INFECTION AND IMMUNITY,0019-9567/97/$04.0010

Oct. 1997, p. 4273–4280 Vol. 65, No. 10

Copyright © 1997, American Society for Microbiology

Identification and Characterization of Three NewPromoter/Operators from Corynebacterium diphtheriae

That Are Regulated by the Diphtheria ToxinRepressor (DtxR) and Iron

JOHN H. LEE,1 TING WANG,2† KRISTEN AULT,2‡ JUAN LIU,2§ MICHAEL P. SCHMITT,2\AND RANDALL K. HOLMES1*

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262,1 andDepartment of Microbiology and Immunology, Uniformed Services University of the Health Sciences,

Bethesda, Maryland 208142

Received 20 May 1997/Accepted 24 July 1997

DtxR is a dimeric, sequence-specific, DNA-binding protein that functions as an iron-dependent, negativeglobal regulator in Corynebacterium diphtheriae. Under high-iron conditions, DtxR represses the synthesis ofdiphtheria toxin, corynebacterial siderophore, and other components of the high-affinity iron uptake system.Three DtxR-regulated promoter/operators designated tox, IRP1, and IRP2 were reported previously. In thisstudy, we identified and characterized three additional DtxR-regulated promoter/operators from C. diphtheriaedesignated IRP3, IRP4, and IRP5. When b-galactosidase was expressed from these three new promoter/operators in Escherichia coli containing dtxR1 on pDSK29, enzyme levels were 5- to 30-fold lower duringhigh-iron growth than during low-iron growth. In gel shift assays, the mobility of DNA fragments containingeach promoter/operator decreased in the presence of purified DtxR and Co21. In footprinting assays, DtxRprotected 36-, 35-, and 30-bp regions of IRP3, IRP4, and IRP5, respectively, from cleavage by DNase I. In the19-bp core of each promoter/operator, 12 or 13 bp matched the consensus for the DtxR-binding site. Theputative polypeptides encoded by the open reading frames (ORFs) downstream from IRP3 and IRP4 werehomologous, respectively, to several bacterial transcriptional regulators and to the deduced polypeptideencoded by an ORF located between the E. coli genes for primosomal replication protein N and adeninephosphoribosyltransferase. The putative polypeptide encoded by the ORF downstream from IRP5 was nothomologous to any sequence in the protein database at the National Center for Biotechnology Information.When the ORFs downstream from IRP3 and IRP4 were expressed under the control of the phage T7 promoterin E. coli, polypeptide products of the predicted sizes were detected in small amounts by sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Iron is essential for a variety of cellular processes in mostliving organisms. For pathogenic bacteria to establish andmaintain an infection, they must be able to obtain iron (56). Atneutral or alkaline pH the concentration of ferric iron in so-lution is extremely low, and most of the iron in mammalianhosts is present in complexes with iron storage or iron trans-port proteins or in prosthetic groups such as heme. Manypathogenic bacteria produce siderophores, low-molecular-weight iron chelators that can solubilize iron from the envi-ronment or remove it from host iron-binding proteins such astransferrin or lactoferrin (25), and they use special uptakesystems to assimilate iron from ferrisiderophore complexes(23, 56). Other pathogenic bacteria can bind directly to trans-ferrin or other iron-binding proteins via cell surface receptors

and extract the iron by siderophore-independent pathways(22).

Pathogenic bacteria often use the low-iron environment ofthe host as a signal to induce iron uptake systems and produceother virulence factors (22). In many gram-negative bacteria,including Escherichia coli, Salmonella typhimurium, Vibrio chol-erae, Yersinia pestis, Pseudodomonas aeruginosa, and Neisseriagonorrhoeae, iron-dependent gene expression is negatively reg-ulated by the ferric uptake regulator protein Fur (22, 27, 45, 46,53). Fur uses ferrous iron as a corepressor, and the activeholorepressor binds to regulatory DNA sequences, called Furboxes, which are located near the promoters of the Fur-regu-lated genes (7, 10). Fur is a global regulatory protein, and in E.coli it controls expression of more than 30 different genes thatconstitute a regulon (22, 46). Fur-regulated genes in gram-negative bacteria determine a wide variety of products or phe-notypes, including bacterial toxins, iron uptake systems, theacid tolerance response, pathways of sugar metabolism, de-fenses against oxygen radicals, and other virulence factors (22,27, 45, 46, 53).

Regulatory cascades appear to be involved frequently inglobal iron-dependent gene regulation in bacteria. Severaltranscriptional regulators are expressed under the negativecontrol of the Fur protein and iron, including PupI of Pseudo-monas putida, which activates genes that encode receptors forthe uptake of two heterologous ferric pseudobactins (19); FecI

* Corresponding author. Mailing address: Department of Microbi-ology, Campus Box B-175, University of Colorado Health SciencesCenter, 4200 East Ninth Ave., Denver, CO 80262. Phone: (303) 315-7903. Fax: (303) 315-6785. E-mail: Randall.Holmes@UCHSC.Edu.

