1 Laboratoire de Chimie Bactérienne, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
2 Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
3 Universidad Autonoma de Madrid, Centro de Biologia Molecular, Cantoblanco, Madrid, Spain
Correspondence
Violaine Bonnefoy
bonnefoy{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the partial iro gene sequences from A. ferrooxidans strains CC1, ATCC 19859 and BRGM-1 are AJ621386AJ621388 and the accession numbers of the iro locus from strains BRGM and ATCC 33020 are AJ621560 and AJ320262.
Details of primers, an alignment of iro gene sequences from Fe-1 and ATCC 23270 and the sequence of the iro locus of ATCC 33020 are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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METHODS |
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DNA manipulations.
General DNA manipulations were performed according to Ausubel et al. (1992). Genomic DNA from A. ferrooxidans was prepared using the NucleoSpin Tissue kit from Macherey-Nagel, according to the manufacturer's instructions for bacterial DNA extraction. The nucleotide sequences of the cloned fragments were determined from both strands by GENOME Express. For PCR amplifications, the oligonucleotides were obtained from Sigma-Genosys and Taq polymerase from Roche. Primers used in this study are given as supplementary material with the online version of this paper (Table S1).
Degenerate oligonucleotide primers (DOP) PCR.
An alignment of nucleotide sequences was made between the iro genes of A. ferrooxidans strains Fe-1 and ATCC 23270 in order to determine the most conserved regions from which two degenerate oligonucleotides, iro-degN and iro-degC, were designed (see Supplementary Fig. S1 and Table S1). In a 10 µl reaction, 1 pmol of each primer was used and the PCR amplifications were carried out as follows: 2 min 30 s at 94 °C, 10 cycles at 94 °C for 30 s, 37 °C for 30 s and 72 °C for 30 s, 10 cycles at 94 °C for 30 s, 40 °C for 30 s and 72 °C for 30 s, 10 cycles at 94 °C for 30 s, 45 °C for 30 s and 72 °C for 30 s and finally 2 min 30 s at 72 °C.
Inverse PCR.
A. ferrooxidans ATCC 33020 genomic DNA was digested with AluI and TaqI restriction enzymes (Biolabs). PCR was realized with the religated DNA as matrix according to Ochman et al. (1990) using the divergent oligonucleotides div-hip1 and div-hip2 deduced from the sequence determined by DOP PCR (Table S1).
Cloning of DOP and inverse PCR fragments.
Amplification products were purified using Wizard PCR Preps (Promega). They were cloned in the EcoRV restriction site of the phagemid (SK+) Bluescript. Ligation products were used to transform E. coli strain TG1. White ApR clones were selected on LB-Ap-XGal-IPTG plates. The insert was amplified by PCR and sequenced with the universal primers T3 and T7.
Southern blotting.
Genomic DNA from A. ferrooxidans ATCC 33020 was digested with the restriction enzymes KpnI, NcoI, PstI and SalI (Biolabs). After electrophoresis, digested DNA was denatured and transferred by semi-dry capillary to a positively charged nylon membrane (Roche). A DIG-labelled probe corresponding to the iro gene was obtained by PCR with the oligonucleotides iro1 and airo3 (Table S1). Hybridization and washings were performed under high- and low-stringency conditions. Detection was accomplished by chemiluminescent reaction with CSPD [disodium 3-(4-methoxyspiro{1, 2-dioxetane-3,2'-(5'-chloro) tricyclo [3.3.1.1.3,7] decan}-4-yl) phenyl phosphate] as described by Roche.
Pulsed field gel electrophoresis (PFGE).
PFGE was performed as previously described (Irazabal et al., 1997) on SpeI-digested ATCC 33020 chromosomal DNA.
Cloning of the ATCC 33020 iro gene.
The iro33020 DNA corresponding to the precursor protein of strain ATCC 33020 was amplified by PCR from the genomic DNA of ATCC 33020 with the oligonucleotides hip-N-BamHI and hip-C-XhoI (Table S1). The PCR fragments obtained were purified with Wizard PCR Preps and digested with BamHI and XhoI. The purified fragments were ligated in the pET21(+) vector digested with BamHI and XhoI. Cloning was done in E. coli strain TG1, which lacks the T7 RNA polymerase structural gene and therefore cannot express the target gene. Screening was performed by PCR with oligonucleotides pET-T7 (5'-GTGAGCGGATAACAATTCCCCTC-3') and T7-ter (5'-CGATCAATAACGAGTCGCC-3'), which flank the multiple cloning site of the pET21 vector, and the oligonucleotides used to amplify the iro33020 DNA. The sequence of the cloned fragments was checked. The recombinant plasmid pET21-iro33020 was then introduced into E. coli BL21(DE3), which carries the T7 RNA polymerase structural gene under the control of the IPTG-inducible lacUV5 promoter.
