School of Biological Sciences1 and School of Chemical Sciences2, University of East Anglia, Norwich NR4 7TJ, UK
Author for correspondence: Stephen Spiro. Tel: +44 1603 593222. Fax: +44 1603 592250. e-mail: s.spiro{at}uea.ac.uk
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ABSTRACT |
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Keywords: Fur, iron regulation
Abbreviations: CD, circular dichroism; Gdn . HCl, guanidinium hydrochloride; MBP, maltose-binding protein
a Present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
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INTRODUCTION |
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Iron acquisition is a particular problem for pathogenic bacteria, since the bioavailability of iron in the host is often especially low. Hence, there is often a need for iron acquisition systems to be switched on when the pathogen encounters a host environment (Litwin & Calderwood, 1993 ; Ratledge & Dover, 2000
). Furthermore, there is good evidence to indicate that iron limitation is used, in some cases, as a signal to activate the expression of virulence genes, and that this regulation can ultimately depend on Fur. One example is the opportunistic pathogen Pseudomonas aeruginosa, in which Fur regulates the expression of the alternative sigma factor encoded by the pvdS gene, which is in turn required for the transcription of some virulence genes, including those encoding the endoprotease PrpL and exotoxin A (Leoni et al., 1996
; Wilderman et al., 2001
; Ochsner et al., 1996
; reviewed by Vasil & Ochsner, 1999
).
Genetic characterization of fur mutants, and biochemical analysis of the Fur protein, suggested that the active species is a dimer with one Fe2+ ion bound to each monomer. Results from proteolysis and gene-fusion experiments indicated that the protein folds into an N-terminal DNA-binding domain and a C-terminal metal-binding and dimerization domain (Coy & Neilands, 1991 ; Stojiljkovic & Hantke, 1995
). Early models for the mode of action of Fur had the protein functioning as a classical repressor, with the Fe2+ ion acting as co-repressor. This simple view has been challenged by two recent developments. The first of these is the identification of a structural Zn2+ ion in the E. coli protein that is apparently required for DNA binding, and is bound to a site distinct from the metal-sensing site (Jacquamet et al., 1998
; Gonzalez de Peredo et al., 1999
; Althaus et al., 1999
). Secondly, it has been suggested that the binding of Fur to DNA is directly inhibited by EDTA, which may require the conclusions of some earlier experiments to be re-evaluated (Althaus et al., 1999
). E. coli Fur containing the structural Zn2+ ion and with a vacant metal-sensing site has been shown to bind to DNA with high affinity, which may even call into question the role of Fe2+ as co-repressor (Althaus et al., 1999
). In this context, it is interesting that the Bacillus subtilis Fur apparently does not require Fe2+ for DNA binding activity in vitro (Bsat & Helmann, 1999
). Ligands to the structural Zn2+ ion are believed to include Cys-92 and Cys-95, which have been shown to be essential for normal Fur activity in E. coli (Coy et al., 1994
; Gonzalez de Peredo et al., 1999
). Indeed, amongst a collection of Fur proteins substituted at all cysteine and histidine residues, those in which Cys-92 or Cys-95 were replaced by serine had much the most severe phenotypes (Coy et al., 1994
). These two cysteines are conserved in, for example, the Fur proteins of Vibrio anguillarum and Bacillus subtilis, which are also thought to contain a structural Zn2+ ion (Bsat & Helmann, 1999
; Zheleznova et al., 2000
). On the other hand, only Cys-92 is conserved in some presumed Fur orthologues, including that from P. aeruginosa, and in other cases (such as Pseudomonas putida), neither cysteine is conserved. This raises the possibility of structural and/or mechanistic diversity in the Fur family, perhaps with some members not requiring a structural Zn2+ ion for activity (Zheleznova et al., 2000
). As a first step in exploring whether sequence diversity is reflected in the biochemical properties of Fur proteins, the Fur from P. aeruginosa has been further characterized. It is shown that this protein does not contain a structural Zn2+ ion, and that its single cysteine residue is not essential for activity in vivo. Two histidine residues essential for P. aeruginosa Fur activity are also identified.
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METHODS |
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DNA manipulations.
