The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans

R. Quatrini1,2, C. Lefimil1,3, D. S. Holmes1,2 and E. Jedlicki3

1 Laboratory of Bioinformatics and Genome Biology, University of Andrés Bello, Santiago, Chile
2 Millennium Institute of Fundamental and Applied Biology, Santiago, Chile
3 Program of Cellular and Molecular Biology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile

Correspondence
D. S. Holmes
dsholmes2000{at}yahoo.com


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acidithiobacillus ferrooxidans is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidate furAF (Ferric Uptake Regulator) gene was identified in the A. ferrooxidans ATCC 23270 genome. FurAF has significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity to Escherichia coli Fur antiserum. The furAF gene is able to complement fur deficiency in E. coli in an iron-responsive manner. FurAF is also able to bind specifically to E. coli Fur regulatory regions (Fur boxes) and to a candidate Fur box from A. ferrooxidans, as judged by electrophoretic mobility shift assays. FurAF represses gene expression from E. coli Fur-responsive promoters fiu and fhuF when expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.


Abbreviations: EMSA, electrophoretic mobility shift assay; Q-PCR, quantitative PCR

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY465905.

A comparison of the predicted amino acid sequence of A. ferrooxidans FurAF with that of other micro-organisms, revealing the conservation of several motifs and structural features typical of the Fur family, is available with Supplementary Fig. S1 with the online version of this paper at http://mic.sgmjournals.org.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acidithiobacillus ferrooxidans is an important component in the consortia of micro-organisms used in biomining operations for metal recovery (Rawlings, 2002). It is an acidophilic, chemolithoautotrophic, {gamma}-proteobacterium capable of aerobic growth via the oxidation of Fe(II) to Fe(III) or reduced inorganic sulfur to sulfuric acid. Direct consequences of this metabolism are the generation of a substantial extracellular proton concentration (pH 1–3) and the release of high concentrations of soluble metals, including iron; the latter can reach values as high as 6 g l–1. Such values are in sharp contrast with the scarcity of soluble iron in near-neutral environments [10–17 M solubility limit for Fe(III) at pH 7].

Because of its redox properties, iron is an essential micronutrient for almost all organisms. However, given its limited bioavailability, most micro-organisms have developed sophisticated mechanisms to scavenge this metal from their environment (Braun & Killmann, 1999). On the other hand, the redox properties of iron also turn it into a biological hazard when acquired in excess, and bacteria have developed tight intracellular homeostatic controls to balance iron uptake and storage against possible oxidative stress (Hantke & Braun, 2000; Touati, 2000).

We are interested in understanding the mechanisms by which A. ferrooxidans copes with iron uptake and homeostasis given the unusually abundant supply of soluble iron in its acid environment. Also, since it uses iron as an energy source, it has presumably developed novel regulatory mechanisms to balance iron requirements for assimilation versus those needed for energy production. Very little is known about these postulated regulatory mechanisms, although evidence is beginning to emerge that implicates the use of alternate electron transport pathways depending on the energy source being used (iron or sulfur) (Brasseur et al., 2004) and a role for Fe(II) in the regulation of the rusticyanin operon, the expression of which is required for the oxidation of iron (Yarzábal et al., 2004).

In most bacteria, the iron-responsive transcriptional regulator Fur is responsible for coordinating the expression of iron uptake and storage functions (Andrews et al., 2003; Hantke, 2001). Although originally thought to be a typical apo-repressor of gene expression, activated in the presence of Fe(II) and binding to a conserved 19 bp sequence element in the promoter region of its target genes (Escolar et al., 1999), it is currently recognized to act as a global control switch adjusting substantial parts of bacterial metabolism in response to iron availability (Baichoo et al., 2002; McHugh et al., 2003; Ochsner et al., 2002).

The function of Fur has been investigated in many micro-organisms, including the dissimilatory ferric iron-reducing bacterium Shewanella oneidensis strain M-1 (Thompson et al., 2002). However, there are no studies regarding its role in A. ferrooxidans or in any other acidophilic, iron-oxidizing micro-organism. This deficiency motivated the investigations reported herein.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bioinformatic analysis.
A candidate fur gene was identified by BLASTP and TBLASTX searches in the partial genome sequence of A. ferrooxidans ATCC 23270 deposited in the TIGR (www.tigr.org) and Integrated Genomics (www.integratedgenomics.com) databases. The predicted amino acid sequence of the candidate fur gene was then used to formulate a BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) search of the non-redundant database at NCBI and bidirectional best hits were accepted as evidence for putative homologues. The candidate gene and the translated protein were further characterized employing the following bioinformatic tools available online: primary structure similarities (www.ebi.ac.uk/ClustalW), secondary structure predictions (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_multalin.html) and domain/motif recognition (www.ebi.ac.uk/InterProScan). The GenBank accession number of FurAF is AY481558.

