interaction of bacillus subtilis fur (ferric uptake repressor) with the

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

July 1999, p. 4299–4307 Vol. 181, No. 14

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Interaction of Bacillus subtilis Fur (Ferric Uptake Repressor)with the dhb Operator In Vitro and In Vivo

NADA BSAT† AND JOHN D. HELMANN*

Section of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received 12 March 1999/Accepted 29 April 1999

Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur)family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction withthe operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon.The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does notrequire added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Furselectively inhibits transcription from the dhb promoter by sA RNA polymerase, even if Fur is added after RNApolymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition offerrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesisof the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought tobe involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand,since a His96Ala mutant mediates repression at 50 mM but not at 5 mM iron. Our data lead us to suggest thatFur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effectof an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

Iron is an essential and often growth-limiting nutrient formicroorganisms. The rapid oxidation of ferrous to ferric iron,which is virtually insoluble at a nearly neutral pH, reduces thelevel of bioavailable iron far below the approximately 1 mMthat most organisms require for optimal growth (4, 18, 28, 29).Consequently, many bacteria synthesize and excrete high-af-finity iron chelators (siderophores) that can solubilize ferriciron and subsequently be imported by a corresponding ferri-siderophore uptake system (29). The expression of genes forsiderophore biosynthesis and transport is repressed by addediron. In many bacteria, this repression is mediated by a metal-sensing DNA-binding protein, the ferric uptake repressor(Fur) protein (22, 23).

Fur has been best characterized from Escherichia coli (des-ignated FurEC), but homologs have been identified in numer-ous other bacteria (22, 23). In vivo, FurEC represses a largeregulon of iron uptake functions when iron is present in excess(6). Most other metals are ineffective at eliciting repression invivo, although Mn(II) leads to the repression of some, but notall, Fur-regulated genes (3, 5, 21, 32). In many gram-negativebacteria, selection for manganese-resistant (Mnr) mutantsleads to mutations in fur, suggesting that the ability of Mn(II)to activate Fur for DNA binding may be a source of manganesetoxicity, perhaps by inappropriately repressing iron uptakefunctions (21).

FurEC is an ;16-kDa dimeric protein with an amino-termi-nal DNA recognition domain and a carboxyl-terminal metal-binding domain (12, 37). In vitro studies have demonstratedthat FurEC binds to a specific DNA target site, the fur box, andthat this binding requires a divalent metal ion as a cofactor (3,14). In the vast majority of studies, Mn(II) is used to activateFur, since this ion, unlike Fe(II), is stable in the presence ofoxygen. It is generally assumed that Mn(II)-Fur is functionally

analogous to the Fe(II) form thought to mediate repression invivo. However, the metal-binding sites of Fur are not yet wellcharacterized. Fur binds two divalent ions per monomer, andbinding is thought to involve a cluster of conserved histidineresidues and two pairs of cysteines in the carboxyl-terminalmetal-binding domain (12, 24). One site is apparently occupiedby a tightly associated (structural) zinc ion, while the secondsite is thought to bind Fe(II) reversibly to regulate DNA bind-ing (24).

Bacillus subtilis contains three Fur homologs (8, 17) thatregulate a peroxide stress response (PerR), zinc uptake (Zur),and iron uptake (Fur). Fur regulates the expression of severaloperons implicated in iron transport (8, 23), including thedihydroxybenzoate siderophore biosynthesis (dhb) operon.Transcription of the dhb operon is initiated from a singlesA-dependent promoter with an overlapping consensus fur box(33). Mutations in this fur box or in fur prevent the iron-mediated repression of dhb (8, 33). However, unlike its ho-mologs from gram-negative organisms, B. subtilis Fur does notrecognize Mn(II) as a corepressor in vivo, and selection forMnr does not generate fur mutants (8, 9).

We have overproduced and purified B. subtilis Fur and ini-tiated a study of its interactions with both DNA and metal ions.Surprisingly, Fur is purified in an active zinc-containing formthat does not require the addition of Fe(II) to bind DNA or torepress transcription. Nevertheless, genetic studies are consis-tent with a model in which iron does interact directly with Furin vivo.

MATERIALS AND METHODS

Bacterial strains, plasmids, and oligonucleotide primers. The strains, plas-mids, and oligonucleotide primers used in this study are listed in Table 1. E. coliDH5a was used for routine cloning experiments, while RK4353 (RecA1) wasused to generate multimeric plasmids for efficient Campbell integration into theB. subtilis genome. To overproduce Fur, the gene was amplified by PCR withprimers 75 and 76. These primers introduce NdeI and KpnI sites on the PCRproduct ends; these sites are then used for cloning into NdeI-KpnI-cut pET17b(Novagen), generating pHB6505. The synthetic oligonucleotide primers usedwere obtained from the DNA Services Facility of the Cornell New York StateCenter for Advanced Technology-Biotechnology.

* Corresponding author. Mailing address: Section of Microbiology,Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607)255-6570. Fax: (607) 255-3904. E-mail: [email protected].

† Present address: Section of Genetics and Development, CornellUniversity, Ithaca, NY 14853.