The P. aeruginosa fur gene was amplified from chromosomal DNA purified from strain PAO1, using a primer (5'-CATATGGTTGAAAATAGCGAACTT-3') that incorporated an NdeI site at the start codon (underlined) of the coding region. The PCR product was cloned into pUC18, and sequenced to check that mutations had not been introduced. The fur gene was then excised from this clone using the vector PvuII sites and was blunt-end cloned into the ScaI site of pBR322 to generate pPAD24. The fur gene in pPAD24 is expressed from the lac promoter derived from pUC18, and the lower-copy-number vector was found to be required for stable maintenance of the clone. Mutations were introduced into a clone of the fur gene in pUC18 by a PCR-based method (Hutchings et al., 2000 ); mutant genes were sequenced twice to confirm that the correct mutation had been introduced, and were then cloned on PvuII fragments into the ScaI site of pBR322. The NdeIEcoRI fragment from the pUC18 fur clone was cloned into pET21a to generate a clone suitable for overexpression in BL21(
DE3). For construction of an MBPFur fusion, fur was amplified by PCR using a 5' primer (5'-GGGAATTCATGGTTGAAAATAGCGAACT-3') incorporating an EcoRI site immediately upstream of the start codon (underlined). PCR products were blunt-end cloned into SmaI-digested pUC18, and, from a clone in the correct orientation, fur was excised on an EcoRIBamHI fragment (using the vector BamHI site) and cloned into pMAL-c2 (New England Biolabs). The intermediate pUC18 clone was sequenced to ensure that no mutations had been introduced. This procedure fused fur in-frame to malE; the gene fusion is predicted to encode a protein that adds the sequence Ile-Ser-Glu-Phe on to the N-terminus of Fur after cleavage with factor Xa.
Protein purification.
Fur was purified from the pET21 clone in BL21(DE3) initially using the method of Ochsner et al. (1995)
. Subsequently a modified procedure was developed, which did not involve metal-affinity chromatography. Cultures (500 ml) were grown to an OD650 of 0·50·6, induced with 1 mM IPTG, then incubated overnight. Cells were harvested, washed in 50 mM Tris/HCl (pH 7·9)-0·5 mM EDTA-50 mM NaCl, then resuspended in the same buffer containing 1 mM PMSF and 1 mM DTT. After sonication, the cell-free extract was applied to a 300 ml DEAE cellulose ion-exchange column equilibrated with 50 mM Tris/HCl (pH 7·9)-50 mM NaCl, and then eluted with a linear 50500 mM NaCl gradient in the same buffer. Fur-containing fractions (as judged by SDS-PAGE) were dialysed overnight in 20 mM Tris/HCl (pH 7·0) then applied to a 20 ml heparin-agarose column equilibrated with 20 mM Tris/HCl (pH 7·0). The column was washed with the same buffer, then Fur was eluted with 20 mM Tris/HCl (pH 7·0)-1 M NaCl.
For purification of the MBP fusion, 500 ml cultures were grown in Lennox broth containing 0·2% (w/v) glucose to an OD650 of 0·50·6, induced with 1 mM IPTG, then incubated overnight. Cells were harvested, washed in 50 mM Tris/HCl (pH 7·9)-50 mM NaCl, then resuspended in the same buffer containing 0·5 mM EDTA, 1 mM PMSF and 1 mM DTT. Cells were disrupted by sonication, clarified, and the cell extract applied to a 15 ml amylose column equilibrated with 50 mM Tris/HCl (pH 7·9)-100 mM NaCl. The column was washed with the same buffer, then the fusion protein eluted with 50 mM Tris/HCl (pH 7·9)-100 mM NaCl-10 mM maltose. The fusion was treated with factor Xa, and then 3 M guanidinium hydrochloride (Gdn.HCl). The partially denatured Fur was purified by FPLC on an S75 gel-filtration column, then renatured by dialysis against 20 mM Tris/HCl (pH 7·0)-50 mM NaCl.
DNA-binding assays.