Bacterial strains and growth conditions.
Bacterial strains and plasmids are described in Table 1. A. ferrooxidans was grown in modified 9K basal salt media [per litre: 0·4 g (NH4)2SO4, 0·1 g K2HPO4, 0·4 g MgSO4.7H2O] containing iron (9K+Fe: 100–400 mM FeSO4; adjusted to pH 1·6 with H2SO4) or sulfur (9K+S: 0·5 % ethanol-sterilized powdered sulfur, adjusted to pH 3·5 with H2SO4) at 30 °C under aerobic conditions on a rotary shaker at 150 r.p.m. E. coli was grown at 37 °C in LB broth with the pertinent antibiotics: ampicillin (Amp, 100 µg ml–1), streptomycin (Sm, 100 µg ml–1) and/or kanamycin (Km, 30 µg ml–1), as indicated in Table 1. LB medium was supplemented with 40 µM FeSO4 to generate iron-plentiful growth conditions or with the iron chelator o-phenanthroline (200 µM) to achieve iron deprivation.


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Table 1. Bacterial strains and plasmids used in this study

 
Growth curves.
9K+Fe or 9K+S media were inoculated with fresh mid-exponential-phase cells of A. ferrooxidans ATCC 23270 pre-grown in 9K+Fe or 9K+S, respectively, and incubated at 30 °C. Samples were removed daily and counted with a Petroff–Hausser counting chamber under a phase-contrast microscope at 400x magnification. The number of cells per millilitre was plotted against the incubation time. All experiments were performed at least twice with two to three replicates per treatment.

Cell collection.
A. ferrooxidans cultures, to be used for nucleic acid or protein purification, were centrifuged at 6000 g to remove solid sulfur or iron precipitates prior to cell harvest. The cell pellet was resuspended in 9K salt solution (ice cold solution in the case of RNA purification) for further washing. Washed cells were collected by centrifugation at 10 000 g for 10 min.

General DNA techniques and cloning procedures.
DNA isolation and routine manipulations were carried out following standard protocols as described by Sambrook et al. (1989) or by the manufacturers of the reagents. Plasmid DNA was prepared with the Wizard Plasmid Miniprep Kit (Promega) or the QIAprep Spin Mini-kit (Qiagen). PCR products for cloning were amplified with proofreading DNA polymerase Elongase (Invitrogen) and were purified from agarose gels with the QiaEx DNA purification kit (Qiagen). Oligonucleotide primers used in this study are listed in Table 2. Each PCR reaction contained 10 ng template DNA, 0·5 µM required primers and 0·2 mM each deoxyribonucleotide in a volume of 25 µl of 1x PCR buffer containing 1·5 mM MgCl2 (Invitrogen). PCR conditions were as follows: initial denaturing step at 95 °C for 5 min followed by 28–30 amplification cycles (denaturation at 95 °C for 30 s, annealing at the appropriate temperature depending on the specific primer pairs for 30 s and elongation at 72 °C) and a final elongation step at 72 °C for 10 min.


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Table 2. Primers used in this study

 
The A. ferrooxidans ATCC 23270 529 bp fur coding region was amplified from chromosomal DNA preparations with primers Fur1 and Fur2 (Table 2). The primer oligonucleotide pair Fur5 and Fur6 (Table 2) was used to amplify the E. coli K-12 fur gene from plasmid pMH15. Forward primers Fur1, Fur3, Fur5 and Fur7 included a consensus Shine–Dalgarno sequence placed at 11 or 10 bp from the start codon in varying sequence contexts (Table 2). PCR products were purified by gel electrophoresis and cloned into the pGEMT-Easy plasmid at the EcoRV site generating plasmids pAFH, pAFL, pECH and pECL, respectively (Table 1). Ligation mixtures were transformed into E. coli DH5{alpha} strain by electroporation and transformants were selected on LB plates containing the appropriate antibiotic. Clones selected for further analysis were validated by PCR analysis followed by DNA sequencing.

RNA preparation, RT-PCR and quantitative PCR.
RNA was isolated from E. coli grown to an OD600 of 0·5, as previously described by Aiba et al. (1981), and from 0·5 l A. ferrooxidans grown to mid-exponential phase in 9K basal salts medium in the presence of iron (100 and 200 mM) or sulfur (0·5 %), as described previously by Guacucano et al. (2000). Contaminating DNA was removed using a High Pure RNA Isolation Kit (Roche). Reverse transcription and coupled PCR amplification (RT-PCR) using the appropriate primers (Table 2) and including controls (Guacucano et al., 2000) were performed, respectively, with Superscript II reverse transcriptase (Gibco-BRL) and Taq Polymerase (Invitrogen), according to the manufacturer's recommendations. One microgram of total cellular RNA was used for each reaction.

Transcriptional expression levels of fur and control genes were estimated through real-time quantitative PCR (Q-PCR) on reversed transcribed cDNA. cDNA was prepared from 2–5 µg total RNA utilizing random hexamers and Superscript II reverse transcriptase. Specific primers (Table 2) amplifiying mean products of 300 bp were used to quantify fur and pertinent control gene transcripts. The Fast Start DNA Master SYBR Green I PCR kit (Roche Applied Biosystems) and the LightCycler System (Roche Applied Biosystems) were utilized according to the manufacturer's protocol. Quantifications were performed twice, with both independent total RNA and cDNA preparations, by the comparative threshold cycle method. The calculated threshold cycle (Ct) for each gene was normalized to Ct of the 16S rRNA gene (which has invariant expression under different growth conditions; Yarzábal et al., 2004) amplified from the corresponding sample before calculating the fold change between growth conditions (S, Fe100, Fe200).

