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Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain
Biochimica et Biophysica Acta 1443 (1998) 99^112
Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain Abderrahmane Bengrine a , Nicolas Guiliani a , Corinne Appia-Ayme a , Eugenia Jedlicki b , David S. Holmes c , Marc Chippaux a , V. Bonnefoy a; * a
Laboratoire de Chimie Bacte¨rienne, Institut de Biologie Structurale et de Microbiologie, Centre National de la Recherche Scienti¢que, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France b Departamento de Bioquimica, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile c Departamento de Ciencias Biologicas, Facultad de Quimica y Biologia, Universidad de Santiago de Chile, Santiago, Chile Received 1 July 1998; received in revised form 5 September 1998; accepted 9 September 1998
Abstract The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Rusticyanin; Copper protein ; Electron transport; Iron oxidation; (Thiobacillus ferrooxidans)
1. Introduction Thiobacillus ferrooxidans is an acidophilic chemolithotrophic Gram-negative eubacterium, well adapted to pH environments as low as pH 1.6. It is able to derive all the energy required for nitrogen and carbon ¢xation through the biological oxidation
* Corresponding author. Fax: +33 (4) 91718914; E-mail: [email protected]
of ferrous iron (Fe2 ) to ferric iron (Fe3 ) [1^3]. The resulting Fe3 is a powerful oxidant in acid media oxidizing insoluble metallic sul¢des to their soluble form. This process, known as bioleaching, is currently used at an industrial scale for the recovery of several metals such as copper, uranium and cobalt from low grade ores [4] and in the bioprocessing of gold ores. Rusticyanin (Rus) is a periplasmic blue copper protein and is generally considered to be an important component in iron oxidation since it is present at high concentration (up to 5% of total soluble proteins) when T. ferrooxidans is grown on iron [1,5]. Its high redox potential (+670 mV) and its acid stability [5^7] have made it the most studied T. ferrooxidans
0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 9 9 - 7
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protein. However, its physiological role in the iron respiratory electron transport chain remains unclear. In di¡erent models, rusticyanin receives electrons from a c-type cytochrome [8] or a cP-type cytochrome [9] and gives electrons to cytochrome c4 [9] or to another unidenti¢ed electron transporter [8]. Other investigators give no signi¢cant role for rusticyanin in the iron respiratory chain [2,10]. Due to the absence of mutants lacking the protein, its exact function awaits con¢rmation. Given the proposed important role of rusticyanin in the iron metabolism of T. ferrooxidans, several laboratories tried to clone its structural gene. However, this cloning was problematic since this gene cannot be found by genetic complementation of an Escherichia coli mutant. Two labs have reported its cloning by screening an expression library with an antibody directed against rusticyanin [11,12], but no sequence has followed up these works suggesting that false positive clones were indeed obtained. This prompted Casimiro et al. [13] to construct a synthetic gene using the E. coli codon usage. It was only recently that fragments of the rus gene were obtained by ampli¢cation in polymerase chain reaction (PCR) with degenerate primers deduced from the known rusticyanin amino acid sequences ([14^17]; this paper). As soon as a marker exchange mutagenesis will be possible in ATCC33020 strain, we wish to construct rus mutants to investigate rusticyanin function in T. ferrooxidans. For this purpose, cloned internal fragments of the gene and an arti¢cial gene would be inadequate. This paper reports the cloning and complete sequencing of the ATCC33020 strain rus gene with its 5P and 3P regions by combining PCR and inverse PCR approaches. Its regulation in T. ferrooxidans and its expression in E. coli are also reported. 2. Materials and methods 2.1. Strains, plasmids and growth conditions T. ferrooxidans ATCC33020 was obtained from the American Type Culture Collection. E. coli TG1 strain (supE hsdv5 thi v(lac-proAB) FP:traD36 proAB lacIq lacZvM15) was used for phagemid propagation. BL21(DE3) strain (F3 ompT hsdSB [VcIts857
ind1 Sam7 nin5 lacUV5-T7 gene1]) was purchased from Novagen. Phagemid Bluescript was purchased from Stratagene and the plasmids pET22b and pET21 from Novagen. E. coli strains were grown in L-broth according to Miller [18]. Conditions to grow T. ferrooxidans on iron medium have been described [19]. Sulfur medium consisted of 25% 9K basal salts pH 3.5, as described in Guiliani et al. [19], and 1% sulfur. The thiosulfate medium is the same as above with 0.07% thiosulfate. 2.2. DNA manipulations General techniques were performed according to Ausubel et al. [20]. Ultrapure plasmid DNA was obtained using the Qiagen plasmid kit. T. ferrooxidans chromosomal DNA was prepared from 200 ml iron medium culture. Cells were harvested and washed several times with 10-fold diluted 9K [19] to remove precipitated iron. The pellet was resuspended in 200 Wl of 10 mM Tris-HCl pH 8, 1 mM EDTA, then gently mixed with 80 Wl 10% SDS, 80 Wl 0.5 M EDTA pH 8 and 10 Wl 10 mg/ml RNase A and incubated for 15 min at room temperature. The suspension was frozen at 320³C for 15 min. After thawing at 55³C for 5 min, 50 Wl 3 mg/ml proteinase K were added. The mixture was incubated for 10 min at 55³C. DNA from lysed cells was submitted to phenol extraction, followed by phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol extractions. It was precipitated with ethanol and resuspended in 150 Wl Tris-HCl pH 8, 1 mM EDTA. Restriction enzyme digestions were performed according to the manufacturer's recommendations. DNA fragment blunting and ligation were performed with the DNA blunting kit from Amersham. Ligations were generally carried out in the presence of the restriction enzyme used to cleave the vector to prevent its recircularization. Southern blotting was performed with T. ferrooxidans total DNA digested with di¡erent restriction enzymes. After electrophoresis, the DNA was denatured and transferred to a positively charged nylon membrane (Hybond-N from Amersham) by the semi-dry capillary method [21]. Prehybridization
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and hybridization were performed under stringent conditions. DIG-labeled probes were obtained by PCR as described by Boehringer Mannheim between the T3 and T7 primers encompassing the rus internal fragment obtained by DOP PCR and cloned in the SmaI site of the Bluescript vector (pINTrus plasmid, see below). Detection was accomplished by a chemiluminescent reaction with Lumigen PPD as described by Boehringer Mannheim. The dideoxy chain termination method was used to sequence DNA using K-35 S-dATP and the T7 Sequence Kit purchased from Pharmacia. 2.3. Cloning of rus fragments by DOP PCR and inverse PCR Five degenerate oligonucleotides (purchased from Appligene) able to form six pairs of convergent primers were designed on the basis of the highly conserved regions of rusticyanin [22^24] and using the most favorable codon usage in strain ATCC33020 [25]. Taq polymerase from Boehringer was used according to the manufacturer's recommendations. Using 400 pmol of oligonucleotides ARus57 and Rus 115 (Table 1) in a 50 Wl reaction, a 195 bp DNA fragment was ampli¢ed from T. ferrooxidans total DNA (30 ng/reaction). PCR ampli¢cations were as follows: 5 min at 94³C followed by 30 cycles at 94³C for 30 s, 37³C for 30 s and 72³C for 30 s, then 5 min at 72³C. The 195 bp fragment was recovered, puri¢ed with the Mermaid kit from Bio101, bluntended with T4 DNA polymerase and cloned at the SmaI site of the Bluescript SK vector to give the pINTrus vector. Ampli¢cation of £anking sequences by inverse PCR was described by Ochman et al. [26]. PCR reactions with Rus2, Rus4, Rus9M and Rus7 primers (Table 1) were performed on total T. ferrooxidans DNA previously digested with HaeIII and religated, as follows: 5 min at 94³C, followed by 30 cycles at 94³C for 30 s, 65³C for 30 s and 72³C for 30 s and a ¢nal incubation of 5 min at 72³C. In the case of inverse PCR with TaqI, a two step protocol was followed: 5 min at 94³C, 30 cycles at 94³C for 30 s and 68³C for 2 min 30 s and a ¢nal incubation of 5 min at 72³C. PCR-ampli¢ed DNA fragments were puri¢ed with Prep-A-Gene from Bio-Rad and cloned at the SmaI site of the Bluescript vector.
