed and the protein purified as a functional dimer of 56.5 kDa molecular mass. Spectral analysis, using absorption, fluorescence emission, near- and far-UV circular dichroism spectroscopy were used to compare the separated Thermotoga enzyme with its homologs from mesophiles. The catalytic properties of the enzyme at approximately 80 degrees C are similar to those of its mesophilic counterparts at their respective physiological temperatures, in accordance with the idea that under in vivo conditions enzymes occupy corresponding states. As taken from chaotropic and thermal denaturation transitions, the separated enzyme exhibits high intrinsic stability, with a half-concentration of guanidinium-chloride at 3.8 M, and a denaturation half-time at 80 degrees C of 2 h. Comparing the properties of the TIM portion of the PGK-TIM fusion protein with those of the isolated recombinant TIM, it is found that the fusion of the two enzymes not only enhances the intrinsic stability of TIM but also its catalytic efficiency.
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octameric enolase from the Hyperthermophilic Bacterium thermotoga maritima purification characterization and image processing
Protein Science, 2008Co-Authors: Hartmut Schurig, Reinhard Rachel, Kerstin Rutkat, Rainer JaenickeAbstract:Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the Hyperthermophilic Bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.
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stability and folding of dihydrofolate reductase from the Hyperthermophilic Bacterium thermotoga maritima
Biochemistry, 1999Co-Authors: Thomas Dams, Rainer JaenickeAbstract:Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding. The enzyme DHFR from the Hyperthermophilic Bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability. The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding. The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C. The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C. There is no flattening of the stability profile. Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability. TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates. The unfolding reaction is much slower (ca. 10(8) times) compared to DHFR from Escherichia coli (EcDHFR). In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable. Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude. Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer.
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recombinant phosphoglycerate kinase from the Hyperthermophilic Bacterium thermotoga maritima catalytic spectral and thermodynamic properties
Journal of Molecular Biology, 1998Co-Authors: Mira Grättinger, Hartmut Schurig, Arnd Dankesreiter, Rainer JaenickeAbstract:Recombinant phosphoglycerate kinase from the Hyperthermophilic Bacterium Thermotoga maritima (TmPGK) has been expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity applying heat incubation of the crude extract at 80 degreesC, ion exchange chromatography and gel filtration. The biochemical, catalytic and spectral properties were compared with those of the natural enzyme and found to be identical. As shown by SDS-PAGE, ultracentrifugal analysis and gel filtration chromatography, the enzyme is a 43 kDa monomer. At neutral pH, the guanidinium chloride (GdmCl) and temperature-induced denaturation transitions reveal two-state behaviour with high cooperativity. As taken from the temperature dependence of the free energy of unfolding at zero GdmCl concentration and pH 7, optimum stability is observed at approximately 30 degreesC. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta DeltaG=85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about -10 and +85 degreesC. This indicates that the stability profile of TmPGK is shifted to higher free energy values and broadened over a wider temperature range, compared to that observed for PGKs from mesophiles or moderately thermophiles. In order to achieve cold or heat-denaturation, GdmCl concentrations of approximately 1.8 or approximately 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long.
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Crystallographic analysis of phosphoglycerate kinase from the Hyperthermophilic Bacterium Thermotoga maritima.
Biological chemistry, 1998Co-Authors: Günter Auerbach, Hartmut Schurig, Uwe Jacob, Mira Grättinger, Rainer JaenickeAbstract:Phosphoglycerate kinase from the Hyperthermophilic Bacterium Thermotoga maritima has been co-crystallized with its substrate 3-phosphoglycerate and the ATP analogue AMP-PNP using the vapour diffusion method. Crystals were obtained from a solution containing polyethylene glycol (MW 3000/8000) as precipitating agent. A complete diffraction data set from orthorhombic crystals was collected up to 2.0 A resolution. The TmPGK crystallizes in the space group P2(1)2(1)2 (cell dimensions: a = 62.0 A, b = 76.9 A, c = 87.5 A) with one molecule in the asymmetric unit. The structure was solved by Patterson search methods using Bacillus stearothermophilus PGK as a search model and was refined to a crystallographic R factor of 22.0%. Compared to the enzyme from B. stearothermophilus, horse, pig and yeast, the Thermotoga enzyme exhibits a drastically reduced interdomain angle, similar to the one reported for PGK from Trypanosoma brucei. Here we present crystallographic data of the first high-resolution structure of a PGK in largely closed conformation, complexed with the two products of the catalyzed reaction, and, at the same time, the first PGK structure from a Hyperthermophilic organism.
Thomas Hansen - One of the best experts on this subject based on the ideXlab platform.
