Regulation of the aerobic respiratory chain in the facultatively aerobic and hyperthermophilic archaeon Pyrobaculum oguniense

Takuro Nunoura1,{dagger}, Yoshihiko Sako1, Takayoshi Wakagi2 and Aritsune Uchida1

1 Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2 Department of Biotechnology, Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo 113-8657, Japan

Correspondence
Takuro Nunoura
takuron{at}jamstec.go.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The aerobic respiratory chain of Pyrobaculum oguniense is expressed constitutively even under anaerobic conditions. The membranes of both aerobically and anaerobically grown cells show oxygen consumption activity with NADH as substrate, bovine cytochrome c oxidase activity and TMPD oxidase activity. Spectroscopic analysis and haem analysis of membranes of aerobically grown cells show the presence of cytochrome b559, cytochrome c551 and haem Op1 containing cytochrome c oxidase in aerobically and anaerobically grown cells, and haem As containing cytochrome c oxidase in aerobically grown cells. The gene clusters of SoxB-type and SoxM-type haem copper oxidase and cytochrome bc complex have been cloned and sequenced and the regulation of these genes was analysed. The Northern blot analysis indicated that the constitutive transcription of the gene cluster of SoxB-type haem-copper oxidase and cytochrome bc complex is observed under both aerobic and anaerobic conditions, and the transcription of the operon of SoxM-type haem-copper oxidase was stimulated under aerobic conditions. Furthermore, the presence of the binding residues for CuA in subunit II of both SoxB- and SoxM-type haem-copper oxidase suggests that these haem-copper oxidases are cytochrome c oxidases.


Abbreviations: MEGA9, MEGA10 n-nonanoyl- n-decanoyl-N-methylglucamide; TMBZ, 3,3',5,5'-tetramethylbenzidine; TMPD, N,N,N',N'-tetramethyl-1,4-phenylendiamine

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB056510 and AB056511.

{dagger}Present address: Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Marine Science and Technology Center, Yokosuka 237-0061, Japan.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An aerobic respiratory chain, especially its terminal oxidase, is indispensable for aerobic organisms. Most terminal oxidases belong to the haem-copper oxidase family; a few of them belong to the cyanide-insensitive alternative oxidase group (Garcia-Horsman et al., 1994). Phylogenetic and biochemical analysis of haem-copper oxidases indicates that the haem-copper oxidase family can be divided into three groups: FixN-, SoxB- and SoxM-type (Castresana et al., 1994; Garcia-Horsman et al., 1994). Mitochondria have one SoxM-type terminal oxidase but a number of bacteria and archaea have multiple terminal oxidases and the regulation of multiple oxidases is the important factor in their adaptation to changes in oxygen tension in their habitat. In general, FixN-type haem-copper oxidase is utilized for low oxygen tension (Castresana et al., 1994) and SoxM-type haem-copper oxidase for atmospheric oxygen tension. SoxB-type haem-copper oxidases are observed in extremely thermophilic or hyperthermophilic bacteria and archaea, and in halophilic archaea, but their general character is still in question.

The regulation of terminal oxidases in mesophilic bacteria such as Paracoccus denitrificans, Escherichia coli and Rhodobacter sphaeroides has been well analysed. In these studies, additional functions of terminal oxidases were observed in a few species of bacteria. For example, Paracoccus denitrificans has three terminal oxidases: cbb3-type cytochrome c oxidase (FixN type) is expressed at a low oxygen concentration, aa3-type cytochrome c oxidase (SoxM type) is expressed at atmospheric oxygen concentration, and the pathway to ba3-type quinol oxidase (SoxM type) becomes active when the reduction state of the Q-pool increases (Otten et al., 1999, 2001). E. coli has a bo3-type quinol oxidase (SoxM-type) that is utilized for atmospheric oxygen concentrations and a bd-type quinol oxidase (alternative oxidase) that is utilized for limited oxygen tension (Kita et al., 1984a, b). Furthermore, the terminal oxidases protect cells from oxidative stress in the form of H2O2 and paraquat (Lindqvist et al., 2000; Skulachev, 1994; Wall et al., 1992). Rhodobacter sphaeroides has an aa3-type cytochrome c oxidase (SoxM-type) for atmospheric oxygen concentration and a cbb3-type cytochrome c oxidase (FixN type) for limited oxygen tension. In addition, cbb3-type cytochrome c oxidase is expressed under anaerobic conditions as the oxygen sensor (O'Gara et al., 1998; Oh & Kaplan, 1999; Shapleigh et al., 1992). The extremely thermophilic bacterium Thermus thermophilus has a caa3-type (SoxM-type) terminal oxidase for high oxygen concentrations and a ba3-type terminal oxidase (SoxB-type) for microaerobic conditions (Fee et al., 1993; Mather et al., 1993; Keightley et al., 1995).

The terminal oxidases of hyperthermophilic archaea have only been analysed biochemically for Aeropyrum pernix and three Sulfolobales species: Sulfolobus acidocaldarius, Sulfolobus tokodaii and Acidianus ambivalens (Ishikawa et al., 2002; Anemüller & Schäfer, 1990; Lübben et al., 1994a, b; Iwasaki et al., 1995a, b; Wakagi et al., 1989; Anemüller et al., 1994). In addition, the genome sequences of Sulfolobus solfataricus, S. tokodaii and Pyrobaculum aerophilum suggest the scheme of an aerobic respiratory chain (Kawarabayasi et al., 2001; She et al., 2001; Fitz-Gibbon et al., 1997, 2002). By these analyses, these hyperthermophilic archaea, except for A. ambivalens, which has one aa3-type quinol oxidase, have multiple haem-copper oxidases as terminal oxidase. However, to our knowledge the regulation of the terminal oxidase of hyperthermophilic archaea has not previously been studied.

Pyrobaculum oguniense is one of the most hyperthermophilic organisms that can grow under both atmospheric air and anaerobic conditions. Under anaerobic conditions, this organism grows with elemental sulfur, thiosulfate, glutathione (oxidized form) or L-cystine as terminal electron acceptor (Sako et al., 2001). From the aspect of respiration, the genus Pyrobaculum is uniquely variable among the hyperthermophilic archaea, which may be closest to the common ancestor of all organisms; P. aerophilum is not only a microaerophile but also a nitrate, nitrite, thiosulfate, arsenate and selenite reducer (Völkl et al., 1993; Huber et al., 2000); Pyrobaculum islandicum reduces sulfur and iron(II) compounds (Huber et al., 1987; Kashefi & Lovely, 2000; Vargas et al., 1998); Pyrobaculum organotrophum reduces sulfur compounds (Huber et al., 1987); Thermoproteus neutrophilus, which is classified in the genus Thermoproteus although the phylogenetic analysis of 16S rDNA indicated that it should be reclassified as a Pyrobaculum species (Ito et al., 1998), is a sulfur reducer (Fischer et al., 1983); Pyrobaculum arsenaticum is arsenite reducer (Huber et al., 2000). This diversity of respiration in close relatives suggests that this genus may show a good example of the evolution of respiration and also show the nature of adaptation to the aerobic environment. In this study, we analysed the elements of the aerobic respiratory chain of P. oguniense in the membranes of both aerobically and anaerobically grown cells. Furthermore, we cloned and sequenced the genes of the aerobic respiratory chain and investigated the regulation of these genes by Northern blot analysis.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strain and growth conditions.
P. oguniense (JCM10595T) was grown in MS medium at 90 °C and pH 7·0–7·5 as described previously (Sako et al., 2001). Aerobic batch cultures were grown in cotton-plugged 2 l Erlenmeyer flasks containing 1 l medium in an air bath rotary shaker (RGS-32.TT; Sanki Seiki, Osaka, Japan) with vigorous rotary shaking (100 r.p.m.). Anaerobic growth conditions were achieved by the technique of Balch et al. (1979). Autoclaved MS medium (500 ml) in a 1 l glass bottle (Schott Glaswerke, Germany) with a butyl rubber plug was reduced by 0·05 % (w/v) Na2S.9H2O and pressurized by N2 (100 %, 200 kPa). Cells were harvested at late exponential growth phase and stored at -80 °C.

Membrane preparation.
Cells were suspended in 20 mM phosphate buffer (pH 7·3) and disrupted with a French press (Otake Works, Tokyo) at 1500 kg cm-2 five times. After 15 min centrifugation at 3000 g, the resulting supernatant was ultracentrifuged at 40 000 r.p.m. in a TET 80.13 rotor (Kontron) for 20 min. The membrane pellets were washed twice with 0·1 M phosphate buffer (pH 7·3) and resuspended in the same buffer.