The iro33020 gene cloned in pET21 (see above) was amplified with primers pET-T7 and T7-ter. The PCR fragment obtained was blunt-ended by T4 DNA polymerase, purified with Wizard PCR Preps and cloned in the vector pJF119EH (Furste et al., 1986) previously digested with SmaI. The sequence of the cloned fragments was checked. The pJF119EH-iro33020 plasmid was then introduced into E. coli strains TG1 and TGC (TG1
tatC).
Protein analysis.
Periplasmic and spheroplastic fractions of E. coli were prepared as described previously (Bengrine et al., 1998). Protein concentration was estimated by the modified Bradford method (Bio-Rad protein assay) in 0·8 M NaOH according to the manufacturer's guidelines. BSA was used as the standard. Equal amounts of proteins were heated at 100 °C for 5 min in Laemmli's sample buffer and electrophoresed in 18 % SDS-PAGE. Proteins were detected using the Phastgel Blue-R staining kit (Amersham Pharmacia Biotech).
Fe assays were performed according to the colorimetric method developed by Lovenberg et al., 1963). [4Fe4S] cluster number per polypeptide was calculated by determining the absorption coefficient of the Iro protein using the A388 and the protein concentration estimated by Bradford assays (Bartsch, 1978
).
Purification of the recombinant Iro protein.
E. coli strain BL21(DE3) carrying the pET21-iro33020 plasmid was grown at 37 °C in LB with ampicillin (50 µg ml1) to an OD600 of 1·0 without induction. The recombinant His-tagged Iro protein was purified from the crude extract fraction using the His Trap kit (Amersham Pharmacia Biotech) according to the manufacturer's guidelines. It was desalted using the HiTrap Desalting kit (Amersham Pharmacia Biotech) and dialysed against 50 mM Na2HPO4, 50 mM NaH2PO4 overnight at 4 °C with benzoylated dialysis tubing (Sigma-Aldrich).
Western blotting.
Following SDS-PAGE, proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech) with the Mini Trans-Blot unit (Bio-Rad). The His-tagged Iro protein was identified by immunodetection with mouse anti-His antibodies (Amersham Pharmacia Biotech) and by using the Supersignal West Pico Trial kit (Pierce) as described in the manufacturer's guidelines.
Optical and EPR spectra.
Room-temperature spectra were recorded using a dual-wavelength DW2000 SLM-Aminco spectrophotometer. The concentration of HiPIP protein was determined using =18 mM1 at 388 nm. EPR spectra were recorded at liquid helium temperature on a Bruker ESP 300E X-band spectrometer equipped with an Oxford Instruments liquid helium cryostat and temperature control system. See Fig. 4(b)
for the EPR instrument settings.
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RESULTS |
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We probed different A. ferrooxidans strains by PCR with oligonucleotides corresponding to the iroFe-1 locus (iro1 and airo3; Table S1) and to the iro33020 gene (hip9 and hip10; Table S1). Oligonucleotides rusNM and RusCXhoI (Table S1), corresponding to the gene encoding rusticyanin, a protein so far described only in A. ferrooxidans, were used as controls. In addition to strain BRGM, we also tested the collection strains ATCC 19859, ATCC 23270 and ATCC 33020 and two private strains, which have been isolated on solid medium and shown unambiguously to belong to the A. ferrooxidans species by the sequences of the 16S rRNA gene and of the intergenic region between the 16S and 23S rRNA genes (Duquesne et al., 2003): CC1 (Duquesne et al., 2003
) and BRGM-1, which is a BRGM clonal derivative (Liu et al., 2000
). DNA was amplified with the oligonucleotides iro1 and airo3 (390 bp fragment) only from strain BRGM while, for all the strains tested, fragments of the expected size were obtained with rusNM and RusCXhoI and with the hip9 and hip10 oligonucleotides (475 and 310 bp, respectively) (Fig. 1
). The 310 bp DNA fragments were sequenced to confirm that they corresponded to iro23270-orthologous genes. Therefore, iro23270-orthologous genes are present in the six A. ferrooxidans strains we have tested, including strains BRGM and BRGM-1, whereas an iroFe-1-orthologous gene was detected only in strain BRGM and not in the BRGM-1 clonal derivative. Repeated attempts to isolate BRGM clonal derivatives containing the iroBRGM gene were unsuccessful.