† Present address: Department of Microbiology and Immunology,University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

‡ Present address: Yale College, New Haven, CT 06520.§ Present address: Peters Pediatric Services of America, Washing-

ton, D.C. 20012.\ Present address: Center for Biologics Evaluation and Research,

Food and Drug Administration, Bethesda, MD 20892.

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of E. coli, which activates a gene that encodes a receptor foruptake of ferric citrate (54); and several genes that encodeputative transcriptional regulators in P. aeruginosa (27). Al-though most Fur-regulated genes are repressed under high-iron conditions, some are activated (46). It is not yet estab-lished whether activation of such genes is controlled directly byFur or indirectly by other regulators that are controlled by Fur.

In Corynebacterium diphtheriae, the diphtheria toxin repres-sor DtxR functions as an iron-dependent global regulatoryprotein in a manner similar to Fur in gram-negative bacteria(2, 5, 39, 48). DtxR coordinately regulates the expression ofdiphtheria toxin and the high-affinity iron uptake system of C.diphtheriae in response to changes in environmental iron con-centrations. DtxR homologs, designated iron-dependent regu-lators (IdeR) (43), have also been found in other gram-positiveand acid-fast bacteria, including Mycobacterium tuberculosisand other Mycobacteria species (12, 43), Streptomyces lividansand Streptomyces pilosus (15), and Brevibacterium lactofermen-tum (28).

DtxR uses ferrous iron as a corepressor; binds to operatorssuch as tox, IRP1, and IRP2; and strongly inhibits expression ofthe iron-regulated genes by preventing their transcription (5,39, 42, 44). Binding to DtxR decreases the electrophoreticmobility of DNA fragments that contain DtxR-regulated pro-moter/operators, and footprinting experiments indicate thatthe DtxR-regulated operator sequences overlap with proven orputative 210 promoter sequences and contain a 9-bp invertedrepeat within a 19-bp core region (42, 44, 50). DtxR bindssymmetrically about the dyad axis of the operator, in a mannersimilar to the binding of several other well-characterized bac-terial repressors (16, 17, 29, 30). Only rudimentary informationconcerning the functions of genes other than tox that are reg-ulated by DtxR and Fe21 in C. diphtheriae is available. Trans-lation of a partial open reading frame (ORF) located down-stream from IRP1 suggested that the product of the ORF is amembrane-associated lipoprotein that may function as a ferri-siderophore receptor, and a partial ORF located downstream

from IRP2 was too short to yield significant information aboutthe putative protein encoded by the ORF (42).

In this study, three additional DtxR-dependent, iron-regu-lated promoter/operators (IRPs), designated IRP3, IRP4, andIRP5, and the ORFs located immediately downstream fromthem were cloned from the C. diphtheriae C7 chromosome andcharacterized. The findings reported here extend significantlythe available information on the family of DtxR-regulated pro-moter/operators in C. diphtheriae and the set of genes thatconstitute the DtxR regulon.

MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli and C. diphtheriae strains and

plasmids used in this study are listed in Table 1. Strains were routinely culturedin Luria-Bertani broth (LB) or terrific broth (21). Heart infusion broth (Difco,Detroit, Mich.) containing 0.2% Tween 80 was used for routine growth of C.diphtheriae C7, a nonlysogenic, nontoxinogenic reference strain of the mitisbiotype. E. coli BL21(DE3) was cultured at 30°C with shaking for protein ex-pression, and all other bacteria were cultured at 37°C with shaking. Antibioticsand chromogenic substrates, when required, were included in the culture me-dium or plates at the following concentrations: ampicillin, 100 mg/liter; kanamy-cin, 150 mg/liter; tetracycline, 12.5 mg/liter; and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal), 40 mg/liter. In order to create iron-limiting growthconditions, the iron chelator ethylenediamine-di(o-hydroxyphenyl)acetic acid(EDDA) was added at 500 mg/liter to LB cultures and at 50 mg/liter to LB agarmedium. Plasmid pQF50 was used for the isolation and analysis of cloned DNAfragments for promoter activity. E. coli strains carrying the dtxR1 plasmidpDSK29 were used as hosts when cloned promoter/operators from C. diphtheriaein pQF50 were tested for iron-dependent regulation by DtxR.

DNA preparation, cloning, and sequencing. Chromosomal DNA was isolatedand purified from C. diphtheriae C7 by cesium chloride-ethidium bromide densitygradient centrifugation (42). Restriction enzymes and other DNA-modifyingenzymes were used as instructed by the manufacturer (GIBCO-BRL, Gaithers-burg, Md.). DNA fragments were separated by electrophoresis in low-melting-point agarose gels, excised, and purified by using Gene Clean kits (Bio101 Inc.,La Jolla, Calif.). Recombinant DNA was introduced into E. coli strains by theCaCl2 transformation method (8) or by electroporation (Bio-Rad, Hercules,Calif.). Promega Miniprep kits (Promega, Madison, Wis.) were used to prepareplasmid DNA for subcloning and sequencing. Nucleotide sequence analysis ofDNA fragments cloned into pBluescript SK2 was performed by the di-deoxynucleotide chain termination method using Sequenase 2.0 (United StatesBiochemical, Cleveland, Ohio) and by use of an automated sequencing facility(Department of Biochemistry, Colorado State University, Fort Collins).

