Vol. 57, No. 10INFECTION AND IMMUNITY, OCt. 1989, p. 3221-32250019-9567/89/103221-05$02.00/0Copyright © 1989, American Society for Microbiology

NOTES

Evidence for Direct Regulation of Diphtheria Toxin Gene

Transcription by an Fe21-~Dependent DNA-Binding Repressor,

DtoxR, in Corynebacterium diphtheriae

GENEVIEVE FOUREL,lt ARMELLE PHALIPON2t AND MICHEL KACZOREK2§

Unite' d'Immunoge'netique, Instfifuf Nafional de la Sante' ef de la Recherche Scienfifique U276,1and Unite' des Applicafions du Ge'nie Ge'ne'tique, Instfifuf Pasteur,2 Paris, France

Received 27 January 1989/Accepted 6 July 1989

Previous studies provided indirect evidence that in Corynebacterium diphtheriae regulation of diphtheriatoxin gene (tox) transcription by iron is mediated by a bacterial repressor. By performing in vitro protein-DNA

binding experiments, we establish that a corynebacterial Fe ~sensitive protein, named DtoxR, can bind to a

palindromic motif present in the tox promoter region. Binding of this factor prevents the interaction of the

transcription initiation machinery with presumptive critical promoter elements, providing evidence that DtoxR

is responsible for the repression of toxinogenesis observed in iron-containing growth medium.

Infection by temperate phages carrying the structural gene(fox) for diphtheria toxin (DT), including bacteriophage P3, isresponsible for the lysogenic conversion of Corynebacte-rium diphtheriae to toxigenicity (13, 14). Maximal expres-sion of toxin occurs only during the decline phase of thebacterial growth cycle, when iron becomes the rate-limitingsubstrate (19, 27). If excess iron is added to cultures of C.diphtheriae that are producing DT, synthesis of toxin-spe-cific mRNA is inhibited and the rate of synthesis of DTrapidly declines to undetectable levels (16). Regulation of toxgene expression via an iron-dependent repressor was firstproposed by Murphy and co-workers (17), who observedthat corynebacterial extracts inhibited fox gene expression inan in vitro Escherichia coli protein-synthesizing system.Genetic evidence for the key role of a bacterial factor hasbeen further provided by the isolation of corynebacterialmutants which constitutively produce high levels ofDT uponphage P lysogenization (11). Moreover, two cis-dominantmutations of phage p (18, 28) responsible for a fox operatorconstitutivelike phenotype very likely map at the targetbinding site for the repressor. Finally, the similarity of thissystem to negatively regulated E. coli operons allowed theproposal of a palindromic sequence which overlaps the foxtranscription initiation site as a possible operator site (9).Here we present evidence that in C. diphtheriae toxin

expression is directly regulated by the iron-dependent bind-ing of a bacterial factor to the fox promoter region. To detectthis transcriptional factor and to define its DNA targetsequence, interaction between protein extracts from C.

* Corresponding author.t Present address: Unit6 de Recombinaison et Expression Gdn-

dtique, Institut National de la Sante et de la Recherche ScientifiqueU163, Centre National de la Recherche Scientifique UA271, Paris,France.

t Present address: Unitd des Ent,6robactdries, Institut Pasteur,75724 Paris Cedex 15, France.

§ Present address: Pasteur Vaccins, BP101, 27101 Val de ReuilCedex, France.

diphtheriae cells, grown either in low-iron or in high-ironmedium, and fox promoter sequences was analyzed in vitro.

Preparation of protein extracts was adapted from Gorskiet al. (7). Briefly, lysogenic C7 (p tox-228) bacteria, grown asdescribed (10), were lysed in buffer I (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.6],100 mM KCI, 3 MM MgCl2, 0.1 mM EDTA, and 10% glycerol,with 1 mM dithiothreitol-0.1 mM phenylmethylsulfonyl fluo-ride-i jig of aprotinin per ml freshly added). The lysate wasadjusted to 0.36 M (NH4)2S04 (9% saturation) to destabilize

