Genetic analyses of the functions of [NiFe]-hydrogenase maturation endopeptidases in the hyperthermophilic archaeon Thermococcus kodakarensis

The maturation of [NiFe]-hydrogenases requires a number of accessory proteins, which include hydrogenase-specific endopeptidases. The endopeptidases carry out the final cleavage reaction of the C-terminal regions of [NiFe]-hydrogenase large subunit precursors. The hyperthermophilic archaeon Thermococcus kodakarensis harbors two [NiFe]-hydrogenases, a cytoplasmic Hyh and a membrane-bound Mbh, along with two putative hydrogenase-specific endopeptidase genes. In this study, we carried out a genetic examination on the two endopeptidase genes, TK2004 and TK2066. Disruption of TK2004 resulted in a strain that could not grow under conditions requiring hydrogen evolution. The Mbh large subunit precursor (pre-MbhL) in this strain was not processed at all whereas Hyh cleavage was not affected. On the other hand, disruption of TK2066 did not affect the growth of T. kodakarensis under the conditions examined. Cleavage of the Hyh large subunit precursor (pre-HyhL) was impaired, but could be observed to some extent. In a strain lacking both TK2004 and TK2066, cleavage of pre-HyhL could not be observed. Our results indicate that pre-MbhL cleavage is carried out solely by the endopeptidase encoded by TK2004. Pre-HyhL cleavage is mainly carried out by TK2066, but TK2004 can also play a minor role in this cleavage.

     

Distinct physiological roles of the three [NiFe]-hydrogenase orthologs in the hyperthermophilic archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H₂) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S⁰) or H₂ generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H₂S- and H₂-evolving conditions. H₂S generation in PHY1 was equivalent to that of the host strain, and H₂ generation was higher in PHY1, suggesting that Hyh functions in the direction of H₂ uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H₂-evolving conditions. This fact, as well as the impaired H₂ generation activity in MHD1, indicated that Mbh is mainly responsible for H₂ evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H₂ transfer (i.e., H₂ evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H₂S-evolving conditions and exhibited a lower H₂S generation activity, indicating the involvement of Mbx in the S⁰ reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.     

Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H₂ in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H₂ during growth without S°. Growth rates and steady‐state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H_2(aq). Differential gene expression analysis using RNA‐Seq showed consistent expression of six hydrogenase operons with and without added H₂. In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H₂. Flux balance analysis showed H₂ oxidation and formate production using FHL became an alternate route for electron disposal during H₂ inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H₂ production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H₂ efflux causes growth inhibition.     

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S⁰) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H₂S from S⁰ and free CoA–SH was observed, and the addition of enzyme and NADPH enhanced H₂S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA–SH acted to solubilize S⁰ by forming CoA persulfides, followed by reduction of an enzyme–S–S–CoA intermediate produced after both enzymatic and non-enzymatic evolution of H₂S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA–SH, implying a direct reaction with S⁰. TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA–SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S⁰-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S⁰, suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S⁰ was absent.     

Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α₂β₂ structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS III_Tk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICS_Tk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICS_Tk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

Phytoene (C₄₀H₆₄) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L⁻¹ medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

     

Thermococcus kodakarensis Genetics: TK1827-Encoded β-Glycosidase,New Positive-Selection Protocol,and Targeted and Repetitive Deletion Technology

