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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB056510 and AB056511.
Present address: Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Marine Science and Technology Center, Yokosuka 237-0061, Japan.
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INTRODUCTION |
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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.
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METHODS |
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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 (0·51 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 50100 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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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.
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RESULTS |
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Visible spectrum of the membranes
The reduced minus oxidized spectrum of membranes of aerobically grown cells showed typical absorbance at and
peaks of cytochrome a at 447 and 605 nm but these peaks were not apparent in anaerobically grown cells. The
peak of cytochrome b or c at 522 nm, and the
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
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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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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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. 6
a).
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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.
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DISCUSSION |
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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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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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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 ironsulfur 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.
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ACKNOWLEDGEMENTS |
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Received 16 September 2002;
revised 15 December 2002;
accepted 16 December 2002.
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