V25-120

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FIG. 1. Schematic representation of tox promoter region andprobes used for protein-DNA binding analyses. Top line, tox pro-moter region cloned into the SmnaI site of pEX+ and adjacentpolylinker sequences. Numbers refer to nucleotide position relativeto the first coding residue, + 1 (8). The putative -35 and -10 E. colipromoter consensus boxes are indicated by the open and stippledcircles; the position of the putative Shine-Dalgarno sequence isshown by a square, and the transcription initiation site is indicatedby a curved arrow. Open arrows illustrate the position of the27-base-pair interrupted palindromic motif. The Apal restrictionfragment contains the first 17 amino acids of the DT signal peptide(5). Hatched area represents polylinker sequences from the pEX+plasmid. Lower lines, Probes used for protein-DNA binding analy-ses. The 12P-labeled 3' end is indicated by a star. Top strand labeledprobe I was generated by Nhel digestion of the double-labeledHindlll fragment extending from -121 to the Hindlll polylinkersite. The bottom strand was labeled at the NheI site, followed bydigestion with HindIII to generate probe II, or at the EcoRI polylinkersite, followed by digestion with BamnHl to generate probe III.

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3222 NOTES

0D 0

dIdC NaCI

0 1 2 3 4 5 6 7 8 9 10

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FIG. 2. Detection of specific tox promoter-protein complex usingC. diphtheriae extracts in gel mobility shift assay. Lanes 1 through 9demonstrate the effects of both nonspecific DNA and salt concentra-tion on complex formation, using protein extract from iron-starved C.diphtheriae cells (active tox promoter, indicated by a plus sign).Probe I was incubated with 7 p.g of extract in the presence of 50 mMNaCl plus 1, 2, 3, 4.5, or 6 ,ug of poly(dI-dC) in lanes 1, 2, 3, 4, and5 respectively, or with 2 ,ug of poly(dI-dC) plus 75, 100, 150, or 200mM NaCl in lanes 6, 7, 8, and 9, respectively. Binding reactions wereperformed for 30 min at room temperature in a volume of 15 ,ul andcontained 20 mM phosphate buffer (pH 6.0), 5 mM MgCl2, 2 mMdithiothreitol, 0.01% Nonidet P-40, and 10% glycerol in addition toprotein extract and probe. Reaction mixes were electrophoresedthrough a 6% polyacrylamide gel under nondenaturing conditions toresolve free (F) and bound (Cl and C2) DNA. Lane 10 was as for lane2 except that the protein extract was 5 ,ug from C. diphtheriae cellsgrown in the presence of iron (inactive tox promoter, indicated by a

minus sign). Lane 0, Control incubation mix without added extract.

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DNA-protein complexes and was then centrifuged. Proteinsfrom the supernatant fluid were precipitated by the addition of0.3 g of (NH4)2SO4 per ml (48.5% saturation), dissolved in aminimal amount of buffer 11 (25 mM HEPES [pH 7.6], 40 mMKCl, and 10% glycerol with 1 mM dithiothreitol-0.1 mMphenylmethylsulfonyl fluoride-1 ,ug of aprotinin per mlfreshly added), usually 0.2 ml/g of bacteria, and dialyzedagainst the same buffer. The resulting protein extract had aconcentration ranging from 5 to 8 j,g/,u; it was frozen andstored at -70°C.An ApaI fragment, extending from nucleotides -256 to

+49 relative to the tox structural gene (Fig. 1), was purifiedfrom plasmid pTD134 (10), blunt ended, and subcloned intothe SmaI site of pEX+ (Bluescribe M13+, Genofit). 3'-End-labeled probes were generated, taking advantage of theEcoRI, BamHI, and Hindlll sites present in the polylinkersequence (Fig. 1). This ApaI fragment contains putativepromoter elements functional in C. diphtheriae as well as inE. coli (10, 12), including -10 and -35 homology boxes andthe palindromic sequence proposed as an operator site.