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.Members of the Thermococcales, hyperthermophilic Euryarchaea, grow readily under laboratory conditions and are the focus of many basic and applied research projects (2, 8). Their investigation has, however, been limited by the lack of genetics, and it was a seminal advance therefore when Thermococcus kodakarensis (formerly Thermococcus kodakaraensis [1]) was shown to be naturally competent and to recombine added DNA into its genome (21, 23). T. kodakarensis is attractive as an experimental system as a fermentative heterotroph that grows rapidly on a variety of different substrates (1), optimally at 85°C, including substrates from which it generates substantial levels of hydrogen (11). The T. kodakarensis genome sequence and genome microarray assays are established (7, 12, 18) and, since the discovery of transformation (21), inactivation and manipulation of chromosomal genes has revealed novel biochemical pathways, facilitated in vivo evaluations of the archaeal gene expression machinery, and simplified enzyme purifications (3, 5, 6, 9, 10, 12, 15-20, 22-24, 28). For complementation assays and to facilitate heterologous gene expression in T. kodakarensis, shuttle plasmids have been constructed that replicate and confer selectable phenotypes in both T. kodakarensis and Escherichia coli (19), and TK1761 expression has been established as a reporter system that can be used to identify and quantify regulatory elements in T. kodakarensis (18, 20).During the development of the TK1761 reporter system, a T. kodakarensis strain, designated TS416, was constructed with a nonsense mutation in TK1761 (18). This mutation had no discernible effects on growth, confirming that TK1761 was not an essential gene, but lysates of T. kodakarensis TS416 retained a low level β-glycosidase activity. T. kodakarensis apparently, therefore, had a second β-glycosidase, but since this activity was very low and remained constant with changes in TK1761 expression, its presence did not compromise the use of TK1761 expression as a reporter system in the laboratory media used. The existence of a second β-glycosidase did, nevertheless, raise a potential concern for TK1761 assays in cells grown under different conditions in which expression of the gene encoding this second β-glycosidase might not be constant. To address this, we purified and characterized the second β-glycosidase and, to eliminate the concern for the reporter assay, we constructed a T. kodakarensis strain with both TK1761 and the gene (TK1827) that encodes the second β-glycosidase deleted.To construct the double-deletion strain, we developed a new selection/counterselection protocol and have extended this into a procedure that can be used to delete any nonessential gene from the T. kodakarensis genome. A two-gene cassette has been constructed that can be integrated into the T. kodakarensis chromosome at any desired locus by homologous recombination of flanking genes. Expression of the cassette provides a positive selection for transformants and confers sensitivity to 6-methyl purine (6MP^s). Mutants then isolated that are spontaneously resistant to 6-methyl purine (6MP^r) have both the cassette and the adjacent target gene(s) precisely deleted.Sato et al. (21, 23) established the T. kodakarensis transformation protocol by selecting transformants of tryptophan (trpE) and uracil (pyrF) auxotrophs that grew on minimal medium without tryptophan or uracil. Overexpression of the hydroxy-methylglutaryl-coenzyme A reductase encoded by PF1848, cloned from Pyrococcus furiosus, was later found to confer to resistance to simvastatin (15) and mevinolin (19), allowing the selection of T. kodakarensis transformants on rich media that contain either antibiotic. Mutants spontaneously resistant to these antibiotics do, however, occur at experimentally significant frequencies, and these are very expensive reagents for routine use and prohibitively expensive for incorporation into large preparative cultures. With this in mind, to develop an alternative selection that might be used in nutrient-rich media, we used the 6MP cassette system to delete TK0149. As predicted (6), the T. kodakarensis ΔTK0149 strain generated was an agmatine auxotroph that only grows in nutrient-rich media when agmatine is added. When transformed with DNA expressing TK0149, transformants of this strain can be selected directly on standard nutrient-rich media, and complementation of the ΔTK0149 mutation can be used to maintain the presence of an expression plasmid in T. kodakarensis cells grown in large-volume, rich-medium cultures for enzyme purification.     

Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.     

Survivin mutant protects differentiated dopaminergic SK-N-SH cells against oxidative stress

Oxidative stress is due to an imbalance of antioxidant/pro-oxidant homeostasis and is associated with the progression of several neurological diseases, including Parkinson''s and Alzheimer''s disease and amyotrophic lateral sclerosis. Furthermore, oxidative stress is responsible for the neuronal loss and dysfunction associated with disease pathogenesis. Survivin is a member of the inhibitors of the apoptosis (IAP) family of proteins, but its neuroprotective effects have not been studied. Here, we demonstrate that SurR9-C84A, a survivin mutant, has neuroprotective effects against H₂O₂-induced neurotoxicity. Our results show that H₂O₂ toxicity is associated with an increase in cell death, mitochondrial membrane depolarisation, and the expression of cyclin D1 and caspases 9 and 3. In addition, pre-treatment with SurR9-C84A reduces cell death by decreasing both the level of mitochondrial depolarisation and the expression of cyclin D1 and caspases 9 and 3. We further show that SurR9-C84A increases the antioxidant activity of GSH-peroxidase and catalase, and effectively counteracts oxidant activity following exposure to H₂O₂. These results suggest for the first time that SurR9-C84A is a promising treatment to protect neuronal cells against H₂O₂-induced neurotoxicity.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.

     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.     

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H₂-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H₂ uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH₃, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.     