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TABLE 1. Strains, plasmids, and oligonucleotide primers used in this study

Strain, plasmid, or primer Characteristicsa Reference or source

StrainsB. subtilis

ZB307A W168 SPbc2D2::Tn917::pSK10D6 42MO1099 amyE::erm pheA1 trpC2 19HB1000 ZB307A attSPb 9HB6543 HB1000 fur::kan 8HB6634 HB1000 amyE::erm This workHB6637 HB6543 amyE::erm This workHB6640 HB6637 amyE::fur This workHB6650 HB6634 amyE::fur H93A This workHB6651 HB6634 amyE::fur H97A This workHB6652 HB6634 amyE::fur H132A This workHB6653 HB6634 amyE::fur C100A This workHB6654 HB6634 amyE::fur C103A This workHB6655 HB6634 amyE::fur C140A This workHB6656 HB6634 amyE::fur C143A This workHB6657 HB6637 amyE::fur H93A This workHB6658 HB6637 amyE::fur H97A This workHB6659 HB6637 amyE::fur H132A This workHB6660 HB6637 amyE::fur C100A This workHB6661 HB6637 amyE::fur C103A This workHB6662 HB6637 amyE::fur C140A This workHB6663 HB6637 amyE::fur C143A This workHB6668 HB6634 amyE::fur H95A This workHB6670 HB6634 amyE::fur H96A This workHB6672 HB6637 amyE::fur H96A This workHB6673 HB6637 amyE::fur H95A This work

E. coliDH5a f80 D(lacZ)M15 D(argF-lac)U169 endA1 recA1 hsdR17 (rK

2 mK1) deoR thi-1 supE44 gyrA96 relA1 34

RK4353 araD139 D(argF-lac)U169 flhD5301 gyrA219 non-9 rpsL150 ptsF25 relA1 deoC1 V. J. StewartBL21(DE3)/pLysE F2 ompT hsdSB (rB

2 mB2) gal dcm(DE3)/pLysE Novagen

PlasmidspBKSII1 pBR322 replicon StratagenepDG1662 Ectopic integration vector 19pET17b Overexpression vector NovagenpGEM-cat pGEM-3zf(1)-cat-1 41pJM114 pBS (Stratagene) with kan gene 31pJPM122 cat-lacZ operon fusion vector for phage SPb 36pHB6502 pGEM-cat with 3.3-kb XbaI-SphI fragment including fur 8pHB6504 pHB6502 with the 0.4-kb BsiWI-NsiI fragment replaced by the 1.4-kb Acc65I-PstI kan gene from pJM114 8pHB6505 pET17b with the 0.47-kb NdeI-KpnI fragment encoding fur (primers 75 and 76) This workpHB6524 pGEM-cat with the fur gene (primers 140 and 139) This workpHB6525 pDG1662 with the fur gene This workpHB6527 pGEM-cat with the fur H93A gene This workpHB6528 pGEM-cat with the fur H97A gene This workpHB6529 pGEM-cat with the fur H132A gene This workpHB6530 pGEM-cat with the fur C100A gene This workpHB6531 pGEM-cat with the fur C103A gene This workpHB6532 pGEM-cat with the fur C140A gene This workpHB6533 pGEM-cat with the fur C143A gene This workpHB6534 pGEM-cat with the fur H95A gene This workpHB6537 pGEM-cat with the fur H96A gene This workpHB6538 pDG1662 with the fur H93A gene This workpHB6539 pDG1662 with the fur H97A gene This workpHB6540 pDG1662 with the fur H132A gene This workpHB6541 pDG1662 with the fur C100A gene This workpHB6542 pDG1662 with the fur C103A gene This workpHB6543 pDG1662 with the fur C140A gene This workpHB6544 pDG1662 with the fur C143A gene This workpHB6545 pDG1662 with the fur H95A gene This workpHB6547 pDG1662 with the fur H96A gene This workpHB6548 pJPM122 with the 0.4-kb HindIII-BamHI fragment encoding dhbA This work

Primersb

75 (forward) 59-GAGGGAAACATATGGAAAACCG-39 This work76 (reverse) 59-TGAGAAAAGGTACCCGCTCG-39 This work139 (reverse) 59-CATACTCTGGATCCACCCATATC-39 This work140 (forward) 59-TTAATGGAAAAGCTTACATCTAGAC-39 This work147 (H93A) 59-GGCGCAGCTGCCTTTCATCAC-39 This work148 (H95A) 59-GCTCACTTTGCTCACCACTTG-39 This work149 (H96A) 59-CACTTTCATGCCCACTTGGTG-39 This work150 (H97A) 59-TTTCATCACGCCTTGGTGTGC-39 This work151 (H132A) 59-ATTAAAGATGCTAGATTGACG-39 This work152 (C100A) 59-CACTTGGTGGCCATGGAGTGC-39 This work153 (C103A) 59-TGCATGGAGGCCGGAGCCGTTG-39 This work154 (C140A) 59-TTCACGGCATTGCGCACCGCTGTAACG-39 This work155 (C143A) 59-TGCCACCGCGCTAACGGAAAAG-39 This work200 (forward) 59-GCGTTTTAAGCTTCACCCTGA-39 This work201 (reverse) 59-GCTTTGAGGATCCTCACAACC-39 This work

a Amino acid changes are shown as, e.g., H93A.b Restriction enzyme cloning sites and relevant mutagenized nucleotides are underlined.

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Reagents, media, and growth conditions. Chemicals and antibiotics were pur-chased from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise indicated.CuSO4 (99.999%), FeSO4 (99.999%), FeCl3 (at least 99.99%), MnCl2(99.999%), NiSO4 (99.999%), and ZnSO4 (99.999%) were obtained from Al-drich Chemical Co. (Milwaukee, Wis.). Radioactive isotopes were purchasedfrom DuPont, NEN Research Products (Boston, Mass.). Manganese- and iron-limited minimal media (MM) were prepared as previously described (7). Ampi-cillin (100 mg/ml) was used for the selection of E. coli strains. Erythromycin (1mg/ml) and lincomycin (25 mg/ml) (for testing macrolide-lincosamide-strepto-gramin B resistance), kanamycin (10 mg/ml), spectinomycin (100 mg/ml), andchloramphenicol (5 mg/ml) were used for the selection of B. subtilis strains.