Catalytic activity.
Oxygen consumption by the respiratory chain in the membranes of P. oguniense was monitored polarographically using NADH and succinate as the reductant. The assay was performed in 20 mM sodium phosphate buffer (pH 7·0) at 50 °C with a Clark-type oxygen electrode (Oxygen analyser MP-1000, Iijima Products, Tokyo, Japan). The membrane suspensions (final concentration 100 µg ml-1) were added to the cuvette and then NADH or succinate was added to a final concentration of 1 mM.

TMPD (N,N,N',N'-tetramethyl-1,4-phenylendiamine) oxidase activity was measured spectrophotometrically at various temperatures by oxidation of TMPD as substrate in 20 mM Tris/HCl buffer (pH 6·9) using a UV-240 spectrophotometer (Shimadzu, Japan) at 560 nm (Schäfer et al., 2001). Cytochrome c oxidase activity was measured spectrophotometrically by oxidation of bovine heart cytochrome c (Sigma) that was reduced by dithionite at 40 °C (Wakagi & Oshima, 1986).

Spectroscopic methods.
Optical spectra of membranes were determined at room temperature with a JASCO V-560 spectrophotometer. The samples (250 µg protein ml-1) were scanned (1) under air-oxidized conditions, (2) after addition of a few grains of sodium dithionite (reduced), and (3) after gassing the reduced sample with carbon monoxide (CO/reduced). The data were stored for calculation of the reduced minus oxidized and CO/reduced minus reduced difference spectra.

The succinate- or NADH-dependent reduction behaviour of cytochromes of the aerobic respiratory chain in the 1·5 % MEGA9 (n-nonanoyl-N-methylglucamide) and MEGA10 (n-decanoyl-N-methylglucamide) extracts of membranes (250 µg protein ml-1) with or without inhibitors (cyanide, azide and antimycin A) was determined at room temperature with a Shimadzu UV 160A spectrophotometer. The MEGA extracts of membranes were scanned (1) under air-oxidized conditions with or without inhibitors, and (2) after addition of 20 mM NADH or succinate, and incubated at 50 °C for 10 min (Iwasaki et al., 1995a).

Pyridine haemochrome spectra were determined as described by Berry & Trumpower (1987). Membrane suspensions (200 µl) were added to stock solution (200 µl) containing 200 mM NaOH, 40 % (v/v) pyridine and 60 µM K3Fe(CN)6 and the samples were scanned. After addition of a few grains of sodium dithionite, the reduced pyridine haemochrome spectra were recorded (Berry & Trumpower, 1987).

Haem extraction and HPLC analysis.
Non-covalently bound haems were extracted from 50 µl of membrane suspensions (0·5–1 mg protein) with 450 µl acetone/HCl (19 : 1, v/v) at room temperature for 20 min. After centrifugation for 5 min at 18 000 g, 1·0 ml ice-cold water and 0·3 ml ethyl acetate were added to the supernatant, and the sample was vortexed and centrifuged. This process was repeated several times. The ethyl acetate phase was recovered and the solvent was removed by vacuum concentrator. The residues were dissolved in 50–100 µl acetonitrile and stored at -20 °C (Lübben & Morand, 1994; Sone & Fujiwara, 1991). The haem composition was analysed using a Navi C18-5 reversed-phase column (2·0 mmx150 mm) (Wako Pure Chemical Industries). Haemoglobin was used as the standard for haem B, membranes from Sulfolobus acidocaldarius were used as the standard for haem As and haem B (Lübben et al., 1994b) and membranes from anaerobically grown cells of P. aerophilum were used as the standard for haem Op1 and haem Op2 (Lübben & Morand, 1994).

Haem staining.
Prior to electrophoresis, the solubilized membranes were denatured for 5 min at 37 °C in SDS loading buffer, which contained 50 mM Tris/HCl (pH 6·8), 1·0 % SDS, 10 % glycerol and 0·01 % bromophenol blue. After electrophoresis, the bands of c-type cytochromes were visualized by 3,3',5,5'-tetramethylbenzidine (TMBZ)/H2O2 staining (Thomas et al., 1976).

Protein analysis.
Protein was measured by the BCA assay (Pierce Chemical Co.) with bovine serum albumin as a standard.

Construction of phage library.
Genomic DNA was isolated from P. oguniense as described by Lauerer et al. (1986). For constructing the genomic phage library, genomic DNA was partially digested with Sau3AI before ligation to the BamHI cloning site of the Lambda Gem11 phage arms (Promega). Packaging was done using Giga Pack III XL Packaging Extract (Stratagene), which preferentially packages large inserts.

Isolation of the genes.
In order to obtain the gene fragment of haem-copper oxidase, PCR was performed using degenerate primers designed from the conserved region of subunit I of haem-copper oxidase (Lübben et al., 1994a): ToxF and ToxR (Table 1). The DNA fragment (294 bp) of the gene of SoxB-type haem-copper oxidase was amplified from the genomic DNA of P. aerophilum using the primers paercox1357F and paercox1357R (Table 1). These amplified gene fragments were directly cloned into pCRII vector (Invitrogen). For Southern analysis and plaque hybridization, DIG (digoxigenin)-labelled nucleotide probes were synthesized with a DIG RNA labelling kit (Roche). Hybridization and detection were carried out using nylon membranes (Hybond-N, Amersham Pharmacia) and a DIG luminescent detection kit (Roche), respectively. The gene locus of SoxM-type haem-copper oxidase and the gene locus of SoxB-type haem-copper oxidase were cloned as a 20 kb and a 16 kb BamHI fragment in the lambda phage vector. The 20 kb gene fragment of the SoxM-type haem-copper oxidase gene locus was digested by XhoI and cloned into pGem-3Zf(+) (Perkin Elmer). The 16 kb fragment of the SoxB-type haem-copper oxidase gene locus was digested by BglII, KpnI, XhoI and XbaI, and cloned into pGem-3Zf(+). These plasmids were deleted with a Kilo sequence deletion kit (TaKaRa) in order to prepare templates for sequencing. DNA sequencing was performed by the deoxynucleotide chain termination method using an ABI 373A automated DNA sequencer (Applied Biosystems). The nucleotide sequences were analysed by DNASIS software (Hitachi Software Engineering).


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Table 1. PCR primers used in this study

 
mRNA analysis.
RNA from aerobically and anaerobically grown cells of P. oguniense was isolated with a Quick Prep Total RNA Extraction kit (Amersham Pharmacia). The mRNAs of both oxidase gene clusters were analysed by Northern blotting with probes specific for each ORF. For Northern hybridization, DNA probes for each ORF were synthesized by PCR using DIG DNA labelling mixture. Table 1 shows the primers for DNA probes. The amount of hybridization signal intensity and area was determined by NIH image v.1.62.

Sequence analysis.
The amino acid sequences of the haem-copper oxidases were aligned with a subset of the amino acid sequences of haem-copper oxidase obtained from the DNA Database of Japan (DDBJ) by CLUSTALX version 1.81. Neighbour-joining analysis (Saitou & Nei, 1987) was done using CLUSTALX version 1.81 (Thompson et al., 1997). Predicted transmembrane segments were analysed by TSEG (http://www.genome.ad.jp/SIT/tseg.html) (Kihara et al., 1998) and SOSUI (http://www.genome.ad.jp/SOSui/index.html) (Hirokawa et al., 1998). Protein motif searching was done by MOTIF (http://motif.genome.ad.jp/MOTIF.html) (Ogiwara et al., 1996).

Nucleotide sequence and accession number.
The sequences of the poxC locus and the poxI locus have been deposited in the DDBJ database under accession nos AB056510 and AB056511.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Catalytic activity in the membranes of P. oguniense
The NADH- and succinate-dependent oxygen consumption rate in the membranes of aerobically grown cells were 93 and 8 pmol min-1 (µg protein)-1, respectively. The NADH-dependent oxygen consumption [225 pmol min-1 (µg protein)-1] was also detected in anaerobically grown cells, but succinate-dependent oxygen consumption was not. Cyanide (1 mM), azide (1 mM) and HOQNO (2-heptyl-4-hydroxyquinoline-N-oxide; 1 µM) did not inhibit the oxygen consumption. The membranes of both aerobically and anaerobically grown cells also showed TMPD oxidase activity and bovine cytochrome c oxidase activity; the TMPD oxidation rate of the two types of membranes was 118 nmol min-1 (µg protein)-1 and 40 nmol min-1 (µg protein)-1 at 70 °C, respectively.