Specific DNA-protein interactions were analyzed by gelelectrophoretic mobility shift (5), and the assays were per-formed as described (8).Probe I (nucleotides -114 to +49) incubated with crude

bacterial extracts yielded a single specific nucleoproteincomplex, Cl (Fig. 2), with extracts from a DT-producing(lanes 2 through 9) or nonproducing (lane 10) culture. A

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CFIG. 3. Footprint analyses of the tox promoter sequences with C. diphtheriae extracts. Direct DNase I protection (A and B) and

methylation interference and indirect DNase I protection (C) analyses of binding to the tox promoter region were performed on either thebottom strand (panel A, probe III) or top strand (panels B and C, probe I). Autoradiographs of 7 M urea-6% polyacrylamide gel. Legend forthe map shown on the left side of each panel and for the numbers is similar to that of Fig. 1. (A and B) Binding reactions for in vitro directDNase I protection analyses were carried out in 30 ,ul of a buffer having the following final composition: 12.5 mM HEPES (pH 7.6), 40 mM

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bottom strand

2- 2- 2-Fe F Fe+ + +

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FIG. 4. Fe2+-inducible binding of a corynebacterial factor (DtoxR) to tox promoter sequences. Top, DNase I footprinting analysis in thepresence of Fe2+. The conditions were as described for Fig. 3A and B. In addition, 500 ,uM Fe(SO4)2 was included in the binding reactionwhen indicated (+ Fe2+), and probe II was used for analysis of the bottom strand. Bottom, Diagrammatic summary of tox promoter sequenceprotection by C. diphtheriae extracts in the presence of Fe21 (F.rep.). Activated DtoxR binding fully prevents DNase I digestion ofdark-stippled sequences and enhances it at residues indicated by a vertical arrow. The significance (see Fig. 3 legend) and Fe2' dependenceof DNase I digestion pattern modifications observed in the light-stippled area are unclear.

KCI, 0.5 mM dithiothreitol, and 5% glycerol. Each reaction contained 5 x 104 cpm (10-2 pmol) ofDNA probe incubated with 45 ,ug of proteinextract either from DT-expressing cells (plus sign, lane 3) or from DT-nonexpressing cells (minus sign, lane 4), or without protein (lane 2 and5) in the presence of 500 ng of poly(dI-dC) at 0°C for 1 h, followed by treatment with DNase I. Lane 1 is a C+T Maxam and Gilbert sequenceladder of the probe (G residues also weakly reacted in panel B, lane 1). Results are summarized on the right side of each panel. Increasingprotein extract amount did not strengthen the protection pattern presented here. Hence, brackets indicate areas where visualization of proteinbinding could readily be assumed as both the observed partial protections and the accompanying sites of enhanced DNase I cleavage(horizontal arrows) were highly reproducible. In contrast, dashed lines denote DNA regions where the very slight observed modifications ofthe DNase I digestion pattern could be due either to protein binding to this sequence or to distortion of DNA structure induced by theabove-mentioned protein binding to neighboring sequences. (C) Preparative-scale binding reaction, as described for Fig. 1, lane 7, was eitherperformed using a G-methylated probe I (lanes 2 and 3, methylation interference analyses) or submitted to DNase I treatment before loadingon the retention gel (indirect DNase I protection analyses, lanes 4 and 5). DNA samples eluted from C2 (lanes 2 and 4) and Cl (lanes 3 and5) were then analyzed (after piperidine treatment for methylation interference analyses). Lane 1 is a G sequence ladder of the probe. Opentriangles indicate G residues underrepresented in the methylation interference study. On the right side, bracket, arrows, and dashed linessummarize Cl complex DNA protection results (F.exp.).

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faster-migrating complex, C2, was also detected with amobility depending on both the concentration of nonspecificDNA (Fig. 2, lanes 1 through 5) and the ionic strength (lanes6 through 9). Since C2 was not detected under more stringentconditions (25) (data not shown), it is likely that C2 resultedfrom a nonspecific DNA-protein interaction.To delineate precisely the site of interaction correspond-