Photosynthesis in the Archean Era

The earliest reductant for photosynthesis may have been H₂. The carbon isotope composition measured in graphite from the 3.8-Ga Isua Supercrustal Belt in Greenland is attributed to H₂-driven photosynthesis, rather than to oxygenic photosynthesis as there would have been no evolutionary pressure for oxygenic photosynthesis in the presence of H₂. Anoxygenic photosynthesis may also be responsible for the filamentous mats found in the 3.4-Ga Buck Reef Chert in South Africa. Another early reductant was probably H₂S. Eventually the supply of H₂ in the atmosphere was likely to have been attenuated by the production of CH₄ by methanogens, and the supply of H₂S was likely to have been restricted to special environments near volcanos. Evaporites, possible stromatolites, and possible microfossils found in the 3.5-Ga Warrawoona Megasequence in Australia are attributed to sulfur-driven photosynthesis. Proteobacteria and protocyanobacteria are assumed to have evolved to use ferrous iron as reductant sometime around 3.0 Ga or earlier. This type of photosynthesis could have produced banded iron formations similar to those produced by oxygenic photosynthesis. Microfossils, stromatolites, and chemical biomarkers in Australia and South Africa show that cyanobacteria containing chlorophyll a and carrying out oxygenic photosynthesis appeared by 2.8 Ga, but the oxygen level in the atmosphere did not begin to increase until about 2.3 Ga.     

Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon

Many genomes of anaerobic hyperthermophiles encode multiple homologs of NAD(P)H oxidase that are thought to function in response to oxidative stress. We investigated one of the seven NAD(P)H oxidase homologs (TK1481) in the sulfur-reducing hyperthermophilic archaeon Thermococcus kodakarensis, focusing on the catalytic properties and roles in oxidative-stress defense and sulfur-dependent energy conservation. The recombinant form of TK1481 exhibited both NAD(P)H oxidase and NAD(P)H:polysulfide oxidoreductase activities. The enzyme also possessed low NAD(P)H peroxidase and NAD(P)H:elemental sulfur oxidoreductase activities under anaerobic conditions. A mutant form of the enzyme, in which the putative redox-active residue Cys43 was replaced by Ala, still showed NADH-dependent flavin adenine dinucleotide (FAD) reduction activity. Although it also retained successive oxidase and anaerobic peroxidase activities, the ability to reduce polysulfide and sulfur was completely lost, suggesting the specific reactivity of the Cys43 residue for sulfur. To evaluate the physiological function of TK1481, we constructed a gene deletant, ΔTK1481, and mutant KUTK1481C43A, into which two base mutations altering Cys43 of TK1481 to Ala were introduced. ΔTK1481 exhibited growth properties nearly identical to those of the parent strain, KU216, in sulfur-containing media. Interestingly, in the absence of elemental sulfur, the growth of ΔTK1481 was not affected by dissolved oxygen, whereas the growth of KU216 and KUTK1481C43A was significantly impaired. These results indicate that although TK1481 does not play a critical role in either sulfur reduction or the response to oxidative stress, the NAD(P)H oxidase activity of TK1481 unexpectedly participates in the oxygen sensitivity of the hyperthermophilic archaeon T. kodakarensis in the absence of sulfur.Recent genetic analyses have determined that hyperthermophiles, which are capable of growing at 90°C and above, occupy the deep branches closest to the root within phylogenetic trees. This suggests that the study of hyperthermophiles will provide valuable perspectives on the evolution of biological systems essential for living organisms. Interestingly, although many hyperthermophiles are strict anaerobes, they are equipped with several systems for responding to oxidative stress. They often employ superoxide reductase, instead of the more common superoxide dismutase, for the removal of the highly toxic superoxide anion (13). It has been proposed that in Pyrococcus furiosus, the reduction of the superoxide anion to H₂O₂ by superoxide reductase is linked to the oxidoreduction of rubredoxin by NAD(P)H:rubredoxin oxidoreductase (10). Although the mechanism used for the removal of the moderately toxic stressor H₂O₂ by hyperthermophiles is unclear, Dps-like protein (27) and rubrerythrin (39), both of which contain nonheme iron, exhibit peroxidase activity and may serve this role. The osmotically inducible protein C (OsmC) identified in Thermococcus kodakarensis also shows peroxidase activity in the presence of reducing agents in vitro (25).Detailed genome surveys have also revealed the presence of multiple NAD(P)H oxidases (NOXs) in a wide range of anaerobic hyperthermophiles, including Thermococcus (14) and Pyrococcus spp. (38), Archaeoglobus fulgidus (17, 28), Methanocaldococcus jannaschii (4), and Thermotoga spp. (41, 42). NOXs catalyze the 2-electron reduction of O₂ to H₂O₂ or the 4-electron reduction of O₂ to H₂O, and they contain a highly conserved domain containing flavin adenine dinucleotide (FAD)-binding and NAD(P)H-binding sites (7). They are members of the pyridine nucleotide disulfide oxidoreductase (PNDOR) class of enzymes, which are, in turn, included in the GR₁ subfamily of the glutathione reductase structural family (1). PNDORs are divided into three groups; NOXs belong to group 3, together with NADH peroxidases and coenzyme A (CoA) disulfide reductases (CoADRs).The reaction mechanisms of a number of group 3 PNDORs have been investigated in detail. For example, the H₂O-forming NOX from the mesophilic organism Lactobacillus sanfranciscensis uses the first equivalent of NAD(P)H to form an enzyme-FADH₂ complex that reacts with O₂ to generate H₂O₂ (18). The H₂O₂ molecule further reacts with a redox-active cysteine via a peroxyflavin intermediate to form cysteine-sulfenic acid (Cys-S-OH) and one water molecule. The second equivalent of NAD(P)H is then used to reduce the sulfenic acid to thiolate via FADH₂, a process accompanied by the production of a second water molecule. The cysteine-sulfenic acid redox center is also important in reactions catalyzed by NADH peroxidases (26). Likewise, the active Cys43 residue in the CoADR from Staphylococcus aureus forms a Cys43-S-SCoA redox center during turnover (23). The NOX homologs in hyperthermophiles are considered to play important roles in the defense against oxidative stress (4, 14, 17, 28, 38, 41, 42). However, the actual physiological functions of NOXs in anaerobic hyperthermophiles have not yet been completely elucidated, mainly due to a lack of useful genetic manipulation systems for these organisms.Members of the genera Pyrococcus and Thermococcus, both of which belong to the hyperthermophilic archaeal order Thermococcales, are obligate heterotrophs that generally prefer proteinaceous compounds as carbon and energy sources and whose growth is strongly associated with the reduction of elemental sulfur (S⁰) to H₂S. Interestingly, unlike the genomes of mesophilic sulfur-reducing bacteria, such as Wolinella succinogenes (6), the available genomes of these hyperthermophiles do not contain any homologs related to S⁰-reducing respiratory systems, such as molybdenum-containing sulfur reductases, quinones, and cytochromes. In Pyrococcus furiosus, cytosolic hydrogenases (21) and a sulfide dehydrogenase (19) were initially reported to exhibit S⁰-reducing activity in vitro. However, the former enzymes are now thought to function in the recycling of H₂ generated by membrane-bound hydrogenase, while the latter has been recharacterized as ferredoxin:NADP oxidoreductase (20). Recently, Schut et al. shed light on the mysterious mechanism of sulfur reduction in the order Thermococcales (34). They proposed that a unique S⁰-dependent energy conservation system operates in P. furiosus, in which membrane-bound hydrogenase-related membrane-bound oxidoreductase first generates a proton motive force by proton pumping via electron transfer from reduced ferredoxin to NAD(P)⁺. CoA-dependent NAD(P)H:elemental sulfur oxidoreductase then reoxidizes NAD(P)H with S⁰ as a terminal electron acceptor. It should be noted that NAD(P)H:elemental sulfur oxidoreductase, which is encoded by PF1186, is one of the NOX homologs in P. furiosus, suggesting that the energetic reduction of sulfur and the defensive reduction of oxygen may share similar catalytic mechanisms mediated by NOX homologs in this order of hyperthermophiles.T. kodakarensis KOD1 (2) is a useful model hyperthermophile, because its whole genome has been sequenced and annotated (8), and a practical genetic manipulation system, the first for hyperthermophiles, has been developed for this archaeon (24, 32, 33). T. kodakarensis exhibits metabolic and energy-conserving mechanisms similar to those of P. furiosus. For example, although T. kodakarensis prefers S⁰ as a terminal electron acceptor, it is able to couple growth with the concomitant reduction of protons to H₂ when either pyruvate or starch is available in the absence of S⁰. One of the major distinctions between T. kodakarensis and P. furiosus is the preferred growth temperature: the former displays a lower and wider range of growth temperatures (60 to 100°C) than the latter (70 to 103°C). Previous comparative-genomic studies of T. kodakarensis and three Pyrococcus spp. have revealed that they share 1,204 proteins, including those involved in information processing and basic metabolism. However, 689 proteins were unique to T. kodakarensis; these are likely responsible for the specific traits displayed by this species and other members of the genus Thermococcus (8). For example, the unique presence of methionine sulfoxide reductase, an enzyme that repairs oxidized proteins, has been suggested to facilitate growth in low-temperature environments, which often contain increased dissolved-oxygen concentrations (9).In this study, we focused on a NOX homolog in T. kodakarensis, TK1481, for which no homologous counterpart was present in Pyrococcus spp. The recombinant protein was purified and characterized in terms of its ability to reduce oxygen and sulfur. In addition, we examined its physiological role by constructing a site-directed mutant of the protein and a gene disruption mutant of T. kodakarensis.