1 Laboratorio de Organización y Expresión del Gen, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela
2 Laboratoire de Chimie Bactérienne, CNRS, IBSM, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Correspondence
Violaine Bonnefoy
bonnefoy{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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INTRODUCTION |
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Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic bacterium, which thrives in harsh environments and exhibits a versatile energy metabolism. Although this obligate chemolithoautotroph obtains its energy mainly from the oxidation of ferrous iron, Fe(II), or various reduced sulfur compounds (Leduc & Ferroni, 1994; Rohwerder et al., 2003
), it can also use hydrogen or formate in oxic conditions (Drobner et al., 1990
; Pronk et al., 1991
). Moreover, under anoxic conditions, it is able to reduce ferric iron, Fe(III), with sulfur (S0) or hydrogen as electron donors (Das et al., 1992
; Ohmura et al., 2002
; Pronk et al., 1992
). The respiratory chains involved in these redox reactions have not been clearly established. Nevertheless, three operons encoding redox proteins have been characterized in A. ferrooxidans (Appia-Ayme et al., 1999
; Levicán et al., 2002
; P. Bruscella and others, unpublished data). One of these, the rus operon, encodes two c-type cytochromes, Cyc1 and Cyc2 (cyc1 and cyc2), an ORF of unknown function (orf), an aa3-type cytochrome oxidase (coxBACD) and rusticyanin (rus) (see Fig. 6
) (Appia-Ayme et al., 1999
). Based on the operon organization, the subcellular localization of the corresponding redox proteins (Yarzábal et al., 2002a
, b
) and interaction studies (Giudici-Orticoni et al., 1999
), we have proposed the following electron transfer pathway: Cyc2
Rus
Cyc1
cytochrome oxidase
O2. Because Rus has been shown to constitute up to 5 % of total soluble proteins in Fe(II)-grown cells (Cobley & Haddock, 1975
; Cox & Boxer, 1978
), this protein, and therefore the electron transfer chain presented above, have been proposed to be involved in Fe(II) oxidation (Appia-Ayme et al., 1999
).
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METHODS |
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Liquid cultures of S0- and/or Fe(II)-adapted cells were carefully washed with the respective basal salts solution, before inoculating a fresh medium. Growth was followed by direct cell counting in a Neubauer chamber. Samples were removed at 1 day intervals from the different cultures and centrifuged at 15 000 g for 5 min. Pelleted cells were stored at 20 °C for further studies.
DNA manipulations.
General DNA manipulations were performed according to standard procedures (Ausubel et al., 1987). A. ferrooxidans genomic DNA was prepared with the NucleoSpin Tissue kit (Macherey-Nagel). YellowStar DNA polymerase (Eurogentec) was used for PCR amplifications. The nucleotide sequences of the cloned fragments were determined by GENOME express. A. ferrooxidans ATCC 23270 preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org.
Plasmid construction.
All the plasmid constructions are summarized in Table 1. For the construction of expression plasmids to produce wild-type polypeptides fused to a hexa-histidine tag at the C-terminus, the DNA fragment corresponding to the sequence of the peptide was amplified by PCR as far as the last codon upstream from the translational termination site. For cyc2, cyc1 and orf, the cloned fragments started upstream from the ShineDalgarno sequence. For coxB, the peptide corresponding to the periplasmic domain of CoxB was fused at its NH2-terminus to the PelB signal sequence from the pET22 vector. In this case, the ShineDalgarno sequence and the translational start site were both provided by the vector. The upstream oligonucleotides contained a BamHI restriction site, and the downstream oligonucleotides a XhoI site (Table 2
), to allow cloning of the amplified products in the correct orientation in BamHI- and XhoI-digested pET21 or pET22 vectors (Table 1
).
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All these constructions were checked by nucleotide sequencing.
Biochemical methods.
Protein analyses were performed as previously described (Yarzábal et al., 2003). The primary antibodies used in immunodetection studies were: (i) mouse anti-His (Invitrogen); (ii) rabbit anti-Rus serum, kindly provided by M. Bruschi; (iii) mouse anti-Omp40 serum, kindly provided by N. Guiliani; and (iv) rabbit anti-Cyc2HisTag, anti-Cyc1HisTag, anti-ORFHisTag and anti-CoxBHisTag sera (see below).