ing to the Cl complex, a preparative-scale binding reactionwas submitted to limited DNase I treatment before separa-tion on a retention gel (indirect DNase I footprinting).Analysis of the digestion products on a sequencing gel didnot reveal any major modification of the digestion patterneither when the probe was eluted from the C2 complex (Fig.3C, lane 4) or was separately processed without bacterialextract (Fig. 3B, lane 5). In contrast, interaction of protein(s)with promoter sequences within the Cl complex resulted ina broad region of protection from DNase I digestion, extend-ing from nucleotides -84 to -5 on the top strand, which isinterrupted by a single sensitive site at -23 (Fig. 3C, lane 5).We will refer to this footprint hereafter as "F.exp." for DTexpression-related footprint. Methylation interference ex-periments (22) further showed that binding of the protein(s)involved in the Cl complex requires essential contacts onthe top strand with -16, -15, -10, and -8 G residues (Fig.3C, lane 3).These techniques did not show any functional difference

between extracts from toxin-expressing or nonexpressingcells. At this point no in vivo function for the Cl complexcan be proposed. We then examined the tox promoter bydirect DNase I footprinting (6), and the assays were per-formed as described (13). A similar protection pattern of theprobe as for the Cl complex, although weaker, was inducedby extracts from cells grown under iron starvation (Fig. 3B,lane 3, and Fig. 3A, lane 3). In contrast, F.exp. was notobtained with extracts from cells grown in iron-containingmedium (Fig. 3B, lane 4). Additionally, the strand asymme-try, the extent, and the localization of F.exp. were reminis-cent ofRNA polymerase footprints on E. coli promoters (21,23, 24). This strongly suggests that F.exp. (and Cl) couldreveal the interaction between a corynebacterial RNA poly-merase (and auxiliary factors) and tox promoter sequences.On the other hand, in the presence of extracts from cells

grown in iron-containing medium, very weak protectionencompassing the previously mentioned palindromic se-quence was observed. This protection could be more clearlyseen on the bottom strand (Fig. 3A, lane 4) than on the topstrand (Fig. 3B, lane 4) and was also visible as changes in theintensity order of the bands corresponding to nicks atnucleotides -49 to -45 on the bottom strand. Moreover, thepresence of the complex revealed by this later footprint(called "F.rep." for DT repression-related footprint) couldbe increased by the addition of ferrous ions to the bindingreaction (Fig. 4A, lanes 5 and 7). In contrast, adding ferricions did not have any effect (data not shown). Hence, aFe2+-activated protein present within DT-nonexpressingcells is able to displace the transcription initiation machineryfrom the tox promoter. We named this factor "DtoxR" forDT repressor. Additionally, this strong Fe2+-inducible pro-tection could be obtained with extracts from cells which didor did not express the tox gene (Fig. 4A, lanes 3 and 8) atsimilar threshold amounts of extract (data not shown).Assuming that the repressor is identically extracted underthe various Fe2+ growth conditions we employed, thisobservation demonstrates that DtoxR is constitutively ex-pressed and able to repress toxin production. The repressioncan be reversed by decreasing the intracellular ferrous ion

concentration. The DtoxR protein showed all of the ex-pected properties of the aporepressor hypothesized by Mur-phy and Bacha (15). In experiments that contained 1 nM ofthe palindromic motif (0.1 ng/4dl of probe) and 1.3 ,ug ofbacterial crude extract per ,ul, we estimated that Fe(SO4)2concentrations as low as 50 ,uM allow in vitro saturation ofthe DtoxR binding site. However, the high redox instabilityof ferric ions hindered precise determination of the effectiveFe2+ concentration in the binding mix. This instability mightalso explain the inability of the gel mobility shift assay todetect this Fe2+-dependent interaction.Together these results demonstrate that C. diphtheriae

constitutively expresses an aporepressor, DtoxR, able tocompete in an Fe2+-dependent fashion with the transcriptioninitiation machinery for binding to the tox promoter region.Thus, DtoxR could directly regulate tox gene expression.The region protected by DtoxR against DNase I digestion isdiagrammed in Fig. 4B. Although the intensity of the ob-tained footprint varies with the amount of extract and Fe2+concentration used, F.rep. closely fits the 27-base-pair inter-rupted palindromic element, which suggests, as in the caseof Fur protein (4), that DtoxR could interact with DNA as adimer.

Iron regulation of the tox promoter in E. coli was recentlyshown to be dependent on the fur gene product (26). Inter-estingly, the expression of the Shiga-like toxin produced bysome isolates of E. coli, lysogenized by a temperate bacte-riophage carrying the structural gene for this toxin, was alsoshown to be iron regulated via the fur gene product (3).Significant homology between the tox palindromic motif andthe consensus sequence for the Fur binding site stronglysuggests that the regulation could involve binding of the Furprotein to the same sequences as DtoxR. In C. diphtheriae,DtoxR could play a role similar to that of Fur in E. coli andmight in the same way regulate the transcription of a wholeregulon (1, 2) including genes for the high-affinity irontransport system in C. diphtheriae (20). Moreover, beyond afunctional similarity between the two proteins, conservationof their target binding sites hints that regulatory systemsinvolved in sensing specific environmental signals werehighly conserved during evolution. Purification ofDtoxR hasnow been undertaken to clone its structural gene and com-pare its peptide sequence with that of the Fur protein.

We are grateful to Tommaso Meo for support at the initial phaseof this work. We thank Isabelle Saint-Girons for many constructivecomments, and Pascale Cossart, Marie-Annick Buendia, and KarlReich for critical reading of the manuscript.

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4. de Lorenzo, V., S. Wee, M. Herrero, and J. B. Neilands. 1987.Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulator (fur) repressor. J.Bacteriol. 169:2624-2630.

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6. Galas, D., and A. Schmitz. 1978. DNase footprinting: a simplemethod for the detection of protein DNA binding specificity.

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12. Leong, D., and J. R. Murphy. 1985. Characterization of thediphtheria tox transcript in Corynebacterium diphtheriae andEscherichia coli. J. Bacteriol. 163:1114-1119.

13. Lichtsteiner, S., J. Wuarin, and U. Schibler. 1987. The interplayof DNA-binding proteins on the promoter of the mouse albumingene. Cell 51:%3-973.

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16. Murphy, J. R., J. L. Michel, and M. Teng. 1978. Evidence thatthe regulation of diphtheria toxin production is directed at thelevel of transcription. J. Bacteriol. 135:511-516.

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Borms. 1974. Synthesis of diphtheria tox gene products inEscherichia coli extract. Proc. Natl. Acad. Sci. USA 71:11-15.

18. Murphy, J. R., J. Skiver, and G. McBride. 1976. Isolation andpartial characterization of a corynebacteriophage 1, tox opera-tor constitutive-like mutant lysogene of Corynebacterium diph-theriae. J. Virol. 18:235-244.

19. Pappenheimer, A. M., Jr. 1977. Diphtheria toxin. Annu. Rev.Biochem. 46:69-94.

20. Russel, L. M., S. J. Cryz, and R. K. Holmes. 1984. Genetic andbiochemical evidence for a siderophore-dependent iron trans-port system in Corynebacterium diphtheriae. Infect. Immun.45:143-149.

21. Schmitz, A., and D. J. Galas. 1979. The interaction of RNApolymerase and lac repressor with the lac control region.Nucleic Acids Res. 6:111-137.

22. Siebenlist, U., and W. Gilbert. 1980. Contacts between Esche-richia coli RNA polymerase and early promoter of phage T7.Proc. Natl. Acad. Sci. USA 77:122-126.

23. Siebenlist, U., R. B. Simpson, and W. Gilbert. 1980. E. coli RNApolymerase interacts homologously with two different promot-ers. Cell 20:269-281.

24. Simpson, R. B. 1979. The molecular topography of RNA poly-merase-promoter interaction. Cell 18:277-285.

25. Singh, H., R. Sen, D. Baltimore, and P. A. Sharp. 1986. Anuclear factor that binds to a conserved sequence motif intranscriptional control elements of immunoglobulin gene. Na-ture (London) 319:154-157.

26. Tai, S. S., and R. K. Holmes. 1988. Iron regulation of the cloneddiphtheria toxin promoter in Escherichia coli. Infect. Immun.56:2430-2436.

27. Uchida, T. 1982. Diphtheria toxin: biological activity, p. 1-31. InP. Cohen and S. Van Heyningen (ed.), Molecular action oftoxins and viruses. Elsevier Biomedical Press, New York.

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