A chaperonin from a thermophilic bacterium,
            Thermus thermophilus

Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a therm ophilic bacterium , Thermus thermophilus , consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex. Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet. We conclude that a single cpn 60-heptam er ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers. The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptam er. The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HCl is different at three temperature ranges. At high temperatures above 55°C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner. At middle temperatures (25-55°C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding. At lower temperatures below 25°C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP. When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10°C by inclusion of Thermus chaperonin in the solution. Furthermore, once the folding intermediate of a protein is captured by Thermus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature. These results indicate that during heat denaturation proteins assume the common structure which is recognizable by the chaperonin. Finally, a ‘folding intermediate reservoir’ model to explain the effect of chaperonin is proposed, and is compared with a ‘marsupium ’ model.

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A Key Enzyme of the NAD+ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

Journal of Bacteriology ◽

10.1128/jb.00359-17 ◽

2017 ◽

Vol 199 (17) ◽

Cited By ~ 7

Author(s):

Hironori Taniguchi ◽

Sathidaphorn Sungwallek ◽

Phatcharin Chotchuang ◽

Kenji Okano ◽

Kohsuke Honda

Keyword(s):

High Temperatures ◽

De Novo ◽

Cell Metabolism ◽

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

High Catalytic Activity ◽

Salvage Pathway ◽

Content Type ◽

Key Enzyme ◽

The Impact

ABSTRACT NAD (NAD+) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD+ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD+ de novo synthesis pathway. Herein, we report the importance of the NAD+ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD+ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (Km of 17 μM, k cat of 50 s−1, k cat/Km of 3.0 × 103 s−1 · mM−1). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD+ salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD+ and NADH. IMPORTANCE NAD+ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD+ decomposition is, in general, more rapid. This study emphasizes that NAD+ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.

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Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27

FEBS Journal ◽

10.1111/j.1742-4658.2006.05416.x ◽

2006 ◽

Vol 273 (18) ◽

pp. 4210-4218 ◽

Cited By ~ 37

Author(s):

Cornelia Schwarzenlander ◽

Beate Averhoff

Keyword(s):

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

Dna Transport

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Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog

Journal of Bacteriology ◽

10.1128/jb.00710-18 ◽

2019 ◽

Vol 201 (14) ◽

Cited By ~ 1

Author(s):

Takeo Tomita ◽

Hajime Matsushita ◽

Ayako Yoshida ◽

Saori Kosono ◽

Minoru Yoshida ◽

...

Keyword(s):

Enzymatic Activity ◽

Glutamate Dehydrogenase ◽

Ternary Complex ◽

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

Regulatory Subunit ◽

Binary Complex ◽

Bacterial Cells ◽

Adenine Phosphoribosyltransferase ◽

Content Type

ABSTRACT Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

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Cloning and in silico study of an endoglucanase from a thermophilic bacterium isolated from a hydrothermal vent of West Kawio, Sangihe‐Talaud waters, North Sulawesi, Indonesia

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.48272 ◽

2020 ◽

Vol 24 (2) ◽

pp. 105

Author(s):

Edvan Arifsaputra Suherman ◽

Maelita Ramdani Moeis ◽

Elvi Restiawaty

Keyword(s):

Amino Acids ◽

High Temperatures ◽

Hydrothermal Vent ◽

Animal Feed ◽

Thermophilic Bacteria ◽

Glycosyl Hydrolase ◽

Thermophilic Bacterium ◽

Lower Percentage ◽

Bacillus Species ◽

North Sulawesi

Endoglucanase is used in industries that apply high temperatures, such as bioethanol, detergent, paper, and animal feed industries. Most available endoglucanases have very low stability at high temperatures. Therefore, this study aimed to identfy a new thermostable endoglucanase that is able to maintain its actvity at high temperatures. Five isolates of thermophilic bacteria were previously isolated from the hydrothermal vent of West Kawio, Indonesia. Among them, the DSI2 isolate showed the highest endoglucanase actvity, and was identfed and named as Bacillus safensis DSI2. The EgDSI2 gene was cloned from B. safensis DSI2. EgDSI2 is 1851 bp long encoding a protein of 616 amino acids. The encoded protein, EgDSI2, has high sequence identty to other B. safensis endoglucanases and was predicted with the Compute pI/Mw tool to be 69.41 kDa. EgDSI2 was high in hydrophobic amino acids. The enzyme had higher percentage of Ala andPro, and lower percentage of Gly compared to thermolabile endoglucanases from two Bacillus species. EgDSI2 harbored a catalytc domain belonging to glycosyl hydrolase family 9 (GH9) and a type 3 cellulose‐binding domain (CBM3). Propertes of endoglucanases with GH9‐CBM3 modular organizaton include actvity over a wide pH range, high optmum temperature, and thermostablity. Therefore, EgDSI2 has potental applicatons in the industries.