Complementation of fur mutations.
Complementation was performed by transformation of fur mutant strains H1780 or H1681 (Hantke, 1987) with the constructs presented in Table 1 and was assessed by three methods: phenotypic analysis on MacConkey-lactose agar plates (Hantke, 1987), {beta}-galactosidase activity determination (Miller, 1972) or survival to H2O2 challenge (Touati et al., 1995).

{beta}-Galactosidase activity was assayed by the ONPG assay as described by Miller (1972) using SDS and chloroform-permeabilized cells. {beta}-Galactosidase activity was calculated in Miller units. For this purpose, freshly inoculated cultures were grown to OD600 0·4–0·5 in LB plus antibiotics and divided into two 15 ml replicate aliquots to be either supplemented with 40 µM FeSO4 or depleted of iron by addition of 200 µM of the iron chelator o-phenanthroline. Treatments were for 3 h at 37 °C with agitation (200 r.p.m.) and were terminated by chilling on ice. Duplicate samples of each culture were assayed in at least three repetitions of each experiment.

H2O2 challenge was performed as described by Touati et al. (1995), with minor modifications. LB-grown cells (OD600 0·4–0·5) were distributed into 5 ml aliquots, and treated with 0, 1, 5, 15, 30 mM H2O2. After 20 min incubation with shaking (200 r.p.m.) at 37 °C, treatment was stopped by chilling on ice and dilution into fresh LB. Dilutions of each treatment were immediately plated on LB agar and colonies were counted after overnight incubation at 37 °C. Killing by H2O2 was assessed as the percentage of surviving colonies compared with unchallenged cells. Experiments were performed twice with duplicate samples.

Construction of vectors expressing low concentrations of Fur.
The furAF gene was cloned into pGEMT-Easy such that its transcription was controlled by the vector promoter but its translational control was exerted by a synthetic ribosome-binding sequence (RBS) introduced upstream of the ATG start site of furAF (Table 2). The new construct was termed pAFL (L, low expression). This sequence contains a translation initiation region with a predicted secondary structure twice as stable as that in the equivalent structure of pAFH (H, high expression) and this modification significantly reduces the efficiency of translation (de Smith & van Duin, 1990; Gross et al., 1990; Hartz et al., 1991). A similar construct containing FurEC under the control of the sequence that reduces translational efficiency was prepared and termed pECL.

Western blotting.
Samples of E. coli cultures (1·5 ml) grown to OD600 0·5 in LB broth were centrifuged, suspended in sample loading buffer (100 µl) and heat denatured (10 min) in the presence of reducing agents (Sambrook et al., 1989). Aliquots of 4–10 µl of the total protein extract were separated by SDS-PAGE in 15 or 18 % acrylamide and transferred onto nitrocellulose filters by standard methods with a Bio-Rad blotting apparatus. The blotted proteins were subsequently screened using a polyclonal rabbit anti-Fur serum generated against Fur from E. coli (obtained from M. L. Vasil, University of Colorado Health Sciences Center). When necessary, antiserum was pre-adsorbed against E. coli H1780 crude extracts as described by Sambrook et al. (1989). Filters were blocked overnight at 4 °C with agitation in blocking solution [5 % skimmed milk, 0·05 % Triton X-100, Tris-buffered saline (TBS)], incubated 1 h with a 1 : 500 dilution of the primary antibody in TBS/Tween 20 (0·05 %) and further incubated in a 1 : 15 000 dilution of peroxidase-conjugated anti-rabbit immunoglobulin in TBS/Tween 20 (0·05 %) for another hour. Immunoreactive proteins were detected using the Supersignal West Pico chemiluminescent substrate (Pierce). Prestained broad-range molecular mass protein standards from Bio-Rad were used. Protein concentrations were determined with Bio-Rad Protein Assay, using BSA as standard.

Immunoprecipitation.
E. coli was grown to OD600 0·8 in LB broth and cells were collected by centrifugation at 12 000 g. Pellets were washed with 25 mM Tris/HCl, pH 8·0, and suspended in 10 vols lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris/HCl, pH 8·0, 1 mM PMSF) containing 4 mg lysozyme ml–1. After 30 min incubation on ice, cells were sonicated for 10 s per pulse (5 pulses alternating with 1 min incubation in ice). Following centrifugation for 10 min at 12 000 g, the supernatant was diluted in 2x radioimmunoprecipitation assay (RIPA) buffer [100 mM Tris/HCl, pH 8·0, 2 mg SDS ml–1, 10 mg deoxycholate ml–1, 300 mM NaCl, 2 % (v/v) Nonidet P40, 2 mM PMSF] and incubated 5 min on ice by the method of Qi et al. (1999). The samples were treated with polyclonal rabbit anti-FurEC antibodies (dilution 1 : 500) for 2 h at 4 °C and immediately incubated with stirring for 1 h at 4 °C with 300 µl protein A–agarose (Invitrogen) pre-equilibrated in 1x RIPA buffer. Immunoprecipitated proteins were released from the collected beads in 8 M urea and resuspended in gel loading buffer for subsequent electrophoresis in 15 % polyacrylamide gels and Western blotting (20 µl per well).