For use in gel retardation assays, a 300 bp fragment of the P. aeruginosa pvdS promoter region was amplified by PCR and cloned into the SmaI site of pUC18. The fragment extended 315 bp upstream of the pvdS start codon, not including the 14 bp immediately 5' to the ATG. Thus, the fragment included the pvdS -35 and -10 sequences, and a predicted Fur-binding site overlapping the -35 sequence (Ochsner et al., 1995 ). This fragment was excised with EcoRI and HindIII (using the vector sites, generating a fragment of total size 350 bp) and was labelled by end-filling with DNA polymerase I in the presence of [35S]dATP
S. The procedure for gel retardation assays was modified from that of Althaus et al. (1999)
. Binding buffer (5x concentrate) contained 100 mM Tris/HCl (pH 7·0), 25% (v/v) glycerol, 10 µg herring sperm DNA µl-1, 5 mM MgCl2, 200 mM KCl and 500 µg bovine serum albumin ml-1, and was supplemented with MnCl2 as required. Binding reactions contained 2 µl 5x binding buffer, 1 µl radiolabelled DNA, Fur to the desired final concentration, and distilled water to a final reaction volume of 10 µl. The binding reaction was left at room temperature for 2030 min before loading on to a 6% polyacrylamide gel. The gel was made up in 20 mM Tris, buffered to pH 7·0 with boric acid, and was run at 120 V for 80 min. The gel was transferred to 3MM Whatman paper, dried, and exposed to a Molecular Dynamics phosphorimager screen overnight. The screen was digitized and quantified using a Storm 840 laser scanner (Molecular Dynamics) and images were visualized and quantified using ImageQuant version 5.0 (Molecular Dynamics).
Spectroscopy.
Fluorescence emission spectra were recorded on a Perkin Elmer LS50 fluorimeter using an excitation wavelength of 290 nm and excitation and emission slits of 5 nm. Scan speed was 1000 nm min-1 and a mean of 10 scans was recorded. Both proteins were 13 µM in 20 mM Tris/HCl (pH 8)-50 mM NaCl, and emission spectra of the buffer were subtracted. Circular dichroism (CD) spectra were collected on a Jasco J-710 spectropolarimeter with a scan speed of 100 nm min-1 and a path length of 0·2 mm. Each sample was scanned three times, and a mean spectrum calculated. Fur samples were at 0·30·9 mg ml-1 in 20 mM Tris/HCl (pH 8·0)-50 mM NaCl. The observed ellipticity in millidegrees () was converted to mean residue molar ellipticity (
)MR in deg cm2 dmol-1 residue-1 according to equation 1:
![]() | (1) |
where Mr is the relative molecular mass of the protein, c is the concentration in g ml-1, l is the path length in cm, and n is the number of residues.
Protein unfolding.
Protein samples (0·14 mg ml-1) were dialysed for 4 h at room temperature against varying Gdn . HCl concentrations, then incubated at 37 °C for a further 1 h before recording CD spectra as above. Baselines collected at the relevant Gdn.HCl concentrations were subtracted. The ellipticity at 222 nm () was used to calculate the fraction of unfolded protein (fu) according to equation 2:
![]() | (2) |
where u is the ellipticity at 222 nm of the fully denatured protein and
n is the ellipticity at 222 nm of the native protein. Data points were corrected for the slopes of the pre- and post-transitional regions of the curves using linear regression (Santoro & Bolen, 1988
; Pace & Scholtz, 1997
). The fraction of unfolded protein (fu) was plotted against [Gdn] and the resultant curves fitted to equation (3) using Fig.P v2.98 (BIOSOFT):
![]() | (3) |
where [Gdn]0·5 is the concentration of Gdn.HCl at which the protein is 50% unfolded, and m is the rate of change of free energy with respect to [Gdn].
Analytical techniques.
Electrospray mass spectrometry was done on a Micromass Platform I mass spectrometer calibrated with horse heart myoglobin. The solvent was 50% (v/v) acetonitrile and 0·1 % (v/v) formic acid in water, and samples were run at a rate of 20 µl min-1. For electrothermal atomic absorption spectroscopy, all glassware was washed in 10% (v/v) nitric acid, and rinsed thoroughly in triple-distilled water followed by Milli-Q water. Fur was dialysed overnight against 50 mM Tris/HCl (pH 8·0)-50 mM EDTA-200 mM NaCl, then dialysed extensively against 50 mM Tris/HCl (pH 8·0)-200 mM NaCl. To assay zinc binding, 1 ml samples containing 10 µM Fur in 50 mM Tris/HCl (pH 8·0)-200 mM NaCl were made to 150 µM ZnCl2, and left overnight at 4 °C, before the dialysis against 200 mM NaCl was repeated. All samples and ZnCl2 standards were passed through a 0·2 µm filter before analysis on a Philips PU9200 atomic absorption spectrophotometer with an electrographite cuvette. Absorption was at 213·9 nm with a 0·5 nm bandpass, and each sample was measured five times. Protein concentrations were determined with a bicinchoninic acid assay kit (Sigma), or using an absorption coefficient (280 nm) of 4218 M-1 cm-1 estimated for the protein cleaved from the MBP fusion according to the method of Gill & von Hippel (1989) . ß-Galactosidase was assayed according to the method of Miller (1992)
. DNA sequences were determined by MWG Biotech (Germany).