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Characteristics Reference or source

E. coliDH5a F2 supE44 DlacU169 (f80 lacZ DM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Bethesda Research LaboratoriesXL1-Blue MRF9 D (macrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE4 thi-1 recA1 gyrA96 relA1

lac (F9 proAB lacIq ZDM15 Tn10 [Tetr])Stratagene

SOLR e14-(mcrA) D(mcrCB-hsdSMR-mrr)171 sbcC recB recJ (umuC::Tn5 [Kanr]) uvrClac gyrA96 relA1 thi-1 endA1 lr (F9 proAB lacIq ZDM15) Su2

Stratagene

BL21(DE3) F2 dcm ompT hsdS(rB2 mB

2) gal::l(DE3) 47

C. diphtheriae C7 (2) tox mutant 4

PlasmidspDSK29 5-kb fragment carrying dtxR1 allele in RSF1010-derived vector 40pQF50 Promoter/probe vector 14pQFtox tox-lacZ fusion in pQF50 42pIRP3 0.6-kb AluI fragment carrying IRP3 in pQF50 This studypIRP4 0.8-kb AluI fragment carrying IRP4 in pQF50 This studypIRP5 2.4-kb AluI fragment carrying IRP5 in pQF50 This studypIRP3-1 0.2-kb HindIII-NotI subclone carrying IRP3 region in pQF50 This studypIRP4-3 0.3-kb HindIII-MspI subclone carrying IRP4 region in pQF50 This studypIRP5-2 0.4-kb HindIII subclone carrying IRP5 region in pQF50 This studypBluescript SK2 T7 promoter expression and cloning vector StratagenepJL3 6.2-kb EcoRI downstream fragment from IRP3 in pSK2 This studypJL4 2.3-kb EcoRI downstream fragment from IRP4 in pSK2 This studypJL5 4.0-kb EcoRI downstream fragment from IRP5 in pSK2 This study

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Gel mobility shift assays and footprinting assays. The Klenow fragment ofDNA polymerase I was used for labeling of the 39 termini of DNA fragmentscarrying the various promoter/operator regions with [a-32P]dCTP (35). Theend-labeled DNA fragments, at approximately 0.5 nM, were incubated withvarious concentrations of purified DtxR in 10-ml reaction volumes in buffercontaining 20 mM Na2HPO4 (pH 7.0), 50 mM NaCl, 5 mM MgCl2, bovine serumalbumin (100 mg/ml), sonicated salmon sperm DNA (10 mg/ml), and 10% glyc-erol. CoSO4 was present at 150 or 300 mM for individual experiments. Formobility shift assays, the reaction mixtures were incubated for 10 to 15 min atroom temperature and then subjected to electrophoresis on 5% nondenaturingpolyacrylamide gels in 20 mM Na2HPO4 buffer (pH 7.0) for 1 to 1.5 h at 70 V and4°C. For footprinting assays, a 1-ml aliquot containing 10 ng of DNase I (GIBCO-BRL) was added at the end of the incubation used for gel mobility shift assays.The reaction was continued for another 1 to 3 min and was terminated byphenol-chloroform extraction. The samples were electrophoresed through 6%denaturing polyacrylamide gels at 70 to 90 V for 2 h at room temperature. Afterelectrophoresis, the gels were dried and analyzed by autoradiography.

Construction of a C. diphtheriae genomic library. C. diphtheriae chromosomalDNA was partially digested with EcoRI, and 2- to 10-kb fragments were purifiedby sucrose gradient centrifugation (35). The DNA fragments were ligated intothe Lambda ZAP II vector (Stratagene, La Jolla, Calif.), and Gigapack II extract(Stratagene) was used to package the DNA clones. E. coli XL1-Blue MRF9(Stratagene) was used for amplifying, plating, screening, and determining thetiter of the library, and E. coli SOLR (Stratagene) with helper phage was used forin vivo excision of pBluescript SK2 phagemid clones of interest.