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2.4. RNA manipulations T. ferrooxidans total RNA was prepared from 500 ml as described previously [27]. Northern blotting was performed using formaldehyde gels as described in Ausubel et al. [20]. RNA was transferred to a positively charged nylon membrane from Boehringer Mannheim by capillary blotting. The DIG-labeled RNA probe was obtained from the SK/RSD2BamHI-RusCXhoI plasmid described in Section 2.5. In vitro transcription with T7 RNA polymerase was performed on this plasmid linearized with EcoRI using DIG-UTP as substrate according to the instructions of the Boehringer DIG RNA labeling kit. Prehybridization and hybridization with DIG-labeled probe were performed under stringent conditions according to Boehringer Mannheim. RNA was detected as described above for Southern experiments. Primer extension has been performed according to the Promega Access RT-PCR system omitting the PCR step. The primers used were Rusv or Rusv2 oligonucleotides (Table 1) labeled with Q-32 P-dCTP. Coupled reverse transcription and PCR ampli¢cation (RT-PCR) were performed with the Promega Access RT-PCR system. Approx. 1 Wg of total RNA from T. ferrooxidans was denatured at 94³C for 2 min in the presence of both oligonucleotides. Immediately, reverse transcription and PCR ampli¢cation were carried out according to Promega recommendations with the following program: 48³C for 50 min; 94³C for 5 min; 30 or 40 cycles of 30 s at 94³C, 30 s at 62³C, 1 min at 72³C; 72³C for 3 min. Oligonucleotides used were: RusC, Rus5, Rusv, RusAmF, ARusv, ARusv3 and RusAvF (Table 1). For each RT-PCR experiment, three controls were performed: one without template as a negative control to detect any contamination, one with genomic DNA as a control for PCR ampli¢cation, and one with RNA but without reverse transcriptase to check that there were no DNA traces in the RNA preparation. In some cases, RNAse was added to the RNA before performing the RT-PCR experiment. 2.5. Construction of pETrus and pCrus plasmids Oligonucleotides RusNm and RusC (Table 1) were used to amplify the DNA fragment encoding the
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mature rusticyanin from T. ferrooxidans total DNA and the product was blunt-ended by T4 DNA polymerase and cloned into the blunt ended XhoI-NcoI sites of the pET22b vector to yield the pETrus plasmid. In this construction, the sequence of the mature rusticyanin was (i) preceded and in frame with the nucleotidic sequence encoding the PelB classical Gram-negative signal sequence; and (ii) followed by an in frame sequence coding for a His-Tag which allows a one-step puri¢cation of the produced polypeptide. An attempt to place directly the rus gene under the control of bacteriophage T7 transcriptional, but not translational, regulatory elements (pCrus), failed. Therefore, the following strategy was followed. DNA encompassing the ribosome binding site to the translational termination site of the rus gene was ampli¢ed from T. ferrooxidans total DNA with RSD2BamHI and RusCXhoI oligonucleotides (Table 1). This ampli¢ed product was then cloned in the EcoRV site of the SK Bluescript phagemid. At the same time, the interposon 6-Smr , obtained by HindIII digestion of pHP456-Smr [28], was cloned in the HindIII site of the pET21 plasmid. This pET216-Smr plasmid was then easily cut by BamHI and XhoI. The fragment corresponding to the pET21 plasmid was puri¢ed and ligated to the BamHIXhoI fragment originating from the RSD2BamHIRusCXhoI ampli¢ed DNA cloned in the SK Bluescript vector. The pETrus and pCrus clonings were done in E. coli TG1 strain which lacks the T7 polymerase structural gene and therefore cannot express the target gene. Screening was performed by PCR with the oligonucleotides used to amplify the rus DNA. In all cases, the sequence of the cloned fragment was checked. These plasmids were then introduced into the E. coli BL21(DE3) strain which carries the T7 RNA polymerase structural gene under the control of the IPTG-inducible lacUV5 promoter. 2.6. Protein analysis The E. coli BL21(DE3) strain carrying the plasmid of interest was grown at 37³C, 30³C or 20³C in L-broth. When the culture reached an optical density at 600 nm of 1, 1 mM, 250 WM or 50 WM IPTG was added. Cells were harvested after 2 h and submitted
to spheroplasting. E. coli spheroplasts were prepared from 1 g of pelleted cells. Cells were resuspended with 10 ml of 0.2 M Tris-HCl, pH 8. This suspension was mixed with 10 ml of 0.2 M Tris pH 8, 34% sucrose. 100 mM EDTA (¢nal concentration) was added at room temperature and under magnetic stirring. After 2.5 min, 100 mg/ml lysozyme was added and 2.5 min later, 20 ml water. Spheroplasts were then isolated by 15 min centrifugation at 1500Ug and resuspended in 20 ml of 0.04 M Tris, pH 7.6. The supernatant was considered as the periplasmic fraction. A sample of 100 Wl of each fraction (total cells, spheroplasts or periplasm) was mixed with 50 Wl TS/TD loading bu¡er (2 vols. of 0.2 M Tris-HCl pH 8.8, 5 mM EDTA, 1 M sucrose, 0.1% bromothymol blue mixed to 1 vol. of 18% SDS, 0.3 M DTT, 5% L-mercaptoethanol). Total cells and spheroplasts were boiled for 10 min before applying 4 Wl or 25 Wl to 20% SDS-PAGE using either Pharmacia PhastSystem or Protean vertical electrophoresis system from Bio-Rad respectively according to the manufacturer's instructions. A 500 ml T. ferrooxidans ATCC33020 culture grown in iron medium was washed several times to remove residual iron and then transferred to 10 l iron, sulfur or thiosulfate medium. Samples were removed all along the growth from the di¡erent cultures. Periplasmic proteins were prepared from all these samples as previously described [29]. Protein quanti¢cation was performed according to the protocol of Lowry et al. [30]. Equal amounts of proteins from iron-, sulfur- or thiosulfate-grown cells were analyzed by SDS-PAGE. Proteins were either stained with Coomassie brilliant blue (PhastSystem Development) or transferred to a nitrocellulose membrane (Schleider and Schuell) with the PhastSystem blot transfer system (Pharmacia) or the Trans-Blot SD Semi-Dry transfer Cell system (Bio-Rad). Rusticyanin was identi¢ed by immunological cross-reactivity to antibody directed against rusticyanin according to the protocol described in Sambrook et al. [21]. 2.7. Sequence analysis The DNA sequences were compiled, analyzed and compared with the data bank through the Nestcape facilities. The EMBL data bank accession number of the rus gene is X95823.
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Table 1 Oligonucleotides used in this study Name
Sequence
Localization and orientationa
ARus57
5P-CAYGAYRWRAARAAYCCNAC-3P
Rus115
5P-TARCCRAAYTTRCCRTCYTT-3P
HDKKNPT (positions 57^63b ) positions 1599^1618 (+) KDGKFGY (positions 116^122b ) positions 1776^1795 (3) positions 1626^1644 (+) positions 1710^1729 (3) positions 1765^1784 (+) positions 1500^1519 (3) positions 1431^1460 (+) positions 1869^1896 (3) positions 1316^1337 (+) positions 1870^1896 (3) positions 1443^1464 (3) positions 1271^1290 (3) positions 1857^1877 (+) to position 15d (+) from position 2148e (3) positions 663^681 (+) positions 1201^1219 (+)
Rus7 Rus2 Rus4 Rus9M RusNm RusC RSD2BamHIc RusCXhoIc Rusv Rusv2 Rus5 RusAmF RusAvF ARusv ARusv3
a The positions refer to the nucleotide sequence submitted to EMBL data bank and to Fig. 1. (+) corresponds to the rus coding strand and (3) to the rus non-coding strand. b The positions indicated here correspond to the sequence of the mature rusticyanin. c Nucleotides have been added between the restriction site and the 5P end oligonucleotide according to the restriction endonuclease requirement. d The sequence of the 5P end of this oligonucleotide has only been determined on one strand and is not given in the sequence submitted to EMBL data bank and in Fig. 1. e The sequence of the 3P end of this oligonucleotide has only been determined on one strand and is not given in the sequence submitted to EMBL data bank and in Fig. 1.
3. Results 3.1. Sequencing of the rus gene Since our long term goal is to construct rus mutants of T. ferrooxidans, we have decided to look for the rus gene in a known pure strain which is genetically stable and in which molecular biology has been shown to be feasible. ATCC33020 strain ¢ts all these conditions. Using degenerate oligonucleotides based upon the amino acid sequences of mature rusticyanin puri¢ed from three T. ferrooxidans strains [22^24], a fragment of the expected size (195 bp) was ampli¢ed from ATCC33020 genomic DNA by PCR. This fragment was cloned and sequenced. The deduced amino acid sequence matched the expected internal fragment of rusticyanin. This DNA fragment was used
as a probe in Southern hybridization experiments with ATCC33020 total DNA. With undigested DNA, the signal corresponded to chromosomal DNA whereas, when the DNA was digested with di¡erent restriction enzymes, only one DNA fragment hybridized with the probe in each case (data not shown), indicating that strain ATCC33020 carries a single chromosomal copy of the rus gene. Two divergent oligonucleotides designed on the basis of the determined sequence were used in inverse PCR experiments. From the sequence of the ampli¢ed fragment, a new pair of divergent oligonucleotides was designed for subsequent use. This approach of chromosome walking on either side of the rus DNA fragment led to the determination of a DNA sequence of 2164 bp encompassing the rus gene (EMBL GenBank accession No. X95823) (Fig. 1).
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Fig. 1.
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Fig. 1. Nucleotide sequence of the rus gene region from T. ferrooxidans. The deduced amino acid sequences of ORFA, ORF1 and ORF2 and rus are shown. Putative Shine and Dalgarno sequences are indicated in bold. Putative transcription termination signals are represented by arrows above the sequence. Potential c70 -speci¢c promoter sequences are underlined. The +1 labeled arrow corresponds to the rus transcriptional start site. The rusticyanin signal peptide is underlined.
3.2. Analysis of the rus gene The sequence corresponding to the known mature rusticyanin is located between positions 1431 and 1896 (Fig. 1). Two possible in frame translation starts (positions 1335 and 1353) are present upstream from this sequence (Fig. 1). However, only the ATG1335 is preceded 12 bp upstream by a good match to the canonical E. coli Shine-Dalgarno sequence (TAAGGAG), indicating that the rus open reading frame (ORF) starts at position 1335. The nucleotide sequence of the rus gene coding for the mature rusticyanin is 92.7% homologous to the rus sequence from strain ATCC23270 [17] and 93.1% to the partial rus sequence from strain ATCC19859 [16], nine out of ten di¡erences being on the third position of the codons. The deduced polypeptide is 92.3^98.7% identical to the sequences of the known mature rusticyanins [14] and almost all the changes are conservative, indicating that the mature rusticyanin is very well conserved among the di¡erent isolates of T. ferrooxidans. For example, only two conservative di¡erences, Ser to Thr and Val to Ile, are observed with the sequence reported by Ronk et al. [23]. Starting from the potential translational initiation ATG (position 1335) down to the codon corresponding to the ¢rst residue of the mature protein (position 1431) the amino acid sequence presents all the features expected for the signal sequence of a periplasmic protein, including a consensus cleavage sequence Ala-Met-Ala situated appropriately just upstream from the start of the mature protein (Fig. 1).