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RESULTS AND DISCUSSION |
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In the first instance, the maltose-binding protein (MBP) was chosen for fusion to Fur, since MBP has previously been used successfully for making fusions to transcriptional regulators (Chai & Stewart, 1998 ; Li et al., 1994
). The fur gene was fused to the malE gene encoding MBP in the vector pMALc2. This procedure fused Fur to the C-terminus of a derivative of MBP from which the signal peptide has been deleted. The fusion protein was purified by affinity chromatography on an amylose resin and was cleaved with factor Xa. Following cleavage with factor Xa (confirmed by SDS-PAGE), Fur and MBP co-eluted in a number of chromatographic separations (amylose affinity, heparin affinity and ion exchange), indicating the formation of a complex between the cleaved Fur and MBP. Some free MBP (but no Fur) was always seen in these preparations, suggesting a stoichiometric excess of Fur in the complex, which is consistent with an oligomeric form of Fur binding to MBP. A similar post-cleavage interaction has been observed with a fusion of the cystic fibrosis transmembrane regulator to MBP (Ko et al., 1993
).
Separation of Fur from MBP was achieved by exploiting their different stabilities in Gdn.HCl. MBP was 50% unfolded with 1·1 M Gdn.HCl, while Fur required 3·2 M Gdn.HCl for 50% unfolding (Fig. 1). The mixture containing Fur and MBP was treated with 3 M Gdn.HCl, and the partially unfolded Fur was separated from the unfolded MBP by gel filtration. Subsequent removal of the Gdn.HCl by dialysis yielded Fur that was active, as judged by its ability to bind to DNA (see below). The yield of protein from this procedure was 30 mg from 1·5 l of culture. Electrospray mass spectrometry gave an estimated mass of 15709 Da, close to the predicted mass of 15711 Da for the protein that has the sequence Ile-Ser-Glu-Phe added to its N-terminus as a result of the cloning procedure. Some of the biochemical experiments reported below were done with material prepared in this way, and protein preparations from the two different sources were indistinguishable by all criteria tested.
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Analysis of as-prepared P. aeruginosa Fur by electrothermal atomic absorption spectroscopy (EAAS) revealed the presence of a significant amount of zinc that varied in different preparations. Unlike the E. coli Fur, treatment with EDTA removed the bulk of this zinc, to yield protein containing 0·19 Zn2+ ions per monomer. Treatment of the as-prepared Fur with a large excess of ZnCl2 followed by dialysis to remove unbound Zn2+ yielded protein containing 0·94 Zn2+ ions per monomer as judged by EAAS, again unlike the E. coli protein, which binds two Zn2+ ions under similar conditions. The simplest interpretation of these results is that the P. aeruginosa Fur binds a single Zn2+ ion in the metal-sensing site, and does not have the structural Zn2+-binding site found in the E. coli protein. This distinction is consistent with the absence of Cys-95 in the P. aeruginosa protein, and is indicative of a degree of structural diversity in the Fur family. Structural diversity does not seem to be reflected in mechanistic diversity, at least to the extent that the P. aeruginosa fur gene can complement an E. coli fur mutant (Prince et al., 1993 ; and see below). Addition of a ZnCl2 solution to metal-free Fur did not produce significant changes to the CD or fluorescence spectra of the protein (data not shown), consistent with Zn2+ binding not causing large-scale conformational changes affecting the chromophores giving rise to the spectroscopic effects.