Fur titration assay (FURTA).
The Fur titration assay was performed according to Stojiljkovic et al. (1994). A pUC18-based randomly cloned genomic library from A. ferrooxidans ATCC 19859 (pGTF) containing Sau3A DNA fragments (averaging 1 kb in size) was electroporated into the E. coli strain H1717 (fur+, fhuF : : lacZ, Table 1). A total of 3·1x105 AmpR clones were recovered on selective MacConkey indicator plates containing 40 µM FeSO4. The cloning vector pGEMT-Easy and the E. coli Fur box upstream of the fhuF gene (FB-FhuF1+2, Table 2) were employed as negative and positive controls, respectively. Clones exhibiting red colour in MacConkey-Fe plates after 24 h incubation at 37 °C were isolated and further restreaked. Inserts were subsequently subjected to DNA sequencing.

Electrophoretic mobility shift assays (EMSA).
Double-stranded DNA probes (33 bp), containing the well-characterized Fur box of the fhuF gene from E. coli and the putative Fur box of the mntH gene from A. ferrooxidans, were generated by annealing two perfectly complementary oligonucleotides designed after de Lorenzo et al. (1988) (Table 2). Equal parts of both oligonucleotides (50 pmol) were heated to 65 °C for 10 min and allowed to cool slowly to room temperature. The probe was end labelled with [{gamma}32P]ATP using T4 polynucleotide kinase (Invitrogen). Unincorporated nucleotides were removed through a Bio-Gel P10 Micro Bio-spin chromatography column (Bio-Rad). E. coli QC-1732 carrying pAFH and pECH was grown to OD600 0·8, harvested at 12 000 g and suspended at 1/10 volume in gel mobility buffer [20 mM Tris/HCl, pH 8·0, 0. 5 % (v/v) glycerol, 1 mM PMSF, 2 µg aprotinin ml–1] containing 100 µg lysozyme ml–1. After 40 min in ice, the cell suspension was sonicated for 10 s per pulse (5 pulses alternating with 1 min incubations in ice). Following centrifugation for 10 min at 12 000 g, the supernatant was stored at –20 °C until use.

EMSA assays were performed as described by de Lorenzo et al. (1988), with the following modifications: 80–100 µg total protein was equilibrated in 20 µl final volume of gel mobility shift buffer [20 mM Tris/HCl, pH 8·0, 40 mM KCl, 1 mM MgCl2, 0·1 mM MnSO4, 0·1 mg BSA ml–1, 5 % (v/v) glycerol]. The labelled probe (50–75 pM; 10 000 c.p.m.) and non-specific competitor salmon sperm DNA (50 µg µl–1) were added, and the reactions incubated for 10 min at 30 °C. A 50-fold excess of cold annealed oligonucleotide was used to challenge the labelled probe. In supershift experiments, the target DNA and a 1 : 500 dilution of Fur-specific antiserum were added to the reaction and incubated for an additional 5 min. Mixtures were resolved by non-denaturing polyacrylamide (4 %, w/v) gel electrophoresis at 200 V for 1 h in Tris/acetate buffer at 4 °C. Retardation was examined by exposing the gel after drying to Kodak X-OMAT film (Sigma) or analysed with a PhosphorImager (Molecular Imagen FX Pro Plus, Bio-Rad).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the fur homologue in A. ferrooxidans
Examination of the partial genomic sequence of A. ferrooxidans ATCC 23270 revealed the presence of a candidate gene encoding a potential Fur protein with 60 % amino acid sequence identity to the predicted Fur of Alteromonas sp. O-7 (gi: BAB13366 and 51 % identity to the experimentally verified Fur of E. coli (gi: P06975). The nucleotide sequence of the candidate furAF and the amino acid sequence of its hypothetical protein product were deposited in GenBank (accession no. AY465905). The candidate FurAF has a predicted length of 158 amino acids (molecular mass 17·9 kDa) and an isoelectric point of 5·0.

Comparison of the predicted amino acid sequence of FurAF with that of other micro-organisms revealed the conservation of several motifs and structural features typical of the Fur family (see Supplementary Fig. S1, available with the online version of this paper at http://mic.sgmjournals.org). FurAF exhibits a conserved helix–turn–helix motif near the N-terminus known to be involved in DNA binding in well-documented Fur proteins (Gonzalez de Perédo et al., 2001; Holm et al., 1994). It also presents conservation of all residues demonstrated or suggested to be involved in iron binding and structural zinc coordination (Adrait et al., 1999; Bsat & Helmann, 1999; Coy et al., 1994; Jacquamet et al., 1998; Pohl et al., 2003). On the basis of these similarities, it is proposed that FurAF is a member of the Fe-responsive subfamily of Fur regulators.

Inspection of the genome sequence of A. ferrooxidans around the candidate furAF gene revealed an ORF potentially encoding the putative outer-membrane protein OmlA, 66 bp upstream from the suggested start site of FurAF, but oriented in the opposite direction. The juxtaposition of this gene pair is conserved in several {alpha}- and {beta}-Proteobacteria, including Burkholderia, Ralstonia, Neisseria, Bordetella, Pseudomonas, Xylella and Xanthomonas (Lowe et al., 2001; Ochsner et al., 1999). Although divergent genes frequently share common regulatory mechanisms (Escolar et al., 1998), experimental evidence suggests that transcription of Pseudomonas aeruginosa omlAfur is not co-regulated (Ochsner et al., 1999). In addition, none of the bacteria where the omlAfur context is conserved are capable of the otherwise frequently observed autoregulation of Fur (Loprasert et al., 1999; Barton et al., 1996; Thomas & Sparling, 1994).

