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J. Biol. Chem., Vol. 279, Issue 31, 32100-32105, July 30, 2004

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The Ferric Uptake Regulator (Fur) Protein from Bradyrhizobium japonicum Is an Iron-responsive Transcriptional Repressor in Vitro^*

Yali E. Friedman and Mark R. O'Brian

From the Department of Biochemistry and Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York, Buffalo, New York 14214

Received for publication, May 3, 2004 , and in revised form, May 13, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Fur protein represses transcription of iron-responsive genes in bacteria. The discovery that Fur is a zinc metalloprotein and the use of surrogate metals for Fe²⁺ for in vitro studies question whether Fur is a direct iron sensor. In the present study, we show that the affinity of Fur from Bradyrhizobium japonicum (BjFur) for its target DNA increases 30-fold in the presence of metal, with a K_d value of about 2 nM. DNase I footprinting experiments showed that BjFur protected its binding site within the irr gene promoter in the presence of Fe²⁺ but not in the absence of metal, showing that DNA binding is Fe²⁺-dependent. BjFur did not inhibit in vitro transcription from the irr promoter using purified components in the absence of metal, but BjFur repressed transcription in the presence of Fe²⁺. Thus, BjFur is an iron-responsive transcriptional repressor in vitro. A regulatory Fe²⁺-binding site (site 1) and a structural Zn²⁺-binding site (site 2) inferred from the recent crystal structure of Fur from Pseudomonas aeruginosa are composed of amino acids highly conserved in many Fur proteins, including BjFur. BjFur mutants containing substitutions in site 1 (BjFurS1) or site 2 (BjFurS2) bound DNA with high affinity and repressed transcription in vitro in an Fe²⁺-dependent manner. Interestingly, only a single dimer of BjFurS2 occupied the irr promoter, whereas the wild type and BjFurS1 displayed one- or two-dimer occupancy. We suggest that the putative functions for metal-binding sites deduced from the structure of P. aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Control of iron homeostasis is essential to most living organisms. Iron availability can be limited because it is predominantly in the ferric form in aerobic environments. On the other hand, excessive intracellular iron concentration can be deleterious because it generates reactive oxygen species that damage cellular components (1, 2). In bacteria, studies on the control of iron homeostasis have focused largely on Fur (ferric uptake regulator), a regulatory protein that responds to cellular iron (reviewed in Ref. 3). Fur represses genes involved in high affinity iron transport under iron-replete conditions. It is also involved in numerous other facets of iron metabolism and in processes not obviously linked to iron (4–6).

The working model for Fur function posits that, when bound by ferrous (Fe²⁺) iron, Fur binds the target operator within the promoter of the regulated gene to repress transcription. However, when iron is limited in the cell, the Fur protein is unbound by iron and no longer binds DNA with high affinity; hence, gene expression is derepressed. It was recently shown that binding of Fur to the promoter element is sufficient to block transcription in vitro, and thus Fur probably does not act to recruit another repressor or exclude an activator protein (7).

Several years ago it was discovered that Escherichia coli Fur is a zinc metalloprotein that binds two Zn²⁺ atoms per monomer (8). One of the zinc atoms is very tightly bound and is likely to have a structural function, whereas the second zinc can be easily removed with chelators. In addition, Fur containing one Zn²⁺ atom is sufficient for DNA binding, and the affinity does not increase when the second Zn²⁺ site is occupied (8). These observations question whether Fur is a direct iron sensor. Furthermore, most in vitro studies use a surrogate for Fe²⁺ such as Mn²⁺ because ferrous iron readily oxidizes to Fe³⁺ in air. Thus, the assumption that the surrogate metal is a substitute for Fe²⁺ is equivocal.