Detection of the products of genes downstream from IRP3 and IRP4. DNAfragments of 1.3 and 2.3 kb carrying the ORFs downstream from IRP3 and IRP4,respectively, were cloned into pBluescript SK2 under the control of the T7promoter. Each recombinant plasmid was transformed into E. coli BL21(DE3).The transformants were grown overnight at 30°C in LB medium with ampicillinat 100 mg/liter and subcultured by inoculating 0.5-ml samples into 10-ml aliquotsof fresh LB medium, which were incubated to an A600 of 0.6 prior to beinginduced by addition of 50-ml samples of 100 mM isopropyl-b-D-thiogalactopyr-anoside (IPTG) and then further incubated for 1 h. A 1-ml sample from eachculture was centrifuged, and each cell pellet was resuspended in 160 ml of a lysisbuffer containing 60 mM Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS),1% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue. The sam-ples were heated for 5 min at 95°C and then subjected to electrophoresis on 12%polyacrylamide gels containing 1% SDS.

Purification of DtxR protein. DtxR was overexpressed in E. coli by use of a T7expression system, and the DtxR was purified from sonic extracts of the bacteriaby affinity chromatography on nickel-nitrilotriacetic acid-agarose (Qiagen Inc.,Chatsworth, Calif.) followed by anion-exchange chromatography on DEAE-cellulose (41, 44). The purified DtxR protein appeared homogeneous by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

b-Galactosidase assays. Bacterial cultures were grown overnight in LB me-dium with the appropriate antibiotics and either 500 mg of EDDA per ml(low-iron conditions) or no added EDDA (high-iron conditions). The reactionwas initiated by adding o-nitrophenyl-b-D-galactopyranoside at 4 mg/ml, absor-bance was measured at 420 and 590 nm, and units of b-galactosidase activity werecalculated according to the method of Miller (24).

Computer analysis. The analysis of DNA sequences was performed withDNASIS-Mac version 2.0 (Hitachi, San Bruno, Calif.), Mac Targ search (Carn-egie Mellon University, Pittsburgh, Pa.), and sequences in GenBank. Amino acidsequences were compared by using the program BLAST at the National Centerfor Biotechnology Information (1).

RESULTS

Cloning of new DtxR-regulated, iron-dependent promoter/operators from the chromosome of C. diphtheriae. Schmitt andHolmes developed methods for expression cloning of IRPs(42). They isolated and characterized IRP1 and IRP2 from alibrary of Sau3AI fragments from partially digested chromo-somal DNA of C. diphtheriae C7 (42). Subsequent screening inour laboratory of additional IRP-positive clones from Sau3AIlibraries yielded no other IRPs. As part of an effort to identifyand characterize the complete set of DtxR-regulated genes ofC. diphtheriae, we searched for additional IRPs in C. diphthe-riae chromosomal libraries prepared with other restriction en-donucleases.

Chromosomal DNA from C. diphtheriae C7 was partiallydigested with AluI, and 0.5- to 3-kb DNA fragments recoveredfrom agarose gels were ligated into SmaI-digested promoterprobe vector pQF50. The recombinant plasmids were trans-formed into E. coli DH5a containing pDSK29 (dtxR1), and thetransformed cells were plated on low-iron LB agar medium

containing X-Gal plus ampicillin and kanamycin to maintainpositive selection for both plasmids. Although most of theantibiotic-resistant colonies were white, we identified approx-imately 800 colonies that varied in color from light blue to darkblue and were presumed to have active promoters in theircloned DNA inserts. When these colonies were restreakedonto high-iron LB agar containing X-Gal, ampicillin, andkanamycin, we initially identified 14 clones that formed whitecolonies under high-iron conditions and were presumed tohave DtxR-repressible promoter/operators. After eliminatingthe clones that were not clearly and consistently repressible byDtxR and iron or were duplicate isolates of IRP1, IRP2, or oneanother, we selected three clones, designated pIRP3, pIRP4,and pIRP5, that contained different inserts of 0.6, 0.8, and 2.4kb, respectively, for further study.

To obtain subclones of these putative DtxR-regulated pro-moters, we excised the inserts from pIRP3, pIRP4, and pIRP5with HindIII and BamHI, digested them with various restric-tion enzymes, ligated the resulting fragments into pQF50 in thesame orientation as they occurred in the parental clones, andidentified the subclones that still contained the active IRPs(Fig. 1). Plasmid pIRP3-1 contains a 0.2-kb HindIII-NotI frag-ment derived from pIRP3. Plasmid pIRP4-3 contains a 0.3-kbHindIII-MspI fragment derived from the 0.5-kb insert ofsubclone pIRP4-1. Plasmid pIRP5-2 contains a 0.4-kbHindIII-HindIII fragment derived from pIRP5. None of thesesubclones exhibited promoter activity when it was ligated intopQF50 in the orientation opposite that of lacZ.