3.3. Analysis of the ORFs present upstream from the rus gene In addition to the rus gene encoding the rusticyanin protein, three open reading frames, ORFA (to position 160), ORF1 (positions 179^728) and ORF2 (positions 771^963), are located upstream from rus (Fig. 1). We have shown that ORF1 and ORF2 are transcribed (see below). They are preceded by a correctly positioned potential Shine-Dalgarno sequence (Fig. 1). ORF1 would encode a polypeptide of 183 residues with a calculated molecular mass of 20 213 Da, ORF2 a 64 residue polypeptide of 7212 Da. No signi¢cant homologies were detected for ORF1 and ORF2 when submitted to a search in protein data banks. An analysis of the hypothetical ORF1 and ORF2 polypeptides using the `TM pred' program [31] reveals that they could potentially encode integral inner membrane proteins, containing ¢ve and one transmembrane helices respectively. 3.4. Analysis of the intergenic regions The untranslated region between ORF2 and rus is 369 bp long and contains two potential E. coli-like c70 -speci¢c promoter sequences with correctly spaced 310 and 335 boxes (Fig. 1). The 3P £anking region of ORF2 has a sequence similar to a rho-independent transcription terminator consisting of a stemloop structure (vG of 320.6 kcal/mole) followed by the sequence TTGTTTT (Fig. 1) suggesting that ORF2 and the rus gene are independently transcribed. Two potential stem-loop structures (vG values of 317.1 and 328.4 kcal/mol) were found down-
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strongly suggests that the rusticyanin protein is constitutively synthesized in strain ATCC33020. 3.6. Transcription of rus gene in T. ferrooxidans ATCC33020 strain
Fig. 2. Western analysis of rusticyanin synthesis in T. ferrooxidans ATCC33020. Periplasmic proteins from T. ferrooxidans cells were prepared after 1, 2, 3 or 4 days of growth on ferrous iron or sulfur. Equal amounts of proteins were applied to a SDS-PAGE and transferred to a nitrocellulose membrane. Rusticyanin is identi¢ed with an antibody directed against rusticyanin.
According to the sequence data, the 369 bp untranslated region between ORF2 and rus gene contains a putative rho-independent transcriptional termination site and two E. coli c70 -type promoters suggesting that ORF2 and rus gene are independently transcribed (Fig. 1). To check this hypothesis, the rus transcripts were studied by Northern blot with total RNAs from T. ferrooxidans ATCC33020 strain and an in vitro synthesized RNA probe. Two major transcripts of approx. 1200 and 850 nucleotides were detected (Fig. 3A). Furthermore, several minor transcripts were also observed whose sizes are comprised between 6000 and 300 nucleotides (Fig. 3B). All the transcripts were shown to be speci¢c to rus since no signals could be detected by Northern blotting with a RNA probe prepared from the same plasmid lacking the rus insert (data not shown). To con¢rm the presence of the large transcripts, we have used the RT-PCR. This extremely sensitive
stream from rus gene, the last one being followed by a T rich region (Fig. 1). These structures could correspond to the rus transcriptional termination site. 3.5. Analysis of rusticyanin synthesis in T. ferrooxidans ATCC32020 strain Equal amounts of T. ferrooxidans periplasmic proteins, obtained from iron-, thiosulfate- or sulfurgrown cells were analyzed by SDS-PAGE using Coomassie blue staining. It was observed that a protein band corresponding to rusticyanin was present in all three samples (data not shown). The same experiment was repeated using Western blot analysis with a polyclonal antibody directed against the rusticyanin. In ATCC33020 strain, rusticyanin is synthesized in iron- as well as in sulfur- (Fig. 2) or thiosulfategrown cells even after 12 days of growth representing approx. 36 cellular divisions (data not shown). This
Fig. 3. Northern blot of total T. ferrooxidans RNA (3.5 Wg) probed for rus mRNA with DIG-UTP labeled rus RNA. (A) 15 min exposure; (B) 2 h exposure.
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Fig. 4. RT-PCR on T. ferrooxidans total RNA. (A) Localization of the oligonucleotides used for RT-PCR experiment. (B) RT-PCR experiments between RusAmF and Rusv (I), Rus5 and RusAvF (II) oligonucleotides with no added template (0), T. ferrooxidans chromosomal DNA (D), total RNA extracted from Fe2 -grown cells (R), (3) as in (R) but without reverse transcriptase. MW, 1 kb molecular weight ladder from Boehringer Mannheim.
system couples the reverse transcription of a speci¢c target RNA from total RNA (RT) and PCR ampli¢cation of this cDNA. Using di¡erent oligonucleotides hybridizing upstream from rus gene and a convergent one inside of the rus open reading frame (see Section 2 and Fig. 4), this approach has permitted the detection of a transcript su¤ciently large to incorporate transcription from ORFA to rus (Fig. 4 and data not shown). By following the same approach, we have shown that no ampli¢cation product was obtained with the oligonucleotides Rus5 and RusAvF located respectively inside rus gene and at the end of the sequence determined (Fig. 4). This suggests that the rus gene is the last gene of an operon comprising at least ORF1 and ORF2. The di¡erent rus transcripts observed can be due either to the degradation of RNA during their preparation, to mRNA processing, or to the presence of several promoters for the rus gene. We tested the third possibility by carrying out primer extension experiments, using total RNAs and several oligonucleotides to prime the reaction. In all the cases, several very minor large bands were observed all along the gel con¢rming that some rus transcripts are initiated far upstream from the rus translational initiation site. Furthermore, with two di¡erent oligonucleotides (Rusv and Rusv2), the same 5P end of a major rus transcript was detected corresponding to a G (position 1171) located 166 bases upstream from
the translation initiation site (Fig. 5). It is noteworthy that, upstream from this putative translation initiation site, an E. coli-like Pribnow box, TATtAT (positions 1157^1162), and a 335 hexamer, TTGAtc (positions 1134^1139) are present (Fig. 1). 3.7. Expression of the rus gene in E. coli One explanation put forward to account for the repeated failures to clone the rus gene was that the Rus signal sequence could be speci¢c for T. ferrooxidans and not be recognized by the E. coli leader peptidase. This could result in the jamming of the inner membrane with concomitant selection against recombinant clones containing an expressed Rus protein. To circumvent this potential problem, the DNA fragment encoding the mature rusticyanin fused to a classical Gram-negative signal sequence (PelB) was ¢rst under the control of T7 transcriptional and translational signals to yield pETrus plasmid (see Section 2). In BL21(DE3)/pETrus IPTG-induced cells, no polypeptide corresponding to free rusticyanin could be detected in the periplasmic £uid (data not shown). However, two polypeptides of about 18 kDa and 20 kDa, absent from the cells before induction, were detected in the spheroplasts, the largest being more abundant (data not shown and [14]). The amino-terminal of the minor 18 kDa polypeptide exactly matched that of the mature aporusticyanin.
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Fig. 5. Primer extension experiment performed with Rusv2 oligonucleotide on 10 Wg of T. ferrooxidans total RNA (E). Lanes A, C, G and T represent the nucleotide sequence of the rus coding strand performed with Rusv2 oligonucleotide on the DNA fragment obtained by PCR between Rusv and ARusv oligonucleotides.
The ¢rst 29 residues of the largest corresponded to the PelB signal sequence still fused to the mature aporusticyanin sequence. The ine¤cient cleavage of the PelB signal sequence has previously been reported for other secreted proteins overexpressed from the T7 system in E. coli. It is likely due to the uncoupling between translation and translocation. The ratio between precursor to mature polypeptide can usually be reversed by lowering the growth rate and/or by decreasing the induction rate. Accordingly, we have compared the ratio of the precursor to the mature aporusticyanin when the BL21(DE3)/ pETrus cells were grown at 37³C, 30³C or 20³C or in the presence of 50 WM, 250 WM or 1 mM IPTG, respectively. As shown in Fig. 6A (lanes 2, 6, 10), 50 WM IPTG was su¤cient for full induction of the pelB-rus gene under T7 RNA polymerase. However, this did not in£uence the ratio between precursor and mature rusticyanin (lanes 2, 6 or 10 versus lanes 3, 7 or 11 or versus lanes 4, 8 or 12, respectively). In contrast, lowering the growth temperature to 20³C dramatically improved and almost inverted the ratio (Fig. 6A, lanes 2, 3 or 4 versus lanes 10, 11 or 12). Unexpectedly, the mature aporusticyanin was detected in the membrane fraction (Fig. 6A) and none in the periplasmic £uid (data not shown). Washing these membrane fractions with 1 M sodium acetate
Fig. 6. Expression of rus gene in E. coli. (A) Membrane proteins from E. coli BL 21 carrying pETrus plasmid (lanes 2^13). Cells were grown at 20³C (lanes 2^5), 30³C (lanes 6^9) or 37³C (lanes 10^14). Cells were induced with 50 WM (lanes 2, 6, 10), 250 WM (lanes 3, 7, 11) or 1 mM (lanes 4, 8, 12) IPTG, or not induced (lanes 5, 9, 13). Lane 14, membrane proteins from E. coli TG1 strain carrying pETrus grown at 37³C and induced with 1 mM IPTG. Lane 1, protein standards. Arrows show the precursor and the mature rusticyanin. (B) Total proteins from E. coli BL21 carrying pCrus plasmid. Cells were grown at 37³C and induced with 1 mM IPTG (+) or not induced (3). M, protein standards. Arrows show the precursor and the mature form of rusticyanin.