Visible spectrum of the membranes
The reduced minus oxidized spectrum of membranes of aerobically grown cells showed typical absorbance at {gamma} and {alpha} peaks of cytochrome a at 447 and 605 nm but these peaks were not apparent in anaerobically grown cells. The {beta} peak of cytochrome b or c at 522 nm, and the {alpha} peaks of cytochrome b or c at 553 nm and cytochrome b at 565 nm were observed for both aerobically and anaerobically grown cells. However, the {gamma} band of cytochrome b of aerobically grown cells was at 430 nm, which was different from that of anaerobically grown cells (428 nm). The CO/reduced minus reduced spectrum of the membranes of anaerobically grown cells indicated CO-reacting species with an absorbance at 432 and 442 nm. On the other hand, in the membranes of aerobically grown cells, the absorbance maximum was at 420 nm and minima were at 432 and 447 nm. The CO-reacting species of 447 nm, which was the same as that of the reduced minus oxidized spectrum, most likely resulted from cytochrome a of haem-copper oxidase (Fig. 1). In addition, the CO-reacting species of 432 nm suggested the presence of cytochrome o as a part of the haem-copper oxidase in the membrane of both aerobically and anaerobically grown cells. The reduced minus oxidized pyridine haemochrome difference spectrum of membranes from aerobically grown cells showed maxima at 551 and 588 nm and that of anaerobically grown cells showed a maximum at 553 nm (not shown). Considering that the typical maxima of haems A, B and C are 588, 557, 550 nm, respectively, maxima at 551 and 553 nm indicated a mixture of cytochrome b and cytochrome c. Thus, both cytochrome b and cytochrome c were in the membrane of both aerobically and anaerobically grown cells but cytochrome a is specific for aerobically grown cells.



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1 Reduced minus oxidized difference spectra (A) and CO/reduced minus reduced difference spectra (B) of the membranes of aerobically (panel a) and anaerobically (panel b) grown cells of P. oguniense.

 
HPLC analysis of haem composition
The HPLC analysis of non-covalently bound haems showed three peaks specific to aerobically grown cells, from haem B (9·5 min), haem As (14·7 min) and an unknown component (16·3 min), and four peaks specific to anaerobically grown cells, from unknown components (9·3, 11·9, 14·8 and 17·5 min) (Fig. 2). On the other hand, haem Op1 (18·8 min) and some unknown components (8·3, 12·9, 13·7, 14·3, 20·3 min) were common to aerobically and anaerobically grown cells (Fig. 2). The unknown components may be modified haems (Lübben & Morand, 1994).



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Fig. 2. Reversed-phase HPLC chromatograms of the non-covalently membrane-bound haems from P. oguniense. (a, b) Haems extracted from the membranes of aerobically grown cells (a) and anaerobically grown cells (b). (c, d) Haem standards extracted from the membranes of anaerobically grown cells of P. aerophilum (haems B, Op1 and Op2) (c) and from S. acidocaldarius (haems B and As) (d).

 
Haem staining
The membrane fractions of both aerobically and anaerobically grown cells were further analysed by TMBZ/H2O2 staining of SDS-PAGE gels to determine the profiles of c-type cytochromes. The membranes from both aerobically and anaerobically grown cells showed a 30 kDa TMBZ-stained band. In addition, the band of 35 kDa was observed in aerobically grown cells and the band of 28 kDa was observed in anaerobically grown cells (Fig. 3). There was no cytochrome c in the cytosol fraction (data not shown).



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Fig. 3. Profiles of c-type cytochromes in membrane fractions. Membranes from aerobically (lane 1) and anaerobically (lane 2) grown cells were analysed by SDS-PAGE and c-type cytochromes were revealed by their peroxidase activity using TMBZ.

 
Red-shift spectra of membrane-bound cytochromes
By adding succinate, cytochrome b559 was reduced in MEGA extracts of the membranes from both aerobically and anaerobically grown cells. Reduction of cytochrome c551 was observed in the presence of succinate with 10 mM cyanide or 100 mM azide in MEGA extracts of membranes of both types of cells. Cytochrome a602 was also reduced by succinate with cyanide or azide in the MEGA extracts of membranes of aerobically grown cells. The red-shift spectrum of cytochrome a was not observed in the membranes from anaerobically grown cells (Fig. 4). Similar reduction of these cytochromes was also observed with NADH as substrate (data not shown).



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Fig. 4. Succinate-reduced minus oxidized difference spectra (A) and succinate-reduced with cyanide minus oxidized difference spectra (B) of the membranes of aerobically (panel a) and anaerobically (panel b) grown cells.

 
Cloning of two oxidase gene loci
With haem-copper oxidase gene specific primers, a 170 bp gene fragment of SoxM-type haem-copper oxidase was amplified. The Southern blot analysis using this 170 bp gene fragment as a probe showed that P. oguniense has one copy of the gene for SoxM-type haem-copper oxidase (data not shown). On the other hand, the gene fragment of SoxB-type haem-copper oxidase was not amplified by haem-copper oxidase gene specific primers. Thus, the gene fragment of SoxB-type haem-copper oxidase of P. aerophilum was used as a probe in Southern hybridization analysis; this analysis showed that P. oguniense also has one copy of the SoxB-type haem-copper oxidase gene (data not shown). To isolate the entire genes of both SoxB and SoxM-type haem-copper oxidase, plaque hybridization was performed against P. oguniense genomic libraries. Phages harbouring 20 kb and 18 kb fragments were obtained; they contained the genes of SoxB-type and SoxM-type haem-copper oxidase, respectively, and will henceforth be termed the poxI and poxC genes.

The poxC locus.
Fig. 5(a) shows the gene structure of the cloned genomic region with poxC and surrounding oxidase genes. The genes of the oxidase complex, poxA, poxB, poxC, poxK and poxL, are present in an operon-like arrangement and each gene is preceded by putative TATA box elements (Bell et al., 1999; Gelfand et al., 2000; Soppa, 1999a, b). PoxA, containing 79 amino acid residues, is a hypothetical membrane-binding protein which is a homologue of SoxI of Sulfolobus acidocaldarius, whose function is unknown (Castresana et al., 1995). PoxB, containing 148 amino acid residues, is a putative haem-copper oxidase subunit II. This protein has one putative transmembrane helix in the N-terminal region. PoxC, containing 804 amino acid residues, putatively represents haem-copper oxidase subunits I + III, with 19 transmembrane helices. PoxK, whose function is unknown, contains 64 amino acid residues and has two transmembrane helices. PoxL, whose function is also unknown, contains 168 amino acid residues and has four membrane helices. The gene structure of this region is similar to that in P. aerophilim but there is no similarity between P. oguniense and P. aerophilum in either the 5' upstream or 3' downstream regions of this gene locus.



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Fig. 5. Schematic representation of the gene structure and the mRNA transcripts (arrows) originating from the poxC (a) and poxI (b) loci. The dotted lines indicate the location of the hybridization probes for Northern blot analysis.

 
The poxI locus.
The physical map and locations of ORFs at the poxI locus are shown in Fig. 5(b). This gene locus encodes eight hypothetical membrane-binding proteins; the gene cluster consists of two gene clusters separated by a 319 bp non-coding region. poxD, poxE, poxF and poxG are genes of the cytochrome bc complex; they are arranged in an operon-like manner and TATA box-like elements are present 5' upstream of each gene. PoxD, containing 177 amino acid residues, has a signature sequence of cytochrome c (-CTACHS-) and has a transmembrane helix in the N-terminal region. PoxE, containing 186 amino acid residues, is a hypothetical Rieske iron–sulfur protein and has a transmembrane helix in the N terminal region. PoxF, containing 521 amino acid residues, has 11 transmembrane helices and is a hypothetical cytochrome b. In addition, the transmembrane helix in the C terminus of PoxF showed a signature sequence of an ABC transporter family (-VAAGQLAAASFIVGI-). PoxG, whose function is unknown, contains 85 amino acid residues and has two transmembrane helices. poxH, poxI and poxJ are genes of an oxidase complex but these genes are interrupted by 49 bp and 28 bp non-coding elements and are not organized in an operon-like manner. poxH, poxI and poxJ have typical TATA box elements and putative transcription termination elements. PoxH, comprising 157 amino acid residues, is a putative haem-copper oxidase subunit II and has one transmembrane helix in the N-terminal region. PoxI, comprising 557 amino acid residues, is a putative haem-copper oxidase subunit I and has 13 transmembrane helices. PoxJ, comprising 206 amino acid residues, has one transmembrane helix in the N-terminal region. This gene is a homologue of prrC of Rhodobacter sphaeroides (Eraso & Kaplan, 1995), which encodes a part of a signal transduction system, and of sco1, which encodes a haem transporter of Saccharomyces cerevisiae (Schulze & Rödel, 1989; Rentzsch et al., 1999). The gene structure of this region is also similar to that in P. aerophilum but there is no similarity between P. oguniense and P. aerophilum in either the 5' upstream or the 3' downstream regions of this gene locus.

