scholarly journals Cold Shock of a Hyperthermophilic Archaeon: Pyrococcus furiosus Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins

2005 ◽  
Vol 187 (1) ◽  
pp. 336-348 ◽  
Author(s):  
Michael V. Weinberg ◽  
Gerrit J. Schut ◽  
Scott Brehm ◽  
Susmita Datta ◽  
Michael W. W. Adams
Keyword(s):  
Growth Temperature ◽  
Cold Shock ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Optimal Growth ◽  
Cold Shock Protein ◽  
Hyperthermophilic Archaea ◽  
Lag Phase ◽  
Cold Response ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95°C, and at the lower end of the temperature range for significant growth, 72°C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72°C. This resulted in a 5-h lag phase, during which time little growth occurred. Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the P. furiosus genome showed that cells undergo three very different responses at 72°C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72°C). Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response. These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins. Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins. Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR. Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins. While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB. These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria. The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea.

scholarly journals Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

2000 ◽  
Vol 182 (9) ◽  
pp. 2559-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Servé W. M. Kengen ◽  
John van der Oost ◽  
Willem M. de Vos
Keyword(s):  
Molecular Mass ◽  
Alanine Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Northern Analysis ◽  
Transcriptional Level ◽  
Significant Activity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s−1mM−1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

scholarly journals Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coliK-12

1999 ◽  
Vol 181 (18) ◽  
pp. 5557-5562 ◽  
Author(s):  
T. Atlung ◽  
F. G. Hansen
Keyword(s):  
Low Temperatures ◽  
Generation Time ◽  
Protein Concentration ◽  
Cold Shock ◽  
Cold Shock Protein ◽  
Lag Phase ◽  
Dnaa Protein ◽  
Temperature Downshift ◽  
Cellular Dna ◽  
Low Activity

ABSTRACT Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14°C than at 37°C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30°C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.

scholarly journals Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic Archaeon Pyrococcus furiosus

2006 ◽  
Vol 189 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Lara S. Madding ◽  
Joshua K. Michel ◽  
Keith R. Shockley ◽  
Shannon B. Conners ◽  
Kevin L. Epting ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Pyrococcus Furiosus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Response Shift ◽  
Transcriptional Analysis ◽  
20S Proteasome ◽  
Significant Activity ◽  
Peptide Hydrolysis ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum)

1992 ◽  
Vol 70 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Randal W. Giroux ◽  
W. Gary Filion
Keyword(s):  
Cold Shock ◽  
Chilling Stress ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Cold Shock Protein ◽  
Relative Mass ◽  
Temperature Decrease ◽  
Cold Shock Response

The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [35S]methionine, both during a gradual temperature decrease (2 °C/day) and also during a rapid cold shock (4 °C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis and revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chloramphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance.Key words: cold-shock protein(s), tomato, chilling stress, acclimation, cold tolerance.

scholarly journals Characterization of a Thermostable DNA Glycosylase Specific for U/G and T/G Mismatches from the Hyperthermophilic ArchaeonPyrobaculum aerophilum

2000 ◽  
Vol 182 (5) ◽  
pp. 1272-1279 ◽  
Author(s):  
Hanjing Yang ◽  
Sorel Fitz-Gibbon ◽  
Edward M. Marcotte ◽  
Jennifer H. Tai ◽  
Elizabeth C. Hyman ◽  
...  
Keyword(s):  
Growth Temperature ◽  
Optimal Growth ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Optimal Growth Temperature ◽  
Sequence Comparisons ◽  
Hyperthermophilic Archaeon ◽  
Sequencing Project ◽  
A Genome

ABSTRACT U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeonMethanobacterium thermoautotrophicum, a thermophile with an optimal growth temperature of 65°C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeonPyrobaculum aerophilum, whose optimal growth temperature is 100°C. The open reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in Escherichia coli and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.

scholarly journals Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Mirko Basen ◽  
Junsong Sun ◽  
Michael W. W. Adams
Keyword(s):  
Protein Expression ◽  
Boiling Point ◽  
Cold Shock ◽  
Pyrococcus Furiosus ◽  
Biofuel Production ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Chemical Inducers ◽  
Moderately Thermophilic ◽  
End Products

ABSTRACT Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100°C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [T opt] of 78°C) controlled by a “cold shock” promoter that is upregulated when cells are transferred from 98°C to 72°C. At 98°C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72°C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation. IMPORTANCE Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100°C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100°C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter for protein expression used in this study is activated at suboptimal growth temperatures of P. furiosus, it is a powerful genetic tool for protein expression with minimal interference of the host’s metabolism and without the need for chemical inducers.

scholarly journals A non-cold-inducible cold shock protein homolog mainly contributes to translational control under optimal growth conditions

FEBS Journal ◽  
2012 ◽  
Vol 279 (6) ◽  
pp. 1014-1029 ◽  
Author(s):  
Toshiko Tanaka ◽  
Ryosuke Mega ◽  
Kwang Kim ◽  
Akeo Shinkai ◽  
Ryoji Masui ◽  
...  
Keyword(s):  
Translational Control ◽  
Cold Shock ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Cold Shock Protein

Study on the transgenic tobacco expressing truncated wheat cold shock protein gene TA3-13 and analysis of disease resistance

2011 ◽  
Vol 33 (5) ◽  
pp. 520-526 ◽  
Author(s):  
Na LI ◽  
Xiu-Zhen DU ◽  
Xiao-Mei PAN ◽  
Jin-Sheng WANG ◽  
Cong-Feng SONG
Keyword(s):  
Disease Resistance ◽  
Transgenic Tobacco ◽  
Cold Shock ◽  
Protein Gene ◽  
Cold Shock Protein

scholarly journals Characteristics of Phomopsis juglandina (Sacc.) Hohn. Associated with Dieback of Walnut in the Climatic Conditions of Southern Romania

Agronomy ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 46
Author(s):  
Cristina Mihaescu ◽  
Daniel Dunea ◽  
Adrian Gheorghe Bășa ◽  
Loredana Neagu Frasin
Keyword(s):  
Growth Temperature ◽  
Potential Evapotranspiration ◽  
Optimal Growth ◽  
Climatic Conditions ◽  
Cultural Characteristics ◽  
Optimal Growth Temperature ◽  
Conidial State ◽  
Optimal Ph

Phomopsis juglandina (Sacc.) Höhn., which is the conidial state of Diaporthe juglandina (Fuckel) Nitschke, and the main pathogen causing the dieback of branches and twigs of walnut was recently detected in many orchards from Romania. The symptomatological, morphological, ultrastructural, and cultural characteristics, as well as the pathogenicity of an isolate of this lignicolous fungus, were described and illustrated. The optimum periods for infection, under the conditions prevailing in Southern Romania, mainly occur in the spring (April) and autumn months (late September-beginning of October). Strong inverse correlations (p < 0.001) were found between potential evapotranspiration and lesion lengths on walnut branches in 2019. The pathogen forms two types of phialospores: alpha and beta; the role of beta phialospores is not well known in pathogenesis. In Vitro, the optimal growth temperature of mycelial hyphae was in the range of 22–26 °C, and the optimal pH is 4.4–7. This pathogen should be monitored continuously due to its potential for damaging infestations of intensive plantations.

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