DNA manipulations and sequencing. Routine molecular biology proceduresand DNA manipulations were carried out as described previously (34). B. subtilistransformation was done by standard procedures (13). E. coli plasmid DNA andDNA fragments were isolated with QIAprep Spin Miniprep and PCR purifica-tion and gel extraction kits, respectively (Qiagen Inc., Chatsworth, Calif.). Re-striction endonucleases, DNA ligase, Vent DNA polymerase, T4 polynucleotidekinase, and calf intestinal alkaline phosphatase (New England Biolabs, Beverly,Mass.), Sequenase (Amersham Life Science Inc.), Pfu DNA polymerase (Strat-agene, La Jolla, Calif.), and RNasin RNase inhibitor (Promega Corporation,Madison, Wis.) were used according to manufacturers’ instructions. DNA se-quencing was performed on both strands for new constructs with AmpliTaq-FSDNA polymerase and dye terminator chemistry at the DNA Services Facility ofthe Cornell New York State Center for Advanced Technology-Biotechnology.

Overproduction and purification of Fur. Wild-type Fur was purified with E.coli BL21(DE3)/pLysE (39) containing pHB6505, a pET17b derivative. A 1-literculture was grown from a fresh transformant at 37°C in Luria broth containing0.4% glucose to enhance plasmid stability. At an optical density at 600 nm(OD600) of 0.4, isopropyl-b-D-thiogalactopyranoside (IPTG; Amersham Life Sci-ence) was added to 1 mM, and growth was continued for 1 h. Rifampin wasadded to 100 mg/ml, incubation was continued for 2 h, and the cells wereharvested. Cell pellets were suspended in 20 ml of disruption buffer (50 mMTris-HCl [pH 8], 2 mM EDTA, 0.1 mM dithiothreitol [DTT], 1 mM 2-mercap-toethanol, 100 mM NaCl, 10% [vol/vol] glycerol, 1 mM phenylmethylsulfonylfluoride [PMSF], 5% [vol/vol] bacterial protease inhibitor cocktail [SigmaP8465]) containing 130 mg of hen egg white lysozyme/ml, and the suspension wasincubated on ice for 10 min. Sodium deoxycholate was added to 0.05% (wt/vol),and the cell suspension was disrupted by pulsed sonication for 2 min. Lysateswere diluted with 20 ml of TEDG buffer (10 mM Tris-HCl [pH 8], 0.1 mMEDTA, 0.1 mM DTT, 5% [vol/vol] glycerol, 1 mM PMSF) and clarified twice bycentrifugation for 15 min each time. The resulting supernatant was applied at 4°Cto a heparin–Sepharose CL-6B column (Pharmacia LKB, Piscataway, N.J.), andFur was eluted with a linear gradient of 0.05 to 1 M NaCl in TEDG buffer (itelutes near 350 mM NaCl). Fractions containing Fur were pooled, precipitatedwith ammonium sulfate (65% saturation), suspended in TEDG buffer, and ap-plied to a Mono-Q (HR5/5) column for Pharmacia fast protein liquid chroma-tography. Fur was eluted with 350 mM NaCl, concentrated by ammonium sulfateprecipitation, and resuspended in TEDG buffer containing 300 mM NaCl and 1mM DTT. Fur was further purified by fast protein liquid chromatography on aSuperdex-75 column with the same buffer. As judged against molecular massstandards, Fur elutes with an apparent molecular mass of 35 kDa, as predictedfor a dimer. Fur was .98% pure, as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The glycerol concentration wasadjusted to 50% (vol/vol), and Fur was stored in aliquots at 220°C. The Furprotein concentration was determined with the Bio-Rad Laboratories (Hercules,Calif.) dye-binding (Bradford) assay and refers in all cases to the dimeric protein.

The percentage of purified Fur active for DNA binding can be estimated fromthe transcription repression experiments. In these studies, between 16 and 32 nMFur was required to completely repress transcription from a 4 nM DNA tem-plate. Since these experiments are performed at concentrations of Fur well abovethe measured dissociation constant, we can estimate the fraction of active Fur asbeing between 0.12 and 0.25 (assuming that the binding of a single dimer issufficient for repression). All Fur concentrations reported in this study are totalprotein rather than active molecules.

To estimate the metal content of Fur, Fur was dialyzed at 4°C three timesagainst TG buffer (5 mM Tris-HCl [pH 8], 10% [vol/vol] glycerol, 1 mM PMSF)and analyzed by flame atomic absorption spectroscopy at the ICP AnalyticalLaboratory of the Cornell Department of Fruit and Vegetable Science. Iron,zinc, and nickel atomic absorption standard solutions were purchased fromSigma.

Treatment of Fur with chelators. Two strategies were used to remove looselyassociated metals from Fur. In one protocol, Fur was dialyzed at 4°C againstTEDG buffer containing 25 mM EDTA and 300 mM NaCl and then dialyzedagainst TEDG buffer containing 1 mM DTT and 300 mM NaCl to reduce theconcentration of EDTA. The glycerol concentration was adjusted to 50% (vol/vol) and Fur was stored in aliquots at 220°C. In the other protocol, Fur waspreincubated with 5% (wt/vol) Chelex 100 (Bio-Rad) in electrophoretic mobilityshift assay (EMSA) buffer (see below) on ice for 1 h with frequent mixing. Theresin was allowed to settle, and the Fur-containing supernatant was removed andused in EMSA.

