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SurR: a transcriptional activator and repressor controlling hydrogen and elemental sulphur metabolism in Pyrococcus furiosus.

ABSTRACT: This work describes the identification and characterization of SurR, Pyrococcus furiosus sulphur (S(0)) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is downregulated in the primary response of P. furiosus to S(0), as revealed by DNA microarray experiments. SurR was validated as a sequence-specific DNA binding protein, and characterization of the SurR DNA binding motif GTTn(3)AAC led to the identification of several target genes that contain an extended motif in their promoters. A number of these were validated to contain upstream SurR binding sites. These SurR targets strongly correspond with open reading frames and operons both up- and downregulated in the primary response to S(0). In vitro transcription revealed that SurR is an activator for its own gene as well as for two hydrogenase operons whose expression is downregulated during the primary S(0) response; it is also a repressor for two genes upregulated during the primary S(0) response, one of which encodes the primary S(0)-reducing enzyme NAD(P)H sulphur reductase. Herein we give evidence for the role of SurR in both mediating the primary response to S(0) and controlling hydrogen production in P. furiosus.

SUBMITTER: Lipscomb GL

PROVIDER: S-EPMC2745277 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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SurR: a transcriptional activator and repressor controlling hydrogen and elemental sulphur metabolism in Pyrococcus furiosus.

Lipscomb Gina L GL Keese Annette M AM Cowart Darin M DM Schut Gerrit J GJ Thomm Michael M Adams Michael W W MW Adams Michael W W MW Scott Robert A RA

Molecular microbiology 20081110 2

This work describes the identification and characterization of SurR, Pyrococcus furiosus sulphur (S(0)) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is downregulated in the primary response of P. furiosus to S(0), as revealed by DNA microarray experiments. SurR was validated as a sequence-specific DNA binding protein, and characterization of the SurR DNA binding motif GTTn(3)AAC led to the identification of several target genes that cont ...[more]

PMID: 19017274

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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

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 100Â°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 â 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. Pyrococcus furiosus (DSM 3638) was grown at 95Â°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 Â´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4Â°C. Total RNA was extracted using acid-phenol and stored at -80Â°C until needed. A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.

Dataset Information

SurR: a transcriptional activator and repressor controlling hydrogen and elemental sulphur metabolism in Pyrococcus furiosus.

Publications

SurR: a transcriptional activator and repressor controlling hydrogen and elemental sulphur metabolism in Pyrococcus furiosus.

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