Several groups have shown that the structural Zn²⁺ atom of E. coli Fur is coordinated by Cys-92 and Cys-95 (9, 10). However, these cysteines are not conserved in many Fur proteins. Very recently, the Fur protein from Pseudomonas aeruginosa was crystallized in the presence of Zn²⁺, revealing two metal-binding sites (11). None of the coordinating amino acids composing these sites are cysteine. One of the Zn²⁺ atoms binds tightly and may be a structural ligand. The second metal-binding site can be occupied by Fe²⁺ and is a presumptive regulatory metal-binding site. Interestingly, the eight amino acids identified in the two metal-binding sites are highly conserved, leading Pohl et al. (11) to extrapolate their conclusions about the Fur protein from P. aeruginosa to other Fur proteins. Alteration of residues corresponding to some of these metal-binding residues in Fur proteins produces mutant bacterial strains with altered phenotypes (4, 12–14).

Fur from Bradyrhizobium japonicum (BjFur)¹ typifies bacterial Fur proteins in that it complements an E. coli Fur mutant, mediates iron-dependent control of gene expression, and binds to a so-called Fur box, a consensus Fur-binding DNA element (7, 15, 16). However, BjFur also binds to DNA dissimilar to the Fur box within the promoter of the irr gene, whereas E. coli Fur does not bind that element in vitro (7). The irr gene from B. japonicum encodes Irr, a protein that mediates iron control of heme biosynthesis (17, 18). Irr expression is controlled both transcriptionally and post-translationally, the former being regulated by BjFur. The amino acids identified in metal binding in P. aeruginosa Fur are conserved in the B. japonicum protein, whereas the cysteines involved in zinc binding in E. coli Fur are not present. In the present study, we demonstrate that BjFur is an iron-responsive DNA-binding protein in vitro and that iron confers on BjFur the ability to repress transcription. Furthermore, we show that the amino acids in BjFur corresponding to the presumptive metal-binding sites of the P. aeruginosa protein are not required for iron-responsive DNA binding or transcriptional repression.

	MATERIALS AND METHODS

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Chemicals and Reagents—All chemicals were reagent grade and were purchased from Sigma, Fisher, VWR Scientific (West Chester, PA), or J. T. Baker Inc. Agar, purified noble agar, and yeast extract were purchased from Difco, and [-³²P]dCTP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. [-³²P]UTP (800 Ci/mmol) was purchased from ICN Biomedicals (Irvine, CA).

Bacterial Strains, Plasmids, Media, and Growth—E. coli strain DH5 was used for the propagation of plasmids and was grown at 37 °C on Luria-Bertani media with appropriate antibiotics. Plasmid pSKiron contains the 19-mer Fur box consensus sequence 5'-GATAATGATAATCATTATC-3' cloned into the ClaI site of pBluescript II SK+ (19). pET14bjFur (Novagen, Madison, WI) contains the B. japonicum fur gene cloned into the NdeI and BamHI sites of pET14b. B. japonicum fur was derived from parental strain USDA I110 (15). pET14ecFur contains the E. coli fur gene cloned into the NdeI and BamHI sites of pET14b.

Overexpression and Purification of Fur—Fur was overexpressed in E. coli strain BL21(DE3)(plysS) cells containing fur in pET14b initially grown on Luria-Bertani media containing chloramphenicol (25 µg/ml) and ampicillin (200 µg/ml). Cells were inoculated from an overnight culture grown in Luria-Bertani media containing chloramphenicol and ampicillin into 1 liter of fresh 2x YT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, pH 7.0) containing antibiotics, 50 µM FeCl_3, and 1 mM ZnCl₂. Overexpression was induced in cells at mid-log phase by adding 20 ml of 95% ethanol and a final concentration of 0.5 mM isopropyl-1-thio--D-galactopyranoside, and incubation followed at 20 °C for 4 h with shaking. Cells were pelleted by centrifugation at 4000 x g, washed in TNG (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 7.4), and resuspended in 15 ml of phosphate binding buffer (5 mM imidazole, 300 mM NaCl, 10% glycerol, 50 mM NaPO₄, pH 8.0), 1 mM phenylmethylsulfonyl fluoride, and 25.5 µg of aprotinin per 5 g of cells. Cells were disrupted by passage twice through a French pressure cell at 1200 p.s.i. and clarified by centrifugation at 37,000 x g for 45 min. 1 ml of 50% Ni-NTA slurry (Qiagen Inc., Valencia, CA) was added to 4 ml of clear lysate and rocked for 60 min at 4 °C.