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Multiple Mechanisms for Degradation of Bacteriophage T4 soc mRNA

Genetics ◽

10.1093/genetics/160.1.5 ◽

2002 ◽

Vol 160 (1) ◽

pp. 5-12

Author(s):

Toshie Kai ◽

Tetsuro Yonesaki

Keyword(s):

Translation Initiation ◽

Mrna Stability ◽

Polypeptide Chain ◽

Bacteriophage T4 ◽

Residual Activity ◽

Chain Elongation ◽

Late Gene ◽

Dependent Manner ◽

Late Genes ◽

Dmd Gene

Abstract The dmd gene of bacteriophage T4 is required for regulation of mRNA stability in a stage-dependent manner during infection. When this gene is mutated, late genes are globally silenced because of rapid degradation of mRNAs. To investigate the mechanism of such mRNA degradation, we analyzed the late gene soc transcripts. The degradation of soc mRNA was remarkably stabilized when its ability to be translated was impaired; either disruption of translation initiation signals or elimination of termination codons was effective in stabilization of soc mRNA and removal of elongation modestly stabilized it. Even in the absence of translation, however, the residual activity was still significant. These results suggested that the degradation of soc transcripts was promoted by two different mechanisms; one is dependent on translation and the other independent of translation. We found several cleavages introduced into soc RNA specifically when the dmd gene was mutated; some of them could be linked to polypeptide chain elongation and termination, suggesting the correlation with ribosomal action, and the others were independent of translation.

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Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.

Journal of Bacteriology ◽

10.1128/jb.175.1.103-110.1993 ◽

1993 ◽

Vol 175 (1) ◽

pp. 103-110 ◽

Cited By ~ 20

Author(s):

K M Borges ◽

P L Bergquist

Keyword(s):

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

Restriction Map ◽

Thermus Thermophilus Hb8

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On the Characterization of the Putative S20-Thx Operon of Thermus thermophilus

Biological Chemistry ◽

10.1515/bc.2001.126 ◽

2001 ◽

Vol 382 (7) ◽

pp. 1001-1006 ◽

Cited By ~ 5

Author(s):

Fotini Leontiadou ◽

Dimitra Triantafillidou ◽

Theodora Choli-Papadopoulou

Keyword(s):

High Temperatures ◽

Genomic Dna ◽

Ribosomal Proteins ◽

Positive Charge ◽

Thermus Thermophilus ◽

Promoter Sequence ◽

30S Subunit ◽

Feature Characteristic ◽

Putative Operon

Abstract A putative operon of the ribosomal proteins S20 and Thx has been determined in a 1.4 kb sequenced region of T. thermophilus genomic DNA. Both genes have a promoter sequence 29 nt upstream of ORF1, possess their own ShineDalgarno motifs (GGAG) and are separated by only 9 nucleotides, a feature characteristic of the compact Thermus thermophilus genome. This is a novel arrangement, since Thx is unique to the Thermus bacteria and in all other prokaryotes the S20 gene is monocistronic. Our results, in conjunction with the recent finding that Thx is located on the top of the head of the 30S subunit in a cavity between multiple RNA elements stabilizing them with its positive charge, corroborate the observation that thermophilic ribosomes require constituents with special features for their stabilization at high temperatures.

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Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

Thrombosis and Haemostasis ◽

10.1160/th16-03-0223 ◽

2016 ◽

Vol 116 (12) ◽

pp. 1022-1031 ◽

Cited By ~ 8

Author(s):

Yuki Takagi ◽

Moe Murata ◽

Toshihiro Kozuka ◽

Yukiko Nakata ◽

Ryo Hasebe ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Residual Activity ◽

Base Substitution ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Missense Mutations ◽

Wild Type ◽

Gene Encoding ◽

Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

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