Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin

Andrés Yarzábal1, Corinne Appia-Ayme2, Jeanine Ratouchniak2 and Violaine Bonnefoy2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The respiratory flexibility found in Bacteria and Archaea, resulting from complex multicomponent branched electron-transfer systems, allows these micro-organisms to colonize a wide range of biotopes. The genes that encode electron-transfer proteins belonging to the same respiratory system are often organized as operons, whose expression is modulated depending on the growth conditions, thus allowing rapid adaptation to environmental changes.

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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Fig. 6. Model of the transcriptional and post-transcriptional regulation of the rus operon. Each gene is shown by an open box. The positions of the promoters (PI, PII and Prus) are indicated as bent arrows. Transcripts detected in RNA blots are shown by horizontal arrows, with thickness indicating their relative abundance. Putative RNase E cleavage sites are represented as vertical arrowheads. Inverted repeats are shown as open stem–loops, while the putative transcriptional Rho-independent termination sites are represented as a closed stem–loop.

 
However, Rus has also been detected in S0-grown cells (Cox & Boxer, 1986; Espejo et al., 1988; Osorio et al., 1993; Bengrine et al., 1995, 1997, 1998; Yarzábal et al., 2001, 2003). Furthermore, the Fe(II) oxidation activity has been reported to be maintained over several generations in cells shifted from Fe(II) to S0 medium (Margalith et al., 1966; Sugio et al., 1986; Espejo et al., 1988; Suzuki et al., 1990; Mansch & Sand, 1992). These apparently contradictory data may reflect (i) a variability among A. ferrooxidans strains, (ii) a role for Rus in S0 metabolism and/or (iii) a complex regulation of rus-operon expression. An argument in favour of the last hypothesis is that at least three putative promoters have been characterized for the rus operon (see Fig. 6), two upstream from cyc2 and one between coxD and rus (Bengrine et al., 1998; Appia-Ayme et al., 1999). Therefore, to gain further insight into its regulation, the expression of the rus operon in the ATCC 33020 strain was studied under various growth conditions, at both the transcriptional and translational levels. Our results show that the expression of the rus operon is regulated at the transcriptional level by ferrous iron. They also suggest that the expression of this operon is regulated at the post-transcriptional level and that, furthermore, transient expression of the rus operon takes place during the early exponential phase of S0-grown cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Strains, plasmids and culture conditions.
A. ferrooxidans ATCC 33020 was obtained from ATCC. Escherichia coli TG1 strain [supE hsd{Delta}5 thi {Delta}(lac–proAB) F': traD36 proAB lacIq lacZ{Delta}M15] was used for plasmid propagation. BL21(DE3) strain [F ompT hsdSB ({lambda}cIts857 ind1 Sam7 nin5 lacUV5-T7 gene1)] and the plasmids pET22b and pET21, used to produce recombinant proteins, were purchased from Novagen. Phagemid Bluescript SK+ was purchased from Stratagene. The plasmid pEC86, which constitutively expresses the E. coli ccm operon encoding the cytochrome c maturation system (Arslan et al., 1998), was kindly provided by L. Thöny-Meyer. E. coli strains were grown in L broth (LB; Ausubel et al., 1987). A. ferrooxidans ATCC 33020 was grown at 30 °C under oxic conditions in Fe(II) medium or S0 medium, as previously described (Yarzábal et al., 2003), and in Fe(II)+S0 medium consisting of 1 % (w/v) elemental S0 and 3·5 % (w/v) FeSO4 in basal salts solution [1·6 g (NH4)2SO4 l–1, 1·6 g K2HPO4 l–1, 1·6 g MgSO4.7H2O l–1], adjusted to pH 1·6 with H2SO4. Usually, 99 % purity minimum S0 (Prolabo) was used. However, in some experiments, extra-pure S0 (Fluka) was used to avoid contamination of the S0 medium with Fe(II) traces. In both cases, no Fe(II) was detected in S0 medium by o-phenanthroline analysis (Muir & Anderson, 1977).