Transcription analysis of the oxidase genes
The transcription patterns of the poxC and poxI gene loci were analysed by Northern hybridization with an equal amount of total RNA isolated from aerobically and anaerobically grown cells. The probes that were used in this study are presented in Fig. 5. In these conditions, aerobically grown cells contained 1·5-fold more rRNA than anaerobically grown cells (Fig. 6a).



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Fig. 6. Northern blot analysis of RNA isolated from aerobically and anaerobically grown cells of P. oguniense. (a) Samples (4 µg) of RNA of aerobically (lanes 1) and anaerobically (lanes 2) grown cells were electrophoresed on 1 % agarose containing 2·2 M formaldehyde. (b–m) Hybridization of probes specific for mRNA from the poxC (b), poxA (c), poxB (d), poxK (e), poxL (f), poxD (g), poxE (h), poxF (i), poxG (j), poxH (k), poxI (l) and poxJ (m). Lanes (c)–(f) are from aerobically grown cells; lanes (h)–(j) are from anaerobically grown cells.

 
The transcription of the poxC locus was stimulated under aerobic conditions: the amount of hybridization signal from aerobically grown cells was 10-fold higher than that of anaerobically grown cells (Fig. 6b). Each probe detected transcripts of 3·9 kb and (except for the poxL probe) 3·5 kb. The transcript of 3·9 kb is the expected size of poxA to poxL and the transcript of 3·5 kb is the expected size of poxA to poxK. The probes for poxA and poxB detected 0·4 kb and 0·5 kb transcripts, respectively, in good agreement with the size of poxA and poxB, with weak signal intensity (Fig. 6b–f).

In contrast to the results with the poxC locus, the amount of hybridization signal of all genes of the poxI locus of anaerobically grown cells was threefold higher than that of aerobically grown cells (Fig. 6g, k, l, m). The probes for poxD, poxE, poxF and poxG detected 3·4 kb mRNA fragments consistent with the size of poxD to poxG. Furthermore, appropriate probes detected a 1·2 kb mRNA fragment that is the size of poxD and poxE and the 0·5 kb mRNA fragment of poxD (Fig. 6g, h, i, j). The probe for poxH, poxI and poxJ detected 0·5 kb, 1·7 kb and 0·6 kb fragments; these mRNAs are in accord with the size of poxH, poxI and poxJ, respectively (Fig. 6k, l, m).

Putative topologies of subunits
PoxC and PoxI, which form the catalytic core of the two different haem-copper oxidases of P. oguniense, exhibit the typical consensus sequences for the haem- and copper-binding sites. These are located in putative transmembrane helices II and X for the low-spin haem (PoxC: H71, H390; and PoxI: H59, H373) and in helices VI, VII and X for the high-spin haem and the copper ion (PoxC: H252, H302, H303, H388; and PoxI: H219, H268, H269, H371) as found for well-investigated terminal oxidases of known structure (Abramson et al., 2000; Chepuri et al., 1990; Iwata et al., 1995; Santana et al., 2001; Soulimane et al., 2000; Tsubaki et al., 1994). The residues for the K channel of proton pumping are conserved in both oxidases (PoxC: Y257, S268, S328, K331; and PoxI: Y223, S234, S295, Y299). On the other hand, the residues necessary for the D channel of haem-copper oxidases are conserved in PoxC (N89, N107, D100, T165, F251, G252 and E255), but are not conserved in PoxI as for other known SoxB-type haem-copper oxidases like those of Sulfolobus acidocaldarius, Acidianus ambivalens, Thermus thermophilus and Geobacillus stearothermophilus (Riistama et al., 1996; Purschke et al., 1997; Keightley et al., 1995; Nikaido et al., 1998; Pereira et al., 2001).

Alignment of subunit II of haem-copper oxidase from different organisms reveals that PoxB and PoxH have two invariant cysteine residues (PoxB: 129, 133; and PoxH: 137, 141), two histidine residues (PoxB: 94, 137; and PoxH: 100, 141) and a methionine residue (PoxB: 140; and PoxH: 148) that are the CuA-binding centre for all cytochrome c oxidases (Van der Oost et al., 1992; Kelly et al., 1993).

poxJ encodes a homologue of scoI of Saccharomyces cerevisiae that is widely conserved in eukarya, proteobacteria and Aquifex aeolicus (Chinenov, 2000). Alignment of ScoI-related proteins showed that PoxJ contained Cu(I)-binding residues (C84, C88 and H169) (Nittis et al., 2001).

Alignment of the cytochrome b subunit of cytochrome bc complex from different organisms showed that PoxF proteins have three invariant haem-binding histidine residues (positions 112, 127, 218) but another invariant histidine residue (position 233) was substituted to lysine (Schütz et al., 2000). This substitution was also observed in P. aerophilum.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aerobic respiratory chain in aerobically grown cells
Oxygen consumption of membrane suspensions with NADH or succinate as substrate indicates that the aerobic respiratory chain has NADH dehydrogenase, succinate dehydrogenase and all elements of the aerobic respiratory chain in the membranes. Succinate- or NADH-induced red-shift of absorption spectrum of cytochrome b559 is a sign of mitochondrial, and some bacterial, cytochromes b of respiratory complex III. Furthermore, the fact that succinate or NADH reduced cytochrome c551 and cytochrome a604 in the presence of inhibitors (10 mM cyanide or 100 mM azide) of terminal oxidase indicates that cytochrome c551 is the electron donor of the terminal oxidase and cytochrome a is a part of the terminal oxidase (Fig. 4). From the reduced minus oxidized and CO/reduced minus reduced spectra of the membranes, there were signatures of cytochrome a-containing haem-copper oxidase and cytochrome o-containing haem-copper oxidase but there was no signals reminiscent of bd-type alternative oxidase (Fig. 1). In addition, HPLC analysis of non-covalently bound haems indicated that the cytochrome a contained haem As and the cytochrome o contained haem Op1 but there was no haem D (Fig. 2). These results strongly suggested that P. oguniense contains no cyanide-resistant alternative oxidase but one or two cyanide-sensitive haem-copper oxidase(s) that contain cytochrome a or cytochrome o.

Aerobic respiratory chain in anaerobically grown cells
In the membranes of anaerobically grown cells, oxygen consumption, TMPD oxidase activity and bovine cytochrome c oxidase activity were detected. Furthermore, cytochrome b559 was reduced by succinate or NADH reduction of MEGA extracts of the membranes of anaerobically grown cells, and cytochrome c551 was also reduced by succinate or NADH in the presence of cyanide or azide. The reduction of cytochrome b559 and cytochrome c551 was also observed in aerobically grown cells. On the other hand, the CO/reduced minus reduced spectrum of the membranes shows a trough at 432 nm that is reminiscent of cytochrome o-containing haem-copper oxidase and is common to aerobically grown cells, but there was no CO-reacting cytochrome a. Furthermore, the HPLC analysis of non-covalently bound haems indicates that both aerobically and anaerobically grown cells have haem Op1. In addition, haem staining of covalently bound haems of both aerobically and anaerobically grown cells showed a 30 kDa TMBZ-stained band that could be cytochrome c, a possible substrate for cytochrome c oxidase. These results suggest that cytochrome o-containing haem-copper oxidase, NADH oxidase, succinate oxidase and cytochrome bc complex are present in the membranes of anaerobically grown cells.

Genes encoding the components of the aerobic respiratory chain
P. oguniense has the genes of SoxB-type and SoxM-type haem-copper oxidases and cytochrome bc complex. The twelve structural genes encoding the oxidase complexes are divided into two gene loci, poxC (SoxM-type) and poxI (SoxB-type and cytochrome bc complex). The gene structures of both the poxC locus and the poxI locus are similar to the genome sequences of P. aerophilum. In the poxC locus, five genes are in an operon structure (Figs 5a and 6). The fusion of subunits I and III of haem copper oxidase is observed in known members of the SoxM-type haem-copper oxidase family of hyperthermophilic archaea: Sulfolobus acidocaldarius (Lübben et al., 1994a) and Aeropyrum pernix (Ishikawa et al., 2002). poxA encodes a homologue of the N-terminal fragment of SoxI of Sulfolobus acidocaldarius. This fact indicates that the functional part of SoxI is located in the N-terminal region and that this gene is not specific for S. acidocaldarius but conserved in aerobic archaea. This gene locus indicates that the SoxM-type haem-copper oxidase of P. oguniense lacks the gene for subunit IV. The poxI locus consisted of two gene clusters: the operon of cytochrome bc complex and the cluster of SoxB-type cytochrome c oxidase (Fig. 5b). poxF encodes the fusion of cytochrome b and a part of an ABC transporter, but the coding sequences for cytochrome b and a part of an ABC transporter are divided into two genes in P. aerophilum. This is the only difference between P. oguniense and P. aerophilum in the poxI locus. This gene locus indicates that SoxB-type haem-copper oxidase of P. oguniense lacks subunits III and IV like other known SoxB-type haem-copper oxidases (Keightley et al., 1995; Purschke et al., 1997; Riistama et al., 1996; Sakamoto et al., 1997) and there is a novel subunit PoxG in the cytochrome bc complex.