Promoter strength and repressibility of new IRPs in E. coli.Expression of b-galactosidase was determined for each pro-moter/operator clone in cultures of E. coli DH5a containing orlacking the dtxR1 plasmid pDSK29 and grown under high-ironor low-iron conditions (Table 2). The pQF50 vector (withoutany insert) and pQFtox (containing the tox promoter/operatorfrom corynebacteriophage b in pQF50) were included as con-trols. The activity of b-galactosidase from pQF50 was barelydetectable under any set of conditions tested and was consid-ered as the baseline for the assay. The promoter/operators onpQFtox, pIRP3, pIRP4, pIRP5, and the various subclones ofthese plasmids were all repressible by iron plus DtxR. The rankordering of these promoter/operators based on promoterstrength, as assessed by the maximum levels of b-galactosidaseactivity observed for each clone under derepressing conditions(low iron, without DtxR, or both), was IRP5 . IRP3 . IRP4 .tox. Under repressing conditions (high iron, with DtxR), thelevel of b-galactosidase activity was significantly greater for

FIG. 1. Restriction maps of original clones and DtxR-regulated promoter-positive subclones in pQF50. The restriction fragments from the original cloneswere separately subcloned in pQF50 in the same orientation as they occur in theparental clones. The arrows indicate the directions of transcription from theDtxR-regulated promoters. B, BamHI; H, HindIII; M, MspI; N, NotI; S, SphI.

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IRP3 than for the other three promoter/operators. This findingsuggested that DtxR might bind to the IRP3 operator withlower affinity than it binds to the other operators or that itslocation relative to the promoter might be suboptimal for ef-fecting repression.

Characterization of new promoter/operators by gel mobilityshift assays, DNase I footprinting, and nucleotide sequencing.DtxR is a sequence-specific DNA-binding protein, and itsbinding activity in vitro is dependent on Fe21 or any of severalother transition metal divalent cations (41, 42, 44). To analyzethe binding of DtxR to DNA fragments containing the opera-tor regions from the newly isolated clones, gel mobility shiftassays were performed (Fig. 2). The 0.2-kb HindIII-NotI frag-ment from pIRP3-1, the 0.3-kb HindIII-MspI fragment frompIRP4-3, and the 0.4-kb HindIII fragment from pIRP5-2 werepurified and end labeled with [a-32P]dCTP. Furthermore, theHindIII fragment from pIRP5-2 was cut into 0.25- and 0.15-kbfragments with MspI before it was used for the gel mobility

shift assays. The mobilities of the 0.2-kb fragment frompIRP3-1, the 0.3-kb fragment from pIRP4-3, and the 0.25-kbHindIII-MspI fragment from pIRP5-2 were retarded only inthe presence of both DtxR and Co21. A 10-fold greater con-centration of DtxR (1 mM versus 0.1 mM) was required toobtain an optimal gel mobility shift with pIRP3-1, supportingthe hypothesis that DtxR has lower affinity for the IRP3 oper-ator than for the other DtxR-dependent operators. The mo-bility of the 0.15-kb DNA fragment from pIRP5-2 was un-changed by the presence of DtxR and Co21, confirming thatthe binding of DtxR to DNA is sequence specific and localizingthe IRP5 operator to the 0.25-kb HindIII-MspI fragment.

DNase I footprinting was used to identify the operator se-quence in each of the newly isolated clones (Fig. 3). PurifiedDtxR at 50 or 200 nM protected a 30-bp sequence in the0.25-kb MspI-HindIII DNA fragment of pIRP5-2 from diges-tion by DNase I (Fig. 3). DtxR at 50 or 200 nM also protecteda 35-bp sequence in the 0.3-kb HindIII-MspI fragment ofpIRP4-3. In contrast, no sequence in the 0.2-kb HindIII-NotIfragment of pIRP3-1 was protected by DtxR at 50 nM (datanot shown) or 200 nM, although a 36-bp sequence was pro-tected by DtxR at the higher concentration of 1 mM. In allcases, protection of the operator sequence from digestion byDNase I required both DtxR and Co21.

The sequences of the IRP3, IRP4, and IRP5 operators de-termined by the DNase I protection experiments are presentedin Fig. 4 and are compared with the previously reported tox,IRPI, and IRP2 operators (42). The consensus sequence forthe palindromic 19-bp core region of the DtxR-binding sitededuced from this larger data set confirmed the previouslyreported consensus sequence (42, 52). Eleven of the 19 bp inthe core region were highly conserved and were present in atleast five of the six DtxR-regulated operators shown in Fig. 4.

Analysis of DtxR-regulated genes located downstream fromIRP3, IRP4, and IRP5. When the sequences of the clonedDNA fragments in pIRP3, pIRP4, and pIRP5 were deter-mined, they were not long enough to include any complete

FIG. 2. Gel mobility shift assays. All of the DNA fragments were less than300 bp. The fragments were end labeled with [a-32P]dCTP and incubated in thepresence (1) or absence (2) of Co21 (300 mM) and DtxR (0.1 or 1 mM, asindicated). The IRP5-2 fragment was cut with MspI before the gel shift assay wasperformed, thereby localizing the DtxR-binding sequence to the larger MspIfragment of IRP5-2.

FIG. 3. DNase I footprinting assays. All the fragments were 39-end labeledwith [a-32P]dCTP on one strand and incubated in the presence (1) or absence(2) of Co21 (300 mM) and DtxR (in nanomoles per liter). Brackets indicate thesequences protected by DtxR from DNase I digestion.