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atp dependent glucokinase from the Hyperthermophilic Bacterium thermotoga maritima represents an extremely thermophilic rok glucokinase with high substrate specificity
Fems Microbiology Letters, 2003Co-Authors: Thomas Hansen, Peter SchonheitAbstract:The gene (open reading frame (ORF) Tm1469, glk) encoding ATP-dependent ROK (repressors, ORFs, sugar kinases) glucokinase (ATP-GLK, EC 2.7.1.2) of the Hyperthermophilic Bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 80 kDa composed of 36-kDa subunits. Rate dependence (at 80°C) on glucose and ATP followed Michaelis–Menten kinetics with apparent Km values of 1.0 and 0.36 mM, respectively; apparent Vmax values were about 370 U mg−1. The enzyme was highly specific for glucose as phosphoryl acceptor. Besides glucose only 2-deoxyglucose was phosphorylated to some extent, whereas mannose and fructose were not used. With a temperature optimum of 93°C the enzyme is the most thermoactive bacterial ATP-GLK described.
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comparative analysis of pyruvate kinases from the Hyperthermophilic archaea archaeoglobus fulgidus aeropyrum pernix and pyrobaculum aerophilum and the Hyperthermophilic Bacterium thermotoga maritima unusual regulatory properties in Hyperthermophilic
Journal of Biological Chemistry, 2003Co-Authors: Ulrike Johnsen, Thomas HansenAbstract:Abstract Pyruvate kinases (PK, EC 2.7.1.40) from three Hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the Hyperthermophilic Bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 °C (A. fulgidus) and above 98 °C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 °C (A. pernix) and showed melting temperatures for thermal unfolding at 93 °C (A. fulgidus) or above 98 °C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of Hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the Bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 °C, was highly thermostable up to 90 °C, and had a melting temperature above 98 °C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of Hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the Hyperthermophilic Bacterium T. maritima.
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comparative analysis of pyruvate kinases from the Hyperthermophilic archaea archaeoglobus fulgidus aeropyrum pernix and pyrobaculum aerophilum and the Hyperthermophilic Bacterium thermotoga maritima unusual regulatory properties in Hyperthermophilic archaea
Journal of Biological Chemistry, 2003Co-Authors: Ulrike Johnsen, Thomas Hansen, Peter SchonheitAbstract:Pyruvate kinases (PK, EC 2.7.1.40) from three Hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the Hyperthermophilic Bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of Hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the Bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of Hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the Hyperthermophilic Bacterium T. maritima.
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glucose 6 phosphate dehydrogenase from the Hyperthermophilic Bacterium thermotoga maritima expression of the g6pd gene and characterization of an extremely thermophilic enzyme
Fems Microbiology Letters, 2002Co-Authors: Thomas Hansen, Bettina Schlichting, Peter SchonheitAbstract:The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the Hyperthermophilic Bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP+ followed Michaelis–Menten kinetics with apparent Km values of 0.15 mM and 0.03 mM, respectively; apparent Vmax values were about 20 U mg−1. The enzyme also reduced NAD+ (apparent Km 12 mM, Vmax 12 U mg−1). The 1000-fold higher catalytic activity (kcat/Km) with NADP+ over NAD+ defines the G6PD as NADP+ specific in vivo. G6PD activity was competitively inhibited by NADPH with a Ki value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described.
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ATP-dependent 6-phosphofructokinase from the Hyperthermophilic Bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme
Archives of microbiology, 2002Co-Authors: Thomas Hansen, Meike MusfeldtAbstract:The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the Hyperthermophilic Bacterium Thermotoga maritima was purified 730-fold to homogeneity. The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits. Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 °C. Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant (S0.5) of 0.7 mM and a Hill coefficient of 2.2. The apparent Km for ATP was 0.2 mM and the apparent Vmax value was about 360 U/mg. The enzyme also catalyzed in vitro the reverse reaction with an apparent Km for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent Vmax of about 13 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could partially be replaced by Mn2+ and Fe2+. Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP. The enzyme had a temperature optimum of 93 °C and showed a significant thermostability up to 100 °C. Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli. The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T. maritima. The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family. In accordance with its allosteric properties, ATP-PFK of T. maritima contained the conserved allosteric effector-binding sites for ADP and PEP.
Robert M Kelly - One of the best experts on this subject based on the ideXlab platform.
