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Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.

ABSTRACT: Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S(0)): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (?pyrF). The growth of all three mutants on maltose was comparable without S(0), but in its presence, the growth of MBX1 was greatly impaired while the growth of NSR1 and SIP1 was largely unaffected. In the presence of S(0), MBX1 produced little, if any, sulfide but much more acetate (per unit of protein) than the parent strain, demonstrating that MBX plays a critical role in S(0) reduction and energy conservation. In contrast, comparable amounts of sulfide and acetate were produced by NSR1 and the parent strain, indicating that NSR is not essential for energy conservation during S(0) reduction. Differences in transcriptional responses to S(0) in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S(0) reduction system. Genes controlled by the S(0)-responsive regulator SurR were not as highly regulated in MBX1 and NSR1. SIP1 produced the same amount of acetate but more sulfide than the parent strain. That SipA is not essential for growth on S(0) indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S(0) reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.

SUBMITTER: Bridger SL

PROVIDER: S-EPMC3232869 | biostudies-other | 2011 Dec

REPOSITORIES: biostudies-other

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Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.

Bridger Stephanie L SL Clarkson Sonya M SM Stirrett Karen K DeBarry Megan B MB Lipscomb Gina L GL Schut Gerrit J GJ Westpheling Janet J Scott Robert A RA Adams Michael W W MW Adams Michael W W MW

Journal of bacteriology 20110930 23

Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S(0)): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (ΔpyrF) ...[more]

PMID: 21965560

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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:The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S(0) is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H(2)S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S(0) reduction activity (0.7 units/mg, 85 degrees 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 (M(r), 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding 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 sevenfold within 10 min of S(0) addition. This primary response to S(0) also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to 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(0) 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(0). A secondary response to S(0) is observed 30 min after S(0) 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(0)-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S(0)-reducing system in autotrophic archaea and bacteria.

Dataset Information

Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.

Publications

Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.

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