Description of the ATCC 33020 iro locus
A potential ribosome-binding site, AAGGAG, was found seven nucleotides upstream from the translational start codon of the iro33020 gene (Fig. S2). No sequence with obvious similarity to the E. coli 70-specific promoter and no obvious rho-independent transcriptional termination site were detected upstream from this gene. A potential hairpin structure (16·1 kcal mol1; 67·4 kJ mol1) followed by a series of thymidines was found downstream from the iro33020 translational stop codon (Fig. S2) and could correspond to a transcriptional termination site. The nucleotide sequence corresponding to the Iro33020 precursor is 95·6 % identical to the iro23270 gene and 56·2 % to the iroFe-1 and iroBRGM genes (Kusano et al., 1992
; this study). A typical signal sequence of the Tat secretion pathway (Berks, 1996
; Cristobal et al., 1999
; Palmer & Berks, 2003
) is present at the amino terminus of Iro33020 (Fig. S2), characterized by (i) a relatively long signal peptide (48 of 106 aa), (ii) an extended positively charged region (23 aa) including, at the boundary, the motif S R R D M L K, where the underlined residues correspond to the Tat signal peptide consensus sequence, (iii) a small hydrophobic h-region (18 aa) rich in glycine and threonine and devoid of leucine residues, (iv) a potentially helix-breaking proline at the end of this h-region and (v) a basic amino acid residue (histidine), known as sec-avoidance, in its hydrophilic c-region. The theoretical molecular mass deduced from the Iro33020 amino acid sequence is 10·7 kDa for the precursor and 5·9 kDa for the mature protein. The isoelectric point of the mature Iro33020 is estimated to be 8·3. The sequence of the deduced mature protein is 58·3 % identical (78·3 % similar) to the Iro protein from A. ferrooxidans strains Fe-1 (Kusano et al., 1992
) and BRGM (this paper), 41 % identical (67·2 % similar) to the HiPIP protein from Rhodopila globiformis (Ambler et al., 1993
) and 31·4 and 28·4 % identical (61·2 and 52·3 % similar) to the HiPIP from Rhodocyclus tenuis strains 2761 and 3761 (Tedro et al., 1979
, 1985
) (Fig. 2
). The four cysteines which bind the iron atoms of the [4Fe4S] cluster are conserved (Fig. 2
). Therefore, the Iro33020 protein belongs to the HiPIP family. Notably, its closest known relative, apart from IroFe-1 and IroBRGM, is the HiPIP from Rhodopila globiformis, which has a growth optimum of pH 5 (Ambler et al., 1993
; Van Driessche et al., 2003
).
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Characterization of recombinant His-tagged Iro33020 protein produced in E. coli
Because of the relative low identity (58·3 %) found between the mature Iro from two different strains (ATCC 33020 and Fe-1) of the same species, we decided to characterize Iro33020. To facilitate this characterization, we overproduced Iro33020 in E. coli. For this purpose, the iro33020 gene was cloned in the pET21 plasmid to produce the Iro33020 precursor fused to a C-terminal hexahistidine tag as described in Methods. In IPTG-induced BL21(DE3)/pET21-iro33020 cells, a polypeptide cross-reacting with anti-His antibodies of apparent molecular mass of about 12 kDa was detected, but not in control TG1/pET21-iro33020 cells, which lack the T7 RNA polymerase structural gene (data not shown). The 12 kDa polypeptide was also detected, but in smaller amounts, in non-induced cells, presumably because of read-through of the T7 RNA polymerase promoter (data not shown). Fractionation revealed that this polypeptide is present mainly in the periplasm (Fig. 3a). In the spheroplast fraction of induced and non-induced cells, another polypeptide of about 16 kDa, probably the Iro33020 precursor form, was detected (Fig. 3a
). The recombinant Iro33020 protein, now referred to as Iro33020-HisTag, was purified on a nickel column from a crude extract of non-induced BL21(DE3)/pET21-iro33020 cells as described in Methods. Two polypeptides, of 16 and 12 kDa, were obtained. The amino-terminal sequences of these polypeptides identified them as the precursor and mature forms of Iro33020. Both migrated in 18 % polyacrylamide gels with an apparent molecular mass higher than those predicted from their amino acid sequence (10·7 and 6 kDa).