or lowering their pH to 3.5 disrupted such non-speci¢c interactions (data not shown). It is concluded that mature aporusticyanin can be overexpressed in the periplasmic space of E. coli without being degraded and without jamming the cytoplasmic membrane. In additional experiments, the DNA fragment corresponding to the entire rus gene was cloned under control of T7 transcriptional signals in the pET21 plasmid to yield plasmid pCrus. Two IPTG-inducible
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polypeptides were also detected in the spheroplast fraction (Fig. 6B). Determination of the NH2-end sequences revealed that they corresponded to the mature and precursor rusticyanin polypeptides respectively, indicating that the rusticyanin signal sequence is functional in E. coli. Utilizing the approach discussed above, it was observed that the processed rusticyanin was bound to the periplasmic side of the cytoplasmic membrane by non-speci¢c interactions (data not shown). One striking feature of cell free extracts containing rusticyanin in which copper is correctly inserted is a deep blue color and an absorbance peak around 600 nm which disappears upon reduction by Fe2 [32]. Membrane, cytoplasmic and periplasmic fractions of BL21(DE3)/pETrus and BL21(DE3)/pCrus cells induced with IPTG were compared to that of non-induced BL21(DE3)/pETrus, BL21(DE3)/pCrus or BL21(DE3)/pET22b cells. No blue color could be observed and no spectroscopic di¡erence could be detected whatever the growth temperature (data not shown), indicating the lack of copper insertion in the rusticyanin polypeptide expressed in the E. coli periplasm. 4. Discussion We have determined all the rus gene sequence together with about 270 bp downstream and 1430 bp upstream from this gene ([14,15]; this paper). As expected, the amino acid sequence deduced from the nucleotide sequence is highly homologous to the rusticyanin sequences reported for various T. ferrooxidans strains ([16,17,22^24]; Ingledew et al. cited in [33]). As the T. ferrooxidans species comprises di¡erent DNA homology groups and is genomically more diverse than usual for a single species [34], this could indicate strong constraints during evolution to maintain a high redox potential and a stability at low pH. The predicted rusticyanin polypeptide includes a classical cleavable leader sequence that is recognized by the E. coli secretion machinery and leader peptidase. The mature polypeptide produced in E. coli remains associated with the periplasmic side of the cytoplasmic membrane through unspeci¢ed interactions that can be broken by acid or salt treatment. A possibility is the likelihood of non-speci¢c attach-
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ments due to incorrect folding and/or lack of appropriate protonation at pH 6.5 in the periplasm of E. coli. Several arguments support this explanation: (i) Cobley and Haddock [35] and Mansch and Sand [36] have shown that rusticyanin remains bound to the T. ferrooxidans membrane when the cells are broken at pH 7 instead of pH 2; (ii) two type I `blue' copper proteins, azurin [37] and plastocyanin [38], from neutrophilic organisms can be overproduced in the E. coli periplasm with the copper correctly inserted; (iii) the rusticyanin expressed in the cytoplasm [13] of E. coli lacks copper but this metal can be incorporated in vitro by lowering the pH to 3.4^5.5 [13]. This recombinant reconstituted holoprotein displayed a tertiary structure identical to that of the protein extracted from T. ferrooxidans [13]; (iv) homology modeling studies, on the basis of the blue copper protein core structures determined by X-ray absorption, indicate that the surface of the rusticyanin shows a large number of polar/charged residues and particularly a high number of uncompensated lysine residues [39^41]. Thus, it is possible that the basic amino acids of rusticyanin interact electrostatically with the negatively charged phospholipid head group of the cytoplasmic membrane of E. coli. In agreement with this hypothesis, a paper of Kar and Dasgupta [42] states that, unlike most of the naturally occurring membranes, the membrane surface in T. ferrooxidans is positively charged at, or near, its physiological pH. This would lead to the repelling of the rusticyanin away from the membrane. Northern, RT-PCR and primer extension analyses of rus gene transcription in T. ferrooxidans ATCC33020 strain have shown that the rus gene can be co-transcribed with the upstream genes, that is at least ORF1 and ORF2. The rus gene is the last gene of this operon as shown by RT-PCR experiments. Several minor (from 6000 to 300 nucleotides) and two major (1200 and 850 nucleotides) transcripts can be detected by Northern experiments. One model that can account for these results is that the rus gene is expressed from alternative internal promoters. Thus, in the 369 bp untranslated region between ORF2 and rus, the 5P end of a transcript has been determined by primer extension. It is noteworthy that this putative transcription initiation site is located 806 bp upstream from the proposed rus transcriptional termination site (Fig. 1). The correspond-
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ing transcript would then have approximately the size of the major rus transcripts detected by Northern experiments. Altogether, this suggest that the rus major transcript originates from an internal promoter located between ORF2 and rus gene. A possible E. coli-type c70 promoter sequence (TTGAtc-17 bpTATtAT) is indeed located upstream from this transcription initiation site. On the other hand, the minor transcripts could correspond to degraded or processed products of a large RNA corresponding to the entire operon. In agreement with this proposal, bands smaller than the size of the rus open reading frame have been detected showing that, indeed, some degradation occurs. In strain ATCC33020, we have found that the rusticyanin is synthesized in iron as well as sulfur, or thiosulfate, growth conditions. Now, one of the major arguments to involve rusticyanin in iron rather than sulfur oxidation [32] is that it constitutes up to 5% of all soluble proteins in iron-grown cells [5,35] whereas it is barely detectable in Torma strain cells grown on sulfur [11]. However, for strains Tf-3 [43], ATCC13661 [12] and ATCC19859 ([44,45]; A. Bengrine, V. Bonnefoy, unpublished data) rusticyanin is synthesized whatever the electron donor present in the growth medium and its level could increase to some extent in the presence of high concentrations of Fe2 . Furthermore, the disappearance [46^48] or the maintenance for many generations [36,44,48^50] of the Fe2 oxidation capacity has been reported on di¡erent strains when the cells were shifted from iron to sulfur growth conditions. As already pointed out, the T. ferrooxidans species constitute a genomically diverse group [34]. Therefore, these contradictory data might just re£ect the variability among T. ferrooxidans strains. It is even possible that these discrepancies may represent actual di¡erences in the electron transfer pathways between T. ferrooxidans strains. If this were the case, rusticyanin could play a pivotal role in the iron and reduced sulfur compounds' energetic metabolism of some strains of T. ferrooxidans as ATCC33020. In that case, the disruption of its structural gene by marker exchange mutagenesis could be detrimental. Preliminary results have already been presented at the Minerals Bioprocessings conference (Snowbird, UT) in 1994 (Guiliani et al. [15]), at the Biohydrometallurgical Processing symposium (Santiago,
Chile) in 1995 (Bengrine et al. [14]) and at the Molecular Genetics of Bacteria and Phages meeting (Cold Spring Harbor, NY) in 1996. Acknowledgements We owe special thanks to V. Me¨jean for helpful advice on PCR experiments and for critical reading of the manuscript. We gratefully acknowledge J. Ratouchniak for excellent technical assistance. We are indebted to F. Nunzi from M. Bruschi lab (B.I.P., I.B.S.M., Marseille) for providing the rusticyanin antibody and the rusticyanin amino acid sequence prior to publication. We are grateful to the Centre de Se¨quenc,age d'ADN and to the Unite¨ de Se¨quence Prote¨ique of the I.B.S.M. (Marseille). NG and AB acknowledge the support of a Graduate Scholarship from l'Agence de l'Environnement et de la Ma|ªtrise de l'Energie (A.D.E.M.E.). This work was supported by A.D.E.M.E., the Bureau de Recherche Ge¨ologique et Minie©re (B.R.G.M.) and the Compagnie Ge¨ne¨rale de Mate¨riaux (CO.GE.MA) and ECOS/CONICYT award to VB, EJ and DH.
