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Hailiang Dong - One of the best experts on this subject based on the ideXlab platform.

  • inhibitory effect of clay mineral on methanogenesis by methanosarcina mazei and Methanothermobacter Thermautotrophicus
    Applied Clay Science, 2016
    Co-Authors: Rajesh Singh, Hailiang Dong, Jing Zhang, Abinash Agrawal, Hongmei Wang
    Abstract:

    Abstract With increased concentration of methane in the atmosphere and its impact on climate, effective mitigation of methane emission is of global importance. Recent studies have shown the inhibition of microbial methanogenesis upon addition of ferruginous clay minerals. To better elucidate the mechanism of the inhibitory effect of clay minerals on methanogenesis, laboratory experiments with Methanosarcina mazei and Methanothermobacter Thermautotrophicus were performed in batch systems in which pristine kaolinite or iron-coated kaolinite were added as representative clay minerals. Soluble Al in solution and production of Fe(II) and methane gas were monitored over the course of the experiments. The mineralogical changes in the kaolinites were characterized with scanning electron microscopy. The results confirmed that both Ms. mazei and Mt. Thermautotrophicus were capable of reducing ferric iron with H2/CO2 as methanogenic reactants. Both pristine and iron-coated kaolinites could act as effective inhibitors of methanogenesis. The overall methane production was similar with pristine kaolinite and iron-coated kaolinite, although at the beginning of the experiment less CH4 was observed in iron-coated kaolinite system. The inhibition of methanogenic activity observed in pristine kaolinite was primarily ascribed to the toxicity effect of aluminum. A more effective inhibition of methanogenesis in iron-coated kaolinite during the first several days could be explained by the combined effects of aluminum toxicity and diversion of electron flow from CO2 to Fe(III). Our results have important implications for mitigating methane emission in natural or anthropogenic settings.

  • cobalt iii edta reduction by thermophilic methanogen Methanothermobacter Thermautotrophicus
    Chemical Geology, 2015
    Co-Authors: Rajesh Singh, Hailiang Dong, Amy R Marts, David L Tierney, Catherine B Almquist
    Abstract:

    Cobalt is a metal contaminant at high temperature radioactive waste disposal sites. In previous studies have largely focused on mesophilic microorganisms to remediate cobalt, despite the presence of thermophilic microorganisms at such sites. In this study,Methanothermobacter Thermautotrophicus, a thermophilic methanogen, was used to reduce Co(III) in the form of [Co(III)–EDTA]-. Bioreduction experiments were conducted in a growth medium with H2/CO2 as a growth substrate at initial Co(III) concentrations of 1, 2, 4, 7, and 10 mM. At low Co(III) concentrations (< 4 mM), a complete reduction was observed within a week. Wet chemistry, X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) analyses were all consistent in revealing the reduction kinetics. But, at higher concentrations (7 and 10 mM) the reduction extents only reached 69.8% and 48.5%, respectively, likely due to the toxic effect of Co(III) to the methanogen cells as evidenced by a decrease in total cellular protein at these Co(III) concentrations. Methanogenesis was inhibited by Co(III) bioreduction, possibly due to impaired cell growth and electron diversion from CO2 to Co(III). Overall, our results demonstrated the ability of M. Thermautotrophicus to reduce Co(III) to Co(II) and its potential application for remediating 60Co contaminant at high temperature subsurface radioactivemore » waste disposal sites.« less

  • [Cobalt(III)–EDTA]− reduction by thermophilic methanogen Methanothermobacter Thermautotrophicus
    Chemical Geology, 2015
    Co-Authors: Rajesh Singh, Hailiang Dong, Amy R Marts, David L Tierney, Catherine B Almquist
    Abstract:

    Cobalt is a metal contaminant at high temperature radioactive waste disposal sites. In previous studies have largely focused on mesophilic microorganisms to remediate cobalt, despite the presence of thermophilic microorganisms at such sites. In this study,Methanothermobacter Thermautotrophicus, a thermophilic methanogen, was used to reduce Co(III) in the form of [Co(III)–EDTA]-. Bioreduction experiments were conducted in a growth medium with H2/CO2 as a growth substrate at initial Co(III) concentrations of 1, 2, 4, 7, and 10 mM. At low Co(III) concentrations (< 4 mM), a complete reduction was observed within a week. Wet chemistry, X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) analyses were all consistent in revealing the reduction kinetics. But, at higher concentrations (7 and 10 mM) the reduction extents only reached 69.8% and 48.5%, respectively, likely due to the toxic effect of Co(III) to the methanogen cells as evidenced by a decrease in total cellular protein at these Co(III) concentrations. Methanogenesis was inhibited by Co(III) bioreduction, possibly due to impaired cell growth and electron diversion from CO2 to Co(III). Overall, our results demonstrated the ability of M. Thermautotrophicus to reduce Co(III) to Co(II) and its potential application for remediating 60Co contaminant at high temperature subsurface radioactivemore » waste disposal sites.« less