EMSAs with the dhb promoter. A DNA fragment containing the promoter, furbox, and partial coding sequence (to codon 53) of dhbA was generated by PCR

with primers 200 and 201, which incorporate HindIII and BamHI sites, respec-tively. The 400-bp product was cleaved with AlwNI, generating a 280-bp fragment(containing the promoter and fur box) and a 120-bp downstream fragment. DNAwas end labeled with T4 polynucleotide kinase. Fur was equilibrated in EMSAbuffer (20 mM Tris-HCl [pH 8], 50 mM KCl, 5% [vol/vol] glycerol, 0.5 mM DTT,0.1 mg of bovine serum albumin per ml) for 10 min at room temperature (RT),50 pM (1 fmol) of end-labeled DNA and 5 mg of competitor salmon testis DNAper ml were added, and incubation was continued for 10 min at RT. Thereactions were analyzed next to a dye marker on a 4% nondenaturing gel (40 mMTris-acetate) that was prerun for 10 min in Tris-acetate buffer containing 0.5 mMDTT. After 2 h at 150 V, the gel was dried and exposed to a PhosphorImagerscreen for analysis (STORM; Molecular Dynamics, Inc.). The data points werefit, by use of the DeltaGraph Professional reiterative curve-fitting algorithm, toan equation of the following form: percent DNA bound equals 100{1/[1 1(K/[Fur])n]}. In this equation, K represents the apparent dissociation constant forFur binding, [Fur] is the concentration of dimeric Fur protein, and n is thecooperativity coefficient. Both K and n were independently optimized. Compe-tition experiments were done with either the same dhb fragment or a PCRproduct containing the fur coding region as a nonspecific control.

In vitro transcription of the dhb promoter fragment. The dhb-containingtemplate and the vector control for in vitro transcription assays were generatedby linearizing pHB6548 and pJPM122, respectively, with BamHI. B. subtilis coreRNA polymerase and sA preparations were described previously (25, 26). RNApolymerase holoenzyme (RNAP) was reconstituted by incubating the core withsA (1:5 molar ratio) on ice for 15 min prior to use. The DNA template (4 nM)was preincubated with RNAP (80 nM, unless otherwise indicated) in transcrip-tion buffer (20 mM Tris-HCl [pH 8], 50 mM KCl, 5% [vol/vol] glycerol, 0.5 mMDTT, 0.1 mg of bovine serum albumin per ml, 10 mM MgCl2, 10 U of RNasinRNase inhibitor per reaction) for 10 min at 37°C. To assay transcriptionalrepression, Fur or RNAP was preincubated with DNA for 5 min at RT or 37°C,respectively, prior to the addition of the other protein. When appropriate, 10 mMfreshly dissolved FeSO4 was added. Transcription was initiated by the addition ofa nucleotide mixture (400 mM each ATP, GTP, and CTP and 60 mM[a-32P]UTP; ;3,000 cpm/pmol), and reaction mixtures were incubated for 8 minat 37°C. The reactions were stopped with 100 ml of stop solution (2.5 M ammo-nium acetate, 20 mM EDTA [pH 8], 0.2 mg of glycogen per ml), and nucleicacids were recovered by phenol extraction and ethanol precipitation prior toresuspension in 10 ml of formamide gel loading buffer (80% formamide, 10 mMEDTA [pH 8], 1 mg of xylene cyanol FF per ml, 1 mg of bromphenol blue perml). The samples were denatured for 4 min at 90°C and loaded on a 6%denaturing polyacrylamide gel. The gel was dried and exposed to a STORMPhosphorImager screen for analysis.

Siderophore assays. B. subtilis cultures were grown overnight at 37°C in MMwith or without added FeCl3 (5 mM). Siderophore levels were determined withthe Arnow assay as previously described (2, 9). Siderophore yields were normal-ized to the cell mass by dividing the measured siderophore level (OD510) by theculture density (OD600). All assays were performed with duplicate samples, andthe values were averaged.

PCR. The PCR mixture contained HB1000 chromosomal DNA, 50 mM eachdeoxynucleoside triphosphate, 100 pmol each of the forward and reverse prim-ers, and 2 U of Vent DNA polymerase (or 1.25 U of Pfu DNA polymerase) in atotal volume of 100 ml. The reaction mixtures were denatured for 2 min at 94°C,followed by 30 cycles of 10 s at 95°C, 30 s at 50°C (or 55°C, depending on theprimers used), and 30 s at 72°C and a final extension of 5 min at 72°C.

Megaprimer site-directed mutagenesis and complementation analysis. Thefur-containing 810-bp DNA fragment (100 bp upstream of the fur transcriptionstart site to 220 bp downstream of the fur translation stop codon) was amplifiedfrom HB1000 chromosomal DNA with primers 140 and 139 by PCR as describedabove with the following modifications: cloned Pfu polymerase and Pfu bufferwere used instead of Vent polymerase, the annealing temperature was 47°C, andthe extension time was 1 min. The resulting product was cloned into pGEM-catas a HindIII-BamHI fragment (pHB6524), sequenced, and then subcloned frompHB6524 into pDG1662 to generate pHB6525. For fur complementation anal-ysis, pHB6525 was transformed into HB6637, and double-crossover recombi-nants were selected with chloramphenicol and screened for Spcs and macrolide-lincosamide-streptogramin B sensitivity, ensuring integration at amyE. Theresulting strain (HB6640) was tested by siderophore assays for complementationof fur::kan. With pHB6524 as a template, the various histidine and cysteinemutants were generated by megaprimer PCR mutagenesis as described previ-ously (35) with a few modifications. Briefly, pHB6524 was cut with HindIII andPCR amplified with the appropriate mutagenic primer (147 to 155) and primer139 at an annealing temperature of 50°C. To enrich for the megaprimer DNAstrand, the first PCR product was used as a template in a subsequent asymmetricPCR with 10-fold less of the mutagenic primer than of primer 139. The asym-metric PCR product was used with primer 140 and BamHI-cut pHB6524 as atemplate in a third PCR at an annealing temperature of 48°C. The final productwas purified, cloned into pGEM-cat as an 810-bp HindIII-BamHI fragment(pHB6527 to pHB6534 and pHB6537), sequenced, and then subcloned intopDG1662 (pHB6538 to pHB6545 and pHB6547). The pDG1662-derived plas-mids were then integrated into HB6637 (fur null mutant) and HB6634 (fur wildtype) (HB6650 to HB6663, HB6668, HB6670, HB6672, and HB6673) to studycomplementation and recessiveness to wild-type Fur, respectively.