References [1] J.G. Cobley, J.C. Cox, Energy conservation in acidophilic bacteria, Microbiol. Rev. 47 (1983) 579^595. [2] T. Yamanaka, Y. Fukumori, Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans, FEMS Microbiol. Rev. 17 (1995) 401^413. [3] T. Yamanaka, T. Yano, M. Kai, H. Tamegai, Y. Fukumori, The electron transport system coupled to the oxidation of Fe2 , in: A.E. Torma, M.L. Apel, C.L. Brierley (Eds.), Biohydrometallurgical Technologies, Vol. II, The Minerals, Metals and Material Society (TMS), Warrendale, PA, 1993, pp. 453^463. [4] C.L. Brierley, Bacterial leaching, Crit. Rev. Microbiol. 6 (1978) 207^262. [5] J.C. Cox, D.H. Boxer, The puri¢cation and some properties of rusticyanin, a blue copper protein involved in iron(II) oxidation from Thiobacillus ferrooxidans, Biochem. J. 174 (1978) 497^502. [6] W.J. Ingledew, J.G. Cobley, A potentiometric and kinetic study on the respiratory chain of ferrous iron grown Thiobacillus ferrooxidans, Biochim. Biophys. Acta 683 (1980) 89^ 117. [7] A.G. Lappin, C.A. Lewis, W.J. Ingledew, Kinetics and
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[35] J.G. Cobley, B.A. Haddock, The respiratory chain of Thiobacillus ferrooxidans: the reduction of cytochromes by Fe2 and the preliminary characterization of rusticyanin a novel `blue' copper protein, FEBS Lett. 60 (1975) 29^33. [36] R. Mansch, W. Sand, Acid-stable cytochromes in ferrous ion oxidizing cell-free preparations from Thiobacillus ferrooxidans, FEMS Microbiol. Lett. 92 (1992) 83^88. [37] B.G. Karlsson, T. Pascher, M. Nordling, R.H.A. Arvidsson, L.G. Lundberg, Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, FEBS Lett. 246 (1989) 211^217. [38] M. Nordling, T. Olausson, L.G. Lundberg, Expression of spinach plastocyanin in E. coli, FEBS Lett. 276 (1990) 98^ 102. [39] J.G. Grossmann, W.J. Ingledew, I. Harvey, R.W. Strange, S.S. Hasnain, X-Ray absorption studies and homology modeling de¢ne the structural features that specify the nature of the copper site in rusticyanin, Biochemistry 34 (1995) 8406^ 8414. [40] M.V. Botuyan, A. Toy-Palmer, J. Chung, R.C. Blake II, P. Beroza, D.A. Case, H.J. Dyson, NMR solution structure of Cu(I) rusticyanin from Thiobacillus ferrooxidans: structural basis for the extreme acid stability and redox potential, J. Mol. Biol. 263 (1996) 752^767. [41] R.L. Walter, S.E. Ealick, A.M. Friedman, R.C. Blake II, P. Proctor, M. Shoham, Multiple wavelength anomalous diffraction (MAD) crystal structure of rusticyanin: a highly oxidizing cupredoxin with extreme acid stability, J. Mol. Biol. 263 (1996) 730^751. [42] N.S. Kar, A.K. Dasgupta, The possible role of surface charge in membrane organization in an acidophile, Indian J. Biochem. Biophys. 33 (1996) 398^402. [43] J.C. Cox, D.H. Boxer, The role of rusticyanin, a blue copper protein, in the electron transport chain of Thiobacillus fer-
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Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain Abderrahmane Bengrine a , Nicolas Guiliani a , Corinne Appia-Ayme a , Eugenia Jedlicki b , David S. Holmes c , Marc Chippaux a , V. Bonnefoy a; * a
Laboratoire de Chimie Bacte¨rienne, Institut de Biologie Structurale et de Microbiologie, Centre National de la Recherche Scienti¢que, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France b Departamento de Bioquimica, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile c Departamento de Ciencias Biologicas, Facultad de Quimica y Biologia, Universidad de Santiago de Chile, Santiago, Chile Received 1 July 1998; received in revised form 5 September 1998; accepted 9 September 1998
Abstract The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Rusticyanin; Copper protein ; Electron transport; Iron oxidation; (Thiobacillus ferrooxidans)
1. Introduction Thiobacillus ferrooxidans is an acidophilic chemolithotrophic Gram-negative eubacterium, well adapted to pH environments as low as pH 1.6. It is able to derive all the energy required for nitrogen and carbon ¢xation through the biological oxidation
* Corresponding author. Fax: +33 (4) 91718914; E-mail: [email protected]
of ferrous iron (Fe2 ) to ferric iron (Fe3 ) [1^3]. The resulting Fe3 is a powerful oxidant in acid media oxidizing insoluble metallic sul¢des to their soluble form. This process, known as bioleaching, is currently used at an industrial scale for the recovery of several metals such as copper, uranium and cobalt from low grade ores [4] and in the bioprocessing of gold ores. Rusticyanin (Rus) is a periplasmic blue copper protein and is generally considered to be an important component in iron oxidation since it is present at high concentration (up to 5% of total soluble proteins) when T. ferrooxidans is grown on iron [1,5]. Its high redox potential (+670 mV) and its acid stability [5^7] have made it the most studied T. ferrooxidans
0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 9 9 - 7
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protein. However, its physiological role in the iron respiratory electron transport chain remains unclear. In di¡erent models, rusticyanin receives electrons from a c-type cytochrome [8] or a cP-type cytochrome [9] and gives electrons to cytochrome c4 [9] or to another unidenti¢ed electron transporter [8]. Other investigators give no signi¢cant role for rusticyanin in the iron respiratory chain [2,10]. Due to the absence of mutants lacking the protein, its exact function awaits con¢rmation. Given the proposed important role of rusticyanin in the iron metabolism of T. ferrooxidans, several laboratories tried to clone its structural gene. However, this cloning was problematic since this gene cannot be found by genetic complementation of an Escherichia coli mutant. Two labs have reported its cloning by screening an expression library with an antibody directed against rusticyanin [11,12], but no sequence has followed up these works suggesting that false positive clones were indeed obtained. This prompted Casimiro et al. [13] to construct a synthetic gene using the E. coli codon usage. It was only recently that fragments of the rus gene were obtained by ampli¢cation in polymerase chain reaction (PCR) with degenerate primers deduced from the known rusticyanin amino acid sequences ([14^17]; this paper). As soon as a marker exchange mutagenesis will be possible in ATCC33020 strain, we wish to construct rus mutants to investigate rusticyanin function in T. ferrooxidans. For this purpose, cloned internal fragments of the gene and an arti¢cial gene would be inadequate. This paper reports the cloning and complete sequencing of the ATCC33020 strain rus gene with its 5P and 3P regions by combining PCR and inverse PCR approaches. Its regulation in T. ferrooxidans and its expression in E. coli are also reported. 2. Materials and methods 2.1. Strains, plasmids and growth conditions T. ferrooxidans ATCC33020 was obtained from the American Type Culture Collection. E. coli TG1 strain (supE hsdv5 thi v(lac-proAB) FP:traD36 proAB lacIq lacZvM15) was used for phagemid propagation. BL21(DE3) strain (F3 ompT hsdSB [VcIts857
ind1 Sam7 nin5 lacUV5-T7 gene1]) was purchased from Novagen. Phagemid Bluescript was purchased from Stratagene and the plasmids pET22b and pET21 from Novagen. E. coli strains were grown in L-broth according to Miller [18]. Conditions to grow T. ferrooxidans on iron medium have been described [19]. Sulfur medium consisted of 25% 9K basal salts pH 3.5, as described in Guiliani et al. [19], and 1% sulfur. The thiosulfate medium is the same as above with 0.07% thiosulfate. 2.2. DNA manipulations General techniques were performed according to Ausubel et al. [20]. Ultrapure plasmid DNA was obtained using the Qiagen plasmid kit. T. ferrooxidans chromosomal DNA was prepared from 200 ml iron medium culture. Cells were harvested and washed several times with 10-fold diluted 9K [19] to remove precipitated iron. The pellet was resuspended in 200 Wl of 10 mM Tris-HCl pH 8, 1 mM EDTA, then gently mixed with 80 Wl 10% SDS, 80 Wl 0.5 M EDTA pH 8 and 10 Wl 10 mg/ml RNase A and incubated for 15 min at room temperature. The suspension was frozen at 320³C for 15 min. After thawing at 55³C for 5 min, 50 Wl 3 mg/ml proteinase K were added. The mixture was incubated for 10 min at 55³C. DNA from lysed cells was submitted to phenol extraction, followed by phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol extractions. It was precipitated with ethanol and resuspended in 150 Wl Tris-HCl pH 8, 1 mM EDTA. Restriction enzyme digestions were performed according to the manufacturer's recommendations. DNA fragment blunting and ligation were performed with the DNA blunting kit from Amersham. Ligations were generally carried out in the presence of the restriction enzyme used to cleave the vector to prevent its recircularization. Southern blotting was performed with T. ferrooxidans total DNA digested with di¡erent restriction enzymes. After electrophoresis, the DNA was denatured and transferred to a positively charged nylon membrane (Hybond-N from Amersham) by the semi-dry capillary method [21]. Prehybridization
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and hybridization were performed under stringent conditions. DIG-labeled probes were obtained by PCR as described by Boehringer Mannheim between the T3 and T7 primers encompassing the rus internal fragment obtained by DOP PCR and cloned in the SmaI site of the Bluescript vector (pINTrus plasmid, see below). Detection was accomplished by a chemiluminescent reaction with Lumigen PPD as described by Boehringer Mannheim. The dideoxy chain termination method was used to sequence DNA using K-35 S-dATP and the T7 Sequence Kit purchased from Pharmacia. 2.3. Cloning of rus fragments by DOP PCR and inverse PCR Five degenerate oligonucleotides (purchased from Appligene) able to form six pairs of convergent primers were designed on the basis of the highly conserved regions of rusticyanin [22^24] and using the most favorable codon usage in strain ATCC33020 [25]. Taq polymerase from Boehringer was used according to the manufacturer's recommendations. Using 400 pmol of oligonucleotides ARus57 and Rus 115 (Table 1) in a 50 Wl reaction, a 195 bp DNA fragment was ampli¢ed from T. ferrooxidans total DNA (30 ng/reaction). PCR ampli¢cations were as follows: 5 min at 94³C followed by 30 cycles at 94³C for 30 s, 37³C for 30 s and 72³C for 30 s, then 5 min at 72³C. The 195 bp fragment was recovered, puri¢ed with the Mermaid kit from Bio101, bluntended with T4 DNA polymerase and cloned at the SmaI site of the Bluescript SK vector to give the pINTrus vector. Ampli¢cation of £anking sequences by inverse PCR was described by Ochman et al. [26]. PCR reactions with Rus2, Rus4, Rus9M and Rus7 primers (Table 1) were performed on total T. ferrooxidans DNA previously digested with HaeIII and religated, as follows: 5 min at 94³C, followed by 30 cycles at 94³C for 30 s, 65³C for 30 s and 72³C for 30 s and a ¢nal incubation of 5 min at 72³C. In the case of inverse PCR with TaqI, a two step protocol was followed: 5 min at 94³C, 30 cycles at 94³C for 30 s and 68³C for 2 min 30 s and a ¢nal incubation of 5 min at 72³C. PCR-ampli¢ed DNA fragments were puri¢ed with Prep-A-Gene from Bio-Rad and cloned at the SmaI site of the Bluescript vector.