Evolutionary aspects
Phylogenetic analyses based on multiple alignment of haem-copper oxidase subunit I and cytochrome b showed that P. oguniense clustered with P. aerophilum in the cluster of archaea (Figs 7 and 8). The position of branches of both types of haem-copper oxidase of Pyrobaculum is consistent with the proposed idea that the SoxB-type branch and the SoxM-type branch of haem-copper oxidases evolved from an early gene duplication preceding the divergence of archaea and bacteria (Fig. 7) (Castresana et al., 1994). The phylogenetic analysis of cytochrome b showed that thermophilic archaea are clearly divided from the bacterial branch. This analysis also showed there are two lineages of cytochrome b in thermophilic archaea – SoxC type and SoxG type – and Pyrobaculum species belong to the SoxG cluster (Fig. 8). These phylogenetic analyses indicate that the aerobic respiratory chain of Pyrobaculum belongs to the archaeal lineage. Therefore, the aerobic respiratory chain of Pyrobaculum may show the adaptation mechanisms to the aerobic environment of the thermophilic archaea except for electron transfer and proton pumping.



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Fig. 7. Phylogenetic tree of the subunit I of terminal oxidases from archaea and bacteria by neighbour-joining methods. Numbers at nodes indicate the bootstrap value. The scale bar indicates 1 substitution per 10 amino acids. The number following the species is the accession number of the database. SwissProt: C. jejuni, N. meningitidis, V. cholerae, P. aeruginosa, B. japonicum, Halobacterium sp. NRC-1, B. halodurans, A. aeolicus, A. pernix, S. acidocaldarius, T. thermophilus CaaB, D. radiodurans, Synechocystis sp. PCC 6803, S. pombe, R. prowazekii, B. subtilis, A. aceti, Buchnera sp. APS, E. coli, X. fastidosa, P. putida and P. stutzeri. GenBank: M. loti, S. solfataricus and M. leprae. DAD: R. etli, T. thermophilus, G. stearothermophilus Boa, A. ambivalens, H. salinarum and R. marinus. DDBJ: N. pharaonis, R. sphaeroides, B. japonicum Caa3 and G. stearothermophilus. PIR: B. firmus. NITE: S. tokodaii. TIGR: C. crescentus, M. turberculosis, S. aureus and S. typhimurium. Institut Pasteur: L. innocua. S. meliloti 1021 Genome Project: S. meliloti. Sanger Centre: Y. pestis. Unpublished genome sequences: M. capsulatus, G. sulfurreducens, D. vulgaris and Wolbachia sp. were obtained from TIGR Microbial Database.

 


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Fig. 8. Phylogenetic tree of cytochrome b from archaea and bacteria by neighbour-joining methods. Numbers indicate the bootstrap value. The scale bar indicates 1 substitution per 20 amino acids. The number following species is the accession number of the database. SwissProt: A. pernix, S. acidocaldarius, T. acidophilum, Halobacterium sp. NRC-1, D. radiodurans, C. tepidum, C. limicola, B. halodurans, B. subtilis, G. stearothermophilus, Synechocystis sp. PCC6803, A. aeolicus, C. jejuni, H. pylori, A. vinosum, X. fastidosa, P. aeruginosa, N. meningitidis, S. pombe, R. prowazekii, R. viridis, B. japonicum and P. denitrificans. GenBank: S. solfataricus, T. volcanicum, M. leprae and M. loti. DAD: C. glutamicum, H. gestii, A. thaliana, A. variabilis, D. discoideum, R. galegae and A. tumefaciens. DDBJ: S. acidocaldarius, S. coelicolor and S. oleracea. NITE: S. tokodaii. TIGR: M. tuberculosis and C. crescentus. S. meliloti 1021 Genome Project: S. meliloti. Unpublished genome sequences (G. sulfurreducens, T. ferrooxidans, L. pneumophila, M. capsulatus, P. putida, B. pseudomallei, Wolbachia sp. and B. suis) were obtained from the TIGR Microbial Database.

 
Transcriptional regulation of the aerobic respiratory chain
The transcriptional analyses of SoxM-type terminal oxidase genes (poxC locus) simply indicated that this type of terminal oxidase was utilized as an aerobic-specific oxidase. In contrast, Northern blot analysis of the genes for SoxB-type terminal oxidase and cytochrome bc complex (poxI locus) showed clear hybridization signals from both aerobically and anaerobically grown cells. In this analysis, interpretation of this data was confused because the amount of rRNAs in aerobically grown cells is 1·5-fold more than that in anaerobically grown cells. The different proportion of mRNA and rRNA is due to the difference of the growth rate and metabolism between the growth conditions (the growth of aerobically grown cells is twice as fast as that of anaerobically grown cells). Therefore, we suppose that the genes for SoxB-type terminal oxidase were transcribed constitutively under both aerobic and anaerobic conditions or slightly stimulated under anaerobic conditions, where the amount of hybridization signal of anaerobically grown cells is threefold more than that of aerobically grown cells (Fig. 6).

Overview of the aerobic respiratory chain
The known haem-copper oxidases, both SoxB-type and SoxM-type oxidases, have a CuA-binding centre in subunit II and the CuA-binding centre indicates that the substrate of these oxidases is cytochrome c but not quinol. In addition, the genes of SoxB-type terminal oxidase and cytochrome bc complex are transcribed under both aerobic and anaerobic conditions and those of SoxM-type terminal oxidase are transcribed only under anaerobic conditions. These data agree with the results of enzymic and spectroscopic analysis of P. oguniense that aerobically grown cells have cytochrome a-containing cytochrome c oxidase and cytochrome o-containing cytochrome c oxidase and anaerobically grown cells have cytochrome o-containing cytochrome c oxidase.

From these analyses of the membranes of P. oguniense and the genome sequence of P. aerophilum (Fitz-Gibbon et al., 1997, 2002), which is the closest relative of P. oguniense, we can tentatively represent the aerobic respiration system of P. oguniense as follows: (NADH oxidase – the respiratory complex I) or [succinate dehydrogenase – respiratory complex II (flavoprotein subunit, Fe/S subunit and cytochrome b subunit)] – quinone (not analysed) – cytochrome b559 – Rieske protein – cytochrome c551 – cytochrome a-containing terminal oxidase and cytochrome o-containing terminal oxidase – molecular oxygen (Fig. 9). The subunit structures of respiratory complexes I and II are suspected from the genome sequence of P. aerophilum (Fitz-Gibbon et al., 1997, 2002). The previous studies of the respiratory chain of thermophilic archaea are for Aeropyrum pernix and Sulfolobales species such as Sulfolobus acidocaldarius, S. tokodaii (Sulfolobus sp. strain 7) and Acidianus ambivalens. The respiratory chain of Sulfolobales species contains type II NADH dehydrogenase (Gomes et al., 2001; Schäfer, 1996; Schäfer et al., 1996) and a novel family of succinate dehydrogenase (Gomes et al., 1999; Iwasaki et al., 1995c; Janssen et al., 1997; Lemos et al., 2001). In addition Sulfolobus acidocaldarius and S. tokodaii have alternative electron carriers for cytochrome c such as sulfocyanin or cytochrome a (Gleißner et al., 1997; Iwasaki et al., 1995b; Schäfer, 1996; Lübben et al., 1992). Acidianus ambivalens lacks complex III or its alternatives (Purschke et al., 1997). On the other hand, the putative respiratory chain of Aeropyrum pernix contains type I NADH dehydrogenase, typical succinate dehydrogenese, cytochrome b, Rieske protein, cytochrome c and two types of cytochrome c oxidase (Kawarabayasi et al., 1999; Ishikawa et al., 2002). The sequence data of P. aerophilum show that its respiratory complex II is not similar to that of Sulfolobales (Fitz-Gibbon et al., 1997, 2002). It is difficult to know from these results and the sequence data of P. oguniense and P. aerophilum whether there are two cytochromes c (as in mitochondria) or one cytochrome c between Rieske protein and the terminal oxidase. Comparing the aerobic respiratory chains of these hyperthermophilic archaea and P. oguniense, the aerobic respiratory chains of Aeropyrum pernix, P. aerophilum and P. oguniense are more similar to that of mitochondria or some bacteria than that of Sulfolobales species.



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Fig. 9. Schematic representation of the aerobic respiratory chain of P. oguniense. White arrows indicate high affinity and dashed arrows indicate low affinity.

