Gerrit J. Schut

The unique features of glycolytic pathways in Archaea

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon: Pyrococcus furiosus Grown on Carbohydrates or Peptides

The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of Pyrococcus furiosus (optimal growth temperature, 100 degrees C). This was used to determine relative transcript levels in cells grown at 95 degrees C with either peptides or a carbohydrate (maltose) used as the primary carbon source. Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition. Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation. Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins. A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products. A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation. Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides. In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold--three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis. The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data. The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that P. furiosus can readily adapt to a change in its primary carbon source.

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(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.

Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

ABSTRACT In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.

(FeFe)- and (NiFe)-hydrogenase diversity, mechanism, and maturation

The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus: Evidence for a New Type of Sulfur-Reducing Enzyme Complex

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

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

The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95 degrees C, and at the lower end of the temperature range for significant growth, 72 degrees C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72 degrees 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 degrees C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72 degrees 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.

The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications.

Hydrogen production is a vital metabolic process for many anaerobic organisms, and the enzyme responsible, hydrogenase, has been studied since the 1930s. A novel subfamily with unique properties was recently recognized, represented by the 14-subunit membrane-bound [NiFe] hydrogenase from the archaeon Pyrococcus furiosus. This so-called energy-converting hydrogenase links the thermodynamically favorable oxidation of ferredoxin with the formation of hydrogen and conserves energy in the form of an ion gradient. It is therefore a simple respiratory system within a single complex. This hydrogenase shows a modular composition represented by a Na(+)/H(+) antiporter domain (Mrp) and a [NiFe] hydrogenase domain (Mbh). An analysis of the large number of microbial genome sequences available shows that homologs of Mbh and Mrp tend to be clustered within the genomes of a limited number of archaeal and bacterial species. In several instances, additional genes are associated with the Mbh and Mrp gene clusters that encode proteins that catalyze the oxidation of formate, CO or NAD(P)H. The Mbh complex also shows extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family. The respiratory-type membrane-bound hydrogenase complex appears to be closely related to the common ancestor of complex I and [NiFe] hydrogenases in general.