Complementation of Fur deficiency in E. coli by FurAF
To determine if FurAF is a functional Fur homologue, complementation of fur deficiency in the E. coli reporter strains H1780 and H1681 was evaluated as described by Hantke (1987). H1780 and H1681 (Table 1) each carry a chromosomally embedded lacZ gene fused to a Fur-dependent promoter such that, in the presence of a functional Fur and the co-repressor Fe(II), {beta}-galactosidase synthesis is significantly inhibited. The candidate furAF from A. ferrooxidans was cloned into the expression vector pGEMT-Easy and the resulting plasmid was termed pAFH (Table 1). Similarly, the experimentally validated E. coli fur gene, furEC, was cloned into pGEMT-Easy and the resulting plasmid was termed pECH (Table 1). pAFH and pECH were each transformed into the E. coli reporter strains H1780 and H1681 and {beta}-galactosidase activity determined in the presence or absence of iron (Fig. 1).



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Fig. 1. Complementation of Fur repression activity by Fur derived from A. ferrooxidans in (a) E. coli H1780 fur and (b) E. coli H1681 fur. Fur activity (mean of three experiments) was determined by {beta}-galactosidase activity in Miller units (Miller, 1972) for cells grown in either the presence of 40 mM FeSO4 (white columns) or 200 µM o-phenanthroline (black columns) and is reported as the percentage activity of E. coli fur. pAFH, construct expressing A. ferrooxidans Fur; pECH, construct expressing E. coli Fur.

 
E. coli fur-host strains H1780 and H1681 express fiu/fhuF-lacZ constitutively in a manner unaffected by iron (200 Miller units). In both hosts, transformation with pAFH resulted in decreased {beta}-galactosidase activity in the presence of iron. Addition of the iron chelator o-phenanthroline diminished this reduction. Similar results were obtained when the positive control pECH was introduced into H1780 and H1681. Under the conditions of the experiment, the activity of FurAF in E. coli is similar to that of FurEC, strongly suggesting that FurAF is a functional Fur homologue capable of iron-dependent repression.

FurAF binds to the E. coli fhuF Fur box
The ability of A. ferrooxidans Fur to bind the E. coli fhuF Fur box in vitro was investigated using EMSA (Fig. 2a). As expected, a whole-cell protein extract from the E. coli host strain QC-1732 ({Delta}fur, Table 2) was unable to retard the radioactively labelled E. coli fhuF Fur box in an EMSA (Fig. 2a, lane 2). In contrast, proteins derived from the same host harbouring pAFH were able to shift the labelled E. coli fhuF Fur box (Fig. 2a, lane 3). Furthermore, the addition of anti-Fur antibodies to the reaction produced a supershift in migration, indicating that the shift reaction was caused specifically by the binding of FurAF to the E. coli fhuF Fur box (Fig. 2a, lane 4). Consistent with this interpretation, a complete loss of the shift was observed when excess unlabelled DNA probe was used to outcompete the labelled probe (Fig. 2a, lane 5). As a positive control, these reactions were repeated using proteins derived from E. coli QC-1732 transformed with pECH (Fig. 2a, lanes 6, 7 and 8), confirming that the homologous FurEC as well as the heterologous FurAF was capable of binding to the labelled fhuF E. coli Fur box.



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Fig. 2. Determination of the ability of FurAF to bind Fur boxes using the EMSA and supershift assay. The probe DNA Fur boxes were: (a) 33 bp oligonucleotide containing the fhuFEC Fur box, (b) 33 bp oligonucleotide containing the mntHAF Fur box and (c) 428 bp PCR product containing the furAF promoter region. Probe DNA was incubated with extracts from E. coli QC-1732 ({Delta}fur) carrying construct pECH or construct pAFH. Abbreviations: S, shift; SS, supershift; P, probe DNA; P*, competing excess unlabelled probe DNA; {lambda}-Fur, anti-Fur antibody; Fur, Fur protein in cell extract; +, addition; –, no addition.

 
FurAF binds to the A. ferrooxidans mntH Fur box
Having demonstrated that FurAF is able to recognize and bind to the heterologous E. coli fhuF Fur box in vitro, we investigated whether it could also recognize and bind a Fur box from A. ferrooxidans (Fig. 2b). A search for functional Fur box sequences in A. ferrooxidans was carried out by subjecting a randomly cloned genomic library of A. ferrooxidans to the Fur titration assay (Stojiljkovic et al., 1994). A candidate target DNA fragment carrying the region upstream of the putative Mn(II)/Fe(II) transporter gene mntH (Makui et al., 2000) was identified and selected for further study based on two lines of evidence: 1) homologues of this gene, including one in E. coli, have been shown to be regulated by upstream Fur boxes (Kehres et al., 2000, 2002) and 2) alignment of the upstream regions of the respective mntH genes of E. coli and A. ferrooxidans reveals a 12 bp region of 100 % identity (AATCATTCTCG) that partially corresponds to the known Fur box of the E. coli mntH gene.