P. aeruginosa Fur forms disulfide-linked dimers
SDS-PAGE analysis of P. aeruginosa Fur showed that in the presence of DTT it behaves as a monomer, while in the absence of DTT a mixture of monomeric and dimeric species was seen (data not shown), with the relative amounts being batch-dependent. These data suggested that Cys-92 (the only cysteine in the protein) forms intermolecular disulfide bonds on air-oxidation, with freshly prepared protein having less of the dimer form than older preparations. This was confirmed by showing that Fur alkylated with iodoacetamide was unable to dimerize in the absence of DTT (data not shown). In a mass spectroscopic analysis of the E. coli Fur, no evidence for the occurrence of disulfide-bridged dimers was found (Michaud-Soret et al., 1997 ).
Mutational analysis of P. aeruginosa Fur
The fur gene of P. aeruginosa is believed to be essential (Hassett et al., 1996 ), so it is difficult to evaluate the phenotypes associated with mutant alleles in a null background. The P. aeruginosa fur gene was originally cloned by complementation of an E. coli mutant (Prince et al., 1993
), and P. aeruginosa Fur can bind to an E. coli Fur box in vitro (Ochsner et al., 1995
). Hence, it was reasoned that it should be possible to examine activity of the P. aeruginosa Fur in the heterologous E. coli background. Therefore, a reporter system was developed in which the P. aeruginosa fur gene was expressed in a strain of E. coli (JRG2653) mutant for the endogenous fur gene and containing a chromosomal fusion of the Fur-regulated bfd promoter to lacZ. The bfd gene is linked to the bfr gene of E. coli, encodes a [2Fe-2S] protein, and is repressed by Fur in response to iron (Andrews et al., 1989
; Garg et al., 1996
; Stojiljkovic et al., 1994
). Measurement of ß-galactosidase activity confirmed that the P. aeruginosa fur gene complements the E. coli fur mutant, and that the complemented strain shows iron-regulated expression of the bfd promoter (Table 1
). Repression is less tight than that seen with the chromosomal E. coli fur gene (in JRG2652), which may reflect either poor expression of the P. aeruginosa fur gene or a reduced activity of the heterologous regulator. The E. coli fur mutant has a pleiotropic phenotype that includes the overproduction of siderophores and an inability to grow on succinate as the sole source of carbon and energy (Hantke, 1987
). Both of these phenotypes were also complemented by the P. aeruginosa gene. The reporter system was then used to assay the consequences of substituting the single cysteine residue of P. aeruginosa Fur with either serine or alanine. Both mutants were able to act as repressors of the bfd promoter (Table 1
), indicating that Cys-92 is dispensable for the activity of P. aeruginosa Fur. This conclusion was supported by the observation that both mutants restored growth on succinate and siderophore production to wild-type patterns (data not shown). Interestingly, the two cysteine mutants both seem to work better as repressors in the presence of the trace amounts of iron present in unsupplemented M9 medium than does the wild-type P. aeruginosa Fur (Table 1
).
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Substitution of each of the three histidines (His-85, His-86 and His-89) with leucine in the E. coli protein had surprisingly little effect on Fur activity in vivo (Coy et al., 1994 ). On the other hand, substitution of His-89 (E. coli numbering) with leucine led to loss of activity of the Vibrio cholerae Fur (Lam et al., 1994
), which is consistent with the phenotype of the H89A mutant of P. aeruginosa Fur (Table 1
). Substitution of His-90 of the S. typhimurium Fur (equivalent to H89 of the E. coli protein) with arginine eliminated iron-mediated repression but had no effect on the role of Fur in the acid tolerance response (Hall & Foster, 1996
). Although the fur gene of P. aeruginosa is thought to be essential (Hassett et al., 1996
), some fur alleles with altered phenotypes have been characterized. Manganese resistance has been used as a screen for the isolation of fur alleles in P. aeruginosa, and amongst several characterized were two mutants with substitutions of arginine and tyrosine at His-86 (Barton et al., 1996
). Strains carrying these mutations showed constitutive expression of siderophores but retained iron-regulation of exotoxin A expression, suggesting only a partial loss of Fur activity (Barton et al., 1996
). This phenotype is consistent with the retention of some activity for the H86A mutant reported here.
Clearly much remains to be learned about the structure and mechanism of the Fur proteins. Future work will be directed towards exploiting the improved expression systems described in this report, with the ultimate goal of establishing the three-dimensional structure of the P. aeruginosa Fur.
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ACKNOWLEDGEMENTS |
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Received 19 April 2002;
revised 3 May 2002;
accepted 13 May 2002.