The Ni-NTA slurry-protein mixture was poured into a column and washed three times with 25 ml of phosphate wash buffer (20 mM imidazole, 300 mM NaCl, 10% glycerol, 50 mM NaPO₄, pH 8.0). Purified His-Fur was eluted with phosphate elution buffer (250 mM imidazole, 300 mM NaCl, 10% glycerol, 50 mM NaPO₄, pH 8.0). The His tag was cleaved by adding 3 units of thrombin (Novagen, Madison, WI) per 2 mg of His-Fur and by incubating at room temperature for 2 h. To remove imidazole, Fur was further purified by retention through a 10-kDa nominal molecular size limit Ultrafree column (Millipore, Billerica, MA). The His tags were removed by resuspending the retentate in 1.2 ml of 50% Ni-NTA/300 µl of purified Fur and loaded on a column. Purified Fur free of His tags was collected in appropriate column flow-through and wash fractions and diluted to 1 µM in electrophoretic gel mobility shift assay (EMSA) binding buffer (10 mM bis-Tris borate, 1 mM MgCl₂, 40 mM KCl, 5% glycerol, 0.1% Nonidet P-40, 1 mM dithiothreitol, pH 7.5).

Construction of Metal-binding Site BjFur Mutants—Nucleotides in the cloned B. japonicum fur corresponding to amino acid residues implicated in structural and metal-sensing metal binding in P. aeruginosa (11) were systematically altered using QuikChange (Stratagene, La Jolla, CA) to code for alanine residues to assess the role of these residues in Fur activity. To construct the site 1 mutant, amino acid residues His-97 (288-CAT-292 288-GCG-292), Asp-99 (294-GAC-298 294-GCG-298), Glu-118 (351-GAG-355 351-GCG-355), and His-135 (402-CAC-406 402-GCG-406) were altered to code for alanine residues. To construct the site 2 (putative Zn²⁺-binding) mutant residues His-43 (126-CAT-130 126-GCG-130), Glu-91 (270-GAG-274 270-GCG-274), His-100 (297-CAC-301 297-GCG-301), and Glu-111 (330-GAG-334 330-GCG-334) were altered to produce alanine residues. A double mutant was constructed containing alanine substitutions for His-43, His-97, Asp-99, His-100, Glu-111, Glu-118, and His-135. The altered fur genes, respectively named BjFurS1, BjFurS2, and BjFurS1S2 were ligated into pET14b and expressed in E. coli strain BL21(DE3)(plysS) cells as described above.

Electrophoretic Mobility Shift Assay—EMSAs were used to determine Fur binding to DNA. Using a protocol modified from de Lorenzo et al. (20), Fur was incubated for 30 min at 4 °C in a 20-µl volume of EMSA binding buffer supplemented with 50 ng of herring sperm DNA, 2 µg of bovine serum albumin, 100 µM MnCl₂ and 1 nM radiolabeled DNA probe. Double-stranded DNA probes were produced by boiling and slowly cooling synthetic DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA) in annealing buffer (150 mM NaCl₂, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) and filled in with [-³²P]dCTP (3000 Ci/mmol) with the Klenow fragment of DNA polymerase (Promega, Madison, WI). Following incubation, EMSA reactions were analyzed on 5% nondenaturing polyacrylamide gels in electrophoresis buffer (20 mM bis-Tris borate, pH 7.5) that were prerun for 30 min at 200-V constant voltage. After electrophoresis at 4 °C for 45 to 90 min at 200 V, gels were dried and autoradiographed. The Fur box probe was isolated as an 83-bp fragment from a NotI and XhoI digestion of pSKIron. A BanII and HpaI digest of pSKSBIrr was used to isolate a 63-bp fragment containing the irr promoter. Autoradiograms were developed on BioMax film (Eastman Kodak Co.), scanned using a GS-700 densitometer (Bio-Rad), and signal intensities were detected and quantified using Quantity One (Bio-Rad). To determine the dissociation binding constant (K_d), binding reactions were titrated with various concentrations of Fur. Bound and unbound DNA were quantified by comparing relative signal intensities. Signal intensities were imported into Excel spreadsheets and analyzed using Graphpad Prism (Graphpad Sofware Inc., San Diego, CA).