  • reduction of hexavalent chromium by the thermophilic methanogen Methanothermobacter Thermautotrophicus
    Geochimica et Cosmochimica Acta, 2015
    Co-Authors: Rajesh Singh, Hailiang Dong, Amy R Marts, David L Tierney, Catherine B Almquist, Linduo Zhao, Erik R Farquhar, Brandon R Briggs
    Abstract:

    Despite the significant progress on iron reduction by thermophilic microorganisms, studies on their ability to reduce toxic metals are still limited, despite their common co-existence in high temperature environments (up to 70°C). In this study, Methanothermobacter Thermautotrophicus, an obligate thermophilic methanogen, was used to reduce hexavalent chromium. Experiments were conducted in a growth medium with H2/CO2 as substrate with various Cr6+ concentrations (0.2, 0.4, 1, 3, and 5 mM) in the form of potassium dichromate (K2Cr2O7). Time-course measurements of aqueous Cr6+ concentrations with the 1, 5-diphenylcarbazide colorimetric method showed complete reduction of the 0.2 and 0.4 mM Cr6+ solutions by this methanogen. However, much lower reduction extents of 43.6%, 13.0%, and 3.7% were observed at higher Cr6+ concentrations of 1, 3 and 5 mM, respectively. These lower extents of bioreduction suggest a toxic effect of aqueous Cr6+ to cells at this concentration range. At these higher Cr6+ concentrations, methanogenesis was inhibited and cell growth was impaired as evidenced by decreased total cellular protein production and live/dead cell ratio. Likewise, Cr6+ bioreduction rates decreased with increased initial concentrations of Cr6+ from 13.3 to1.9 μM h₋1. X-ray absorption near-edge structure (XANES) spectroscopy revealed a progressive reduction of soluble Cr6+ to insoluble Cr3+more » precipitates, which was confirmed as amorphous chromium hydroxide by X-ray diffraction and selected area electron diffraction pattern. However, a small fraction of reduced Cr occurred as aqueous Cr3+. Scanning and transmission electron microscope observations of M. Thermautotrophicus cells after Cr6+ exposure suggest both extra- and intracellular chromium reduction mechanisms. Results of this study demonstrate the ability of M. Thermautotrophicus cells to reduce toxic Cr6+ to less toxic Cr3+ and its potential application in metal bioremediation, especially at high temperature subsurface radioactive waste disposal sites, where the temperature may reach ~70°C.« less

  • microbial reduction of fe iii in smectite minerals by thermophilic methanogen Methanothermobacter Thermautotrophicus
    Geochimica et Cosmochimica Acta, 2013
    Co-Authors: Hailiang Dong, Jing Zhang, Abinash Agrawal
    Abstract:

    Abstract Clay minerals and thermophilic methanogens can co-exist in hot anoxic environments, including the continental subsurface, geysers, terrestrial hot springs, and deep-sea hydrothermal vent systems. However, it is unclear whether thermophilic methanogens are able to reduce structural Fe(III) in clay minerals. In this study, the ability of a thermophilic methanogen Methanothermobacter Thermautotrophicus to reduce structural Fe(III) in iron-rich and iron-poor smectites, (nontronite NAu-2 and Wyoming montmorillonite SWy-2) and the relationship between iron reduction and methanogenesis were investigated. M. Thermautotrophicus reduced Fe(III) in nontronite NAu-2 and montmorillonite SWy-2 with H2/CO2 as substrate. The extent of bioreduction was 27% for nontronite and 13–15% for montmorillonite. Anthraquinone-2,6-disulfonate (AQDS) did not enhance the extent of bioreduction, but accelerated the rate. When methanogenesis was inhibited via addition of 2-bromoethane sulfonate (BES), the extent of bioreduction decreased to 16% for NAu-2 and 9% for SWy-2. These data suggest that Fe(III) bioreduction and methanogenesis were mutually beneficial. The likely mechanism was that Fe(III) bioreduction lowered the reduction potential of the system so that methanogenesis became favorable, and methanogenesis in turn stimulated the growth of the methanogen, which enhanced Fe(III) bioreduction. NAu-2 was partly dissolved and high charge smectite and biogenic silica formed as a result of bioreduction.

Peter Smigaň - One of the best experts on this subject based on the ideXlab platform.