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Immunoprecipitation of Fur from 35S-Met-labeled cells. Cells were grown tothe late logarithmic phase in MM without iron supplementation. Aliquots (1.6ml) were labeled with L-[35S]methionine (20 mCi; 1,175 Ci/mmol) for 45 min,chased with nonradioactive methionine (2.5 mM), cooled on ice, and centrifuged.Cell pellets were washed with 0.8 ml of glucose buffer (50 mM glucose, 25 mMTris-HCl [pH 8]), resuspended in 100 ml of lysis buffer (glucose buffer with 0.1 mgof lysozyme per ml), and incubated on ice for 10 min. One hundred microlitersof detergent solution (2% Nonidet P-40, 1% sodium deoxycholate) was added,and the samples were vortexed and incubated for 10 min at 37°C. After centrif-ugation, the supernatant was preadsorbed to 50 ml of protein G-agarose suspen-sion (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) for 3 h. Aftercentrifugation for 20 s, the supernatant was incubated with polyclonal rabbitanti-FurEC antibodies for 1 h, 50 ml of protein G-agarose was added, andincubation was continued overnight. The pellets were washed in the buffersrecommended by the manufacturer (Boehringer). All of the previous incubationswere performed at 4°C on a rocking platform, unless otherwise indicated. Thepellets were resuspended in 50 ml of gel loading buffer, boiled for 5 min, andcentrifuged, and the immunoprecipitated proteins in the supernatant were sep-arated by SDS–12% PAGE. The gel was dried and exposed to a PhosphorImagerscreen for analysis.

RESULTS

Overproduction, purification, and physical characterizationof Fur. The fur gene was cloned into pET17b, and Fur wasoverproduced in E. coli BL21(DE3)/pLysE (39). Since the furgene was toxic for E. coli, it was necessary to use pLysE tostabilize the pET transformants (38). The overproduced Furwas almost equally distributed between inclusion bodies andthe extract supernatant and was purified to homogeneity fromthe supernatant. Purification was achieved by chromatographyon heparin-Sepharose, Mono-Q, and Superdex-75. Fur elutesfrom the Superdex-75 column with an apparent molecularmass of 35 kDa, in agreement with its calculated dimeric mass,and with the dimeric state of FurEC in solution (4, 12). Frompromoter titration experiments (see Materials and Methods

and below), we estimate that between 12 and 25% of theisolated Fur is active for DNA binding.

As purified, Fur migrates on SDS-polyacrylamide gels as adoublet, with the predominant and larger product (A) corre-sponding to the expected molecular mass of 17.4 kDa (Fig. 1).The identities of the purified proteins (A and B, separately)were confirmed by amino-terminal sequencing of the first 10amino acid residues, which matched the predicted Fur se-quence (8). Unlike FurEC (40), B. subtilis Fur retains its N-terminal methionine. The faster-migrating band (B) could beeither an isoform of Fur with altered mobility or a degradationproduct. This band was observed consistently despite the ad-dition of a cocktail of protease inhibitors during cell lysis. Sincethe two isoforms persist even after reduction with freshly pre-pared b-mercaptoethanol or DTT, it is unlikely that there is anintramolecular disulfide bond. Therefore, we tentatively con-clude that the smaller band is truncated at its C terminus. Aprotease-sensitive region has also been observed in the C-terminal region of FurEC mutants altered in either of twoconserved cysteines (11). Preliminary atomic absorption spec-troscopy indicates that zinc but neither iron nor manganesecopurifies with Fur (calculated molar ratio of Zn to Fur ofbetween 2 and 3).

Fur binds specifically to the dhb promoter region. PurifiedFur was assayed for DNA binding in EMSAs with a 400-bp furbox-containing dhb promoter fragment cleaved with AlwNI togenerate a 280-bp fur box-containing fragment and a 120-bpcontrol fragment (Fig. 2). When incubated with the dhb pro-moter fragment, 10 nM Fur caused an electrophoretic mobilityshift of the larger, fur box-containing fragment but not thefragment lacking a fur box (Fig. 3A, lane 2). The specificity ofthe interaction of Fur with the dhb promoter fragment wastested in competition assays with either the same dhb DNAfragment or an unrelated DNA fragment generated by PCR.Fur at 10 nM was first incubated with 50 pM end-labeled dhbfragment before the addition of 10 or 100 nM cold competitorDNA, and incubation was continued for another 10 min at RT(Fig. 3B). While the dhb promoter region efficiently competedfor binding, the nonspecific DNA did not compete, even whenpresent in a 10-fold molar excess (100 nM) over Fur. Thisresult demonstrates that Fur specifically interacts with the dhbfur box-containing promoter element. The inability of 10 nMFur to efficiently shift labeled DNA in the presence of 10 nMdhb competitor is consistent with our estimation of the fractionof active Fur as being between 12 and 25%.

Effects of Fe(II) on the binding of Fur to the dhb promoterregion. DNA binding by Fur is thought to require an activatingmetal ion. We attempted to remove loosely associated metalions from Fur by dialysis against buffer containing 25 mMEDTA followed by dialysis against buffer lacking EDTA. Sur-prisingly, the EDTA-treated Fur was just as active as the nativeFur in EMSAs (Fig. 3A, lane 3). Similarly, treatment of Furwith Chelex did not inhibit DNA binding (data not shown).

FIG. 1. Purification of Fur and SDS–12% PAGE analysis of fractions fromthe purification of Fur. Lanes: M, molecular mass standards; 1 and 2, totalproteins from uninduced and IPTG-induced BL21(DE3)/pLysE/pHB6505, re-spectively; 3, pooled fractions from heparin-Sepharose; 4, pooled fractions fromMono-Q; 5, fraction from Superdex-75. Fur runs just below the 21.5-kDa marker,and the Fur doublet is indicated by A and B on the right.