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Optical spectra of purified Iro33020-HisTag showed changes in absorbance depending upon oxidation or reduction states. The optical spectrum of reduced Iro33020-HisTag exhibited a significant decrease in absorbance in the region of 450550 nm compared with the oxidized Iro33020-HisTag (Fig. 4a), and the oxidized minus reduced spectrum exhibited a characteristic maximum absorption at 490 nm (Fig. 4a
inset), suggesting that Iro33020-HisTag behaves as a correctly folded [4Fe4S]-cluster-containing protein. A purity ratio (A278/A388) of 1·9 was obtained for the reduced HiPIP. The sample showed no contaminants that stained with Coomassie blue on SDS-PAGE (data not shown). As observed with other HiPIPs, the reduced state of Iro33020-HisTag exhibited no EPR signal (Fig. 4b
). Upon oxidation by hexachloroiridate (IrCl6), a typical HiPIP axial EPR signal characterized by g=2·043, g=2·115 appeared (Fig. 4b
), indicating the presence of the [4Fe4S] cluster. These results confirm that the Iro33020 of A. ferrooxidans belongs to the HiPIP family and that, when produced in E. coli, this protein has incorporated a [4Fe4S] cluster into a folded native form that is correctly processed and translocated to the periplasm.
The electrochemical behaviour of Iro33020-HisTag was investigated. Typical cyclic and square-wave voltammograms obtained at the MPG electrode are shown in Fig. 5(a, b) for a 40 µM Iro33020-HisTag solution entrapped between the dialysis membrane and the pyrolytic graphite electrode (see Methods). A quasi-reversible behaviour was obtained and the redox potential either measured as the square-wave voltammogram peak potential (Fig. 5b
) or calculated as the mean of the cathodic and anodic cyclic voltammogram peaks (Fig. 5a
) was 510±5 mV at pH 7·4. This very high redox potential confirms that Iro33020 belongs to the HiPIP family. To our knowledge, this is one of the highest redox potentials described for HiPIP [4Fe4S] clusters (Capozzi et al., 1998
). It has been suggested previously that the major determinant for the variation of redox potential within HiPIP series (from 100 to 500 mV) was the difference in charged residues. The more positive the charge of the protein, the higher the redox potential of the cluster. In this work, a good correlation between the global charge of the protein and its high redox potential is obtained, since the pI of Iro33020 is estimated to be 8·3.
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The recombinant Iro33020-HisTag protein is translocated in the E. coli periplasm by the Tat system
The Iro33020 protein has a typical signal sequence of the Tat secretion pathway (see above). To test whether this signal sequence is recognized by the Tat pathway of the neutrophilic E. coli, iro33020 expression was compared between a wild-type and a tat-deficient mutant of E. coli. Because we were unable to construct a BL21(DE3) tat mutant, we cloned iro33020 in the pJF119EH expression vector and introduced the recombinant plasmid (pJF119EH-iro33020) in the isogenic TG1 and TGC (tatC) strains of E. coli. The mature form of the Iro33020 protein was detected by immunodetection with antibodies directed against the His-tag in the periplasmic fraction of the wild-type E. coli carrying the construct pJF119EH-iro33020 (Fig. 3b
). In contrast, no mature form of Iro33020 was detected in the periplasmic fraction of the
tatC mutant of E. coli. Furthermore, a small amount of the precursor form of Iro33020 was detected in the spheroplastic fraction of the wild-type and of the
tatC mutant after a longer exposure (Fig. 3c
). These results indicated that, in spite of the periplasm pH difference, the A. ferrooxidans Iro33020 protein was processed and translocated to the periplasm via the Tat secretion pathway of E. coli.
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DISCUSSION |
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When the iro33020 gene was expressed in E. coli, the recombinant Iro33020-HisTag protein had all the expected properties of a HiPIP with a correctly incorporated [4Fe4S] cluster. As shown by optical and EPR spectra analyses (Fig. 4), the recombinant Iro33020-HisTag protein contains a typical HiPIP [4Fe4S] cluster. Furthermore, the redox potential of the protein was high (+550 mV at low pH), as expected for a HiPIP from an acidophile (Fig. 5
). Because recombinant HiPIPs produced in E. coli exhibit properties very similar to the native proteins (Agarwal et al., 1993
; Eltis et al., 1994
; Brüser et al., 1998
; Caspersen et al., 2000
), the described properties of the recombinant Iro33020-HisTag produced in E. coli likely reflect those of the native Iro33020 protein. Indeed, they are similar to those of IroFe-1 and IroBRGM (Fukumori et al., 1988
; Yamanaka & Fukumori, 1995
; Cavazza et al., 1995
).