Proteins derived from E. coli QC-1732, transformed with either pAFH (Fig. 2b, lane 3) or pECH (Fig. 2b, lane 6), were able to shift the DNA probe containing the postulated A. ferrooxidans mntH Fur box. These reactions were demonstrated to be Fur specific by supershifting in the presence of anti-Fur antibody (Fig. 2b, lanes 4 and 7) and by competition with cold probe DNA (Fig. 2b, lanes 5 and 8). No effect on the migration of the mntHAF probe could be detected when utilizing the host strain QC-1732 protein extracts (Fig. 2b, lane 2) that do not contain Fur.

Given the frequently observed capacity of Fur to affect its own expression, we also evaluated the migration shift of a probe carrying the olmAfurAF intergenic region, containing the divergent promoters for both genes. No retardation could be detected using either the pAFH or the pECH protein extracts (Fig. 2c, lanes 2 and 3). The inability of FurAF to shift its own promoter region in vitro is in agreement with previously reported evidence indicating that none of the bacteria sharing the conserved gene context omlAfur are capable of autoregulation (Barton et al., 1996; Loprasert et al., 1999; Thomas & Sparling, 1994).

Expression of FurAF in A. ferrooxidans in different growth conditions
FurAF was detected by Western blotting in whole-cell extracts of A. ferrooxidans grown on either S0 or on standard 9K+Fe media [100 mM Fe(II); Fig. 3b, lanes 2 and 3]. However, increasing the Fe(II) concentration to 200 mM significantly decreased FurAF (Fig. 3b, lane 4) and growth in 400 mM Fe(II) almost abolished it (Fig. 3b, lane 5). This decrease was not paralleled by a general reduction in total cell protein yield or by an observable change in the distribution of molecular masses of the proteins as determined by SDS-PAGE (data not shown).



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Fig. 3. (a) Fe(II) concentration remaining (dotted lines) in the media at each time point plotted against the growth (solid lines) of A. ferrooxidans (cells ml–1). A mid-exponential-phase starting culture grown at 100 mM FeSO4 with 9K salts was inoculated into fresh 9K basal media containing increasing concentrations of FeSO4 ({bullet}, 100 mM; {blacktriangleup}, 200 mM; {blacksquare}, 400 mM) and incubated at 30 °C with agitation (150 r.p.m.). (b) Western blot (SDS-PAGE, 18 %) with antiserum raised against E. coli Fur of A. ferrooxidans total proteins from cells cultured either in sulfur-containing medium or in medium containing different Fe(II) concentrations. Arrows mark the migration of proteins with molecular masses of approximately 17 kDa and 19 kDa. Lanes: 1, FurEC expressed from pECH construct as positive control; 2, A. ferrooxidans total protein extracts from 9K+S grown cells; 3, A. ferrooxidans total protein extracts from 100 mM Fe(II)-grown cells; 4, A. ferrooxidans total protein extracts from 200 mM Fe(II)-grown cells; 5, A. ferrooxidans total protein extracts from 400 mM Fe(II)-grown cells; 6, FurAF expressed from pAFH construct as a cross-reactivity control.

 
Quantification of Fe(II) by the o-phenanthroline method in the culture medium at the time of cell harvest (Fig. 3a) revealed that cultures initiated with 100 mM Fe(II) lacked detectable amounts of Fe(II) after 50 h growth, indicating that all the available Fe(II) had been oxidized to Fe(III). In contrast, samples expressing diminished amounts of FurAF protein obtained from cultures grown at either 200 or 400 mM Fe(II) had oxidized only part of the initial Fe(II) content of the medium (5 and 20 %, respectively). The results are thus consistent with a pattern in which Fur levels diminish in cells exposed to increasingly higher concentrations of Fe(II) and this is consistent with the observed presence of Fur in S0-grown cells.

Having demonstrated that Fur concentrations diminished with increasing Fe(II) concentration, we wanted to evaluate whether the decrease resulted from a reduction of transcription of furAF. Quantitative RT-PCR experiments were designed to measure fur mRNA levels. Table 3 shows the mean fold difference in fur mRNA levels between iron and sulfur (Fe100/S0) and between the two iron concentrations employed (Fe200/Fe100). No significant decrease in fur mRNA was detected with increasing Fe(II) concentration and levels of fur mRNA were similar (±twofold) in both Fe(II)- and S0-grown cells, indicating that the observed variation in Fur concentrations does not result from substantial differences in transcriptional activity. Control genes pgm (no variation) and sdrAI (iron induced) were included for comparative purposes.


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Table 3. Fold change in gene expression levels determined by Q-PCR

Values shown are mean±SD.

 
Effect of Fur titre variation on (heterologous) gene expression
In contrast to what has been described in other systems where Fur levels are known to be regulated but do not drop below a threshold (Delany et al., 2002, 2003), FurAF levels varied dramatically in A. ferrooxidans. This prompted the question whether varying FurAF concentration would differentially affect gene expression in E. coli. To address this issue, expression of lacZ from the reporter strain H1780 was monitored in E. coli to provide insight into the effect of FurAF on an established promoter fhuF. In addition, E. coli cell survival was measured as a function of increasing concentrations of H2O2. This latter assay has been used as evidence that E. coli Fur provides protection against iron-induced oxidative damage both by binding iron in a direct manner and also by repressing the transcription of genes involved in iron uptake and homeostasis (Touati et al., 1995).