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2.4. RNA manipulations T. ferrooxidans total RNA was prepared from 500 ml as described previously [27]. Northern blotting was performed using formaldehyde gels as described in Ausubel et al. [20]. RNA was transferred to a positively charged nylon membrane from Boehringer Mannheim by capillary blotting. The DIG-labeled RNA probe was obtained from the SK/RSD2BamHI-RusCXhoI plasmid described in Section 2.5. In vitro transcription with T7 RNA polymerase was performed on this plasmid linearized with EcoRI using DIG-UTP as substrate according to the instructions of the Boehringer DIG RNA labeling kit. Prehybridization and hybridization with DIG-labeled probe were performed under stringent conditions according to Boehringer Mannheim. RNA was detected as described above for Southern experiments. Primer extension has been performed according to the Promega Access RT-PCR system omitting the PCR step. The primers used were Rusv or Rusv2 oligonucleotides (Table 1) labeled with Q-32 P-dCTP. Coupled reverse transcription and PCR ampli¢cation (RT-PCR) were performed with the Promega Access RT-PCR system. Approx. 1 Wg of total RNA from T. ferrooxidans was denatured at 94³C for 2 min in the presence of both oligonucleotides. Immediately, reverse transcription and PCR ampli¢cation were carried out according to Promega recommendations with the following program: 48³C for 50 min; 94³C for 5 min; 30 or 40 cycles of 30 s at 94³C, 30 s at 62³C, 1 min at 72³C; 72³C for 3 min. Oligonucleotides used were: RusC, Rus5, Rusv, RusAmF, ARusv, ARusv3 and RusAvF (Table 1). For each RT-PCR experiment, three controls were performed: one without template as a negative control to detect any contamination, one with genomic DNA as a control for PCR ampli¢cation, and one with RNA but without reverse transcriptase to check that there were no DNA traces in the RNA preparation. In some cases, RNAse was added to the RNA before performing the RT-PCR experiment. 2.5. Construction of pETrus and pCrus plasmids Oligonucleotides RusNm and RusC (Table 1) were used to amplify the DNA fragment encoding the
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mature rusticyanin from T. ferrooxidans total DNA and the product was blunt-ended by T4 DNA polymerase and cloned into the blunt ended XhoI-NcoI sites of the pET22b vector to yield the pETrus plasmid. In this construction, the sequence of the mature rusticyanin was (i) preceded and in frame with the nucleotidic sequence encoding the PelB classical Gram-negative signal sequence; and (ii) followed by an in frame sequence coding for a His-Tag which allows a one-step puri¢cation of the produced polypeptide. An attempt to place directly the rus gene under the control of bacteriophage T7 transcriptional, but not translational, regulatory elements (pCrus), failed. Therefore, the following strategy was followed. DNA encompassing the ribosome binding site to the translational termination site of the rus gene was ampli¢ed from T. ferrooxidans total DNA with RSD2BamHI and RusCXhoI oligonucleotides (Table 1). This ampli¢ed product was then cloned in the EcoRV site of the SK Bluescript phagemid. At the same time, the interposon 6-Smr , obtained by HindIII digestion of pHP456-Smr [28], was cloned in the HindIII site of the pET21 plasmid. This pET216-Smr plasmid was then easily cut by BamHI and XhoI. The fragment corresponding to the pET21 plasmid was puri¢ed and ligated to the BamHIXhoI fragment originating from the RSD2BamHIRusCXhoI ampli¢ed DNA cloned in the SK Bluescript vector. The pETrus and pCrus clonings were done in E. coli TG1 strain which lacks the T7 polymerase structural gene and therefore cannot express the target gene. Screening was performed by PCR with the oligonucleotides used to amplify the rus DNA. In all cases, the sequence of the cloned fragment was checked. These plasmids were then introduced into the E. coli BL21(DE3) strain which carries the T7 RNA polymerase structural gene under the control of the IPTG-inducible lacUV5 promoter. 2.6. Protein analysis The E. coli BL21(DE3) strain carrying the plasmid of interest was grown at 37³C, 30³C or 20³C in L-broth. When the culture reached an optical density at 600 nm of 1, 1 mM, 250 WM or 50 WM IPTG was added. Cells were harvested after 2 h and submitted
to spheroplasting. E. coli spheroplasts were prepared from 1 g of pelleted cells. Cells were resuspended with 10 ml of 0.2 M Tris-HCl, pH 8. This suspension was mixed with 10 ml of 0.2 M Tris pH 8, 34% sucrose. 100 mM EDTA (¢nal concentration) was added at room temperature and under magnetic stirring. After 2.5 min, 100 mg/ml lysozyme was added and 2.5 min later, 20 ml water. Spheroplasts were then isolated by 15 min centrifugation at 1500Ug and resuspended in 20 ml of 0.04 M Tris, pH 7.6. The supernatant was considered as the periplasmic fraction. A sample of 100 Wl of each fraction (total cells, spheroplasts or periplasm) was mixed with 50 Wl TS/TD loading bu¡er (2 vols. of 0.2 M Tris-HCl pH 8.8, 5 mM EDTA, 1 M sucrose, 0.1% bromothymol blue mixed to 1 vol. of 18% SDS, 0.3 M DTT, 5% L-mercaptoethanol). Total cells and spheroplasts were boiled for 10 min before applying 4 Wl or 25 Wl to 20% SDS-PAGE using either Pharmacia PhastSystem or Protean vertical electrophoresis system from Bio-Rad respectively according to the manufacturer's instructions. A 500 ml T. ferrooxidans ATCC33020 culture grown in iron medium was washed several times to remove residual iron and then transferred to 10 l iron, sulfur or thiosulfate medium. Samples were removed all along the growth from the di¡erent cultures. Periplasmic proteins were prepared from all these samples as previously described [29]. Protein quanti¢cation was performed according to the protocol of Lowry et al. [30]. Equal amounts of proteins from iron-, sulfur- or thiosulfate-grown cells were analyzed by SDS-PAGE. Proteins were either stained with Coomassie brilliant blue (PhastSystem Development) or transferred to a nitrocellulose membrane (Schleider and Schuell) with the PhastSystem blot transfer system (Pharmacia) or the Trans-Blot SD Semi-Dry transfer Cell system (Bio-Rad). Rusticyanin was identi¢ed by immunological cross-reactivity to antibody directed against rusticyanin according to the protocol described in Sambrook et al. [21]. 2.7. Sequence analysis The DNA sequences were compiled, analyzed and compared with the data bank through the Nestcape facilities. The EMBL data bank accession number of the rus gene is X95823.
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Table 1 Oligonucleotides used in this study Name
Sequence
Localization and orientationa
ARus57
5P-CAYGAYRWRAARAAYCCNAC-3P
Rus115
5P-TARCCRAAYTTRCCRTCYTT-3P
HDKKNPT (positions 57^63b ) positions 1599^1618 (+) KDGKFGY (positions 116^122b ) positions 1776^1795 (3) positions 1626^1644 (+) positions 1710^1729 (3) positions 1765^1784 (+) positions 1500^1519 (3) positions 1431^1460 (+) positions 1869^1896 (3) positions 1316^1337 (+) positions 1870^1896 (3) positions 1443^1464 (3) positions 1271^1290 (3) positions 1857^1877 (+) to position 15d (+) from position 2148e (3) positions 663^681 (+) positions 1201^1219 (+)
Rus7 Rus2 Rus4 Rus9M RusNm RusC RSD2BamHIc RusCXhoIc Rusv Rusv2 Rus5 RusAmF RusAvF ARusv ARusv3
a The positions refer to the nucleotide sequence submitted to EMBL data bank and to Fig. 1. (+) corresponds to the rus coding strand and (3) to the rus non-coding strand. b The positions indicated here correspond to the sequence of the mature rusticyanin. c Nucleotides have been added between the restriction site and the 5P end oligonucleotide according to the restriction endonuclease requirement. d The sequence of the 5P end of this oligonucleotide has only been determined on one strand and is not given in the sequence submitted to EMBL data bank and in Fig. 1. e The sequence of the 3P end of this oligonucleotide has only been determined on one strand and is not given in the sequence submitted to EMBL data bank and in Fig. 1.
3. Results 3.1. Sequencing of the rus gene Since our long term goal is to construct rus mutants of T. ferrooxidans, we have decided to look for the rus gene in a known pure strain which is genetically stable and in which molecular biology has been shown to be feasible. ATCC33020 strain ¢ts all these conditions. Using degenerate oligonucleotides based upon the amino acid sequences of mature rusticyanin puri¢ed from three T. ferrooxidans strains [22^24], a fragment of the expected size (195 bp) was ampli¢ed from ATCC33020 genomic DNA by PCR. This fragment was cloned and sequenced. The deduced amino acid sequence matched the expected internal fragment of rusticyanin. This DNA fragment was used
as a probe in Southern hybridization experiments with ATCC33020 total DNA. With undigested DNA, the signal corresponded to chromosomal DNA whereas, when the DNA was digested with di¡erent restriction enzymes, only one DNA fragment hybridized with the probe in each case (data not shown), indicating that strain ATCC33020 carries a single chromosomal copy of the rus gene. Two divergent oligonucleotides designed on the basis of the determined sequence were used in inverse PCR experiments. From the sequence of the ampli¢ed fragment, a new pair of divergent oligonucleotides was designed for subsequent use. This approach of chromosome walking on either side of the rus DNA fragment led to the determination of a DNA sequence of 2164 bp encompassing the rus gene (EMBL GenBank accession No. X95823) (Fig. 1).