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 Shine–Dalgarno 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 Shine–Dalgarno 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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Table 1. Plasmids constructed in this study

See Table 2 for description of oligonucleotides.

 

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Table 2. Oligonucleotides used in this study

Restriction sites are shown in italics. Nucleotides have been added between this site and the 5' end of the oligonucleotide according to the restriction endonuclease requirement. The translational start site is underlined with a solid line, and the Shine–Dalgarno sequence is underlined with a dotted line. Position refers to EMBL nucleotide sequence AJ006456 for the rus operon, X95571 for the alaS gene and AJ278719 for the rrs (16S rRNA) gene. (+), Coding strand orientation; (–), non-coding strand orientation.

 
For the construction of the plasmids from which RNA probes were synthesized, an internal fragment of the gene of interest was amplified by PCR (Table 2) and cloned between the T7 and the T3 promoters in the EcoRV site of the Bluescript SK+ vector (Table 1).

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-Cyc2–HisTag, anti-Cyc1–HisTag, anti-ORF–HisTag and anti-CoxB–HisTag 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 ml–1 and 25 µg chloramphenicol ml–1, 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{Delta}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.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Expression of the rus operon is dependent on the energetic substrate
To determine the relative abundance of products encoded by the rus operon during growth, Fe(II) or S0 cells were used to inoculate Fe(II), S0 and/or Fe(II)+S0 medium. Aliquots were removed every day and cells pelleted. Time-course analysis of protein levels was monitored by immunodetection (Figs 1 and 2, and data not shown).



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Fig. 1. Effect of Fe(II)- and S0-dependent growth on rus-operon product levels in A. ferrooxidans ATCC 33020. (a) Cells were grown on iron ({circ}; F->F) or sulfur medium ({bullet}; S->S) for several generations. Ferrous iron concentrations are shown ({square}). (b) Cells were switched from iron to sulfur medium ({bullet}; F->S) and vice versa ({circ}; S->F). Ferrous iron concentrations are shown ({square}). Samples were removed every 24 h to measure cell densities. The samples were subjected to SDS-PAGE and transferred to a PVDF membrane. Western immunoblots were performed with antisera raised against rusticyanin (Rus), the periplasmic domain of subunit II of cytochrome oxidase (CoxB), and the periplasmic membrane-bound cytochrome c4 (Cyc1). The same results were obtained with antibodies directed against the product of the orf gene (data not shown). i, Inoculum.

 
No change in electron donor (Fe(II)- or S0-adapted cells)
When exponentially Fe(II)-grown cells were inoculated into Fe(II) medium, Cyc1, CoxB and Rus were detected throughout the growth cycle, and their levels remained almost unchanged after complete oxidation of Fe(II), that is after 2–3 days of culture (Fig. 1a), and even after 16 days (data not shown). Because the Fe(II) medium used was buffered to slow down the precipitation of ferric salts, the pH of the medium did not vary significantly (1·6–1·8 after 4 days of growth). As expected, growth stopped when no more Fe(II) was available, though the proteins tested were still present, suggesting that they are very stable. When exponentially S0-grown cells (3–4 days) were inoculated into S0 medium, Rus was detected only during the early exponential phase, though its concentration was significantly lower than the level detected in Fe(II)-grown cells (Fig. 1a). Indeed, semi-quantitative analysis from Western blot experiments showed that the highest amount of Rus in S0-grown cells (i.e. cells from day 1 in S->S cultures in Fig. 1a) never exceeded 20 % of the amount observed in Fe(II)-grown cells. Cyc1 and CoxB were also detected during the exponential phase in S0-grown cells, but higher exposure times were needed to visualize them clearly (data not shown). The three proteins disappeared towards the late-exponential phase. The pH of the medium decreased gradually from 3·5 to 1·0, due to H2SO4 production during S0 oxidation. The levels of an outer-membrane protein used as reference, Omp40, whose synthesis is independent of the oxidizable substrate (Osorio et al., 1993), showed no significant variation under any of the conditions tested (data not shown). These results show that rus-operon expression depends on the electron donor available.