For this purpose, a construct was designed expressing low levels of FurAF (pAFL) or FurEC (pECL), to be compared with the previously described high-expression counterparts (pAFH and pECH). The four constructs were transformed into the E. coli fur strain H1780, and Fur expression at both protein and mRNA levels was monitored by Western blotting and RT-PCR, respectively (Fig. 4a, b). A comparison of protein and mRNA expression demonstrated that the absence of detectable Fur protein, in cells containing the low-expression constructs pAFL or pECL, results from reduced translational (Fig. 4a, lanes 4 and 7) and not transcriptional efficiency (Fig. 4b, lanes 4 and 7). Complete absence of FurAF in H1780 containing pAFL was discounted because Fur could be detected in the extracts after immunoprecipitation (Fig. 4a, lane 6).



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Fig. 4. Effect of FurAF protein expression levels in E. coli H1780 (fur), as indicated by (a) Western blot detection of Fur in E. coli crude extracts, (b) RT-PCR detection of fur mRNAs in E. coli total RNA preparations, (c) the expression of lacZ in the E. coli reporter strain H1780 and (d) the survival of E. coli cells as a function of increasing concentrations of H2O2. In (c), the quantitative determination of {beta}-galactosidase ({beta}-Gal) activity for clones grown in either the presence (white columns) or absence (black columns) of Fe(II) is shown. The mean (±SD) {beta}-galactosidase activity (OD420/OD600 ml min) from at least three independent experiments is expressed as a percentage of the derepressed condition for strain H1780. In (d), aerobically grown cultures were challenged for 20 min with 10 mM H2O2. The mean (±SD) c.f.u. ml–1 after overnight incubation from at least three independent experiments is expressed as a percentage of the counts for wild-type E. coli K-12. Abbreviations: FurEC, Fur from E. coli K-12 (fur+); fur, E. coli H1780; FurAF, Fur from A. ferrooxidans grown in S0. pAFL, pAFH, pAFLIPP, pECL and pECH refer to E. coli H1780 carrying plasmids of the same names (described in text).

 
{beta}-Galactosidase activity assays showed that the high-expression constructs pAFH and pECH (Fig. 4c) reduced expression of the reporter by 60 and 90 %, respectively (with respect to basal expression of the host strain H1780 in the absence of Fe), supporting the observations of Fig. 1. Strikingly, while the construct expressing low levels of Fur from E. coli pECL reduced reporter gene expression by nearly 90 % in the presence of iron, pAFL increased expression of the lacZ reporter gene by more than 70 %. Similar results were obtained with the host H1681 containing the fhuF–lacZ fusion (data not shown). The positive effect on the expression of lacZ from these E. coli promoters does not appear to be significantly influenced by the presence or absence of iron. One possible explanation for this observation is that at such low Fur concentrations, all the Fur regulator is in an iron-complexed state.

Results consistent with this interpretation were obtained in the H2O2 survival tests. Whereas nearly 90 % of E. coli K-12 cells containing wild-type fur survived exposure to H2O2 up to 10 mM, growth of E. coli H1780 lacking fur was severely impaired (less than 20 % survival) (Fig. 4d). Partial restoration of survival was achieved by complementation of fur deficiency with either FurEC (pECH) or FurAF (pAFH). However, the incorporation of pAFL into E. coli H1780 rendered it appreciably more sensitive to oxidative stress by H2O2, with no cells surviving exposure above 10 mM. In contrast, low-expression levels of FurEC (pECL) restored survival to the same levels as the high-expression construct pECH.

Taken together, these results raise the possibility that FurAF is able to act as a positive gene regulator of Fur targets in E. coli, inducing expression from E. coli fiu (H1780) and fhuF (H1681) promoters and probably that of other iron-uptake genes, thereby disturbing iron homeostasis and rendering the organism more sensitive to H2O2. The mechanism by which these effects are exerted will only be clarified by the development of appropriate genetic tools for A. ferrooxidans.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper, we report the identification and preliminary functional characterization of the first Fur orthologue from the Fe(II)-oxidizing acidophilic bacterium Acidithiobacillus ferrooxidans. Despite considerable effort, A. ferrooxidans is recalcitrant to standard genetic manipulation techniques, although a promising report for establishing a conjugation system for genetic exchange with E. coli has been published (Liu et al., 2000). This experimental deficiency seriously limits the exploration of the functional role of the postulated Fur by mutation analysis in A. ferrooxidans.

To address this limitation, we analysed a potential fur gene and a fur-responding Fur box in the available A. ferrooxidans genome and experimentally tested predictions of the bioinformatic analysis, both in vitro (EMSA experiments) and in heterologous E. coli mutant strains (Fig. 1).