TABLE 2. Expression of DtxR-regulated promoter/operators inE. coli DH5a

Plasmid Ironconcentration

b-Galactosidase activity (Millerunits 6 SD)a

With dtxR Without dtxR

pQF50 High 0.3 6 0.2 0.2 6 0.1Low 0.2 6 0.2 0.3 6 0.2

pQFtox High 0.5 6 0.2 8.5 6 0.7Low 9.6 6 0.8 10.1 6 0.7

pIRP3 High 5.7 6 1.1 42.8 6 2.5Low 48.7 6 1.7 40.0 6 3.8

pIRP3-1 High 5.3 6 1.3 NDb

Low 47.2 6 4.5 ND

pIRP4 High 1.3 6 0.4 14.2 6 0.8Low 15.2 6 0.9 15.7 6 2.1

pIRP4-3 High 1.3 6 0.6 NDLow 19.6 6 2.0 ND

pIRP5 High 1.8 6 0.4 57.5 6 6.6Low 55.9 6 4.6 58.1 6 5.6

pIRP5-2 High 3.2 6 0.6 NDLow 95.4 6 6.2 ND

a Values are averages of at least three determinations.b ND, not determined.

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ORF downstream from the DtxR-regulated promoter/opera-tors. To obtain additional downstream sequences, a library ofchromosomal DNA fragments from C. diphtheriae C7 was con-structed in Lambda ZAP II, and plaques were screened byhybridization with 32P-labeled DNA probes from pIRP3-1,pIRP4-3, and pIRP5-2. Clones that hybridized well with eachprobe were isolated, and the presence of the expected promot-er/operator sequence in each clone was confirmed by PCR.Clones with 6.2-, 2.3-, and 4.0-kb DNA inserts carrying IRP3,IRP4, and IRP5 and sequences downstream from them, re-spectively, were selected for further analysis. These DNA in-serts were recloned into pBluescript SK2 and designated pJL3,pJL4, and pJL5, respectively. Nucleotide sequences that weresufficiently long to enable us to place each of the three newDtxR-regulated promoter/operators in context with the firstcomplete ORF located downstream from it were determined(Fig. 5). Putative 210 and 235 promoter regions were identi-fied for each clone by computer analysis (18). Each of theoperators overlapped with the 210 sequence of the corre-sponding promoter.

Translation of the ORF downstream from IRP3 led to theprediction of a 126-amino-acid polypeptide of approximately15 kDa (Fig. 5A). A BLAST search (1) of GenBank sequencesrevealed that the deduced amino acid sequence exhibited ho-mology to a family of bacterial transcriptional regulators thatincludes Rns from E. coli (6), the transcriptional activatorCSVR from E. coli (9), and the urease operon transcriptionalactivator from Proteus mirabilis (26). Comparison of the de-duced sequence of the 51 N-terminal amino acids from thisORF with the homologous sequences of the bacterial tran-scriptional regulators listed above revealed 42, 44, and 34%identity and 60, 63, and 51% similarity, respectively.

Translation of the ORF downstream from IRP4 led to theprediction of a 125-amino-acid polypeptide with a mass ofabout 15 kDa (Fig. 5B). A BLAST search of the predictedpolypeptide revealed that it exhibited homology to a deduced15-kDa polypeptide from E. coli that is encoded by an ORF(GenBank accession no. g1786674) located between the genesfor primosomal replication protein N and adenine phosphori-bosyltransferase. A comparison of the sequences of the 64N-terminal amino acids of these predicted polypeptides fromC. diphtheriae and E. coli revealed 35% identity and 51%similarity. The functions of these hypothetical proteins are notknown.

Translation of the ORF downstream from IRP5 led to the

FIG. 4. Comparison of the known DtxR-regulated promoter/operator se-quences from C. diphtheriae. The 19-bp core region consensus sequence wasderived by comparing the sequences of the tox, IRP1, IRP2, IRP3, IRP4, andIRP5 promoter/operators. The identity score below each nucleotide in the coresequence indicates the number of times that nucleotide was found among the sixpromoter/operators included in the analysis. The arrows indicate inverted re-peats within the 19-bp consensus sequence.

FIG. 5. Nucleotide sequences of the promoter/operators and nucleotide anddeduced amino acid sequences of the ORFs downstream from IRP3 (A), IRP4(B), and IRP5 (C). The putative 235 and 210 promoter sequences are under-lined and labeled, and the DtxR-binding sites defined by DNase I footprintingare indicated by arrows below the nucleotide sequences.

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prediction of a short, 67-amino-acid polypeptide (Fig. 5C).However, a BLAST search of the predicted amino acid se-quence revealed no significant homology with known bacterialproteins.