In spite of the fact that E. coli is a neutrophile and encodes no apparent HiPIP (Blattner et al., 1997), the correctly processed HiPIP Iro from the acidophilic A. ferrooxidans can be successfully produced in this bacterium. In E. coli, complex systems are required for the assembly of ironsulfur clusters (Takahashi & Nakamura, 1999
; Takahashi & Tokumoto, 2002
). Putative genes involved in the formation of ironsulfur centres were identified in the genome sequence of A. ferrooxidans (Valdés et al., 2003
). Nevertheless, reconstitution of the [4Fe4S] cluster in vitro into a HiPIP precursor has been reported (Brüser et al., 2003
), suggesting that loading of the ironsulfur centre in HiPIP may also take place without accessory proteins.
The production of periplasmic redox holoproteins from the acidophilic A. ferrooxidans in the neutrophilic E. coli has presented some experimental problems. Rusticyanin, a blue-copper protein, has been produced in the cytoplasm (Casimiro et al., 1995) or in the periplasm (Bengrine et al., 1998
) but, in both cases, the copper was not incorporated and reconstitution in vitro was required. Similarly, copper insertion in subunit II of the aa3-type cytochrome oxidase from A. ferrooxidans did not occur when produced in the periplasm of E. coli (J. A. DeMoss, K. Lund, J. Ratouchniak and V. Bonnefoy, unpublished results). In the case of cytochromes c, a plasmid constitutively expressing the E. coli ccm operon encoding the cytochrome c maturation system was required in order to obtain holocytochromes (Appia-Ayme, 1998
). In all these examples, the recombinant protein was correctly translocated to the periplasm of E. coli, however. Why was no cofactor loaded in these proteins, while Iro33020-HisTag had its [4Fe4S] cluster incorporated? The simplest explanation is that cytochromes c, rusticyanin and subunit II of the aa3-type cytochrome oxidase are translocated by the Sec system, while Iro33020-HisTag is translocated by the Tat pathway. In the former case, the cofactor is inserted in the periplasm and, in the latter case, it takes place in the cytoplasm before translocation (Palmer & Berks, 2003
). Although there is a significant difference in the pH of the periplasm between E. coli (pH 6·5) and A. ferrooxidans (pH 1·53), the pH of the cytoplasm of the two organisms is approximately the same (see Ingledew, 1982
and references therein). We have shown in this paper that translocation of the Iro33020-HisTag protein is Tat-dependent in E. coli, in spite of the fact that the cytoplasmic membranes of A. ferrooxidans and E. coli are facing different pH. First, it has a characteristic twin-arginine signal sequence. Second, recombinant Iro33020-HisTag protein failed to be translocated to the periplasm in a tat mutant of E. coli (Fig. 3b
). These data indicate furthermore that the twin-arginine signal sequence of Iro from the acidophile A. ferrooxidans is recognized by the Tat system of the neutrophile E. coli. Computer searches have revealed a locus containing tatA, tatB and tatC genes (Wu et al., 2000
), a paralogous tatA gene and two tatD genes in the genome of A. ferrooxidans ATCC 23270.