FIG. 2. dhb promoter-operator region. The 235 and 210 regions of the dhb sA-dependent promoter (33) are indicated by underlining. The fur box required foriron-mediated repression of dhb transcription (33) is indicated by double underlining, and the start codon (ATG) for dhbA is shown in uppercase letters. The fragmentused in the EMSA experiments extends from ;90 bp upstream of the 235 element (as indicated) to the right bracket. The AlwNI site used to bisect the dhb fragmentfor EMSA experiments is indicated.

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Thus, purified Fur binds to the dhb promoter fragment in theabsence of any added metal ions, and any copurifying metalions required for binding (e.g., zinc) appear to be tightly asso-ciated and not easily removed by chelating agents.

B. subtilis Fur represses dhb expression in vivo in response toiron (8, 33). However, as purified, Fur is able to bind to DNAspecifically in the absence of added iron. To clarify the dis-crepancy between the in vitro and in vivo data, we wished todetermine if Fe(II) increases the affinity of Fur for the dhbpromoter fragment. EMSAs were conducted over a range ofFur concentrations (0.06 to 128 nM) in the presence and ab-sence of added ferrous ion (Fig. 4). In the absence of addediron, the best-fit binding curve corresponded to a Kd (appar-ent) of 4.6 nM (corresponding to a Kd of ;0.8 nM whencorrected for active protein) and a cooperativity coefficient of2, perhaps indicating oligomerization of Fur concomitant withDNA binding. In the presence of added iron, the calculatedaffinity of Fur for DNA was enhanced twofold (apparent Kd,2.3 nM) and cooperativity was slightly increased (cooperativity

coefficient, ;3). These results suggest that Fe(II) does in-crease, albeit only modestly, the interaction of Fur with the furbox-containing fragment.

Repression of dhb transcription by Fur in vitro. Despite thelack of a dramatic effect on DNA-binding affinity, we hypoth-esized that Fe(II) might nevertheless stabilize a conformationof Fur necessary for efficient transcriptional repression. To testthis idea, purified B. subtilis sA holoenzyme was used to tran-scribe a linearized plasmid DNA template to produce a 268-bprunoff dhb transcript together with a smaller, vector-derivedtranscript that serves as a control for nonspecific effects of Fur(Fig. 5A, lane 2). As expected, when the vector alone was usedas a template, the larger, dhb transcript was not observed (Fig.5A, lane 1). Fur (4 to 80 nM) was preincubated with the dhbtemplate (4 nM) prior to the addition of 80 nM RNAP. Re-pression of the dhb transcript was evident at 16 nM Fur andwas complete at 32 nM Fur. Fur did not repress the vector-derived transcript at any of the concentrations tested. Thus,Fur specifically represses dhb transcription.

To determine if Fe(II) affects the ability of Fur to represstranscription, we measured transcriptional repression in thepresence and absence of freshly prepared 10 mM Fe(II). Inthese studies, we altered the order of addition such that theDNA template was preincubated first with RNAP and thenwith Fur for 5 min prior to initiation of the transcriptionreactions. Once again, 16 nM Fur led to significant repression,which was complete at 32 nM Fur (Fig. 5B). The addition ofFe(II) did not affect the ability of Fur to repress the transcrip-tion of dhb. However, there was some repression of the vector-derived transcript at the highest Fur concentration tested (256nM), and this effect did require iron.

Repression of dhb transcription by Fur in vivo: role of con-served histidine and cysteine residues in repression. Whileiron is required for the in vivo repression of siderophore bio-synthesis, our in vitro studies indicate that Fe(II) addition isnot required for the specific binding and repression of dhbtranscription. In contrast, in other studies of FurEC, the addi-

FIG. 3. Specific binding of Fur to the dhb regulatory region. (A) The dhbpromoter fragment (50 pM) was cut with AlwNI, end labeled, and incubated withnative or EDTA-treated Fur. Lanes: 1, no Fur; 2, 10 nM Fur; 3, 10 nM EDTA-treated Fur. The positions of the unbound fur box and the shifted complex areindicated by unbound and bound, respectively. (B) The dhb promoter fragment(50 pM) was incubated with Fur for 10 min prior to the addition of coldcompetitor DNA. Lanes: 1, no Fur; 2, 10 nM Fur; 3, 10 nM Fur and 10 nM dhbcompetitor DNA; 4, 10 nM Fur and 100 nM dhb competitor DNA; 5, 10 nM Furand 10 nM nonspecific competitor DNA; 6, 10 nM Fur and 100 nM nonspecificcompetitor DNA. The shifted complex is displaced only by the addition of thespecific dhb competitor DNA.

FIG. 4. Effect of added Fe(II) on the affinity of Fur for the dhb promoterregion. A curve for the binding of Fur to the dhb promoter fragment in thepresence and absence of Fe(II) is shown. Fur (0.5, 1, 2, 4, 8, 16, and 32 nM) inthe presence of Fe(II) (■) and Fur (0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and128 nM) in the absence of Fe(II) (F) were incubated with 50 pM end-labeledtemplate and separated by nondenaturing PAGE, and the percentages of boundand unbound DNA were determined by PhosphorImager analysis with the pro-gram ImageQuant (Molecular Dynamics, Inc.).



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tion of divalent cations is typically required to observe DNAbinding (3, 14, 22). Unfortunately, little is known about theactual mechanism of iron sensing by Fur proteins, although itis thought that binding involves a subset of the conservedhistidine and cysteine residues in the carboxyl-terminal do-main.