  • Harmaline-resistant mutant of Methanothermobacter Thermautotrophicus with a lesion in Na + /H + antiport
    General Physiology and Biophysics, 2020
    Co-Authors: Monika Vidova, Janette Bobaľova, Peter Smigaň
    Abstract:

    A spontaneous mutant of Methanothermobacter Thermautotrophicus resistant to the Na(+)/H(+) antiporter inhibitor harmaline was isolated. The Na(+)/H(+) exchange activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Na(+)/H(+) antiport activity of wild-type cells grown in the high Na(+) concentration (125 mmol/l) was significantly increased as compared to the cells grown under low Na(+) concentration (6.25 mmol/l) conditions. In contrast, harmaline resistant mutant showed almost the same Na(+)/H(+) antiport activity under both these conditions. While harmaline profoundly inhibited methanogenesis in the wild-type, increased methanogenesis was observed both in the presence and absence of harmaline in the mutant strain. ATP synthesis driven by methanogenic electron transport was significantly enhanced in the mutant cells. The experimental data revealed the differential expression of A flavoprotein and molybdenum-containing formylmethanofuran dehydrogenase 1 subunit C in harmaline-resistant mutant. The overexpression of these proteins might contribute to harmaline resistance. Taken together the results indicate that harmaline resistance in this mutant has arisen as a consequence of mutation(s) in antiporter gene(s) or protein(s) linked to antiporter activity. Moreover this work provides the evidence that Na(+)/H(+) exchanger deficiency in harmaline-resistant mutant can induce overexpression of several proteins participating in methanogenesis.

  • harmaline resistant mutant of Methanothermobacter Thermautotrophicus with a lesion in na h antiport
    General Physiology and Biophysics, 2011
    Co-Authors: Monika Vidova, Janette Bobaľova, Peter Smigaň
    Abstract:

    A spontaneous mutant of Methanothermobacter Thermautotrophicus resistant to the Na(+)/H(+) antiporter inhibitor harmaline was isolated. The Na(+)/H(+) exchange activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Na(+)/H(+) antiport activity of wild-type cells grown in the high Na(+) concentration (125 mmol/l) was significantly increased as compared to the cells grown under low Na(+) concentration (6.25 mmol/l) conditions. In contrast, harmaline resistant mutant showed almost the same Na(+)/H(+) antiport activity under both these conditions. While harmaline profoundly inhibited methanogenesis in the wild-type, increased methanogenesis was observed both in the presence and absence of harmaline in the mutant strain. ATP synthesis driven by methanogenic electron transport was significantly enhanced in the mutant cells. The experimental data revealed the differential expression of A flavoprotein and molybdenum-containing formylmethanofuran dehydrogenase 1 subunit C in harmaline-resistant mutant. The overexpression of these proteins might contribute to harmaline resistance. Taken together the results indicate that harmaline resistance in this mutant has arisen as a consequence of mutation(s) in antiporter gene(s) or protein(s) linked to antiporter activity. Moreover this work provides the evidence that Na(+)/H(+) exchanger deficiency in harmaline-resistant mutant can induce overexpression of several proteins participating in methanogenesis.

  • effects of 3 hydroxy 3 methylglutaryl coenzyme a reductase inhibitor pravastatin on membrane lipids and membrane associated functions of Methanothermobacter Thermautotrophicus
    Folia Microbiologica, 2010
    Co-Authors: Zuzana Nováková, Jaroslav Blasko, Ivan Hapala, Peter Smigaň
    Abstract:

    The role of archaeal membrane and its lipid constituents was investigated in bioenergetic functions of Methanothermobacter Thermautotrophicus. The effects were determined of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, pravastatin, on lipid composition, and its impact on some bioenergetic functions of treated cells. Pravastatin remarkably inhibited the growth of M. Thermautotrophicus. On membrane level, pravastatin treatment modulated the composition of the mixture of squalene and hydrosqualene derivatives as well as the activities of ATPase, A1Ao-ATP synthase and Na+/H+ antiporter. SDS-PAGE of chloroform-methanol extracts of membranes from control and pravastatin-treated cells revealed changes in the amount of AtpK proteolipids, which suggests that pravastatin modifies cell-membrane composition, hereby modulating the properties of some membrane-bound enzymes participating in energy transformation in methanoarchaea.