The iro23270 gene is present not only in A. ferrooxidans strain ATCC 33020, but also in ATCC 19859 and in two private pure strains. The genetic organization of the iro locus differs depending on the strain: iro is located downstream from purA in strains BRGM and Fe-1 (Kusano et al., 1992; this study) and upstream from a putative leucyl-tRNA gene in strain Fe-1 (Kusano et al., 1992
), while it is located downstream from the petII operon encoding the second bc1 complex of A. ferrooxidans in ATCC 23270 and ATCC 19859 (Brasseur et al., 2002
; P. Bruscella, G. Levicàn, J. Ratouchniak, E. Jedlicki, D. S. Holmes and V. Bonnefoy, unpublished results) and ATCC 33020 (this study). Downstream, no tRNA gene was detected, but instead a putative gene encoding a protein with no similarity with proteins in the databases was found. The amino acid sequence of the mature Iro33020 has 58·3 % identity to the mature IroFe-1 while, for example, the mature rusticyanin from strain ATCC 33020 has 92·9 and 92·3 % identity to the mature rusticyanin from strains Fe-1 and BRGM, respectively (Guiliani et al., 1995
). Therefore, we can wonder whether the iroFe-1 and iro33020 are orthologous genes or encode HiPIPs with different functions. One possibility we cannot exclude is that IroBRGM was not purified from A. ferrooxidans but from another micro-organism, as strain BRGM, from which the iroBRGM gene and IroBRGM protein have been analysed (Cavazza et al., 1995
and this paper), is in fact a bacterial consortium (Battaglia-Brunet et al., 2002
; Collinet & Morin, 1990
). However, A. ferrooxidans strains have been reported by several authors to be physiologically and genomically diverse (Leduc & Ferroni, 1994
; Harrison, 1982
, 1984
) and to diverge in different phylogenetic groups (Goebel & Stackebrandt, 1994
; Karavaiko et al., 2003
; Novo et al., 1996
; Selenska-Pobell et al., 1998
). In this case, the simplest explanation is that strains Fe-1 and BRGM belong to a different group from the A. ferrooxidans strains analysed in this paper. The phylogenetic tree constructed from HiPIP amino acid sequences (Van Driessche et al., 2003
; data not shown) shows that IroFe-1, Iro23270 and Iro33020 are very close and supports the second hypothesis, according to which these two proteins are encoded by orthologous genes of A. ferrooxidans strains belonging to two different phylogenetic groups. Furthermore, the recombinant Iro33020-HisTag produced in E. coli has properties similar to those of IroFe-1 and IroBRGM (Fukumori et al., 1988
; Yamanaka & Fukumori, 1995
; Cavazza et al., 1995
).
Based on the current results, does Iro play the role proposed by Fukumori et al. (1988) and Yamanaka & Fukumori (1995)
or does it have another function? All the HiPIPs for which a role has been determined, except Iro, act as an electron shuttle between the bc1 complex and either the photosynthetic reaction centre complex (Schoepp et al., 1995
; Hochkoeppler et al., 1995
, 1996
; Menin et al., 1997
; Nagashima et al., 2002
; Verméglio et al., 2002
) or a terminal oxidase (Bonora et al., 1999
; Pereira et al., 1999
). Yamanaka and co-workers (Fukumori et al., 1988
; Yamanaka & Fukumori, 1995
) have proposed that the Iro protein is the first electron carrier in the ferrous iron respiratory chain, from which its name is derived. The high molecular mass cytochrome Cyc2 is a better candidate to play this role, as already discussed in our previous papers (Appia-Ayme et al., 1999
; Yarzábal et al., 2002
). Indeed, this cytochrome is an outer-membrane protein (Yarzábal et al., 2002
) and, because ferrous oxidation has been inferred to take place outside the cell, it is a more likely candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen than the periplasmic HiPIP. Furthermore, the cyc2 gene encoding this cytochrome belongs to the same operon as the genes encoding the cytochrome c4 Cyc1, rusticyanin and the aa3-type cytochrome oxidase (Appia-Ayme, 1998
; Appia-Ayme et al., 1999
), which have been proposed to be involved in ferrous iron oxidation (Yarzábal et al., 2004
). Interestingly, the petII-paralogous operon encoding the second cytochrome bc1 complex of A. ferrooxidans (Brasseur et al., 2002
) has been detected upstream from iro in several strains (this study; P. Bruscella, G. Levicàn, J. Ratouchniak, E. Jedlicki, D. S. Holmes and V. Bonnefoy, unpublished results). Furthermore, preliminary results have shown that iro belongs to the petII operon in strains ATCC 33020 and ATCC 19859 (P. Bruscella, G. Levicàn, J. Ratouchniak, E. Jedlicki, D. S. Holmes and V. Bonnefoy, unpublished results). Interestingly, the Iro redox potential (550 mV) determined in this paper is between that proposed for the bc1 complex of A. ferrooxidans (Elbehti et al., 1999
) and that for the terminal oxidase (Kai et al., 1992
). Therefore, we suggest that Iro is involved in an electron transfer chain between a cytochrome bc1 complex functioning in the forward direction and a terminal oxidase, as are other known HiPIP (Bonora et al., 1999
; Pereira et al., 1999
). We therefore propose to refer to this HiPIP protein as Hip, until its physiological role is determined, and to the corresponding gene as hip.
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ACKNOWLEDGEMENTS |
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Received 9 July 2004;
revised 25 October 2004;
accepted 17 January 2005.
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