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Fig. 1.
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Fig. 1. Nucleotide sequence of the rus gene region from T. ferrooxidans. The deduced amino acid sequences of ORFA, ORF1 and ORF2 and rus are shown. Putative Shine and Dalgarno sequences are indicated in bold. Putative transcription termination signals are represented by arrows above the sequence. Potential c70 -speci¢c promoter sequences are underlined. The +1 labeled arrow corresponds to the rus transcriptional start site. The rusticyanin signal peptide is underlined.
3.2. Analysis of the rus gene The sequence corresponding to the known mature rusticyanin is located between positions 1431 and 1896 (Fig. 1). Two possible in frame translation starts (positions 1335 and 1353) are present upstream from this sequence (Fig. 1). However, only the ATG1335 is preceded 12 bp upstream by a good match to the canonical E. coli Shine-Dalgarno sequence (TAAGGAG), indicating that the rus open reading frame (ORF) starts at position 1335. The nucleotide sequence of the rus gene coding for the mature rusticyanin is 92.7% homologous to the rus sequence from strain ATCC23270 [17] and 93.1% to the partial rus sequence from strain ATCC19859 [16], nine out of ten di¡erences being on the third position of the codons. The deduced polypeptide is 92.3^98.7% identical to the sequences of the known mature rusticyanins [14] and almost all the changes are conservative, indicating that the mature rusticyanin is very well conserved among the di¡erent isolates of T. ferrooxidans. For example, only two conservative di¡erences, Ser to Thr and Val to Ile, are observed with the sequence reported by Ronk et al. [23]. Starting from the potential translational initiation ATG (position 1335) down to the codon corresponding to the ¢rst residue of the mature protein (position 1431) the amino acid sequence presents all the features expected for the signal sequence of a periplasmic protein, including a consensus cleavage sequence Ala-Met-Ala situated appropriately just upstream from the start of the mature protein (Fig. 1).
3.3. Analysis of the ORFs present upstream from the rus gene In addition to the rus gene encoding the rusticyanin protein, three open reading frames, ORFA (to position 160), ORF1 (positions 179^728) and ORF2 (positions 771^963), are located upstream from rus (Fig. 1). We have shown that ORF1 and ORF2 are transcribed (see below). They are preceded by a correctly positioned potential Shine-Dalgarno sequence (Fig. 1). ORF1 would encode a polypeptide of 183 residues with a calculated molecular mass of 20 213 Da, ORF2 a 64 residue polypeptide of 7212 Da. No signi¢cant homologies were detected for ORF1 and ORF2 when submitted to a search in protein data banks. An analysis of the hypothetical ORF1 and ORF2 polypeptides using the `TM pred' program [31] reveals that they could potentially encode integral inner membrane proteins, containing ¢ve and one transmembrane helices respectively. 3.4. Analysis of the intergenic regions The untranslated region between ORF2 and rus is 369 bp long and contains two potential E. coli-like c70 -speci¢c promoter sequences with correctly spaced 310 and 335 boxes (Fig. 1). The 3P £anking region of ORF2 has a sequence similar to a rho-independent transcription terminator consisting of a stemloop structure (vG of 320.6 kcal/mole) followed by the sequence TTGTTTT (Fig. 1) suggesting that ORF2 and the rus gene are independently transcribed. Two potential stem-loop structures (vG values of 317.1 and 328.4 kcal/mol) were found down-
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strongly suggests that the rusticyanin protein is constitutively synthesized in strain ATCC33020. 3.6. Transcription of rus gene in T. ferrooxidans ATCC33020 strain
Fig. 2. Western analysis of rusticyanin synthesis in T. ferrooxidans ATCC33020. Periplasmic proteins from T. ferrooxidans cells were prepared after 1, 2, 3 or 4 days of growth on ferrous iron or sulfur. Equal amounts of proteins were applied to a SDS-PAGE and transferred to a nitrocellulose membrane. Rusticyanin is identi¢ed with an antibody directed against rusticyanin.
According to the sequence data, the 369 bp untranslated region between ORF2 and rus gene contains a putative rho-independent transcriptional termination site and two E. coli c70 -type promoters suggesting that ORF2 and rus gene are independently transcribed (Fig. 1). To check this hypothesis, the rus transcripts were studied by Northern blot with total RNAs from T. ferrooxidans ATCC33020 strain and an in vitro synthesized RNA probe. Two major transcripts of approx. 1200 and 850 nucleotides were detected (Fig. 3A). Furthermore, several minor transcripts were also observed whose sizes are comprised between 6000 and 300 nucleotides (Fig. 3B). All the transcripts were shown to be speci¢c to rus since no signals could be detected by Northern blotting with a RNA probe prepared from the same plasmid lacking the rus insert (data not shown). To con¢rm the presence of the large transcripts, we have used the RT-PCR. This extremely sensitive
stream from rus gene, the last one being followed by a T rich region (Fig. 1). These structures could correspond to the rus transcriptional termination site. 3.5. Analysis of rusticyanin synthesis in T. ferrooxidans ATCC32020 strain Equal amounts of T. ferrooxidans periplasmic proteins, obtained from iron-, thiosulfate- or sulfurgrown cells were analyzed by SDS-PAGE using Coomassie blue staining. It was observed that a protein band corresponding to rusticyanin was present in all three samples (data not shown). The same experiment was repeated using Western blot analysis with a polyclonal antibody directed against the rusticyanin. In ATCC33020 strain, rusticyanin is synthesized in iron- as well as in sulfur- (Fig. 2) or thiosulfategrown cells even after 12 days of growth representing approx. 36 cellular divisions (data not shown). This
Fig. 3. Northern blot of total T. ferrooxidans RNA (3.5 Wg) probed for rus mRNA with DIG-UTP labeled rus RNA. (A) 15 min exposure; (B) 2 h exposure.
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Fig. 4. RT-PCR on T. ferrooxidans total RNA. (A) Localization of the oligonucleotides used for RT-PCR experiment. (B) RT-PCR experiments between RusAmF and Rusv (I), Rus5 and RusAvF (II) oligonucleotides with no added template (0), T. ferrooxidans chromosomal DNA (D), total RNA extracted from Fe2 -grown cells (R), (3) as in (R) but without reverse transcriptase. MW, 1 kb molecular weight ladder from Boehringer Mannheim.
system couples the reverse transcription of a speci¢c target RNA from total RNA (RT) and PCR ampli¢cation of this cDNA. Using di¡erent oligonucleotides hybridizing upstream from rus gene and a convergent one inside of the rus open reading frame (see Section 2 and Fig. 4), this approach has permitted the detection of a transcript su¤ciently large to incorporate transcription from ORFA to rus (Fig. 4 and data not shown). By following the same approach, we have shown that no ampli¢cation product was obtained with the oligonucleotides Rus5 and RusAvF located respectively inside rus gene and at the end of the sequence determined (Fig. 4). This suggests that the rus gene is the last gene of an operon comprising at least ORF1 and ORF2. The di¡erent rus transcripts observed can be due either to the degradation of RNA during their preparation, to mRNA processing, or to the presence of several promoters for the rus gene. We tested the third possibility by carrying out primer extension experiments, using total RNAs and several oligonucleotides to prime the reaction. In all the cases, several very minor large bands were observed all along the gel con¢rming that some rus transcripts are initiated far upstream from the rus translational initiation site. Furthermore, with two di¡erent oligonucleotides (Rusv and Rusv2), the same 5P end of a major rus transcript was detected corresponding to a G (position 1171) located 166 bases upstream from
the translation initiation site (Fig. 5). It is noteworthy that, upstream from this putative translation initiation site, an E. coli-like Pribnow box, TATtAT (positions 1157^1162), and a 335 hexamer, TTGAtc (positions 1134^1139) are present (Fig. 1). 3.7. Expression of the rus gene in E. coli One explanation put forward to account for the repeated failures to clone the rus gene was that the Rus signal sequence could be speci¢c for T. ferrooxidans and not be recognized by the E. coli leader peptidase. This could result in the jamming of the inner membrane with concomitant selection against recombinant clones containing an expressed Rus protein. To circumvent this potential problem, the DNA fragment encoding the mature rusticyanin fused to a classical Gram-negative signal sequence (PelB) was ¢rst under the control of T7 transcriptional and translational signals to yield pETrus plasmid (see Section 2). In BL21(DE3)/pETrus IPTG-induced cells, no polypeptide corresponding to free rusticyanin could be detected in the periplasmic £uid (data not shown). However, two polypeptides of about 18 kDa and 20 kDa, absent from the cells before induction, were detected in the spheroplasts, the largest being more abundant (data not shown and [14]). The amino-terminal of the minor 18 kDa polypeptide exactly matched that of the mature aporusticyanin.
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Fig. 5. Primer extension experiment performed with Rusv2 oligonucleotide on 10 Wg of T. ferrooxidans total RNA (E). Lanes A, C, G and T represent the nucleotide sequence of the rus coding strand performed with Rusv2 oligonucleotide on the DNA fragment obtained by PCR between Rusv and ARusv oligonucleotides.