DNase I Footprint Analysis—DNase I footprint analysis examined the DNA region protected by Fur binding. 15 nM Fur was incubated for 30 min at 4 °C in a 50-µl volume containing EMSA binding buffer, 125 ng of herring sperm DNA, 5 µg of bovine serum albumin, 100 µM MnCl₂ or 100 µM freshly prepared FeSO₄, and 1 nM irr promoter probe end-labeled with [-³²P]dCTP with Klenow. 50 µl of room temperature solution of 5 mM CaCl₂ and 10 mM MgCl₂ was added and after 1 min was followed by the addition of 0.45 unit of RQ1 RNase-free DNase (Promega, Madison, WI) in 18 µl of 40 mM Tris-HCl (pH 7.0). The reaction was incubated for 2 min at room temperature. Reactions were stopped by the addition of 90 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml yeast RNA), and DNA was extracted with phenol: choloroform (1:1) followed by ethanol precipitation. G + A ladders of the labeled DNA were produced as described (21). Digested probe products were separated on a 15% denaturing polyacrylamide gel containing 7 M urea in 1x Tris borate-EDTA electrophoresis buffer. Autoradiograms were developed on BioMax film. The DNA probe used for DNase I footprint analysis was 3'-end-labeled; therefore the top of the gel represents the 5'-end.

In Vitro Transcription Assays—To directly examine the influence of B. japonicum Fur on transcription from the irr promoter, in vitro transcription assays were performed on irr promoter DNA. irr promoter fragments were generated by PCR amplification of an irr sequence in pSKSBIrr using the primers 5'-TTTGAATTCGTGACCAAAATCGGCTAAG-3' and 5'-TTTCTGCAGGGACGTCGTCGTCGTGAT-3', producing a 272-bp DNA fragment that terminated 80 base pairs downstream of the irr transcription start site determined previously (16). Fur was incubated for 30 min at 4 °C in EMSA buffer without glycerol, 1 µg of bovine serum albumin, 20 mM MgCl₂, 10mM 2-mercaptoethanol, 4 nM irr promoter template DNA, and either no added metal, 100 µM MnCl_2, or 100 µM freshly prepared FeSO₄. The final volume was 10 µl. The samples were flushed with N₂ to remove air. 0.5 unit of RNA polymerase (Epicenter, Madison, WI) was added, reactions were incubated at 37 °C for 10 min, after which 10 µl of a preheated NTP mixture (100 µM metal where appropriate, 250 µM each of ATP, CTP, and GTP, 20 µM UTP, 8 µM [-³²P]UTP (800 Ci/mmol in 10 µl of H₂O)) was added, and reactions were incubated for an additional 20 min at 37 °C. Transcription products were run on a 15% denaturing polyacrylamide gel containing 7 M urea in 1x Tris borate-EDTA electrophoresis buffer alongside a Low Range RNA ladder (Fermentas Inc., Hanover, MA) labeled with ATP [-³²P]ATP with polynucleotide kinase (Promega, Madison, WI).