Detection of polypeptides encoded by the ORFs downstreamfrom IRP3 and IRP4. The nucleotide sequences in Fig. 5A andB showed that the ORFs located immediately downstreamfrom IRP3 and IRP4 were homologous to known gene prod-ucts from other bacteria. To determine whether the productsof these ORFs could be produced in E. coli, a 1.3-kb EcoRIfragment of pJL3 carrying the ORF downstream from IRP3and a 2.3-kb EcoRI fragment of pJL4 carrying the ORF down-stream from IPR4 were cloned into pBluescript SK2 under thecontrol of the T7 promoter. These newly constructed pBlue-script SK clones were transformed into E. coli BL21 (whichcarries the lysogenic DE3 derivative of coliphage l with the T7polymerase gene under the control of the lac promoter [47]),and expression of genes under T7 promoter control was in-duced by addition of IPTG. The results of SDS-PAGE analysisof the whole-cell extracts are shown in Fig. 6. Faint bands(indicated by arrows) were seen in heavily loaded gels at thepositions predicted for the 15-kDa products of these ORFs,and no bands were seen at the corresponding positions in thevector controls.

DISCUSSION

Iron regulation of gene expression in procaryotes has beenstudied most extensively in gram-negative bacteria that expressthe iron-dependent global regulatory protein Fur (reviewed inthe introduction and in reference 22). Fur-regulated productsin bacteria usually include components of high-affinity ironuptake systems, but they may also include toxins, other viru-lence factors, regulatory proteins, and factors that participatein defense against oxygen radicals, acidic environments, catab-olism of sugars, etc. (22, 53).

Within the last several years, DtxR from C. diphtheriae wasrecognized as the prototype for a second family of bacterialiron-dependent global regulatory proteins that function in amanner similar to Fur. Thus far, homologs of DtxR have beenidentified primarily in coryneform bacteria and species of My-cobacterium and Streptomyces (12, 15, 28, 43). DtxR and Fur donot exhibit significant amino acid homology, and they do notcomplement one another in vivo (5, 39). They also bind to

different 19-bp consensus core operator sequences, namely59-GATAATGATAATCATTATC-39 for Fur (10, 21) and59-TTAGGTTAGCCTAACCTAA-39 for DtxR (42, 52).

The structure and function of DtxR have been investigatedin detail. Crystal structures have been determined for dimericDtxR in complexes with various divalent metals and for theaporepressor (11, 32, 33, 36), and the structure of a Co-DtxRcomplex at 100°K was recently refined to a resolution of 1.85 Å(31). Each 226-amino-acid polypeptide in DtxR has three do-mains: an amino-terminal domain with a helix-turn-helixDNA-binding motif, a central dimer interface domain with onebinding site for a divalent cation-anion pair and a secondbinding site for a divalent cation, and a third domain withunknown function that has an SH3-like fold. The effects ofmany different amino acid substitutions on the repressor activ-ity of DtxR have also been characterized (11, 51, 55). Never-theless, the mechanisms by which binding of Fe21 or otherdivalent cations activates the sequence-specific DNA-bindingactivity of DtxR remain controversial. Factors that contributeto this controversy include the following: many different aminoacid substitutions have effects on DtxR activity; there are fewdifferences in the reported crystal structures of DtxR-metalcomplexes and the aporepressor; cysteine 102 appears to beoxidized or modified in most reported crystal structures ofwild-type DtxR, which may affect the structure of metal-bind-ing site 2; and no crystal structures are currently available forDtxR complexed with any of its operators.

The DtxR regulon of C. diphtheriae has not yet been char-acterized as well as the Fur regulon of gram-negative bacteria(27, 46, 53). Early observations concerning differences betweenthe high-iron and low-iron phenotypes of C. diphtheriae werereviewed by Barksdale (3). More recently, additional aspects ofthe low-iron phenotype have been identified, including thecoordinate production of diphtheria toxin and corynebacterialsiderophore (48), the activation of a high-affinity siderophore-dependent iron uptake pathway (34), and the production of 14different iron-regulated polypeptides, the most abundant ofwhich is a putative alkyl hydroperoxide reductase encoded bydirA (49). Only three DtxR-regulated promoter/operatorsfrom C. diphtheriae were characterized before the presentstudy.

The findings reported here expand significantly the availableinformation on the DtxR regulon. We cloned and character-ized IRP3, IRP4, IRP5, and the ORFs located immediatelydownstream from them on the chromosome of C. diphtheriaeC7. All three of these new promoters were stronger than thetox promoter in an E. coli expression system (Table 2). Thebinding of DtxR to each promoter/operator was characterizedby gel mobility shift assays (Fig. 2) and DNase I footprints (Fig.3), and the consensus sequence for the 19-bp core of theoperator was confirmed on the basis of the larger data set nowavailable (Fig. 4). The IRP3 promoter/operator was not asstringently repressed under high-iron conditions, and higherconcentrations of DtxR were needed to shift mobility or obtainDNase I footprints with DNA fragments containing IRP3.These findings provided strong evidence that the affinity ofIRP3 for DtxR was lower than that of the other IRPs.