Amino acid sequence determinations were carried out with an Applied Biosystems gas-phase sequenator (model 473A) at the Protein Sequencing Unit of the IBSM.
Production and purification of His-tagged recombinant proteins.
The BL21(DE3) strains carrying pET22-coxBP-HisTag, pET21orf-HisTag, pET21cyc2-HisTag and pEC86, or pET21cyc1-HisTag and pEC86, were grown at 37 °C in LB with 50 µg ampicillin ml1 and 25 µg chloramphenicol ml1, when required, to an OD600 of 1·0. IPTG was then added to the cultures to a final concentration of 1 mM, and the cultures were grown for a further 2 h. The cells were harvested, washed once with 40 mM sodium phosphate, pH 7·2, and stored at 20 °C until use. The cell pellets from 400 ml culture were suspended in 40 ml freshly prepared buffer I [20 mM sodium phosphate, pH 7·2; 0·15 mM phenyl-methyl-sulfonyl fluoride (PMSF); 5 mM benzamidine], and passed three times through a French Press. Cyc2, Cyc1, ORF and CoxB were mainly present in inclusion bodies. Inclusion bodies, unbroken cells and cellular debris were pelleted by centrifugation at 10 000 g for 20 min, suspended in 8 ml freshly prepared buffer I containing 2 % Triton X-100, incubated on ice for 2 h, and centrifuged for 20 min at 10 000 g. The pellet was suspended in 20 ml freshly prepared buffer I containing 1 % Triton X-100, and immediately centrifuged at 10 000 g for 20 min. The pellet corresponding to the inclusion bodies was kept at 20 °C. Solubilization of inclusion bodies with urea and purification of the His-tagged recombinant proteins were performed with the HisTrap kit (Amersham Pharmacia), according to the manufacturer's instructions. The fractions eluted from Ni2+ columns were analysed by immunodetection with antibodies directed against the HisTag and by NH2-terminus sequencing.
Production of specific antisera.
The purified His-tagged recombinant proteins were electrophoresed in 15 % acrylamide gels. The bands corresponding to the His-tagged proteins were detected by CuCl2 staining (Lee et al., 1987) and excised from the gel. Gel strips were crushed and used to immunize New Zealand SSC rabbits, following standard procedures.
Analytical methods.
Fe(II) concentrations were determined by the o-phenanthroline method (Muir & Anderson, 1977).
RNA manipulations.
A. ferrooxidans total RNA was prepared from 500 ml of S0 or Fe(II) culture, as described previously (Guiliani et al., 1997). The Northern blotting protocol was as described in Bengrine et al. (1998)
. The DIG-labelled RNA probes hybridizing to rus, cyc2 and coxB mRNA were obtained from the SK-rus, SK-cyc2 and SK-coxB plasmids described in Table 1
. Following linearization with EcoRI or HindIII, in vitro transcription with T7 or T3 RNA polymerase was performed on these plasmids, using DIG-UTP as substrate, according to the instructions of the Strip-EZ kit (Ambion) (Table 1
). RNA was detected by a chemiluminescent reaction with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3·3·1·13,7]decan}-4-yl) phenyl phosphate (CSPD; Roche), following the manufacturer's instructions.
Primer extension experiments were performed with the Superscript II RNase H reverse transcriptase (InVitro Life Technologies), as previously described (Bengrine et al., 1998; Appia-Ayme et al., 1999
), with Ainv3, Ainv6, orf-inv, R
2 and Porf-inv oligonucleotides (Table 2
). The experiments were performed in duplicate, using RNA samples from independent cultures.
Real-time PCR.
The relative abundances of cyc2, cyc1, orf, coxB, coxA, coxC and rus transcripts in Fe(II)- and S0-grown cells were determined by real-time PCR with the LightCycler instrument and the LightCycler-FastStart DNA Master SYBR Green 1 kit (Roche), with external standards as described in Roche Molecular Biochemicals technical note no. LC 11/2000. The alaS gene, encoding alanyl tRNA synthetase (Guiliani et al., 1997), and rrs (16S rRNA) genes were shown to be expressed at the same (constitutive) level under both conditions of growth (data not shown), and were therefore used as reference standards. Primer pairs are given in Table 2
. The real-time PCR quantification was performed twice, using RNA samples from independent cultures.