 
Functional implications
The similarity of subunits I and II of SoxB-type cytochrome c oxidase between P. oguniense and P. aerophilum is 90 % and 86 %, respectively, while those of SoxM-type cytochrome c oxidase are 80 % and 70 % similar, respectively. These results indicate that there is different evolutionary pressure on these two types of haem-copper oxidases. From the comparison of the physiological features of these two species, the remarkable difference is adaptation capacity to oxygen. The optimum aerobic growth condition of P. oguniense is under atmospheric air with vigorous shaking and that of P. aerophilum is microaerobic, with 0·1 % oxygen in artificial air (Völkl et al., 1993). This difference indicates that SoxB-type haem-copper oxidase is used for low-oxygen conditions, which is a common habitat for both species, and SoxM-type is used for high-oxygen conditions, where P. aerophilum cannot grow. Furthermore, the fact that the transcription level of the genes of SoxB-type haem-copper oxidase does not increase under aerobic conditions also indicates that SoxB-type haem-copper oxidase is not used for high oxygen concentrations. These facts agree with the high-level expression of SoxB-type ba3-cytochrome c oxidase of Thermus thermophilus (Keightley et al., 1995) under microaerobic conditions. In addition, this hypothesis is consistent with the interesting data from enzymic and spectroscopic analyses of membranes of P. oguniense. In these analyses, oxygen consumption with succinate as substrate was not observed, whereas succinate-driven reductions of cytochromes were observed. We suppose that the succinate dehydrogenase activity of anaerobically grown cells was too low for oxygen consumption to be detected because the oxygen consumption rate of the membranes from aerobically grown cells with NADH as substrate was 11·6-fold higher than that of membranes with succinate. However, the oxygen consumption rate with NADH as the substrate of anaerobically grown cells was 2·4-fold higher than that of aerobically grown cells. We speculate that this may arise from the difference of affinity of electron donor and acceptor of the two terminal oxidases. If the affinity to cytochrome c of the terminal oxidase containing cytochrome a is higher than that of the terminal oxidase containing cytochrome o and the affinity to oxygen of the terminal oxidase containing cytochrome a is lower than that of the terminal oxidase containing cytochrome o, most of the electrons flow through the terminal oxidase containing cytochrome a in the aerobically grown cells that show the relatively low oxygen consumption rate (Fig. 9).

We have shown that the aerobic respiratory chain is present in anaerobically grown cells of P. oguniense and this is the first example in archaea. Acidianus ambivalens, whose respiratory chain has been well studied, is a facultative aerobe (Zillig et al., 1986) but there was no report of the aerobic respiratory chain in anaerobically grown cells. P. aerophilum is the species most closely related to P. oguniense and the anaerobic respiratory chain of this species has been well studied (Afshar et al., 1998, 2001) but the demonstration of an aerobic respiratory chain was only from spectroscopic analysis of membranes, HPLC analysis of haems (Lübben & Morand, 1994) and Rieske iron–sulfur protein (Henninger, 1999). On the other hand, there are examples of haem-copper oxidase being expressed under anaerobic conditions in bacteria. The purple non-sulfur bacterium Rhodobacter sphaeroides (O'Gara et al., 1998; Oh & Kaplan, 1999; Shapleigh et al., 1992) has cbb3-type cytochrome c oxidase (FixN type) as oxygen sensor. cbb3-type cytochrome c oxidase in R. sphaeroides forms a complex with a Sco-related protein called PrrC (Toledo-Cuevas et al., 1998). The Sco-related protein (which is the homologue of poxJ in P. oguniense) in the purple non-sulfur photosynthetic bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodovulum sulfidophilum is located within a gene cluster of a trans-acting regulatory protein that controls photosynthetic gene expression in response to oxygen tension and light intensity (Buggy & Bauer, 1995; Masuda et al., 1999). The role of this protein is less clear, but available evidence indicates it is part of sensory transducing pathway (Buggy & Bauer, 1995; Eraso & Kaplan, 1995, 2000; Masuda et al., 1999). In the mitochondria of Saccharomyces cerevisiae, and in Bacillus subtilis, Sco1 (S. cerevisiae) and YpmQ (B. subtilis) is required for biogenesis of the CuA centre of cytochrome c oxidase (Schulze & Rödel, 1989; Rentzsch et al., 1999; Mattatall et al., 2000). Thus, the function of PoxJ in P. oguniense, which is expressed under both aerobic and anaerobic conditions and has Cu(I)-binding residues, is still in question. However, the presence of Sco-related protein in the gene cluster of SoxB-type haem-copper oxidase suggests that it may function as a terminal oxidase under anaerobic conditions.

Concluding remarks
We have demonstrated that the aerobic respiratory chain is expressed in anaerobically grown cells of P. oguniense. We suspect three functions of the aerobic respiratory chain, especially for SoxB-type terminal oxidase under anaerobic conditions: (i) utilizing a small amount of oxygen without any delay; (ii) protection of oxygen-sensitive metabolites in order to reduce any trace of oxygen to water immediately; and (iii) sensing oxygen as a trigger to change the cells' metabolism, as, for example, in Rhodobacter sphaeroides. On the other hand, a small amount of transcription of SoxM-type haem-copper oxidase under anaerobic conditions can be thought of as the fastest way to adapt to an aerobic environment or flush of oxygen. The unique regulation of the aerobic respiratory chain is likely to be an important function of adaptation to the extremely thermal and almost anoxic environment that is believed to be the place where life first originated (Stetter, 1996). The habitat of hyperthermophiles contains many reductive gases such as H2 and H2S. The supply of oxygen may be very changeable and oxygen must be reduced immediately. These functions are very effective for organisms that can utilize oxygen in such high-temperature environments.

P. oguniense is in one of the deepest and shortest branches within the phylogenetic tree, and further study of this archaeon's aerobic respiratory chain and its function in adaptation to aerobic environment may provide insight into the strategy for adaptation to oxygen of the first aerobic organism.


   ACKNOWLEDGEMENTS
 
This work was partially supported by a Grant-in-Aid for Scientific Research (no. 12460093) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, L. A., Iwata, S. & Wikström, M. (2000). The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nat Struct Biol 7, 910–917.[CrossRef][Medline]

Afshar, S., Kim, C., Monbouquette, H. G. & Schroder, I. (1998). Effect of tungstate on nitrate reduction by the hyperthermophilic archaeon Pyrobaculum aerophilum. Appl Environ Microbiol 64, 3004–3008.[Abstract/Free Full Text]

Afshar, S., Johnson, E. , de Vries, S. & Schroder, I. (2001). Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum. J Bacteriol 183, 5491–5495.[Abstract/Free Full Text]

Anemüller, S. & Schäfer, G. (1990). Cytochrome aa3 from Sulfolobus acidocaldarius. A single-subunit, quinol-oxidizing archaebacterial terminal oxidase. Eur J Biochem 191, 297–305.[Abstract]

Anemüller, S., Schmidt, C. L., Pacheco, I., Schäfer, G. & Teixeira, M. (1994). A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon. FEMS Microbiol Lett 117, 275–280.

Balch, W. E., Fox, G. E., Magrum, L. J., Woese, R. C. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol Rev 43, 260–296.

Bell, S. D., Kosa, P. L., Sigler, P. B. & Jackson, S. P. (1999). Orientation of the transcription preinitiation complex in Archaea. Proc Natl Acad Sci U S A 96, 13662–13667.[Abstract/Free Full Text]

Berry, E. A. & Trumpower, B. L. (1987). Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem 161, 1–15.[Medline]

Buggy, J. & Bauer, C. E. (1995). Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus. J Bacteriol 177, 6958–6965.[Abstract]

Castresana, J., Lübben, M., Saraste, M. & Higgins, D. G. (1994). Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen. EMBO J 13, 2516–2525.[Abstract]

Castresana, J., Lübben, M. & Saraste, M. (1995). New archaebacterial genes coding for redox proteins: implications for the evolution of aerobic metabolism. J Mol Biol 250, 202–210.[CrossRef][Medline]

Chepuri, V., Lemieux, L., Au, D. C.-T. & Gennis, R. B. (1990). The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases. J Biol Chem 265, 11185–11192.[Abstract/Free Full Text]

Chinenov, Y. V. (2000). Cytochrome c oxidase assembly factors with a thioredoxin fold are conserved among prokaryotes and eukaryotes. J Mol Med 78, 239–242.[CrossRef][Medline]

Eraso, J. M. & Kaplan, S. (1995). Oxygen-insensitive synthesis of photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase. J Bacteriol 177, 2695–2706.[Abstract]

Eraso, J. M. & Kaplan, S. (2000). From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2.4.1. Biochemistry 39, 2052–2062.[CrossRef][Medline]