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stationary phase and nutrient levels trigger transcription of a genomic locus containing a novel peptide tm1316 in the Hyperthermophilic Bacterium thermotoga maritima
Applied and Environmental Microbiology, 2013Co-Authors: Andrew D Frock, Clemente I Montero, Sara E Blumerschuette, Robert M KellyAbstract:The genome of the Hyperthermophilic Bacterium Thermotoga maritima encodes numerous putative peptides/proteins of 100 amino acids or less. While most of these open reading frames (ORFs) are transcribed during growth, their corresponding physiological roles are largely unknown. The onset of stationary phase in T. maritima was accompanied by significant morphological changes and upregulation of several ORFs located in the TM1298-TM1336 genome locus. This region contains putative HicAB toxin-antitoxin pairs, hypothetical proteins, radical S-adenosylmethionine (SAM) enzymes, and ABC transporters. Of particular note was the TM1315-TM1319 operon, which includes a putative 31-amino-acid peptide (TM1316) that was the most highly transcribed gene in the transcriptome during stationary phase. Antibodies directed against a synthetic version of TM1316 were used to track its production, which correlated closely with transcriptomic data. Immunofluorescence microscopy revealed that TM1316 was localized to the cell envelope and prominent in cell aggregates formed during stationary phase. The only functionally characterized locus with an organization similar to that of TM1315-TM1319 is in Bacillus subtilis, which contains subtilosin A, a cyclic peptide with Cys-to-α-carbon linkages that functions as an antilisterial bacteriocin. While the organization of TM1316 resembled that of the Bacillus peptide (e.g., in its number of amino acids and spacing of Cys residues), preparations containing high levels of TM1316 affected the growth of neither Thermotoga species nor Pyrococcus furiosus, a Hyperthermophilic archaeon isolated from the same locale as T. maritima. Several other putative Cys-rich peptides could be identified in the TM1298-TM1336 locus, and while their roles are also unclear, they merit examination as potential antimicrobial agents in Hyperthermophilic biotopes.
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colocation of genes encoding a trna mrna hybrid and a putative signaling peptide on complementary strands in the genome of the Hyperthermophilic Bacterium thermotoga maritima
Journal of Bacteriology, 2006Co-Authors: Clemente I Montero, Shannon B Conners, Matthew R. Johnson, Derrick L Lewis, Elizabeth Nance, Jason D Nichols, Robert M KellyAbstract:In the genome of the Hyperthermophilic Bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.
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an expression driven approach to the prediction of carbohydrate transport and utilization regulons in the Hyperthermophilic Bacterium thermotoga maritima
Journal of Bacteriology, 2005Co-Authors: Shannon B Conners, Clemente I Montero, Donald A Comfort, Swapnil R Chhabra, Keith R. Shockley, Matthew R. Johnson, Robert M KellyAbstract:Comprehensive analysis of genome-wide expression patterns during growth of the Hyperthermophilic Bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixed-model analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, β-xylan, and β-glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.
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population density dependent regulation of exopolysaccharide formation in the Hyperthermophilic Bacterium thermotoga maritima
Molecular Microbiology, 2004Co-Authors: Matthew R. Johnson, Shannon B Conners, Clemente I Montero, Keith R. Shockley, Stephanie L Bridger, Robert M KellyAbstract:Co-cultivation of the hyperthermophiles Thermotoga maritima and Methanococcus jannaschii resulted in fivefold higher T. maritima cell densities when compared with monoculture as well as concomitant formation of exopolysaccharide and flocculation of heterotroph-methanogen cellular aggregates. Transcriptional analysis of T. maritima cells from these aggregates using a whole genome cDNA microarray revealed the induction of a putative exopolysaccharide synthesis pathway, regulated by intracellular levels of cyclic diguanosine 3',5'-(cyclic)phosphate (cyclic di-GMP) and mediated by the action of several GGDEF proteins, including a putative diguanylate cyclase (TM1163) and a putative phosphodiesterase (TM1184). Transcriptional analysis also showed that TM0504, which encodes a polypeptide containing a motif common to known peptide-signalling molecules in mesophilic bacteria, was strongly upregulated in the co-culture. Indeed, when a synthetically produced peptide based on TM0504 was dosed into the culture at ecologically relevant levels, the production of exopolysaccharide was induced at significantly lower cell densities than was observed in cultures lacking added peptide. In addition to identifying a pathway for polysaccharide formation in T. maritima, these results point to the existence of peptide-based quorum sensing in Hyperthermophilic bacteria and indicate that cellular communication should be considered as a component of the microbial ecology within hydrothermal habitats.
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transcriptional analysis of biofilm formation processes in the anaerobic Hyperthermophilic Bacterium thermotoga maritima
Applied and Environmental Microbiology, 2004Co-Authors: Marybeth A. Pysz, Shannon B Conners, Clemente I Montero, Keith R. Shockley, Matthew R. Johnson, Donald E. Ward, Robert M KellyAbstract:Thermotoga maritima, a fermentative, anaerobic, Hyperthermophilic Bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.
Peter Schonheit - One of the best experts on this subject based on the ideXlab platform.