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RESULTS AND DISCUSSION |
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Cells switched from Fe(II) to S0 and vice versa (non-adapted cells)
When switching exponentially growing cells from Fe(II) to S0 medium, cell growth was observed after a 1 day lag (Fig. 1b). Cyc1 and CoxB, already present in the inoculum, disappeared completely after 2 days of growth. The Rus level decreased more slowly as growth proceeded (Fig. 1b
). When exponentially growing cells were transferred from S0 to Fe(II) medium, after a 1 day lag we observed simultaneous cell growth, Fe(II) oxidation, and a rapid increase in Cyc1, CoxB and Rus levels, relative to the levels observed in the inoculum (Fig. 1b
). Again, the levels of Omp40 did not show any significant variation (data not shown). These results suggest a regulation of rus-operon expression in response to the electron donor.
Cells grown in the presence of both Fe(II) and S0
When A. ferroxidans cells were grown in the presence of both energetic substrates for several generations, i.e. in Fe(II)+S0 medium, Fe(II) was oxidized rapidly, and the pH increased concomitantly (Fig. 2a). This pH increase is likely to be due to proton consumption when Fe(II) is oxidized, suggesting that Fe(II) is used immediately in preference to S0. Cyc1, Rus and CoxB levels increased during Fe(II) oxidation, reaching a maximum at day 3, and then decreased after complete oxidation of Fe(II) (day 4), as growth proceeded, to become undetectable in late-exponential-phase cells (day 8) (Fig. 2b
, and data not shown). This decrease, presumably due to dilution of the proteins during subsequent cell growth and division, coincided with a drop in pH, from 1·8 to 1·0 (Fig. 2a
), which is probably due to H2SO4 production during S0 oxidation and to ferric hydroxide formation, which leads to proton liberation. In conclusion, the rus operon is expressed as long as Fe(II) is present in the medium, even in the presence of S0, indicating that rus-operon expression is not repressed by S0 itself in the presence of Fe(II). However, repression of rus-operon expression by sulfur oxidation product(s), as soon as S0 is metabolized, cannot be excluded.
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To confirm that transcription of the rus operon is dependent on the energetic substrate, we employed real-time PCR to quantify the amount of transcript corresponding to each gene of the rus operon. The results are shown in Table 3. We found that, in contrast with the two genes used as reference standards, alaS and rrs, which did not show any significant differences (data not shown), all the transcripts of the rus operon were more abundant in Fe(II)- than in S0-grown cells by 4·9-fold (cyc2) to 24·5-fold (rus) (Table 3
). In comparison with the other transcripts from the rus operon, rus transcripts were the most abundant, a result that matches the abundance of rusticyanin in whole-cell extracts (Cobley & Haddock, 1975
; Cox & Boxer, 1978
).
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We propose that the expression of the rus operon is positively regulated by Fe(II), based on the following arguments: (i) the expression of the operon is higher in Fe(II)- than in S0-grown cells (Fig. 3 and Table 3
); (ii) a rapid increase in the concentrations of the proteins encoded by the rus operon was observed when switching cells from S0 to Fe(II) medium (Fig. 1b
); (iii) a drastic decrease of rus-operon protein levels was observed when cells were transferred from Fe(II) to S0 medium (Fig. 1b
); (iv) when both substrates were present, the rus-operon-encoded proteins were detected in large amounts only when Fe(II) was still available, and decreased rapidly once it was completely oxidized (Fig. 2
), indicating that the rus operon is not repressed by S0 itself. Furthermore, the levels of the transcripts from the rus operon decreased drastically when Fe(II) was no longer available (Figs 3 and 4
). Note that, in the presence of both Fe(II) and S0, Fe(II) is immediately oxidized, and that the pH decrease due to H2SO4 production occurs after complete Fe(II) oxidation (Fig. 2
), suggesting that Fe(II) is apparently preferred to S0 as an electron donor.