Fee, J. A., Yoshida, T., Surerus, K. K. & Mather, M. W. (1993). Cytochrome caa3 from the thermophilic bacterium Thermus thermophilus: a member of the heme-copper oxidase superfamily. J Bioenerg Biomembr 25, 103–114.[Medline]

Fischer, F., Zillig, W., Stetter, K. O. & Schreiber, G. (1983). Chemolithoautotrophic metabolism of anaerobic extremely thermophilic archaebacteria. Nature 301, 511–513.[Medline]

Fitz-Gibbon, S., Choi, A. J., Miller, J. H., Stetter, K. O., Simon, M. I., Swanson, R. & Kim, U.-J. (1997). A fosmid-based genomic map and identification of 474  genes of the hyperthermophilic archaeon Pyrobaculum aerophilum. Extremophiles 1, 36–51.[CrossRef][Medline]

Fitz-Gibbon, S. T., Ladner, H., Kim, U. J., Stetter, K. O., Simon, M. I. & Miller, J. H. (2002). Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Proc Natl Acad Sci U S A 99, 984–989.[Abstract/Free Full Text]

Garcia-Horsman, J. A., Barquera, B., Rumbley, J., Ma, J. & Gennis, R. B. (1994). The superfamily of heme-copper respiratory oxidases. J Bacteriol 176, 5587–5600.[Medline]

Gelfand, M. S., Koonin, E. V. & Mironov, A. A. (2000). Prediction of transcription regulatory sites in Archaea by a comparative genomic approach. Nucleic Acids Res 28, 695–705.[Abstract/Free Full Text]

Gleißner, M., Kaiser, U., Antonopoulos, E. & Schäfer, G. (1997). The archaeal SoxABCD complex is a proton pump in Sulfolobus acidocaldarius. J Biol Chem 272, 8417–8426.[Abstract/Free Full Text]

Gomes, C. M., Lemos, R. S., Teixeira, M., Kletzin, A., Huber, H., Stetter, K. O., Schäfer, G. & Anemüller, S. (1999). The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex. Biochim Biophys Acta 1411, 134–141.[Medline]

Gomes, C. M., Bandeiras, T. M. & Teixeira, M. (2001). A new type-II NADH dehydrogenase from the archaeon Acidianus ambivalens: characterization and in vitro reconstitution of the respiratory chain. J Bioenerg Biomembr 33, 1–8.[Medline]

Henninger, T., Anemüller, S., Fitz-Gibbon, S., Miller, J. H., Schäfer, G. & Schmidt, C. L. (1999). A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum: sequencing of the gene, expression in E. coli and characterization of the protein. J Bioenerg Biomembr 31, 119–128.[CrossRef][Medline]

Hirokawa, T., Boon-Chieng, S. & Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378–379.[Abstract]

Huber, R., Kristjansson, J. K. & Stetter, K. O. (1987). Pyrobaculum gen. nov. a new genus of neutrophilic, rod-shaped archaebacteria from continental solfataras growing optimally at 100 °C. Arch Microbiol 149, 95–101.

Huber, R., Sacher, M., Vollmann, A., Huber, R. & Dieter, R. (2000). Respiration of arsenate and selenate by hyperthermophilic archaea. Syst Appl Microbiol 23, 305–314.[Medline]

Ishikawa, R., Ishido, Y., Tachikawa, A., Kawasaki, H., Matsuzawa, H. & Wakagi, T. (2002). Aeropyrum pernix K1, a strictly aerobic and hyperthermophilic archaeon, has two terminal oxidases, cytochrome ba3 and cytochrome aa3. Arch Microbiol 179, 42–49.[CrossRef][Medline]

Itoh, T., Suzuki, K. & Nakase, T. (1998). Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus. Arch Microbiol 170, 155–161.[CrossRef][Medline]

Iwasaki, T., Matsuura, K. & Oshima, T. (1995a). Resolution of the aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. I. The archaeal terminal oxidase supercomplex is a functional fusion of respiratory complexes III and IV with no c-type cytochromes. J Biol Chem 270, 30881–30892.[Abstract/Free Full Text]

Iwasaki, T., Wakagi, T., Isogai, Y., Iizuka, T. & Oshima, T. (1995b). Resolution of the aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. II. Characterization of the archaeal terminal oxidase supercomplexes and implication for the intermolecular transfer. J Biol Chem 270, 30893–30901.[Abstract/Free Full Text]

Iwasaki, T., Wakagi, T. & Oshima, T. (1995c). Resolution of the aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. III. The archaeal novel respiratory complex II (succinate : caldariella quinone oxidoreductase complex). J Biol Chem 270, 30902–30908.[Abstract/Free Full Text]

Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995). Structure at 2·8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376, 660–669.[CrossRef][Medline]

Janssen, S., Schäfer, G., Anemüller, S. & Ralf, M. (1997). A succinate dehydrogenase with a novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization. J Bacteriol 179, 5560–5569.[Abstract]

Kashefi, K. & Lovely, D. R. (2000). Reduction of Fe(III), Mn(IV), and toxic metals at 100 °C by Pyrobaculum islandicum. J Bacteriol 66, 1050–1056.

Kawarabayasi, Y., Hino, Y., Horikawa, H. & 27 other authors (1999). Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1. DNA Res 6, 83–101.[Medline]

Kawarabayasi, Y., Hino, Y., Horikawa, H. & 27 other authors (2001). Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7. DNA Res 8, 123–140.[Medline]

Keightley, J. A., Zimmermann, B. H., Mather, M. W., Springer, P., Pastuszyn, A., Lawrence, D. M. & Fee, J. A. (1995). Molecular genetic and protein chemical characterization of cytochrome ba3 from Thermus thermophilus HB8. J Biol Chem 270, 20345–20358.[Abstract/Free Full Text]

Kelly, M., Lappalainen, P., Talbo, G., Haltia, T., Van der Oost, J. & Saraste, M. (1993). Two cysteines, two histidines, and one methionine are ligands of a binuclear purple copper center. J Biol Chem 268, 16781–16787.[Abstract/Free Full Text]

Kihara, D., Shimizu, T. & Kanehisa, M. (1998). Prediction of membrane proteins based on classification of transmembrane segments. Protein Eng 11, 961–970.[Abstract]

Kita, K., Konishi, K. & Anraku, Y. (1984a). Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purification and properties of cytochrome b562–o complex from cells in the early exponential phase of aerobic growth. J Biol Chem 259, 3368–3374.[Abstract/Free Full Text]

Kita, K., Konishi, K. & Anraku, Y. (1984b). Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome b558–d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems. J Biol Chem 259, 3375–3381.[Abstract/Free Full Text]

Lauerer, G., Kristjansson, J. K., Langworthy, T. A., König, H. & Stetter, K. O. (1986). Methanothermus sociabilis sp. nov., a second species within the Methanothermaceae growing at 97 °C. Syst Appl Microbiol 8, 100–105.

Lemos, R. S., Gomes, C. M. & Teixeira, M. (2001). Acidianus ambivalens complex II typifies a novel family of succinate dehydrogenases. Biochem Biophys Res Commun 281, 141–150.[CrossRef][Medline]

Lindqvist, A., Membrillo-Hernandez, J., Poole, R. K. & Cook, G. M. (2000). Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress. Antonie Van Leeuwenhoek 78, 23–31.[CrossRef][Medline]

Lübben, M. & Morand, K. (1994). Novel prenylated hemes as cofactors of cytochrome oxidases. Archaea have modified hemes a and o. J Biol Chem 269, 21473–21479.[Abstract/Free Full Text]

Lübben, M., Kolmerer, B. & Saraste, M. (1992). An archaebacterial terminal oxidase combines core structure of two mitochondrial respiratory complexes. EMBO J 11, 805–812.[Abstract]

Lübben, M., Arnaud, S., Castresana, J., Warne, A., Albracht, S. P. J. & Saraste, M. (1994a). A second terminal oxidase in Sulfolobus acidocaldarius. Eur J Biochem 224, 151–159.[Abstract]

Lübben, M., Warne, A., Albracht, S. P. J. & Saraste, M. (1994b). The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem. Mol Microbiol 13, 327–335.[Medline]

Masuda, S., Matsumoto, Y., Nagashima, K. V. P., Shimada, K., Inoure, K., Bauer, C. E. & Matsuura, K. (1999). Structural and functional analyses of photosynthetic regulatory genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus. J Bacteriol 181, 4205–4215.[Abstract/Free Full Text]

Mather, M. W., Springer, P., Hensel, S., Buse, G. & Fee, J. A. (1993). Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence of the fused gene and analysis of the deduced primary structures for subunits I and III of cytochrome caa3. J Biol Chem 268, 5395–5408.[Abstract/Free Full Text]