Atomic Absorption—B. japonicum and E. coli Fur proteins were purified as described above and were analyzed directly or reconstituted using a procedure modified from Althaus et al. (8). 70–100 µM Fur protein was incubated with a 4-fold excess of ZnCl₂ and a 10-fold excess of dithiothreitol in 20 mM Tris-HCl (pH 7.0) for 4 h at 4 °C. Unbound metal was removed by passage through a Sephadex G-25 column (Roche Applied Science). Flameless atomic absorption analyses were performed on a PerkinElmer model 1100B spectrophotometer equipped with a model 700 graphite furnace and an AS-70 autosampler. Fur protein concentrations were determined in Bradford assays using bovine serum albumin standards, adjusted for a 120% overestimation as described previously (8).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Metal Increases the Affinity of B. japonicum Fur for DNA— Fur proteins mediate iron-dependent control of gene expression in vivo and bind metals in vitro. Thus, we wanted to determine the effects of metal on B. japonicum Fur (BjFur) binding to a cognate Fur-binding site. The irr gene from B. japonicum is transcriptionally regulated by BjFur and binds to an element within the irr promoter containing three imperfect direct repeat hexamers (7). The binding of recombinant BjFur to DNA corresponding to the BjFur binding site on the irr promoter was measured in electrophoretic mobility shift assays (EMSA) using Mn²⁺ as a surrogate metal. Titration of DNA with an increasing concentration of BjFur in the presence of Mn²⁺ revealed two complexes in EMSA, a high mobility complex (HMC) at lower protein concentrations and a low mobility complex (LMC) at higher concentrations (Fig. 1A). Previous work shows that the HMC and the LMC correspond to a Fur dimer and to two dimers or a tetramer, respectively (7). In the absence of Mn²⁺, Fur bound DNA much less well than in the presence of metal at each protein concentration tested (Fig. 1B). Furthermore, only the HMC was detected at 50 nM BjFur in the absence of Mn²⁺, whereas the LMC was predominant in the presence of Mn²⁺ at that protein concentration.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Effect of metal on BjFur binding to the irr promoter in EMSA analysis. EMSA was carried out using purified recombinant BjFur and 32P-labeled irr promoter DNA. Samples were run on 5% nondenaturing polyacrylamide gels in the presence (A) or absence (B) of 100 µM MnCl2 and visualized by autoradiography. 0.1 pM DNA and 0–100 nM BjFur protein were used. C, autoradiograms were quantified and plotted as bound DNA versus BjFur concentration in the presence (closed squares) or absence (open squares) of MnCl2.

BjFur Binds DNA in an Iron-responsive Manner in Vitro— We and others routinely use Mn²⁺ or another divalent metal as a presumed surrogate for Fe²⁺ for in vitro analysis of Fur (7, 23). However, the discovery that Fur is a zinc metalloprotein in at least some organisms calls into question the role of these other metals, and one report questions whether a regulatory Fe²⁺ is required for activity (8). EMSA analysis using Fe²⁺ is problematic because the DNA-protein complex must remain intact throughout the electrophoresis where ferrous iron can oxidize to the ferric form. Therefore, we assessed the dependence of Fe²⁺ on DNA binding activity by DNase I footprinting analysis, maintaining anaerobic conditions in the binding and digestion reactions (Fig. 2).

View larger version (135K): [in this window] [in a new window]   FIG. 2. Metal-dependent protection of the irr promoter by wild type and Fur proteins in DNase I footprinting analysis. Protection of DNA from DNase I digestion by bound Fur was carried out as described under "Materials and Methods." The DNA was radiolabeled at the 3'-end; thus the 5'-end of the non-template strand of DNA (with respect to the irr gene) is at the top of the gel. Bars on the left indicate protection regions consistent with formation of high (HMC) and low mobility (LMC) complexes. 15 nM wild type BjFur, site 1 mutant BjFurS1, site 2 mutant BjFurS2, and site 1 and 2 mutant BjFurS1S2 proteins were used in the absence of metal (–) or presence of 100 µM MnCl2 (Mn) or FeSO4 (Fe). Digestions in the absence of Fur are marked NP.