Transient expression of the rus operon in S0-growing cells
As shown in Fig. 1, Cyc1, CoxB and Rus were always detected during the early exponential phase on S0-grown cells, even after many subcultures on S0, suggesting a de novo synthesis of these proteins. To test this hypothesis, cells from S0 cultures (day 10 in Fig. 1a
), in which Cyc1, CoxB and Rus could not be detected by Western blot analysis, were used to inoculate fresh S0 medium. Aliquots were removed every day and analysed by immunodetection, as described above. After 1 day of growth, CoxB, Rus and Cyc1 were clearly detected by immunoassay, although their levels were lower than in Fe(II)-grown cells (Fig. 5
, and data not shown). The same results were obtained using extra-pure S0 (Fluka; data not shown). This demonstrates unambiguously a de novo synthesis of these proteins in S0-grown cells. As noted previously (see Fig. 1a
), Cyc1, CoxB and Rus levels rapidly decreased towards the end of the exponential phase, to become undetectable in the stationary phase, a pattern consistent with dilution of the proteins through growth and multiple cell divisions in the absence of any further synthesis.
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Transcription from PI, PII and Prus promoters was studied further by reverse-transcriptase-mediated primer-extension experiments with RNA extracted from one-day (early exponential phase) and three-day (mid-exponential phase) S0-grown-cells. Transcription from PI, PII and Prus promoters in S0-grown cells was not detected in mid-exponential S0-growing cells (Fig. 4, lanes SM), as already mentioned. However, in contrast to PII and Prus transcription, transcription from PI was higher in early exponential S0-growing cells than in Fe(II)-growing cells (Fig. 4
, lanes F and SE). Thus, the transient expression of the rus operon during the early exponential phase of S0-grown cells, in the absence of any detectable Fe(II), is mainly due to transcription from PI.
As mentioned above, there are contradictory reports in the literature concerning the presence of rusticyanin and iron-oxidizing activity in S0- or thiosulfate-grown cells: in some studies, neither were detected, while in others, both were detected (Landesman et al., 1966; Margalith et al., 1966
; Cox & Boxer, 1986
; Hazeu et al., 1986
; Jedlicki et al., 1986
; Kulpa et al., 1986a
, b
; Sugio et al., 1986
, 1988
; Espejo & Romero, 1987
; Espejo et al., 1988
; Suzuki et al., 1990
; Mansch & Sand, 1992
; Osorio et al., 1993
; Bengrine et al., 1995
, 1997
, 1998
; Yarzábal et al., 2003
). Suzuki et al. (1990)
proposed that such conflicting results were due to strain differences. We have shown here that the rus operon, clearly involved in Fe(II) oxidation, is in fact transiently expressed in S0-grown ATCC 33020 cells. Indeed, transcription of the rus operon and the de novo synthesis of the corresponding products occurred at the beginning of growth in S0 medium (Figs 1a, 4 and 5
). These results could explain the contradictory reports found in the literature: if samples were taken from exponentially growing cells, expression would be detected, but would be absent or undetectable in late-exponential or in stationary-phase samples.
Interestingly, Sugio et al. (1988) noticed that the iron-oxidizing activity of S0-growing cells increased during early exponential phase and decreased later. These authors proposed that this decrease was due to the acidification of the medium. This hypothesis could also explain the rus-operon expression pattern under S0-growth conditions. Indeed, the synthesis of several proteins of A. ferrooxidans has been reported to be pH dependent (Amaro et al., 1991
). De novo expression of the rus operon was observed when stationary-phase bacteria cultured in S0 medium (pH 1) were shifted to fresh S0 medium (pH 3·5). Furthermore, when both Fe(II) and S0 were present in the medium, Rus and CoxB levels first increased and then decreased with the pH (Fig. 2
). Therefore, the rus-operon expression observed in S0-grown cells could be the consequence of a pH upshift. However, when cells of A. ferrooxidans grown at pH 1·5 are shifted to pH 3·5, no significant changes are observed in SDS-PAGE profiles, except the increase of a 36 kDa outer-membrane protein (Amaro et al., 1991
). An alternative hypothesis could explain the transient expression of the rus operon in S0-grown cells. Indeed, the rapid increase in rus-operon transcription, after inoculation into fresh medium, followed by a steep decrease (Fig. 5
), is reminiscent of the transcription pattern of genes regulated by a nutritional upshift (Nilsson et al., 1990
, 1992
). The histone-like Fis protein of E. coli has been proposed to communicate the nutritional quality of the environment to various cellular processes (Ball et al., 1992
), including electron transfer (Green et al., 1996
; Wackwitz et al., 1999
). It is therefore possible that rus-operon expression in S0-growing cells is nutrient-upshift dependent, and is mediated, directly or indirectly, by a Fis-like protein. Interestingly, an orthologous fis gene is present in the A. ferroxidans ATCC 23270 genome. We cannot exclude, however, repression of rus-operon expression during growth on S0 by accumulation of an oxidized S0 compound.