  • Isolation and characterization of a N,N′-dicyclohexylcarbodiimide-resistant mutant of Methanothermobacter Thermautotrophicus with alterations to the ATP synthesis machinery
    Folia Microbiologica, 2009
    Co-Authors: Zuzana Nováková, Alan Majernik, J. Bobáľová, M. Vidová, Peter Smigaň
    Abstract:

    A spontaneous mutant of Methanothermobacter Thermautotrophicus resistant toward the ATP-synthase inhibitor N , N′ -dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 μmol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A_1 and A_o subcomplexes of the A_1A_o-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes ( atp ) for the A_1A_o ATP synthase. The results imply that DCCD resistance is a consequence of increased A_1A_o ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

  • isolation and characterization of a n n dicyclohexylcarbodiimide resistant mutant of Methanothermobacter Thermautotrophicus with alterations to the atp synthesis machinery
    Folia Microbiologica, 2009
    Co-Authors: Zuzana Nováková, Alan Majernik, Monika Vidova, J. Bobáľová, Peter Smigaň
    Abstract:

    A spontaneous mutant of Methanothermobacter Thermautotrophicus resistant toward the ATP-synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 μmol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A1 and Ao subcomplexes of the A1Ao-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A1Ao ATP synthase. The results imply that DCCD resistance is a consequence of increased A1Ao ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

Zvi Kelman - One of the best experts on this subject based on the ideXlab platform.

  • mutational analysis of conserved aspartic acid residues in the Methanothermobacter Thermautotrophicus mcm helicase
    Extremophiles, 2011
    Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi Kelman
    Abstract:

    Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter Thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.

  • ATP Hydrolysis and DNA Binding Confer Thermostability on the MCM Helicase
    Biochemistry, 2009
    Co-Authors: Nozomi Sakakibara, Frederick P. Schwarz, Zvi Kelman
    Abstract:

    The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter Thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 °C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 °C higher in the presence of DNA and either ATP or ADP-AlF4−, a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPγS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA b...

  • the Methanothermobacter Thermautotrophicus mcm helicase is active as a hexameric ring
    Journal of Biological Chemistry, 2009
    Co-Authors: Jaeho Shin, Zvi Kelman
    Abstract:

    Abstract The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukarya. The structure of the single MCM protein homologue from the archaeon Methanothermobacter Thermautotrophicus is not yet clear, and hexameric, heptameric, octameric, and dodecameric structures, open rings, and filamentous structures have been reported. Using a combination of biochemical and structural analysis, it is shown here that the M. Thermautotrophicus MCM helicase is active as a hexamer.

  • the Methanothermobacter Thermautotrophicus cdc6 2 protein the putative helicase loader dissociates the minichromosome maintenance helicase
    Journal of Bacteriology, 2008
    Co-Authors: Jaeho Shin, Zvi Kelman
    Abstract:

    The Cdc6-1 and -2 proteins from the archaeon Methanothermobacter Thermautotrophicus were previously shown to bind the minichromosome maintenance (MCM) helicase. It is shown here that Cdc6-2 protein dissociates the MCM complex. This observation supports the hypothesis that the Cdc6-2 protein functions as a helicase loader.

  • Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein
    Nucleic Acids Research, 2008
    Co-Authors: Nozomi Sakakibara, Eugene Melamud, Rajesh Kasiviswanathan, Frederick P. Schwarz, Zvi Kelman
    Abstract:

    Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter Thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.

J A Gerlt - One of the best experts on this subject based on the ideXlab platform.

  • Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.
    Biochemistry, 2012
    Co-Authors: B. Desai, John P. Richard, Tina L. Amyes, B. Mc Kay Wood, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo, Bogdana Goryanova, J A Gerlt
    Abstract:

    The binding of a ligand to orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the Ec·S⧧ complex) are enforced. Focusing on the OMPDC from Methanothermobacter Thermautotrophicus, we find the “remote” 5′-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5′-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the “mobile” and “fixed...

  • mechanism of the orotidine 5 monophosphate decarboxylase catalyzed reaction effect of solvent viscosity on kinetic constants
    Biochemistry, 2009
    Co-Authors: Mc Kay B Wood, John P. Richard, Tina L. Amyes, K K Chan, J A Gerlt
    Abstract:

    Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/KM)/knon] is 4.8 × 1022 M−1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter Thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent visc...

  • Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization.
    Biochemistry, 2009
    Co-Authors: K K Chan, John P. Richard, Tina L. Amyes, B. Mc Kay Wood, Alexander A. Fedorov, Elena V. Fedorov, Heidi Imker, Steven C. Almo, J A Gerlt
    Abstract:

    The reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (kcat/knon, 7.1 × 1016) and proficiency [(kcat/KM)/knon, 4.8 × 1022 M−1] is uncertain. That the OMPDCs from Methanothermobacter Thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and its flanking transition states (≥1010). The origin of the “missing” contribution, ≤107 (∼1017 total − ≥1010), is of interest. Based on structures of liganded complexes, unfavorable electrostatic interactions between the substrate carboxylate group and a proximal Asp (Asp 70 in MtOMPDC and Asp 91 in ScOMPDC) have been proposed to contribute to the catalytic efficiency [Wu, N., Mo, Y., Gao, J., and Pai, E. F. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2017−2022]. ...