Genome analysis and experimental evidence support the tentative conclusion that the fur gene of A. ferrooxidans encodes a functional Fur orthologue FurAF. FurAF exhibits significant sequence similarity to well-characterized Fur proteins, including conserved motifs involved in DNA recognition and binding. As demonstrated for other Fur orthologues sharing more than 50 % sequence identity with Fur from E. coli, FurAF displays cross-reactivity with E. coli Fur antiserum, which allowed informative supershift EMSA (Fig. 2) and Western blot experiments (Fig. 4) to be carried out. FurAF overcomes fur deficiency in E. coli mutant strains by downregulating the expression of fiu and fhuF genes in an iron-dependent manner, as has been described for well-characterized Fur orthologues (Fig. 1). This suggests that FurAF is capable of recognizing and binding in vivo to the fiu and fhuF Fur boxes. Both FurAF from A. ferrooxidans and FurEC from E. coli are capable of binding in vitro to a proposed A. ferrooxidans Fur box postulated to reside upstream of the putative gene mntH that potentially encodes a Mn(II)/Fe(II) transporter (Fig. 2). This supports the conclusion from sequence analysis that the DNA binding helices and DNA sequence-recognition motifs of FurAF and FurEC are similar. Such similarity has been documented for conserved Fur proteins from many other bacteria that generally also share well-conserved Fur regulons.

Despite their similarities, FurAF and FurEC exhibit an unexpected and important difference in their ability to control transcription in E. coli. Low cellular concentrations of FurAF stimulated transcription not only from reporter genes regulated by two well-established E. coli Fur boxes, fiu and fhuF, but also promoted H2O2-related cell death (Fig. 4d), an observation that is consistent with the idea that FurAF can upregulate Fur target genes that contribute to increased intracellular oxidative stress.

Several reports have identified conditions in which Fur promotes gene expression rather than represses it. These results have been largely attributed to indirect mechanisms involving RNA antisense regulation (Massé & Gottesman, 2002) and regulatory cascades that do not implicate the involvement of Fur boxes (Hall & Foster, 1996; Touati, 2000; Vasil & Ochsner, 1999). In Helicobacter pylori, Fur regulates Fe-activated promoters by a mechanism of derepression (Fe-dependent derepression) (Delany et al., 2001). Only recently was evidence found for the direct participation of Fur in iron-responsive transcriptional activation in Neisseria meningitidis (Delany et al., 2004). In this organism, Fur-activated genes have been shown to have canonical Fur operators centred upstream of the –35 promoter signature. This mechanism is likely to be shared by other Fur orthologues, and among them possibly FurAF.

The fact that FurEC at equivalently low intracellular titres remained a repressor of target genes raises an intriguing question: is this property of dual regulatory activity inherent to A. ferrooxidans FurAF or is it a result of heterologous mixing of FurAF with E. coli Fur-dependent promoters? The answer to this question awaits the development of genetic tools for A. ferrooxidans.

A dual role for FurAF is appealing in light of the measurements showing that Fur levels vary in A. ferrooxidans depending on the concentration of Fe(II) (Fig. 3). Typically, Fur is expressed in other bacteria at constitutively high levels, reaching protein titres that can amount to as many as 10 000 molecules per cell (Barton et al., 1996; Watnick et al., 1997; Zheng et al., 2001). Such abundance, presumably beyond the need for stoichiometric binding to Fur-box promoters, constitutes a distinguishing feature of the regulatory strategy of Fur that may be explained by the need to control the large number of genes of the Fur regulon and by its polymerization-dependent mechanism of repression that requires the cooperative binding of several Fur molecules per regulatory site (Escolar et al., 2000). Small variations in the level of Fur protein that respond to variations in iron availability have previously been documented in other bacteria (Hernández et al., 2002; de Lorenzo et al., 1988; Delany et al., 2002) but in no case do these drop below protein detection limits as occurred in A. ferrooxidans.

The possibility that Fur exhibits a dual capacity as a gene activator or repressor in E. coli, depending on its intracellular concentration, suggests a mechanism by which A. ferrooxidans can simultaneously control the expression of genes involved in iron uptake, which need to be down-regulated in the presence of unusually high iron loads to diminish oxidative stress, and genes involved in the energy-producing oxidation of iron, which need to be upregulated in these conditions, such as the rusticyanin operon (Yarzábal et al., 2004) and the alternate cytochrome bc1 complex thought to be involved in reverse electron flow (Brasseur et al., 2004). These suggestions are currently under experimental investigation.

Although our results are based on in vitro assays (Fig. 2) and measurements of the activity of FurAF in a heterologous system (Figs 2 and 4), the evidence obtained suggests that in addition to classical Fe-dependent repression, Fe-sensitive derepression, as reported by Delany et al. (2001), and Fe-dependent indirect activation exerted by Fur in other systems (Hall & Foster, 1996; Massé & Gottesman, 2002), variations in the levels of Fur might add to the complexity of regulatory strategies of the ferric uptake regulator in A. ferrooxidans.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from FONDECYT 1010623 and a Deutscher Akademischer Austausch Dienst (DAAD) scholarship to R. Quatrini. We thank the Institute of Genome Research (TIGR) and Integrated Genomics (IG) for the use of their partial sequence of the A. ferrooxidans genome. Sequencing of A. ferrooxidans at TIGR was accomplished with support from the USA Department of Energy (DOE). We are indebted to K. Hantke and D. Touati for providing the E. coli host strains used in this study and M. Vasil for kindly providing anti FurEC antibody. Q-PCR was performed at the Transcriptome Platform, CNRS, Marseille, France.


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Received 23 August 2004; revised 7 March 2005; accepted 10 March 2005.



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