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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Fig. 2. Expression of the rus operon in A. ferrooxidans ATCC 33020 cells grown on medium containing both ferrous iron and sulfur. (a) Cell density ({bullet}), ferrous iron concentration ({square}) and pH ({lozenge}) were monitored in daily harvested samples. (b) Western immunodetection of rusticyanin (Rus) and the periplasmic domain of subunit II of cytochrome oxidase (CoxB). Samples of iron- (F) and sulfur-grown cells (S) were included as controls. The same results were obtained with antibodies directed against the products of the orf and cyc1 genes (data not shown).

 
Transcription of the rus operon is dependent on the energetic substrate
The data presented above show that the level of the rus-operon products depends primarily on the electron donor available. To determine whether the protein levels reflected the corresponding mRNA levels, antisense RNA probes complementary to cyc2, coxB and rus were hybridized to total RNA extracted from Fe(II)- and S0-grown cells in mid-exponential phase (see Fig. 1a), as described in Methods. Comparison of the transcript profiles revealed that more transcripts are detected in Fe(II) than in S0 exponentially growing cells, whatever the probes used (Fig. 3). Since the patterns obtained were reproducible in at least three independent experiments, these differences probably reflect regulation at the transcriptional level in response to the energetic substrate. Furthermore, we noticed that in Fe(II) medium, rus transcripts are more abundant in cells after 1 day than after 3 days of growth (Fig. 3, lanes Fs and Fe), that is before and after complete oxidation of Fe(II) to Fe(III) (see Fig. 1a). Similar results were obtained with the cyc1 probe (data not shown). These results explain the data obtained previously (Bengrine et al., 1995; Yarzábal et al., 2001), in which the level of rus transcripts was apparently higher in S0- than in Fe(II)-grown cells, since the RNA was extracted from a Fe(II) culture at day 3, when all the Fe(II) was oxidized. The possibility of rus-operon transcripts being degraded by Fe(III) during the extraction procedure was rejected, because: (i) in this experiment, Fe(III) was already present at day 1 (Fig. 1a); (ii) no obvious RNA degradation was observed in the ethidium bromide-stained gel (data not shown); and (iii), when RNA samples were prepared from S0-grown cells in the presence or absence of the filter-sterilized supernatant of a five-day Fe(II)-grown culture, the rus transcripts presented the same pattern (data not shown). Therefore, the most likely explanation is the induction of rus operon transcription by Fe(II).



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Fig. 3. Northern analysis. Total A. ferrooxidans RNA was extracted from ATCC 33020 cells grown on iron medium for 1 day (Fe) or 3 days (Fs), and on sulfur for 4 days (S). RNA (10 µg) was subjected to agarose-formaldehyde gel electrophoresis, transferred to a positively charged nylon membrane and hybridized to DIG-UTP-labelled antisense-RNA probes complementary to rus, coxB and cyc2 transcripts. Arrows show major transcripts (5100, 1300 and 850 nt), and the largest transcripts detected (7500 nt), following overnight exposure (ON). rRNA, which appeared as white bands, is indicated with bent arrows.

 
Several bands were detected, whatever the RNA probe used, as previously reported with a rus probe (Bengrine et al., 1995, 1998; Yarzábal et al., 2003). The largest transcript observed, 7500 nt long (Fig. 3), was detected only with the cyc2 probe (see Fig. 6). Transcripts of 5100 nt were the largest detected with rus and coxB probes (Fig. 3). Two major bands of 850 and 1300 nt were detected with a rus probe only (Fig. 3). These results, which were reproducible in several independent experiments, suggest the presence of internal promoter(s) and/or the processing by endo- and exoribonucleases of rus-operon transcripts (see the model presented below).