The ¢rst 29 residues of the largest corresponded to the PelB signal sequence still fused to the mature aporusticyanin sequence. The ine¤cient cleavage of the PelB signal sequence has previously been reported for other secreted proteins overexpressed from the T7 system in E. coli. It is likely due to the uncoupling between translation and translocation. The ratio between precursor to mature polypeptide can usually be reversed by lowering the growth rate and/or by decreasing the induction rate. Accordingly, we have compared the ratio of the precursor to the mature aporusticyanin when the BL21(DE3)/ pETrus cells were grown at 37³C, 30³C or 20³C or in the presence of 50 WM, 250 WM or 1 mM IPTG, respectively. As shown in Fig. 6A (lanes 2, 6, 10), 50 WM IPTG was su¤cient for full induction of the pelB-rus gene under T7 RNA polymerase. However, this did not in£uence the ratio between precursor and mature rusticyanin (lanes 2, 6 or 10 versus lanes 3, 7 or 11 or versus lanes 4, 8 or 12, respectively). In contrast, lowering the growth temperature to 20³C dramatically improved and almost inverted the ratio (Fig. 6A, lanes 2, 3 or 4 versus lanes 10, 11 or 12). Unexpectedly, the mature aporusticyanin was detected in the membrane fraction (Fig. 6A) and none in the periplasmic £uid (data not shown). Washing these membrane fractions with 1 M sodium acetate
Fig. 6. Expression of rus gene in E. coli. (A) Membrane proteins from E. coli BL 21 carrying pETrus plasmid (lanes 2^13). Cells were grown at 20³C (lanes 2^5), 30³C (lanes 6^9) or 37³C (lanes 10^14). Cells were induced with 50 WM (lanes 2, 6, 10), 250 WM (lanes 3, 7, 11) or 1 mM (lanes 4, 8, 12) IPTG, or not induced (lanes 5, 9, 13). Lane 14, membrane proteins from E. coli TG1 strain carrying pETrus grown at 37³C and induced with 1 mM IPTG. Lane 1, protein standards. Arrows show the precursor and the mature rusticyanin. (B) Total proteins from E. coli BL21 carrying pCrus plasmid. Cells were grown at 37³C and induced with 1 mM IPTG (+) or not induced (3). M, protein standards. Arrows show the precursor and the mature form of rusticyanin.
or lowering their pH to 3.5 disrupted such non-speci¢c interactions (data not shown). It is concluded that mature aporusticyanin can be overexpressed in the periplasmic space of E. coli without being degraded and without jamming the cytoplasmic membrane. In additional experiments, the DNA fragment corresponding to the entire rus gene was cloned under control of T7 transcriptional signals in the pET21 plasmid to yield plasmid pCrus. Two IPTG-inducible
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polypeptides were also detected in the spheroplast fraction (Fig. 6B). Determination of the NH2-end sequences revealed that they corresponded to the mature and precursor rusticyanin polypeptides respectively, indicating that the rusticyanin signal sequence is functional in E. coli. Utilizing the approach discussed above, it was observed that the processed rusticyanin was bound to the periplasmic side of the cytoplasmic membrane by non-speci¢c interactions (data not shown). One striking feature of cell free extracts containing rusticyanin in which copper is correctly inserted is a deep blue color and an absorbance peak around 600 nm which disappears upon reduction by Fe2 [32]. Membrane, cytoplasmic and periplasmic fractions of BL21(DE3)/pETrus and BL21(DE3)/pCrus cells induced with IPTG were compared to that of non-induced BL21(DE3)/pETrus, BL21(DE3)/pCrus or BL21(DE3)/pET22b cells. No blue color could be observed and no spectroscopic di¡erence could be detected whatever the growth temperature (data not shown), indicating the lack of copper insertion in the rusticyanin polypeptide expressed in the E. coli periplasm. 4. Discussion We have determined all the rus gene sequence together with about 270 bp downstream and 1430 bp upstream from this gene ([14,15]; this paper). As expected, the amino acid sequence deduced from the nucleotide sequence is highly homologous to the rusticyanin sequences reported for various T. ferrooxidans strains ([16,17,22^24]; Ingledew et al. cited in [33]). As the T. ferrooxidans species comprises di¡erent DNA homology groups and is genomically more diverse than usual for a single species [34], this could indicate strong constraints during evolution to maintain a high redox potential and a stability at low pH. The predicted rusticyanin polypeptide includes a classical cleavable leader sequence that is recognized by the E. coli secretion machinery and leader peptidase. The mature polypeptide produced in E. coli remains associated with the periplasmic side of the cytoplasmic membrane through unspeci¢ed interactions that can be broken by acid or salt treatment. A possibility is the likelihood of non-speci¢c attach-
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ments due to incorrect folding and/or lack of appropriate protonation at pH 6.5 in the periplasm of E. coli. Several arguments support this explanation: (i) Cobley and Haddock [35] and Mansch and Sand [36] have shown that rusticyanin remains bound to the T. ferrooxidans membrane when the cells are broken at pH 7 instead of pH 2; (ii) two type I `blue' copper proteins, azurin [37] and plastocyanin [38], from neutrophilic organisms can be overproduced in the E. coli periplasm with the copper correctly inserted; (iii) the rusticyanin expressed in the cytoplasm [13] of E. coli lacks copper but this metal can be incorporated in vitro by lowering the pH to 3.4^5.5 [13]. This recombinant reconstituted holoprotein displayed a tertiary structure identical to that of the protein extracted from T. ferrooxidans [13]; (iv) homology modeling studies, on the basis of the blue copper protein core structures determined by X-ray absorption, indicate that the surface of the rusticyanin shows a large number of polar/charged residues and particularly a high number of uncompensated lysine residues [39^41]. Thus, it is possible that the basic amino acids of rusticyanin interact electrostatically with the negatively charged phospholipid head group of the cytoplasmic membrane of E. coli. In agreement with this hypothesis, a paper of Kar and Dasgupta [42] states that, unlike most of the naturally occurring membranes, the membrane surface in T. ferrooxidans is positively charged at, or near, its physiological pH. This would lead to the repelling of the rusticyanin away from the membrane. Northern, RT-PCR and primer extension analyses of rus gene transcription in T. ferrooxidans ATCC33020 strain have shown that the rus gene can be co-transcribed with the upstream genes, that is at least ORF1 and ORF2. The rus gene is the last gene of this operon as shown by RT-PCR experiments. Several minor (from 6000 to 300 nucleotides) and two major (1200 and 850 nucleotides) transcripts can be detected by Northern experiments. One model that can account for these results is that the rus gene is expressed from alternative internal promoters. Thus, in the 369 bp untranslated region between ORF2 and rus, the 5P end of a transcript has been determined by primer extension. It is noteworthy that this putative transcription initiation site is located 806 bp upstream from the proposed rus transcriptional termination site (Fig. 1). The correspond-
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ing transcript would then have approximately the size of the major rus transcripts detected by Northern experiments. Altogether, this suggest that the rus major transcript originates from an internal promoter located between ORF2 and rus gene. A possible E. coli-type c70 promoter sequence (TTGAtc-17 bpTATtAT) is indeed located upstream from this transcription initiation site. On the other hand, the minor transcripts could correspond to degraded or processed products of a large RNA corresponding to the entire operon. In agreement with this proposal, bands smaller than the size of the rus open reading frame have been detected showing that, indeed, some degradation occurs. In strain ATCC33020, we have found that the rusticyanin is synthesized in iron as well as sulfur, or thiosulfate, growth conditions. Now, one of the major arguments to involve rusticyanin in iron rather than sulfur oxidation [32] is that it constitutes up to 5% of all soluble proteins in iron-grown cells [5,35] whereas it is barely detectable in Torma strain cells grown on sulfur [11]. However, for strains Tf-3 [43], ATCC13661 [12] and ATCC19859 ([44,45]; A. Bengrine, V. Bonnefoy, unpublished data) rusticyanin is synthesized whatever the electron donor present in the growth medium and its level could increase to some extent in the presence of high concentrations of Fe2 . Furthermore, the disappearance [46^48] or the maintenance for many generations [36,44,48^50] of the Fe2 oxidation capacity has been reported on di¡erent strains when the cells were shifted from iron to sulfur growth conditions. As already pointed out, the T. ferrooxidans species constitute a genomically diverse group [34]. Therefore, these contradictory data might just re£ect the variability among T. ferrooxidans strains. It is even possible that these discrepancies may represent actual di¡erences in the electron transfer pathways between T. ferrooxidans strains. If this were the case, rusticyanin could play a pivotal role in the iron and reduced sulfur compounds' energetic metabolism of some strains of T. ferrooxidans as ATCC33020. In that case, the disruption of its structural gene by marker exchange mutagenesis could be detrimental. Preliminary results have already been presented at the Minerals Bioprocessings conference (Snowbird, UT) in 1994 (Guiliani et al. [15]), at the Biohydrometallurgical Processing symposium (Santiago,
Chile) in 1995 (Bengrine et al. [14]) and at the Molecular Genetics of Bacteria and Phages meeting (Cold Spring Harbor, NY) in 1996. Acknowledgements We owe special thanks to V. Me¨jean for helpful advice on PCR experiments and for critical reading of the manuscript. We gratefully acknowledge J. Ratouchniak for excellent technical assistance. We are indebted to F. Nunzi from M. Bruschi lab (B.I.P., I.B.S.M., Marseille) for providing the rusticyanin antibody and the rusticyanin amino acid sequence prior to publication. We are grateful to the Centre de Se¨quenc,age d'ADN and to the Unite¨ de Se¨quence Prote¨ique of the I.B.S.M. (Marseille). NG and AB acknowledge the support of a Graduate Scholarship from l'Agence de l'Environnement et de la Ma|ªtrise de l'Energie (A.D.E.M.E.). This work was supported by A.D.E.M.E., the Bureau de Recherche Ge¨ologique et Minie©re (B.R.G.M.) and the Compagnie Ge¨ne¨rale de Mate¨riaux (CO.GE.MA) and ECOS/CONICYT award to VB, EJ and DH.
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