Figure 4 reveals that 10 of 19 nucleotides in the operatorcore sequence were conserved in five of the six known DtxR-regulated operators from C. diphtheriae, and the C residue atthe fourth position from the 39 end was conserved in all ofthem. In IRP3, which exhibited poor affinity for DtxR, a T-to-Csubstitution at the third position from the 39 end of the coresequence was the only difference among these 11 highly con-served residues of the consensus core sequence. Krafft et al.reported that the partially operator-constitutive mutants of

FIG. 6. Detection of the gene products encoded by ORFs downstream fromIRP3 (A) and IRP4 (B) by SDS-PAGE. Employed were extracts fromBL21(DE3) containing the following constructs: lane 1, pBluescript SK; lane 2,pJL3; and lane 3, pJL4. Polypeptides of the predicted sizes that are produced bystrains containing the appropriate clones, but not by strains containing thevectors, are indicated by the arrows.

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corynephage b designated tox-202 and tox-201 had single G-to-A substitutions at the highly conserved fourth and fifthpositions, respectively, from the 59 end of the 19-bp core se-quence (20), and tox-201 also exhibited poorer binding to DtxRthan did the wild-type tox operator (44). In contrast, IRP4 wasfully repressible by DtxR despite the fact that 6 of 9 nucleo-tides in the 59 arm of the core sequence, including three of thehighly conserved residues at the third, fourth, and fifth posi-tions from the 59 end, differed from the consensus. Finally, theconsensus sequences for the core regions of the DtxR and Furoperators have only four identical nucleotides, and only two ofthem (A at positions 8 and 14 from the 59 end) represent highlyconserved residues in the DtxR operator. Taken together,these findings suggest that combinations of the highly con-served nucleotides in the 19-bp core region consensus se-quence of DtxR are important for binding of the operator toactivated DtxR, but the contributions of the individual nucle-otides are not yet well defined.

Computer analysis of the sequences contiguous to IRP3,IRP4, and IRP5 identified putative 235 promoter sequenceslocated 59 to the operators, putative 210 promoter sequencesoverlapping the operators, and nearby ORFs located 39 to theoperators (Fig. 5). These findings are consistent with the cur-rent theory that the binding of DtxR to the operator interfereswith the binding of RNA polymerase to the promoter, therebyblocking initiation of transcription by RNA polymerase. Be-sides diphtheria toxin, the only proven products of the DtxRregulon for which functions can presently be inferred are thetranscriptional regulator homolog identified in the presentstudy that is encoded by the gene downstream from IRP3, theputative ferrisiderophore receptor located downstream fromIRP1 that is homologous to FhuD from Bacillus subtilis (42),and the recently described homolog of eukaryotic heme oxy-genases that is encoded by hmuO and is essential for theacquisition of iron from heme and hemoglobin by C. diphthe-riae (37, 38). In addition, the homolog of Salmonella typhi-murium alkyl hydroperoxide reductase encoded by dirA wasshown to be iron repressible in C. diphtheriae, but direct reg-ulation of dirA by DtxR has not yet been established (49).Identification of the transcriptional regulator homolog en-coded by the gene downstream from IRP3 provides the firstindication that regulatory cascades may control some compo-nents of the low-iron and high-iron phenotypes of C. diphthe-riae, but the genes in C. diphtheriae that may be regulated bythis transcriptional regulator homolog have not yet been iden-tified. The proteins encoded by the ORFs downstream fromIRP3 and IRP4 were produced in small amounts in E. coliwhen they were expressed under the control of the strong T7promoter (Fig. 6). Possible reasons why the genes encodingthese proteins were not expressed efficiently in E. coli includeinefficient ribosome-binding sites, suboptimal codon usage,high susceptibility of the encoded proteins to degradation byintracellular proteases, toxicity of the gene products to E. coli,or any combination of the above.

In Mycobacterium smegmatis, insertional inactivation ofideR (a homolog of dtxR) was recently shown to cause a pleio-tropic phenotype characterized by derepression of siderophorebiosynthesis under high-iron conditions, decreased productionof manganese superoxide dismutase and catalase/peroxidase(KatG), and increased susceptibility to killing by hydrogenperoxide (13). It is not yet known whether the DtxR regulonaffects the responses of C. diphtheriae to oxidizing stresses.Additional studies will be required to define more completelythe phenotypes determined by DtxR in C. diphtheriae, the fullset of genes that constitute the DtxR regulon, and possible

virulence factors other than diphtheria toxin that may be reg-ulated by DtxR.

ACKNOWLEDGMENTS

This research was supported in part by Public Health Service grantAI14107 from the National Institute of Allergy and Infectious Dis-eases.

We thank Joanne G. Siekierke for helpful comments concerning themanuscript.

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Editor: J. T. Barbieri

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