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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Table 3. Quantification of rus transcripts by RT-PCR

All values (except rrs) are expressed as n-fold relative to 16S rRNA (x104). Values shown are the mean of three independent experiments±SD. Total RNA was extracted from mid-exponential-phase cells, after 1 day for Fe(II) and 4 days for S0 cultures.

 
We have shown previously that the rus operon is transcribed from at least three promoters (see Fig. 6): PI and PII upstream from cyc2 (Appia-Ayme et al., 1999), and Prus upstream from rus (Bengrine et al., 1998). Transcription from these promoters was further studied by primer extension experiments with RNA extracted from mid-exponentially growing Fe(II) and S0 cells. The results obtained clearly show that transcription from the three promoters depends on the electron donor present in the medium. Indeed, transcription from PI, PII and Prus was higher in Fe(II) than in S0 mid-exponentially growing cells (Fig. 4, lanes F and SM).



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Fig. 4. Reverse transcriptase-mediated primer-extension experiments. Reverse transcription (RT) reactions were performed using the primers indicated, and total RNA was extracted from mid-exponential iron-growing cells (F) and from early exponential (SE) or mid-exponential (SM) sulfur-growing cells. RT products were resolved by urea-acrylamide gel electrophoresis and detected by autoradiography. Lanes M, [{gamma}32P]ATP-labelled 25 bp DNA ladder (Promega). RT products corresponding to transcription from the indicated promoters are shown with an arrow.

 
Altogether, these results agree with those obtained at the protein level in that they demonstrate higher rus-operon expression in Fe(II)- than S0-grown cells. Therefore, rus-operon expression is not constitutive, but likely regulated at the transcriptional level, depending on the energy source, as previously proposed for the rus gene alone in the ATCC 19859 strain (Pulgar et al., 1993). Moreover, these data suggest that rus-operon products are involved in Fe(II) oxidation rather than in S0 oxidation.

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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Fig. 5. De novo expression of the rus operon in A. ferrooxidans ATCC 33020 cells grown on sulfur. (a) Adapted cells from stationary cultures were grown on sulfur medium ({bullet}). Transcription of the rus gene was monitored by real-time PCR (bars). (b) Samples were harvested from sulfur cultures and monitored by Western immunodetection for the presence of rusticyanin (Rus) and the periplasmic domain of subunit II of cytochrome oxidase (CoxB). Samples from iron-grown cells (F) were included as controls. i, Inoculum.

 
To determine whether this de novo synthesis reflected transcription of the rus operon, real-time PCR experiments were performed. As can be seen in Fig. 5a, rus transcripts were detected 24 h after inoculation, and then decreased rapidly as growth proceeded. The same results were obtained with cyc2 (data not shown). These results strongly suggest a transient early-exponential-phase expression of the rus operon in S0-grown cells in the absence of any detectable Fe(II).

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 stem–loop structure ({Delta}G=–38·9 kJ mol–1). 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 stem–loop structures ({Delta}G=–45·2 and –118·5 kJ mol–1), 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.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge V. Mejean, M. Foglino, M. Chippaux, J. A. DeMoss (Laboratoire de Chimie Bactérienne, Marseille, France) and A. Manvell for many helpful comments and for critical reading of the manuscript. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org. Sequencing of A. ferrooxidans was accomplished with support from the US Department of Energy. We express our gratitude to L. Thöny-Meyer from the Mikrobiologisches Institut (Zürich, Switzerland) for her gift of pEC86 plasmid. We gratefully acknowledge M. Bruschi's laboratory (Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France) for antibodies directed against rusticyanin, and N. Guiliani (Laboratory of Molecular Microbiology and Biotechnology, Santiago, Chile) for antibodies directed against A. ferrooxidans Omp40 protein. We are grateful to R. Lebrun (IBSM Protein Sequencing Unit, Marseille, France) for performing the NH2-terminal sequence determination, and the staff from the animal care facilities of the IBSM (Marseille, France) for rabbit immunizations.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 5 December 2003; revised 9 March 2004; accepted 25 March 2004.



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