We used site-directed mutagenesis of the fur gene to indi-vidually alter nine cysteine and histidine residues to alanine.We inserted the wild-type and mutant fur genes in single copiesat amyE and tested for complementation of a fur null mutant(Fig. 6). As an indirect measure of the transcriptional activityof the dhb locus, we monitored the levels of siderophores(dihydroxybenzoic acid and dihydroxybenzoyl serine) presentin cell supernatants after growth in the presence of variousconcentrations of iron. In this assay, the H93A-, H95A-, andH132A-expressing strains all displayed a pattern of iron-medi-ated repression not significantly different from that of the wildtype. In contrast, the strains expressing mutant Fur proteinsaltered in any of the four Cys residues were completely dere-pressed for dhb expression, similar to the fur null mutant. TheH96A and H97A Fur mutants appeared to have altered func-tion. While the H97A mutant protein displayed very little ironresponsiveness, it did not yield a completely derepressed nullphenotype. The H96A mutant protein had the most interesting

phenotype: it displayed a level of basal activity similar to thatof the wild type and mediated full repression of dhb at 50 mMbut not 5 mM added iron.

To determine whether the null phenotypes observed forsome of the mutants (Fig. 6) were due to instability of themutant proteins, we labeled the various B. subtilis mutantstrains with L-[35S]methionine, immunoprecipitated Fur fromwhole-cell extracts by using polyclonal antibodies againstFurEC, and analyzed the precipitates by SDS-PAGE (Fig. 7).These antibodies cross-react with purified B. subtilis Fur in dotblot assays (data not shown) and allow visualization of a pro-tein with the expected size for Fur (17.4 kDa) in the wild-typestrain but not in a fur mutant strain. In addition to full-lengthFur, a smaller protein is also detected. This is likely to be adegradation product of Fur, since it is missing from the furmutant strain.

The immunoprecipitation experiment revealed nearly wild-type levels of Fur in the strains expressing the H95A, H96A,and H97A proteins. The presence of essentially wild-type lev-els of H96A Fur and H97A Fur is significant, since theseproteins had altered responses to iron in vivo. The H93A andH132A proteins were present at very low levels in this assay,despite their nearly normal biological activity. We do not knowwhether the reduced levels of these proteins resulted frominstability in vivo or during the immunoprecipitation proce-dure. The lack of signal for the four Cys mutant proteinssuggests that these proteins may fail to accumulate in vivo,consistent with their null phenotypes, with the stability defectsof two FurEC Cys mutant proteins (11), and with the findingthat these mutant proteins are unstable when overproduced inE. coli (data not shown). Although it seems unlikely, we cannotyet rule out the possibility that one or more of these mutationsmay affect the ability of these antisera to recognize Fur.

DISCUSSION

We report the purification and initial characterization of theDNA- and metal-binding properties of B. subtilis Fur, the firstmember of the Fur family to be biochemically characterizedfrom a gram-positive organism.

In vitro repression of the dhb promoter. B. subtilis Fur actsas an iron-dependent repressor of the dhb operon in vivo (8,33). However, in vitro Fe(II) failed to significantly affect eitherthe affinity of Fur for the dhb operator (Fig. 4) or the ability ofFur to repress transcription (Fig. 5). We conclude that in vitroFur is an iron-independent repressor of dhb transcription. In-terestingly, Fur efficiently represses transcription even ifRNAP is incubated with the dhb promoter region prior to theaddition of Fur. This finding suggests either that Fur can bindto the promoter region downstream of prebound RNAP orthat Fur can displace RNAP. Further studies are needed todistinguish between these possibilities. For comparison, invitro studies have demonstrated that neither FurEC nor E. coliRNAP can displace the other (15, 16).

Interactions of Fur with metal ions. FurEC is the prototypefor a large family of metal-dependent repressors (22), includ-ing three functionally distinct B. subtilis homologs (8, 17). It isgenerally accepted that Fur binds to Fe(II) in vivo and isthereby activated to bind DNA, leading to the repression ofiron uptake functions. However, the interaction of Fur withmetal ions is not well understood.

The most unexpected finding from this work is the lack of arequirement for added iron for either DNA binding or tran-scriptional repression in vitro. As purified, B. subtilis Fur con-tains zinc, as reported for FurEC (24), but little or no iron.Moreover, DNA binding is insensitive to treatment of Fur with

FIG. 5. Fur specifically represses dhb transcription. (A) Addition of Furbefore RNAP. The linearized vector (lanes 1 and 3) or the dhb-containingtemplate DNA (lanes 2, 4, 5, 6, 7, 8, and 9) (4 nM) was incubated with 80 nMRNAP for 10 min at 37°C (lanes 1 and 2) or with Fur for 5 min at RT prior tothe addition of 80 nM RNAP and incubation for another 5 min at 37°C (lanes 3to 9). Lanes: 1 and 2, no Fur; 3 and 4, 80 nM Fur; 5, 64 nM Fur; 6, 32 nM Fur;7, 16 nM Fur; 8, 8 nM Fur; 9, 4 nM Fur. The positions of the dhb transcript andthe vector transcript are indicated on the right. (B) Addition of Fur after RNAP.The linearized vector (lane 1) or the dhb-containing template DNA (lanes 2 to10) (4 nM) was incubated with 80 nM RNAP for 10 min at 37°C (lanes 1 and 2)or preincubated with 80 nM RNAP for 5 min at 37°C prior to the addition of Furalone (lanes 3, 5, 7, and 9) or Fur and Fe(II) (lanes 4, 6, 8, and 10) and incubationfor another 5 min at RT. Lanes: 1 and 2, no Fur; 3, 4 nM Fur; 4, 4 nM Fur and10 mM Fe(II); 5, 16 nM Fur; 6, 16 nM Fur and 10 mM Fe(II); 7, 64 nM Fur; 8,64 nM Fur and 10 mM Fe(II); 9, 256 nM Fur; 10, 256 nM Fur and 10 mM Fe(II).The positions of the dhb transcript and the vector transcript are indicated on theright.