John N. Reeve - One of the best experts on this subject based on the ideXlab platform.

  • Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.
    Acta crystallographica. Section F Structural biology and crystallization communications, 2010
    Co-Authors: Jacquelyn Cafasso, Babu A Manjasetty, Elizabeth A Karr, Mark R Chance, Kathleen Sandman, John N. Reeve
    Abstract:

    TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter Thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Å resolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V(M)) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein.

  • Archaeal chromatin proteins histone HMtB and Alba have lost DNA-binding ability in laboratory strains of Methanothermobacter Thermautotrophicus
    Extremophiles, 2008
    Co-Authors: Kathleen Sandman, Hélène Louvel, Rachel Y. Samson, Suzette L. Pereira, John N. Reeve
    Abstract:

    Alignments of the sequences of the all members of the archaeal histone and Alba1 families of chromatin proteins identified isoleucine residues, I19 in HMtB and I39 in MtAlba, in Methanothermobacter Thermautotrophicus , at locations predicted to be directly involved in DNA binding. In all other HMfB family members, residue 19 is an arginine (R19), and either arginine or lysine is present in almost all other Alba1 family members at the structural site equivalent to I39 in MtAlba. Electrophoretic mobility shift assays revealed that recombinant HMtB and MtAlba do not bind DNA, but variants constructed with R19 and R39, respectively, bound DNA; and whereas MtAlba(I19) did not bind RNA, MtAlba(R19) bound both single stranded RNA and tRNA. Amplification and sequencing of MT0254 (encodes HMtB) and MT1483 (encodes MtAlba) from several Methanothermobacter Thermautotrophicus lineages has revealed that HMtB and MtAlba had arginine residues at positions 19 and 39, respectively, in the original isolate and that spontaneous mutations must have occurred, and been fixed, in some laboratory lineages that now have HMtB(I19) and MtAlba(I39). The retention of these variants suggests some continuing functions and fusion of the HMtB(I19) sequence to HMtA2 resulted in a protein that folds to form a histone fold heterodimer that binds and compacts DNA. The loss of DNA binding by HMtB(I19) does not therefore prevent HMtB from participating in DNA interactions as one partner of an archaeal histone heterodimer.

  • trpy regulation of trpb2 transcription in Methanothermobacter Thermautotrophicus
    Journal of Bacteriology, 2008
    Co-Authors: Elizabeth A Karr, Kathleen Sandman, Rudi Lurz, John N. Reeve
    Abstract:

    TrpY binds specifically to TRP box sequences upstream of trpB2, but the repression of trpB2 transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.

  • Spontaneous trpY Mutants and Mutational Analysis of the TrpY Archaeal Transcription Regulator
    Journal of Bacteriology, 2007
    Co-Authors: Lubomira Cubonova, Elizabeth A Karr, Kathleen Sandman, Andrew J. Cochran, John N. Reeve
    Abstract:

    Over 90% of Methanothermobacter Thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan had mutations in trpY. Most were single-base-pair substitutions that identified separate DNA- and tryptophan-binding regions in TrpY. In vivo and in vitro studies revealed that DNA binding was sufficient for TrpY repression of trpY transcription but that TrpY must bind DNA and tryptophan to assemble a complex that represses trpEGCFBAD.

  • Archaeal minichromosome maintenance (MCM) helicase can unwind DNA bound by archaeal histones and transcription factors.
    Journal of Biological Chemistry, 2006
    Co-Authors: Jaeho Shin, John N. Reeve, Thomas J. Santangelo, Zvi Kelman
    Abstract:

    Abstract Protein-DNA complexes must be disassembled to facilitate DNA replication. Replication forks contain a helicase that unwinds the duplex DNA at the front of the fork. The minichromosome maintenance helicase from the archaeon Methanothermobacter Thermautotrophicus required only ATP to unwind DNA bound into complexes by the M. Thermautotrophicus archaeal histone HMtA2, transcription repressor TrpY, or into a transcription pre-initiation complex by M. Thermautotrophicus TATA-box-binding protein, transcription factor B, and RNA polymerase. In contrast, the minichromosome maintenance helicase was unable to unwind DNA bound by this archaeal RNA polymerase in a stalled transcript-elongating complex.