Effects of Mutations in Putative Metal-binding Sites of Fur on DNA Binding in EMSA—The structure of Fur from P. aeruginosa crystallized in the presence of Zn²⁺ revealed two metal-binding sites (11). Each metal coordinates with amino acids that are conserved in many Fur homologs, including B. japonicum (Fig. 3), leading Pohl et al. (11) to extrapolate their findings for P. aeruginosa to Fur proteins from other organisms. Site 1 is proposed to be the regulatory Fe²⁺-binding site and is composed of amino acids corresponding to His-97, Asp-99, Glu-118, and His-135 in B. japonicum (Fig. 3). The mutant BjFurS1 lacking site 1 was constructed by changing these residues to alanine. Surprisingly, this mutant bound DNA in a metal-responsive manner similar to the wild type EMSA analysis (Fig. 4, also see below). Titration of DNA with BjFurS1 in the presence of Mn²⁺ showed a concentration-dependent formation of a HMC and an LMC similar to the wild type, and both proteins had similar K_d values of 2.2 and 4.5 nM for the wild type and the mutant, respectively (Fig. 4, A and B). These data indicate that conserved amino acids attributed to metal site 1 are not required for metal-dependent DNA binding activity.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Alignment of Fur proteins from E. coli, P. aeruginosa, and B. japonicum. The closed circles and open triangles, respectively, refer to residues that comprise the putative regulatory Fe2+-binding site (site 1) and the structural Zn2+-binding site (site 2) derived from crystallographic and spectroscopic analysis of P. aeruginosa Fur and conserved in many Fur proteins, including those from E. coli and B. japonicum.

View larger version (36K): [in this window] [in a new window]   FIG. 4. EMSA analysis of BjFur mutants binding to irr promoter DNA. A, EMSA analysis was carried out essentially as described in the legend to Fig. 1. Either 0, 5, or 50 nM protein was used. The Fur proteins used were wild type BjFur, BjFurS1 altered in site 1 amino acids, BjFurS2 altered in site 2 amino acids, or BjFurS1S2 altered in both sites. B, EMSA gels using protein concentrations from 0 to 100 nM were visualized by autoradiography, quantified, and plotted as bound DNA versus protein concentration for wild type (squares), BjFurS1 (diamonds), BjFurS2 (circles), and BjFurS1S2 (triangles).

Although BjFurS2 bound DNA with high affinity, it formed only a high mobility complex on EMSA (Fig. 4A) even with 50 nM protein, whereas the wild type protein formed a low mobility complex. Thus, one or more of the amino acids mutated in BjFurS2 is necessary for two BjFur dimers to occupy the irr promoter. We have no explanation for this observation, but the metal-responsive, high affinity binding of this mutant is not consistent with a disruption in a portion of the protein necessary to maintain the structural integrity of the monomer.

We also constructed BjFurS1S2, a mutant in which amino acids corresponding to both sites 1 and 2 were changed to alanine. This mutant did not bind DNA in EMSA analysis, even up to 100 nM DNA (Fig. 4, A and B). Wild type BjFur binds DNA in the absence of metal but with lower affinity (Fig. 1). Thus, BjFurS1S2 does not behave as an unmetalated Fur protein but rather as an inactive one. Although no structural analysis of BjFurS1S2 was carried out, it is likely that the extensive mutagenesis compromised the structural integrity of the protein.

The irr promoter element to which BjFur binds is dissimilar to the Fur box consensus sequence. E. coli binds the Fur box only, whereas BjFur binds both elements with similar affinity (7). The irr promoter element was used in the EMSA analysis in Figs. 1 and 4. It seemed plausible that this unique activity of BjFur has different structural requirements for binding than does the conventional Fur box binding activity that does not require sites 1 or 2. To address this further, we carried out EMSA analysis with the wild type and mutant proteins using Fur box consensus DNA as the radiolabeled probe (Fig. 5). The mutant proteins BjFurS1 and BjFurS2 retained DNA binding activity similar to the wild type, further supporting the conclusion that these proposed metal-binding sites are not necessary for activity. Furthermore, no DNA shifts were observed on EMSA gels with BjFurS1S2; thus this mutant did not bind to either DNA element.

