Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.
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ABSTRACT:
SUBMITTER: Heltzel A
PROVIDER: S-EPMC196307 | biostudies-other | 1994 Aug
REPOSITORIES: biostudies-other
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Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.
Heltzel A A Smith E T ET Zhou Z H ZH Blamey J M JM Adams M W MW
Journal of bacteriology 19940801 15
The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic ...[more]
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Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs.
2010-02-25 | GSE20470 | GEO
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2010-05-14 | E-GEOD-20470 | biostudies-arrayexpress
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Project description:Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.
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Project description:Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-alpha-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of alpha-glucan substrates by P. furiosus.
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Project description:Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A-Dependent NAD(P)H Sulfur Oxidoreductase The hyperthermophilic archaeon Pyrococcus furiosus, uses carbohydrates as a carbon source and produces acetate, CO2 and H2 as end products. When S° is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After one hour cells contain high NADPH- and coenzyme A-dependent S° reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (Mr 100 kDa) and is encoded by PF1186. This was previously assigned to an enzyme that reduces coenzyme A disulfide, which is a side-reaction of NSR. Whole genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to 7-fold within 10 min of S° addition. This primary response to S° also involves the up-regulation (> 16-fold) of a 13 gene cluster encoding a membrane-bound oxidoreductase (MBX). MBX is proposed replace the homologous 14 gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S° addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S°. A secondary response to S° is observed 30 min after S° addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins, termed SipA and SipB. This novel S°-reducing system involving NSR and MBX is so far unique to the heterotrophic Thermococcales, and is in contrast to the cytochrome- and quinone-based S°-reducing system in autotrophic archaea and bacteria. Keywords: time course, kinetic, sulfur metabolism, archaea, Pyrococcus furiosus, hyperthermophile
2007-05-25 | GSE7022 | GEO