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chelating agents, including dialysis against EDTA or incuba-tion with Chelex, and is not appreciably enhanced by the ad-dition of Fe(II). Thus, if metal ions are required to activate Furfor DNA binding, they appear to be tightly bound under ourpurification conditions.

FurEC binds at least two metal ions per monomer in a car-boxyl-terminal metal-binding domain that contains severalconserved histidine and cysteine residues (11, 12, 24). FurEChas recently been found to be a zinc metalloprotein (24) witha tightly associated zinc ion thought to play a structural role.This ion is coordinated with two sulfur ligands, perhaps Cys92and Cys95 (equivalent to Cys100 and Cys103 in B. subtilis Fur),and two nitrogen or oxygen ligands. Further analyses haverevealed that Fur purified in the presence of chelators retainsa single zinc ion (Zn1Fur), while in the absence of chelators

each monomer binds two zinc ions (Zn2Fur) (1). Remarkably,both forms bind DNA with similar affinities (1), consistent withour findings that Fur from B. subtilis contains associated zincand binds DNA even in the absence of added iron.

The second, lower-affinity metal-binding site is presumed tobe the regulatory site. In vivo, FurEC maintains a level of free(chelatable) iron near 10 mM (27). Thus, the in vivo dissocia-tion constant for the functional interaction of Fe with Fur isprobably near 10 mM. In vitro studies have suggested thatFurEC can be activated for DNA binding by several divalentions, including Co(II), Cu(II), Cd(II), Fe(II), and Mn(II) (14).Similar results have been obtained with Pseudomonas aerugi-nosa Fur (30). The origins of the in vivo metal selectivity ofFur-mediated repression, in view of the promiscuous metal-binding properties of the purified protein, are not yet clear.

FIG. 6. Complementation of fur::kan by the various histidine-to-alanine and cysteine-to-alanine fur mutations. The mutations were inserted at amyE in HB6637(fur::kan) by a double recombination event, the strains were grown in MM in the absence (dark gray bar) or presence of 5 mM Fe(III) (hatched bar) or 50 mM Fe(III)(light gray bar), and catechol siderophore yields were expressed as OD510/OD600 6 standard deviations. WT, wild type.

FIG. 7. Fur is destabilized by all four cysteine mutations and two of the histidine mutations. Pulse-labeled fur mutants were lysed, and Fur was immunoprecipitatedfrom the extracts with polyclonal antibodies against E. coli Fur and protein-G agarose beads. The immunoprecipitated proteins in the supernatant were separated bySDS–12% PAGE, and the gel was analyzed by PhosphorImager analysis. Arrows show full-length Fur (upper band) and a Fur degradation product (lower band). WT,wild type.



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Presumably, levels of free metal ions in the cell are tightlyregulated, and only iron (and under some conditions, manga-nese) (5, 21, 32) achieves a sufficient level to effect repression.

A model for interactions of B. subtilis Fur with metal ions.We currently favor a model for Fur that includes roles for botha structural zinc-binding site and a regulatory site that sensesthe presence of iron. Our in vivo studies of Fur-mediatedrepression indicate that two histidines (H96 and H97, corre-sponding to D89 and H90 in both FurEC and Salmonella typhi-murium Fur) are likely candidates for Fe(II) ligands. A role forthese residues in binding iron is consistent with the finding thatS. typhimurium H90 mutants are iron blind (20). Curiously,mutation of these residues in FurEC failed to affect iron-de-pendent regulation (11).

How can we reconcile the apparent lack of a requirement foran activating metal ion in vitro with the well-documented ef-fects of iron on in vivo activity? We suggest that in vivo Fur isbound to an inhibitor of DNA binding that is antagonized byiron. In principle, this inhibitor might be another protein, alow-molecular-weight ligand, or another metal ion. The lastpossibility is particularly attractive, as it is easy to imagine howanother metal ion might compete for the Fe(II)-binding site. Acorollary of this hypothesis is that this alternate form of Furmight be active for DNA binding at some sites, albeit not thoseassociated with the regulation of iron uptake functions.

This model is consistent with several observations. First, theability of S. typhimurium H90A Fur to function in the acidtolerance response, despite an inability to repress siderophorebiosynthesis genes, suggests that Fur lacking bound Fe(II) maystill have DNA-binding activity (20). Second, Fur proteins con-taining different metal ions have distinct DNA-binding speci-ficities. For example, while most PerR-regulated genes can berepressed by either iron or manganese, the fur gene is re-pressed only by manganese (7–10 and unpublished data). Sim-ilarly, repression of the sodA gene by FurEC is effected only byiron, while either iron or manganese can lead to repression ofthe aerobactin genes (32). By extension, it is easy to imaginethat in vivo a divalent metal ion binds Fur and stabilizes aconformation inactive for binding to iron-regulated target sites(although not, perhaps, to other target sites) and that onlyupon displacement by Fe(II) is the ability to bind to iron-regulated promoter regions recovered. Indeed, previous stud-ies indicated that the supplementation of growth medium withsome divalent cations can derepress siderophore biosynthesisand the expression of Fur-regulated genes (9).

In summary, our work confirms the notion that iron regula-tion in B. subtilis is mediated by a Fur homolog that bindsdirectly to a fur box, as inferred from previous genetic analyses.However, we have not yet been able to demonstrate iron-responsive DNA binding in vitro because the protein bindstightly to DNA in the absence of added iron. We suggest thatin vivo there is an inhibitory factor, perhaps a metal ion, absentfrom our in vitro system. We are presently exploring this pos-sibility by using crude extracts.

ACKNOWLEDGMENTS

We thank M. Vasil for providing the anti-E. coli Fur antibodies.This work was supported by NSF grant MCB9630411.

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