View larger version (66K): [in this window] [in a new window]   FIG. 5. EMSA analysis of BjFur and mutants binding to Fur box consensus DNA. An 83-bp fragment including the Fur box consensus radiolabeled with 32P was run in a 5% native polyacrylamide gel either in the presence (A) or absence (B) of 100 µM MnCl2. The DNA was run in the absence of protein (F) or in the presence of 10 nM wild type BjFur (Wt), BjFurS1 (S1), BjFurS2 (S2) or BjFurS1S2 (S1S2). The gels were visualized by autoradiography.

BjFur Is an Iron-responsive Transcriptional Repressor in Vitro—Recently we provided direct evidence that binding of a bacterial Fur protein to the cis-acting promoter element is sufficient to repress transcription using an in vitro system composed of purified components (7). A promoter mutation that compromised BjFur binding also mitigated transcriptional repression. Here we sought to address whether repression of the irr promoter by BjFur was iron-responsive using this in vitro system (Fig. 6). In the absence of BjFur, an 80-nucleotide RNA was synthesized from a double-stranded DNA template. When BjFur was included in the absence of metal, there was no repression of RNA synthesis. However, in the presence of Fe²⁺, transcription was strongly repressed by BjFur, with a similar result using Mn²⁺ as the regulatory metal. Mutant protein BjFurS1 also showed iron-dependent transcriptional repression and to a lesser extent with Mn²⁺ as well. These data show that BjFur is an iron-responsive transcriptional repressor in vitro and that the site 1 amino acids are not necessary for this regulated activity. BjFurS1S2 was unable to repress transcription from the irr promoter, which is consistent with an inability to bind DNA. These observations also show that Fe²⁺ or Mn²⁺ do not in themselves inhibit transcription.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Metal-dependent repression of transcription initiated from the irr promoter by BjFur mutants. In vitro transcription from the irr promoter in the absence (NP) or presence (other lanes) of 20 nM wild type Fur (BjFur), site 1 mutant (S1), site 2 mutant (S2), and site 1 and 2 mutant (S1S2) proteins in the absence (–) or presence of 100 µM MnCl2 (Mn) or FeSO4 (Fe). Transcription of the DNA templates was carried out using 0.5 unit of commercial E. coli RNA polymerase holoenzyme as described under "Materials and Methods."

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we demonstrate that Fe²⁺ activates B. japonicum Fur (BjFur) with respect to DNA binding and transcriptional repression activities in vitro. Therefore, iron-dependent regulation of genes mediated by Fur in vivo is most likely because of a direct effect of Fe²⁺ on Fur. E. coli Fur binds DNA with a K_d value of 20 nM when bound by one structural Zn²⁺ atom only or when the second Zn²⁺ site is also occupied (8). Those observations suggest either that E. coli Fur does not have a regulatory metal-binding site that enhances activity or else the second Zn²⁺ is not a surrogate for Fe²⁺ in vitro. It is plausible that Fe²⁺ would have increased the affinity of the protein for the cis-acting element in the protein preparations, as has been deduced in earlier work (24). In the present study, BjFur bound DNA in the absence of added metal, but the affinity increased substantially in the presence of added Fe²⁺ or Mn²⁺ (Figs. 1 and 2).

In a previous study, we provided direct evidence that BjFur binding to the cognate cis-acting element is sufficient to block transcription in vitro without the need for additional regulatory factors (7). Here, we showed that Fe²⁺ controls transcriptional repression in vitro using purified components (Fig. 6), and this regulation can be attributed to the effects of Fe²⁺ on DNA binding affinity. BjFur binding to the irr promoter probably inhibits occupation by RNA polymerase or else it prevents the formation of an open complex needed to initiate transcription. Neither mechanism would require the addition or exclusion of another regulatory protein. Collectively, the in vitro experiments fit a relatively simple model of regulation whereby Fur senses Fe²⁺ directly to effect affinity for a promoter element of iron-controlled genes. Recently, iron was shown to increase the affinity of the Helicobacter pylori Fur protein for some operator DNA elements but decrease the affinity for other elements (25). In that case, a model must accommodate a Fur protein with activity when unbound by iron but with different DNA sequence specificity. Similarly, a B. japonicum fur mutant displays a phenotype with respect to expression of the heme biosynthesis gene hemA under iron limitation (16), indicating that the protein has an activity under those conditions as well.