Model for rus-operon regulation
In this paper, we have demonstrated that the regulation of A. ferroxidans ATCC 33020 rus-operon expression at the transcriptional level depends on the electron donor present in the medium. Furthermore, a transient expression during the early-exponential phase of S0-grown cells suggests a nutritional- or a pH-upshift-dependent regulation. Transcription from the PI promoter seems to depend on the growth phase or pH, while transcription from PII and Prus seems to be regulated mainly by the presence of Fe(II).
In addition, the rus operon seems to be subject to post-transcriptional mRNA processing. The largest transcript, 7500 nt, observed with the cyc2 probe (the first gene of the rus operon, see Fig. 6), corresponds to a transcript covering the whole operon. The 5100 nt transcript detected with the coxB and rus probes may be due to the processing of larger transcripts or may arise from an internal promoter. Assuming that this 5100 nt transcript stops at the putative rus transcriptional termination site (Bengrine et al., 1998
), it would start in the 110 bp intergenic region between cyc1 and orf. In agreement with this hypothesis, transcripts starting approximately 87 nt from the orf translation initiation site were detected by primer extension with the Porf oligonucleotide (data not shown). Although no obvious 35 and 10 regions were detected upstream from that position, there is a putative RNase E recognition site (Fritsch et al., 1995
), followed by a potential stemloop structure (
G=38·9 kJ mol1). Therefore, we propose that the 5100 nt transcript originates from RNase E cleavage of larger transcripts at this site. The two major transcripts, 1300 and 850 nt, were detected only with the rus probe. The 850 nt rus transcript could correspond to transcripts starting from the internal promoter Prus, previously detected between coxD and rus (Bengrine et al., 1998
). Since a putative RNase E recognition site (Fritsch et al., 1995
), followed by two potential stemloop structures (
G=45·2 and 118·5 kJ mol1), lies upstream from Prus, the 1300 nt rus transcripts may also arise from the processing of larger transcripts at this site. The model of rus-operon regulation we propose is summarized in Fig. 6
.
It seems clear from the data presented in this paper that the rus operon is involved in Fe(II) rather than in S0 respiration. Therefore, why is the rus operon expressed at the onset of exponential growth in S0-grown cells? One hypothesis, proposed by Sugio et al. (1985), is that the iron-oxidation pathway allows the recycling of the Fe(III) that is required for S0 oxidation (the ferric ion-reducing system, FIR). However, since the iron-oxidation system is expressed only at the early exponential phase, it is clearly not necessary for subsequent growth in S0 medium. An alternative hypothesis is that the bacterium induces the expression of both the S0- and the Fe(II)-oxidizing system as soon as it detects either one of these substrates in the medium, the natural substrate for A. ferrooxidans being metal sulfides, such as pyrite (FeS2). Furthermore, this hypothesis is in agreement with the model proposed by Rohwerder et al. (2003)
, according to which metal sulfides are leached by Fe(III) and/or by protons, produced by Fe(II) and S0 oxidation, respectively. We would expect this hypothesis also to hold true for the S0-oxidizing system, in which case we would expect the expression of this system in Fe(II)-grown cells to be early-exponential-phase dependent. Similar experiments with the S0-oxidizing system await the detailed elucidation of the genes and gene products of that system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 December 2003;
revised 9 March 2004;
accepted 25 March 2004.
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