Mattatall, N. R., Jazairi, J. & Hill, B. C. (2000). Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis. J Biol Chem 275, 28802–28809.[Abstract/Free Full Text]

Nikaido, K., Noguchi, S., Sakamoto, J. & Sone, N. (1998). The cbaAB genes for bo3-type cytochrome c oxidase in Bacillus stearothermophilus. Biochim Biophys Acta 1397, 262–267.[Medline]

Nittis, T., George, G. N. & Winge, D. R. (2001). Yeast Sco1, a protein essential for cytochrome c oxidase function, is a Cu(I)-binding protein. J Biol Chem 276, 42520–42526.[Abstract/Free Full Text]

O'Gara, J. P., Eraso, J. M. & Kaplan, S. (1998). A redox-responsive pathway for aerobic regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J Bacteriol 180, 4044–4050.[Abstract/Free Full Text]

Ogiwara, A., Uchiyama, I., Takagi, T. & Kanehisa, M. (1996). Construction and analysis of a profile library characterizing groups of structurally known proteins. Protein Sci 5, 1991–1999.[Abstract/Free Full Text]

Oh, J.-I. & Kaplan, S. (1999). The cbb3 terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation. Biochemistry 38, 2688–2696.[CrossRef][Medline]

Otten, M. F., Rejinders, W. N. M., Bedaux, J. J. M., Westerhoff, H. V., Krab, K. & Van Spanning, R. J. M. (1999). The reduction state of the Q-pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans. Eur J Biochem 261, 767–774.[Abstract/Free Full Text]

Otten, M. F., Stork, D. M., Rejinders, W. N. M., Westerhoff, H. V. & Van Spanning, R. J. M. (2001). Regulation of expression of terminal oxidases in Paracoccus denitrificans. Eur J Biochem 268, 2486–2497.[Abstract/Free Full Text]

Pereira, M. M., Santana, M. & Teixeira, M. (2001). A novel scenario for the evolution of haem-copper oxygen reductases. Biochim Biophys Acta 1505, 185–208.[Medline]

Purschke, W. G., Schmidt, C. L., Petersen, A. & Schäfer, G. (1997). The terminal quinol oxidase of hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J Bacteriol 179, 1344–1353.[Abstract]

Rentzsch, A., Krummeck-Weiß, G., Hofer, A., Bartuschka, A., Ostermann, K. & Rödel, G. (1999). Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase. Curr Genet 35, 103–108.[CrossRef][Medline]

Riistama, S., Puustinen, A., Garcia-Horsman, J. A., Iwata, S., Michel, H. & Wikström, M. (1996). Channelling of dioxygen into the respiratory enzyme. Biochim Biophys Acta Bioenerg 1275, 1–4.[Medline]

Saitou N & Nei, M. (1987).The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sakamoto, J., Honda, Y. & Sone, N. (1997). A novel cytochrome b(o/a)3-type oxidase from Bacillus stearothermophilus catalyzes cytochrome c-551 oxidation. J Biochem 122, 764–771.[Abstract]

Sako, Y., Nunoura, T. & Uchida, A. (2001). Pyrobaculum oguniense sp. nov., a novel facultatively aerobic and hyperthermophilic archaeon growing at up to 97 °C. Int J Syst Evol Microbiol 51, 303–309.[Abstract]

Santana, M., Pereira, M. M., Elias, N. P., Soares, C. M. & Teixeira, M. (2001). Gene cluster of Rhodothermus marinus high-potential iron-sulfur protein : oxygen oxidoreductase, a caa3-type oxidase belonging to the superfamily of heme-copper oxidase. J Bacteriol 183, 687–699.[Abstract/Free Full Text]

Schäfer, G. (1996). Bioenergetics of the archaebacterium Sulfolobus. Biochim Biophy Acta 1277, 163–200.[Medline]

Schäfer, G., Purschke, W. G., Gleissner, M. & Schmidt, C. L. (1996). Respiratory chain of archaea and extremophiles. Biochim Biophys Acta 1275, 16–20.[Medline]

Schäfer, G., Moll, R. & Schmidt, C. L. (2001). Respiratory enzymes from Sulfolobus acidocaldarius. Methods Enzymol 331, 369–410.[Medline]

Schulze, M. & Rödel, G. (1989). Accumulation of the cytochrome c oxidase subunits I and II in yeast requires a mitochondrial membrane-associated protein, encoded by the nuclear SCO1 gene. Mol Gen Genet 216, 37–43.[Medline]

Schütz, M., Brugna, M., Lebrun, E. & 9 other authors (2000). Early evolution of cytochrome bc complexes. J Mol Biol 300, 663–675.[CrossRef][Medline]

Shapleigh, J. P., Hill, J. J., Alben, J. O. & Gennis, R. B. (1992). Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides. J Bacteriol 174, 2338–2343.[Abstract]

She, Q., Singh, R. K., Confalonieri, F. & 28 other authors (2001). The complete genome of the Crenarchaeote Sulfolobus solfataricus P2. Proc Natl Acad Sci U S A 98, 7835–7840.[Abstract/Free Full Text]

Skulachev, V. P. (1994). Decrease in the intracellular concentration of O2 as a special function of the cellular respiratory system. Biokhimiia 59, 1910–1912.[Medline]

Sone, N. & Fujiwara, Y. (1991). Effects of aeration during growth of Bacillus stearothermophilus on proton pumping activity and change of terminal oxidase. J Biochem 110, 1016–1021.[Abstract]

Soppa, J. (1999a). Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal base-specific TFB contacts upstream of the TATA box. Mol Microbiol 31, 1589–1592.[CrossRef][Medline]

Soppa, J. (1999b). Transcription initiation in Archaea: facts, factors and future aspects. Mol Microbiol 31, 1295–1305.[CrossRef][Medline]

Soulimane, T., Buse, G., Bourenkov, G. P., Bartunik, H. D., Huber, R. & Than, M. E. (2000). Structure and mechanism of the aberrant ba3-cytochrome c oxidase from Thermus thermophilus. EMBO J 19, 1766–1776.[Abstract/Free Full Text]

Stetter, K. O. (1996). Hyperthermophilic prokaryotes. FEMS Microbiol Rev 18, 149–158.[CrossRef]

Thomas, P. E., Ryan, D. & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75, 168–176.[Medline]

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.[CrossRef]

Toledo-Cuevas, M., Barquera, B., Gennis, R. B., Wikström, M. & Garcia-Horsman, J. A. (1998). The cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides, a proton-pumping heme-copper oxidase. Biochim Biophy Acta 1365, 421–434.[Medline]

Tsubaki, M., Mogi, T., Hori, H., Hirota, S., Ogura, T., Kitagawa, T. & Anraku, Y. (1994). Molecular structure of redox metal centers of the cytochrome bo complex from Escherichia coli. Spectroscopic characterizations of the subunit I histidine mutant oxidases. J Biol Chem 269, 30861–30868.[Abstract/Free Full Text]

Van der Oost, J., Lappalainen, P., Musacchio, A. & 8 other authors (1992). Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of E. coli cytochrome o quinol oxidase complex. EMBO J 11, 3209–3217.[Abstract]

Vargas. M., Kashefi, K., Blunt-Harris, E. L. & Lovely, D. R. (1998). Microbiological evidence for Fe(III) reduction on early earth. Nature 395, 65–67.[CrossRef][Medline]

Völkl, P., Huber R., Drobner, E., Rachel, R., Burggraf, S., Trincone, A. & Stetter, K. O. (1993). Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum. Appl Environ Microbiol 59, 2918–2926.[Abstract]

Wakagi, T. & Oshima, T. (1986). Membrane-bound ATPase and electron transport system of Sulfolobus acidocaldarius. Syst Appl Microbiol 159, 425–426.

Wakagi, T., Yamauchi, T., Oshima, T., Mueller, M. & Azzi, A. (1989). A novel a-type terminal oxidase from Sulfolobus acidocaldarius with cytochrome c oxidase activity. Biochem Biophys Res Commun 165, 1110–1114.[Medline]

Wall, D., Delaney, J. M., Fayat, O., Lipinska, B., Yamamoto, T. & Georgopoulos, C. (1992). arc-dependent thermal regulation and extragenic suppression of Escherichia coli cytochrome d operon. J Bacteriol 174, 6554–6562.[Abstract]

Zillig, W., Yeats, S., Holz, I., Bock, A., Rettenberger, M., Gropp, F. & Simon, G. (1986). Desulfurolobus ambivalens, gen. nov., sp. nov., an autotrophic archaebacterium facultatively oxidizing or reducing sulfur. Syst Appl Microbiol 8, 197–203.

Received 16 September 2002; revised 15 December 2002; accepted 16 December 2002.



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