The recent crystal structure of the P. aeruginosa Fur protein and accompanying spectroscopic data indicate a high affinity Zn²⁺-binding site with a putative structural function and a lower affinity site that can also be occupied by Fe²⁺. The coordinating amino acids are highly conserved among Fur proteins from many organisms, including B. japonicum, suggesting that generalizations can be made regarding these metal sites. However, the present work shows clearly that such extrapolations cannot be made prima facie. The BjFurS1 mutant containing substitutions in all four amino acids corresponding to the putative regulatory iron site of P. aeruginosa is iron-responsive with respect to DNA binding and transcriptional repression activities (Figs. 2 and 6). Furthermore, BjFurS1 formed both a high mobility complex and a low mobility complex in EMSA analysis and had a similar binding affinity for the cognate DNA elements as did the wild type (Figs. 4 and 5). From this we suggest that the conclusions drawn for P. aeruginosa Fur do not represent bacterial Fur proteins as a whole. It will be important to analyze the effects of similar mutations on function in the P. aeruginosa protein.

The BjFurS2 mutant containing amino acid substitutions corresponding to putative structural Zn²⁺-binding residues also bound DNA with high affinity, was iron-responsive, and was able to repress transcription. Therefore, this mutant is a functional protein. It is interesting to note that purified recombinant P. aeruginosa Fur bound to zinc in one study (11) but not in another (22). Therefore the role of zinc and the site 2 residues remains to be elucidated in that protein. We cannot rule out that zinc binds BjFur with low affinity that is removed by dialysis or desalting, but a structural zinc atom should bind with high affinity as was found for E. coli (11).

Although BjFurS2 binds DNA with high affinity and is functional, the substitutions in the mutant protein are not completely innocuous. BjFurS2 occupied the irr promoter only as a dimer as indicated by the HMC in EMSA and did not form the LMC at higher protein concentrations (Fig. 4). However, this trait is very different from that expected of a protein that cannot coordinate a tightly bound metal necessary for the integrity of the monomer. From this we conclude that BjFurS2 is not involved in binding an essential structural Zn²⁺ as envisioned for P. aeruginosa Fur. We do not know whether the inability of BjFurS2 to form an LMC is related to metal or some other feature of the protein. The ability of BjFurS2 to repress transcription agrees with our previous study showing that dimer occupancy is sufficient to inhibit transcription (7). In that work, dimer occupancy was created by mutation of the promoter or by using a low concentration of protein with the normal promoter, whereas in the present study it was the result of an altered protein.

We suggest that a unified model for Fur function cannot be assumed from studies of relatively few organisms and that Fur is a functionally and structurally more diverse protein within the eubacterial kingdom than has been realized previously.

	FOOTNOTES

 
^* This work was supported by National Science Foundation Grant MCB-0089928, United States Department of Agriculture Grant 2003-35319-13269, and National Institutes of Health Grant GM-067966 (to M. R. O'B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, 140 Farber Hall, State University of New York, Buffalo, NY 14214. Tel.: 716-829-3200; Fax: 716-829-2725; E-mail: mrobrian{at}buffalo.edu.

¹ The abbreviations used are: BjFur, B. japonicum Fur; EMSA, electrophoretic mobility shift assay; HMC, high mobility complex; LMC, low mobility complex; Ni-NTA, nickel-nitrilotriacetic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

	ACKNOWLEDGMENTS

 
We thank Dr. M. L. Vasil for the plasmid pSKiron containing the 19-mer Fur box consensus sequence 5'-GATAATGATAATCATTATC-3' cloned into the ClaI site of pBluescript II SK+.

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