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atp dependent glucokinase from the Hyperthermophilic Bacterium thermotoga maritima represents an extremely thermophilic rok glucokinase with high substrate specificity
Fems Microbiology Letters, 2003Co-Authors: Thomas Hansen, Peter SchonheitAbstract:The gene (open reading frame (ORF) Tm1469, glk) encoding ATP-dependent ROK (repressors, ORFs, sugar kinases) glucokinase (ATP-GLK, EC 2.7.1.2) of the Hyperthermophilic Bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 80 kDa composed of 36-kDa subunits. Rate dependence (at 80°C) on glucose and ATP followed Michaelis–Menten kinetics with apparent Km values of 1.0 and 0.36 mM, respectively; apparent Vmax values were about 370 U mg−1. The enzyme was highly specific for glucose as phosphoryl acceptor. Besides glucose only 2-deoxyglucose was phosphorylated to some extent, whereas mannose and fructose were not used. With a temperature optimum of 93°C the enzyme is the most thermoactive bacterial ATP-GLK described.
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comparative analysis of pyruvate kinases from the Hyperthermophilic archaea archaeoglobus fulgidus aeropyrum pernix and pyrobaculum aerophilum and the Hyperthermophilic Bacterium thermotoga maritima unusual regulatory properties in Hyperthermophilic archaea
Journal of Biological Chemistry, 2003Co-Authors: Ulrike Johnsen, Thomas Hansen, Peter SchonheitAbstract:Pyruvate kinases (PK, EC 2.7.1.40) from three Hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the Hyperthermophilic Bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of Hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the Bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of Hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the Hyperthermophilic Bacterium T. maritima.
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glucose 6 phosphate dehydrogenase from the Hyperthermophilic Bacterium thermotoga maritima expression of the g6pd gene and characterization of an extremely thermophilic enzyme
Fems Microbiology Letters, 2002Co-Authors: Thomas Hansen, Bettina Schlichting, Peter SchonheitAbstract:The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the Hyperthermophilic Bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP+ followed Michaelis–Menten kinetics with apparent Km values of 0.15 mM and 0.03 mM, respectively; apparent Vmax values were about 20 U mg−1. The enzyme also reduced NAD+ (apparent Km 12 mM, Vmax 12 U mg−1). The 1000-fold higher catalytic activity (kcat/Km) with NADP+ over NAD+ defines the G6PD as NADP+ specific in vivo. G6PD activity was competitively inhibited by NADPH with a Ki value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described.
Wolfgang Liebl - One of the best experts on this subject based on the ideXlab platform.
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xylanase attachment to the cell wall of the Hyperthermophilic Bacterium thermotoga maritima
Journal of Bacteriology, 2008Co-Authors: Wolfgang Liebl, Christoph Winterhalter, Wolfgang Baumeister, Martin Armbrecht, Michael ValdezAbstract:The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative Bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.
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identification and characterization of a novel intracellular alkaline α amylase from the Hyperthermophilic Bacterium thermotoga maritima msb8
Applied and Environmental Microbiology, 2006Co-Authors: Meike Ballschmiter, Ole Futterer, Wolfgang LieblAbstract:The gene for a novel α-amylase, designated AmyC, from the Hyperthermophilic Bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 α-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an α-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.
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pullulanase from the Hyperthermophilic Bacterium thermotoga maritima purification by β cyclodextrin affinity chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 2000Co-Authors: Gernot Kriegshauser, Wolfgang LieblAbstract:This is the first report about the isolation of a type I pullulanase from a Hyperthermophilic Bacterium, Thermotoga maritima strain MSB8. Purification of the enzyme from a cleared cell-free extract was achieved by anion-exchange chromatography and β-cyclodextrin affinity chromatography. Using this convenient two-step method we have purified the pullulanase 406-fold with a 26% yield. The purified enzyme displayed maximum pullulan hydrolysis at pH 5.9 and 90°C (15-min assay) and was remarkably resistant against thermoinactivation, having a half-life at 90°C of about 3.5 h. To our knowledge, the T. maritima pullulanase is the most thermostable type I pullulanase known to date. The affinity-based purification protocol described here may be useful for the efficient isolation of other pullulanases.
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xylanase xyna from the Hyperthermophilic Bacterium thermotoga maritima structure and stability of the recombinant enzyme and its isolated cellulose binding domain
Protein Science, 1997Co-Authors: Doris Wassenberg, Hartmut Schurig, Wolfgang Liebl, Rainer JaenickeAbstract:The Hyperthermophilic Bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the Hyperthermophilic Bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.
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glutamate dehydrogenase from the Hyperthermophilic Bacterium thermotoga maritima molecular characterization and phylogenetic implications
Extremophiles, 1997Co-Authors: Remco Kort, Wolfgang Liebl, Bernard Labedan, Patrick Forterre, Rik I L Eggen, Willem M De VosAbstract:The Hyperthermophilic Bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the Hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these Hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota.