Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7576-7580, August 1992 Microbiology Purification and characterization of the diphtheria repressor MICHAEL P. SCHMITT, EDDA M. TWIDDY, AND RANDALL K. HOLMES* Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4977 Communicated by R. John Collier, May 11, 1992 (received for review January 31, 1992)

ABSTRACT The repressor gene (dExR) region of the tox gene was postulated to be the diphtheria encodes a protein (DtxR) that regulates transcription of the toxin repressor (25), but the factor was not purified. diphtheria toxin gene (tox) by an iron-dependent mechnism. The diphtheria toxin repressor gene dtxR from C. diphthe- Cloned dtR was expressed in Escherichia coil from the phage riae C7 encodes a Mr 25,316 polypeptide that has slight 17 gene 10 promoter, and DtxR was purified. Specific binding homology with the E. coli Fur protein (24). The cloned dtxR of DtxR to the tox+ operator was dependent on reduction of gene represses expression from the wild-type tox promoter in DtxR and the presence of ferrous ions. DtxR protected a E. coli by an iron-dependent mechanism (17, 24). It also sequence of =30 nucleotide pairs, partially overlapping the tox restores iron-dependent repression of siderophore and diph- promoter and containing a region of dyad symmetry, from theria toxin synthesis when it is introduced into C. diphthe- digestion by DNase I. DtxR exhibited very little binding to the riae C7((B)hm723 (17), a strain that has a missense mutation mutant tox-201 operator region and failed to bind to the in dtxR and makes a defective DtxR protein with very little promoter/operator region ofthe ferric uptake regulation (fur) repressor activity (26). gene of E. coli. In this study, the cloned dtxR gene was expressed from the phage T7 gene 10 promoter in E. coli, and the DtxR protein was purified. The DtxR protein was shown to have iron- Corynebacterium diphtheriae is the causative agent of diph- dependent, sequence-specific DNA-binding activity for the theria. Diphtheria toxin is a Mr 58,342 secreted protein tox operator. produced by C. diphtheriae that kills susceptible eukaryotic cells by catalyzing ADP ribosylation of elongation factor 2, causing inhibition of protein synthesis (for reviews, see refs. MATERIALS AND METHODS 1 and 2). Diphtheria toxin is encoded by the tox gene, which , Phage, Plasmids, and Production of DtUR. E. coli is found in several corynebacteriophages, including the well- DH5a (Bethesda Research Laboratories) was the host strain characterized phage (3(3-5). Only C. diphtheriae infected by used to produce DtxR protein. Plasmid pMS298 carries dtxR a tox+ makes diphtheria toxin. under T7 gene 10 promoter control (17), and plasmid pGP1-2 Expression of tox in C. diphtheriae is regulated by iron, is temperature inducible for T7 RNA polymerase (27). Cory- with maximal toxin synthesis occurring in low-iron environ- nephage ,pox-201 was maintained and propagated as described ments (1, 6). Other bacterial repressed by iron include (28). The tox-201 promoter/operator mutation increases tox Shiga toxin from dysenteriae type I (7), Shiga-like promoter strength and also causes resistance to inhibition by toxin I from (8), and A from iron of diphtheria toxin production (14). Pseudomonas aeruginosa (9). Iron-uptake systems in many An extract containing DtxR was obtained by the following bacterial are regulated by iron-dependent mecha- procedure: E. coli DH5a carrying pMS298 and pGP1-2 was nisms. In E. coli, the ferric uptake regulation gene (fur) grown overnight with aeration at 300C in rich medium [2% encodes an iron-dependent repressor that controls a regulon tryptone/1% yeast extract/0.5% NaCl/0.2% glycerol/50 mM involving at least 30 genes (10, 11). K2HPO4, pH 7.2/ampicillin and kanamycin (50 Hg/ml)]. An iron-dependent diphtheria toxin repressor encoded by Samples (80 ml) were inoculated into fresh medium (800 ml) the of C. diphtheriae was postulated almost 2 in six flasks (4 liters) and incubated until A590 reached 2.0. decades ago (5). Subsequently, mutants ofC. diphtheriae that Prewarmed medium (600 ml) was added to each flask to fail to repress toxin production in high-iron medium (12, 13) increase the temperature rapidly to 420C. Cultures were and tox mutants of phage (B that are insensitive to iron- incubated at 420C for 30 min and then shifted to 3TC for an dependent repression (14, 15) were isolated, and corynebac- additional 90 min. Bacteria [36 g (wet weight)] were harvested terial siderophore was shown to be coordinately regulated by centrifugation at 10,000 x g for 10 min, and subsequent with diphtheria toxin (16, 17). procedures were performed at 40C. Bacteria were resus- The tox gene of C. diphtheriae and its promoter/operator pended in 160 ml of buffer 1 (10 mM Tris, pH 7.2/0.1 mM region have been cloned and sequenced (18-20). The tox EDTA/0.02% NaN3) containing phenylmethylsulfonyl fluo- promoter contains -10 and -35 transcription initiation se- ride (50 zg/ml) and disrupted by sonication. Insoluble debris quences similar to E. coli o,70 promoters (21), and identical was removed by centrifugation at 25,000 x g for 45 min. transcriptional start sites were identified for expression oftox Cloning of the tox-201 Allele. A 230-base-pair (bp) DNA in C. diphtheriae and E. coli (22). An A+T-rich DNA fragment carrying the tox-201 promoter/operator region was sequence containing a region ofdyad symmetry that overlaps obtained by PCR amplification of DNA from phage ftox-201 the -10 sequence of the tox promoter was proposed as a using oligonucleotide primers (DT-1 and DT-2) (29). PCR recognition site for regulation of tox (19, 23). In E. coli, the procedures were done as described (30) and with a DNA fur gene product caused little (23) or no (24) iron-dependent thermal cycler (Perkins-Elmer/Cetus). The 230-bp fragment regulation of reporter genes expressed under control of the contains a unique HindIII site upstream from the tox pro- tox promoter. A factor in crude extracts ofC. diphtheriae that moter and a unique Hae III site downstream from the bound in an iron-dependent manner to the promoter/operator promoter. The 175-bp HindIII/Hae III fragment was excised and ligated into the HindIII/Sma I site of pBluescript KS to The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: DTT, dithiothreitol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7576 Downloaded by guest on September 28, 2021 Microbiology: Schmitt et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7577 construct plasmid pPO201. The nucleotide sequence of the the samples were treated with 2 jud of DNase I (Bethesda 175-bp HindIII/Sma I fragment in pPO201 was determined Research Laboratories) at 10 pg/ml for 30 sec at room and shown to be identical with the reported sequence of temperature. Reactions were terminated by extracting with tox-201 (29). Plasmid pPO201 was the source of DNA for the phenol (buffered with Tris at pH 8.0), and then DNA was promoter/operator fragment used in the gel-shift and DNase precipitated with ethanol and dried. Samples were resus- I protection assays. pended in formamide buffer containing tracking dye, and Purification of a MalE-DtxR Fusion Protein, Preparation of electrophoresis was performed through a 6% denaturing Antiserum, and SDS/PAGE and Western Blot Analysis. A polyacrylamide gel. After electrophoresis, the gel was dried gene fusion between malE and dtxR was constructed in and exposed to x-ray film. To determine precisely the DNA pMAL-c, which has a multiple cloning site at the 3' end ofthe sequences that were protected from DNase I digestion by malE coding region (New England Biolabs). The complete DtxR, Maxam and Gilbert G+A reactions (35) were also dtxR gene in pMS298 was amplified by PCR using primers performed on the 158-bp HindIII/Hha I fragment carrying 5'-ATGAAGGACTTAGTCGATACCACAG-3' and 5'- the wild-type tox promoter/operator region. In addition, a CTACGAATTCGGGCGCACCGGTAAC-3'. The amplified synthetic oligonucleotide that contained at its 5' terminus the DNA was treated with EcoRI, which cuts at the underlined same nucleotide labeled with 32p in the 158-bp HindIII/Hha GAATTC sequence in -the second primer, and dtxR was I fragment was used as a primer to generate a sequencing cloned into Stu I/EcoRI-cut pMAL-c to create an in-frame ladder by using the dideoxynucleotide chain-termination malE-dtxR gene fusion. Production of MalE-DtxR was in- method (36). duced in a mid-logarithmic culture of E. coli DH5a by isopropyl 3-D-thiogalactopyranoside (0.3 mM) for 2 hr at 370C, and the fusion protein was purified from bacterial sonic RESULTS extracts by absorption onto amylose resin and elution into Purification of To DtxR for the buffer containing 10 mM maltose according to methods DtxR. produce purification, provided with the pMAL-c plasmid (New England Biolabs). cloned dtxR gene of pMS298 was expressed in E. coli DH5a The purified MalE-DtxR fusion protein, which migrated as a from the phage T7 gene 10 promoter, after thermal induction single band in SDS/PAGE (data not shown), was used to of T7 RNA polymerase encoded by pGP1-2. Radiolabeled immunize rabbits according to published procedures (31). DtxR was prepared by uniquely expressing the dtxR gene of Preparation of samples for SDS/PAGE, electrophoresis con- pMS298 under T7 promoter control in E. coli DH5a in the ditions, and transfer of proteins from SDS gels to nitrocel- presence of T7 RNA polymerase, rifampicin, and [35S]me- lulose for Western blot analysis has been described (32). thionine (17). A small amount of 35S-labeled DtxR was added Radiolabeling Ends of DNA Fragments. The Klenow frag- to unlabeled extract to serve as a radioactive tracer during ment of DNA polymerase I was used to label the 3' termini purification of DtxR. of DNA fragments (30). A 210-bp HindIII fragment, which extract containing DtxR was fractionated with ammo- carries the wild-type tox promoter/operator region, was nium sulfate. The sample that precipitated between 20%o and excised from pPOB (17), end-labeled, digested with Hha I, 55% of saturation contained most ofthe 35S-labeled DtxR and and separated by electrophoresis on a 2% agarose gel to was dialyzed against buffer 1. A small precipitate was re- isolate a 158-bp HindIII/Hha I fragment labeled at the moved by centrifugation at 100,000 x g for 30 min, and the HindIII site. The analogous 158-bp labeled HindIII/Hha I supernatant was subjected to chromatography on DEAE- fragment carrying the tox-201 allele was isolated from cellulose (Fig. 1A). Samples containing 35S-labeled DtxR pPO201. The 158-bp HindIII/Hha I fragments were used in were pooled, dialyzed, and fractionated by chromatography both the gel mobility-shift assays and the DNase I protection on heparin-Sepharose (Fig. 1B). Fractions containing 35S- experiments. To analyze DtxR binding to the complementary labeled DtxR were combined and subjected to zinc iminodi- strand of the wild-type tox operator, a 325-bp EcoRI/Xba I acetate-agarose chromatography (37) (Fig. 2). fragment from pPOB was uniquely labeled at its Xba I end. Samples of the crude bacterial extracts and the purified An -200-bp Ssp I/Dra I fragment carrying the E. coli fur DtxR were analyzed by SDS/PAGE (Fig. 3A). In the pres- operator region (33) was labeled at both ends with the Klenow ence of 2-mercaptoethanol the major polypeptide in the fragment of DNA polymerase I. purified DtxR migrated at Mr 28,000 (lane 6) and comi- Gel-Shift Assays. Binding of DtxR to 32P-end-labeled DNA grated with 35S-labeled DtxR (data not shown), consistent fragments was performed by a PAGE mobility-shift assay with the size predicted (Mr 25,316) from the dtxR sequence using a modification of a published method (34). End-labeled (24) and with published data concerning the electrophoretic DNA fragments at :0.1 nM were incubated with various mobility of 35S-labeled DtxR (17). In the absence of 2-mer- concentrations of DtxR in 10-,ul reaction volumes in buffer captoethanol, the major polypeptide migrated at Mr 56,000 containing 20 mM Na2HPO4 (pH 7.0), 50 mM NaCl, 2 mM (lane 3), corresponding to a dimer consisting of two DtxR dithiothreitol (DTT), bovine serum albumin (100 ,g/ml), 5 polypeptides linked by a disulfide bond. The purified DtxR mM MgCl2, 10 ,g of sonicated salmon sperm DNA per ml, contained small amounts of polypeptides Mr < 56,000 in the and 10% (vol/vol) glycerol. Freshly prepared ferrous sulfate absence of 2-mercaptoethanol and Mr < 28,000 in the pres- or salts of other divalent cations were added as indicated. ence of 2-mercaptoethanol. Separation of DtxR from these Reaction mixtures were incubated for 10 min at room tem- smaller polypeptides was not accomplished by chromatofo- perature and then immediately loaded (without tracking dye) cusing, gel filtration on Biogel P-100, or chromatography on onto a 5% nondenaturing polyacrylamide gel that contained octyl Sepharose (data not shown). All of the smaller poly- 20 mM Na2HPO4 (pH 7.0) and 1 mM DTT. Electrophoresis peptides in purified DtxR were antigenically related to DtxR was performed in 20 mM Na2HPO4 (pH 7.0) and 1 mM DTT (Fig. 3B, lanes 3 and 6). DtxR in E. coli DH5a (pMS298, at 75 V for 1 hr using the modular minielectrophoresis system pGP1-2) extracts migrated as a Mr 28,000 monomer even in from Bio-Rad. After electrophoresis, the gel was dried and the absence of2-mercaptoethanol (lanes 2 and 5). Extracts of analyzed by autoradiography. E. coli DH5a carrying only the pBluescript KS vector did not DNase I Protection Experiments. Labeled DNA fragments, contain immunoreactive DtxR (lanes 1 and 4). We conclude at -0.5 nM, carrying either tox+ or tox-201, were incubated that purified DtxR was slightly degraded by proteolysis and with various concentrations of purified DtxR. Incubation was oxidized to the disulfide-linked dimeric form during conditions were identical to the gel mobility-shift assays, purification, but it contained no demonstrable contaminating except that reaction volumes were 50 A.l. After incubation, proteins. Downloaded by guest on September 28, 2021 7578 Microbiology: Schmitt et A Proc. Natl. Acad. Sci. USA 89 (1992)

A 2.00 25 £ E E E c i 0 20 W co0 E Go 1.50 CM 04 z 5 0 0 0 I- 15 0 0 _CO co z I cM 0 1.00 0.-, w 10 z z 0 E LU x Cm 0.50 cc c: 0 x 0) 0c C/) 0 2 CD)ax cn (L 0.0c 50 100 150 200 250 0 20 40 60 80 1C )o FRACTION NUMBER FRACTION NUMBER

B FIG. 2. Chromatography of DtxR on zinc-iminodiacetate aga- rose. An iminodiacetate agarose column (0.9 x 14 cm) was washed E 3 400 with 900 ml of20 mM ZnSO4 and then with 200 ml of20 mM Tris HCl E buffer at pH 8.0. A 15-ml sample from the heparin-Sepharose pool CM0 was dialyzed against 20 mM TrisHCI buffer at pH 8.0 and loaded 300 53 onto the column. Adsorbed proteins were eluted with a 0-25 mM 0 N0 2 z gradient of DL-histidine in the same buffer starting at the arrow. co Samples collected were 8.7 ml (fractions 1-24) and 0.95 ml (fractions 0 25-100). Fractions 54-58 from the first peak of eluted proteins, TCD 200 E m E containing -100 jig ofpurifiedDtxR, were pooled, concentrated, and z used for subsequent experiments. A, A280; o, A260; e, cpm X 10-1 per

x ml; *, DL-histidine (mM).

B wild type tox-201 fur 1 2 3 4 5 6 7 8 9 10 11

FIG. 4. (A) Endonuclease restriction map and genetic organiza- tion of the tox+ promoter/operator region of plasmid pPOB. Loca- tion of the tox promoter is indicated by -10 and -35 sequences; inverted arrows show a region of dyad symmetry; hatched line represents 5' portion of the tox gene coding region; thick line represents pUC19 vector sequences. DNA fragments I and H shown below the restriction map were radiolabeled at a 3' terminus (aster- isk) and were used in the gel-shift and DNase I protection assays. E, EcoRI; H, HindIlI; Ha, Hae III; Hh, Hha I; S, Sma I; X, Xba I. (B) PAGE mobility-shift assay. Various concentrations ofpurified DtxR, in either the presence (+Fe) or absence (-Fe) of 500 AM FeSO4, were tested for binding to -0.1 nM 32P-labeled DNA fragments carrying promoter/operator regions from the tox+ gene (158-bp HindIII/Hha I; lanes 1-6), tox-201 allele (158-bp HindIII/Hha I; -35 -10 ATAATTAGGATAGCTTTACCTAATTATTTTATGA -3 lanes 7-9), andfur gene (200-bp Ssp I/Dra I; lanes 10 and 11). Lanes: 5'- CATTGATTTCAGAGCACCCT 1, 0 DtxR (+Fe); 2, 500 ng of DtxR (+Fe); 3, 200 ng of DtxR (+Fe); 3'- GTAACTAAAGTCTCGTGG AATATTAATCCTATCGAAATGGATTAA AAAATACT -5' 4, 100 ng of DtxR (+Fe); 5, 100 ng of DtxR (-Fe); 6, 500 ng of DtxR (-Fe); 7, 0 DtxR (+Fe); 8, 500 ng of DtxR (+Fe); 9, 500 ng of DtxR (-Fe); 10, 0 DtxR (+Fe); 11, 500 ng of DtxR (+Fe). FIG. 5. (A) DNase I protection assays. Various concentrations of purified DtxR, in either the presence or absence of 500 AM FeSO4, in reaction mixtures containing Fe2+, Ni2+, or Co2+ (data not were examined for the ability to protect from DNase I digestion shown). Binding of DtxR to the DNA fragment with the 32P-labeled 158-bp HindIII/Hha I fragments (see Fig. 4A, fragment or tox-201 (lanes 6-9) tox-201 allele was very limited and was detected only at the I) containing either the tox+ (lanes 1-5) the promoter/operator sequence. DNA concentration was -0.5 nM. highest concentration of DtxR and in the presence of Fe2+ Lanes: 1, 2.5 Ag of DtxR (+Fe); 2, 1.0 pg of DtxR (+Fe); 3, 500 ng (lanes 7-9). DtxR showed no detectable iron-dependent bind- of DtxR (+Fe); 4, 1.0 tug of DtxR (-Fe); 5, 0 DtxR (+Fe); 6, 2.5 Pg ing to the DNA fragment with the fur promoter/operator of DtxR (+Fe); 7, 1.0 ,ug of DtxR (+Fe); 8, 1.0 tug of DtxR (-Fe); region of E. coli (lanes 10 and 11). Gel mobility-shift assays 9, 0 DtxR (+Fe). Maxam-Gilbert G+A reactions and dideoxynu- were not effective for measuring DtxR activity in crude cleotide C, T, A, and G reactions are also shown. (B) DNase I extracts ofE. coli DH5a (pMS298, pGP1-2), because DNase protection assay with a 325-bp EcoRI/Xba I tox+ promoter/operator in the extracts degraded the radiolabeled DNA fragments. fragment (see Fig. 4A, fragment II). Lanes: 1, 1.0 Zg of DtxR (+Fe); 2, 0 DtxR (+Fe). Shown below sequencing gel is the nucleotide Localization of DtxR Binding to the tox Operator/Promoter sequence of the tox promoter/operator region. Boxed sequences Region. Purified DtxR interacted with a 158-bp HindIII/Hha indicate regions on each strand of the tox promoter/operator regions I DNA fragment and a 325-bp EcoRI/Xba I DNA fragment protected by DtxR from DNase I digestion; arrows show region of labeled with 32p on opposite strands and protected an =30-bp dyad symmetry; asterisk indicates nucleotide pair mutated from G-C sequence of the tox+ operator on each strand from digestion in the tox+ sequence to A-T in the tox-201 mutant. by DNase I (Fig. 5). This protection occurred in the presence of Fe2+ (500 or 150 ,uM) but not in the absence of Fe2+. from the tox promoter. Crude extracts of C. diphtheriae Identical results were obtained when Co2+ or Ni2+ at 150 ,uM contained a factor, tentatively identified as DtxR, that binds was substituted for Fe2+ (data not shown). The protected to a specific DNA sequence upstream of the tox gene in an sequence contained the region of dyad symmetry that over- iron-dependent interaction (25). In this study, highly purified, laps the -10 sequence of the tox promoter. DtxR exhibited recombinant DtxR from E. coli was shown to bind to DNA only a weak, iron-dependent ability to protect the corre- fragments carrying the tox+ operator; DNase I protection sponding sequence of the tox-201 promoter/operator region. experiments demonstrated that purified DtxR in the presence The tox-201 allele has a single transition mutation that ofFe2+, Co2+, or Ni2+ protects an -30-bp sequence ofthe tox partially disrupts the dyad symmetry of the tox promoter/ operator. The protected region, including the -10 sequence operator region by changing the last G-C pair in the left of the tox promoter and the region of dyad symmetry previ- inverted repeat to an A-T pair (ref. 29; see Fig. 5). ously postulated to be involved in regulation of the tox gene, was nearly identical to the sequence protected by the factor from crude extracts of C. diphtheriae described above (25). DISCUSSION DtxR bound very weakly to the tox-201 promoter/operator Studies in C. diphtheriae and E. coli (17, 24) demonstrated sequence. These findings confirmed and extended previous that DtxR causes iron-dependent repression of transcription analysis of the tox regulatory region and demonstrated that Downloaded by guest on September 28, 2021 7580 Microbiology: Schmitt et al. Proc. Natl. Acad. Sci. USA 89 (1992) purified DtxR binds to the tox operator in a manner that is 7. Van Heyningen, W. E. & Gladstone, G. P. (1953) Br. J. Exp. both iron dependent and sequence specific. Pathol. 34, 221-229. Although DtxR was purified as a disulfide-linked dimer, 8. Calderwood, C. B. & Mekalanos, J. J. (1987) J. Bacteriol. 169, reduction of the disulfide bond was required for its iron- 4759-4764. dependent, sequence-specific DNA binding activity. This 9. Bjorn, M. J., Iglewski, B. H., Ives, S. K., Sadoff, J. C. & wag not surprising, since DtxR is a cytoplasmic protein that Vasil, M. L. (1978) Infect. Immun. 19, 785-791. 10. Bagg, A. & Neilands, J. B. (1987) Microbiol. Rev. 51, 509-518. normally functions in an intracellular environment with low 11. Braun, V. (1985) Trends Biochem. Sci. 10, 76-77. redox potential. By analogy with several other repressor 12. Cryz, S. J., Jr., Russell, L. M. & Holmes, R. K. (1983) J. proteins and their interactions with palindromic sequences in Bacteriol. 154, 245-252. operators (38), DtxR is presumed to function as a dimer; 13. Kanei, C., Uchida, T. & Yoneda, M. (1977) Infect. Immun. 18, however, direct evidence that functional DtxR is dimeric is 203-209. not yet available. 14. Welkos, S. L. & Holmes, R. K. (1981) J. Virol. 37, 936-945. DtxR is believed to function as an iron-dependent repres- 15. Murphy, J. R., Skiver, J. & McBride, G. (1976) J. Virol. 18, sor by binding to the tox operator site, thereby either pre- 235-244. venting RNA polymerase from binding or displacing it from 16. Tai, S.-P. S., Krafft, A. E., Nootheti, P. & Holmes, R. K. the tox promoter. This mechanism is the same as that (1990) Microb. Pathogen. 9, 267-273. established for interaction of Fur with its cognate operators 17. Schmitt, M. P. & Holmes, R. K. (1991) Infect. Immun. 59, in E. coli. Nevertheless, DtxR and Fur differ in specificity, 1899-1904. 18. Greenfield, L., Bjorn, M. J., Horn, G., Fong, D., Buck, G. A., since Fur has little or no ability to regulate transcription from Collier, R. J. & Kaplan, D. A. (1983) Proc. Nati. Acad. Sci. the tox promoter in vivo (23, 24), and DtxR has no effect on USA 80, 6853-6857. expression of Fur-regulated outer membrane proteins (24) or 19. Kaczorek, M., Delpeyroux, F., Chenciner, N., Streek, R. E., siderophore in E. coli (M.P.S., unpublished observation). Murphy, J. R., Boquet, P. & Tiollais, P. (1983) Science 221, DtxR did not bind to the Fur-binding site upstream from the 855-858. fur gene, in spite of the fact that the fur operator has higher 20. Ratti, G., Rappuoli, R. & Giannini, G. (1983) Nucleic Acids homology with the inverted repeats of the tox operator than Res. 11, 6589-6595. do other well-characterized Fur-regulated operators (23). 21. Boyd, J. & Murphy, J. R. (1988) J. Bacteriol. 170, 5949-5952. However, the tox operator has 9 nucleotides separating its 22. Leong, D. & Murphy, J. R. (1985) J. Bacteriol. 163,1114-1119. inverted repeats, whereas thefur operator has 5 nucleotides, 23. Tai, S.-P. S. & Holmes, R. K. (1988) Infect. Immun. 56, and the consensus binding site for Fur has only 2 nucleotides 2430-2436. separating their inverted repeats (39, 40). Differences in the 24. Boyd, J., Oza, M. N. & Murphy, J. R. (1990) Proc. Natl. Acad. Sci. USA 87, 5968-5972. inverted repeat sequences, in the spacing between them, or 25. Fourel, G., Phalipon, A. & Kaczorek, M. (1989) Infect. Immun. both, must determine the differences between dtxR-specific 57, 3221-3225. and fur-specific operators. It is also striking that the single 26. Schmitt, M. P. & Holmes, R. K. (1991) Infect. Immun. 59, G-C to A-T base-pair substitution in the tox-201 allele is 3903-3908. sufficient to interfere dramatically with binding of DtxR to 27. Tabor, S. & Richardson, C. C. (1985) Proc. Nati. Acad. Sci. the tox operator. USA 82, 1074-1078. The dtxR gene was recently shown to regulate synthesis of 28. Holmes, R. K. & Barksdale, L. (1969) J. Virol. 3, 586-598. the C. diphtheriae siderophore as well as diphtheria toxin 29. Krafft, A. E., Tai, S.-P. S., Coker, C. & Holmes, R. K. (1992) (16). These findings suggest that DtxR serves as a global Microb. Pathogen., in press. regulator of gene expression in C. diphtheriae, just as Fur 30. Maniatis, T., Fritsch, E. & Sambrook, J. (1989) Molecular does in E. coli. Additional studies are needed to characterize Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold more fully the dtxR regulon of C. diphtheriae and to identify Spring Harbor, NY). are its 31. Bramucci, M. G., Twiddy, E. M., Baine, W. B. & Holmes, the functional domains of DtxR that responsible for R. K. (1981) Infect. Immun. 32, 1034-1044. iron-binding activity, its sequence-specific DNA-binding ac- 32. Holmes, R. K. & Twiddy, E. M. (1983) Infect. Immun. 42, tivity, and its presumed ability to form biologically active, 914-923.30. noncovalently associated dimers. 33. Schaffers, S., Hantke, K. & Braun, V. (1985) Mol. Gen. Genet. 200, 110-113. This research was supported in part by U.S. Public Health Service 34. Fried, M. & Crothers, D. (1983) Nucleic Acids Res. 11, Grant AI-14107 from the National Institute ofAllergy and Infectious 141-158. Diseases. 35. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 449-560. 1. Pappenheimer, A. M., Jr. (1977) Annu. Rev. Biochem. 46, 36. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. 69-94. Acad. Sci. USA 74, 5463-5467. 2. Ward, W. H. J. (1987) Trends Biochem. Sci. 12, 28-31. 37. Wee, S., Neilands, J. B., Bittner, M. L., Hemming, B. C., 3. Groman, N. (1984) J. Hyg. 93, 405-417. Haymore, B. L. & Seetharam, R. (1988) Biol. Met. 1, 62-68. 4. Uchida, T., Gill, D. M. & Pappenheimer, A. M., Jr. (1971) 38. Harrison, S. C. & Aggarwal, A. K. (1990) Annu. Rev. Bio- Nature (London) New Biol. 233, 8-11. chem. 59, 933-969. 5. Murphy, J. R., Pappenheimer, A. M., Jr., & Tayart de Borms, 39. De Lorenzo, V., Herrero, M., Giovannini, F. & Neilands, J. B. S. (1974) Proc. Nati. Acad. Sci. USA 71, 11-15. (1988) Biochemistry 173, 537-546. 6. Pappenheimer, A. M., Jr., & Johnson, S. J. (1936) Br. J. Exp. 40. De Lorenzo, V., Wee, S., Herrero, M. & Neilands, J. B. (1987) Pathol. 17, 335-341. J. Bacteriol. 169, 2624-2630. Downloaded by guest on September 28, 2021