11
JOURNAL OF BACTERIOLOGY, Apr. 2009, p. 2638–2648 Vol. 191, No. 8 0021-9193/09/$08.000 doi:10.1128/JB.01784-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in Corynebacterium diphtheriae Courtni E. Allen and Michael P. Schmitt* Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 19 December 2008/Accepted 27 January 2009 Many human pathogens, including Corynebacterium diphtheriae, the causative agent of diphtheria, use host compounds such as heme and hemoglobin as essential iron sources. In this study, we examined the Coryne- bacterium hmu hemin transport region, a genetic cluster that contains the hmuTUV genes encoding a previously described ABC-type hemin transporter and three additional genes, which we have designated htaA, htaB, and htaC. The hmu gene cluster is composed of three distinct transcriptional units. The htaA gene appears to be part of an iron- and DtxR-regulated operon that includes hmuTUV, while htaB and htaC are transcribed from unique DtxR-regulated promoters. Nonpolar deletion of either htaA or the hmuTUV genes resulted in a reduced ability to use hemin as an iron source, while deletion of htaB had no effect on hemin iron utilization in C. diphtheriae. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they share some sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembrane regions. Protein localization studies with C. diphtheriae showed that HtaA is associated predominantly with the cell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-rich broth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growth medium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting that these proteins may function as cell surface hemin receptors in C. diphtheriae. Corynebacterium diphtheriae is a gram-positive bacterium and the etiological agent of diphtheria. Studies of C. diphthe- riae have focused primarily on the structural characterization and genetic regulation of diphtheria toxin, a secreted exotoxin that is responsible for much of the morbidity and mortality associated with human infection by this pathogen (9). Expres- sion of diphtheria toxin is repressed by iron, and transcription of the structural gene, tox, is regulated by the diphtheria toxin repressor protein, DtxR, in association with iron (4, 36). DtxR is a global iron-dependent repressor in C. diphtheriae that controls the expression of at least 50 genes at more than 20 different promoters (7, 14, 15, 29, 34, 38). While iron-depen- dent regulation of bacterial virulence factors has been well studied, it is also known that acquisition of iron from the extracellular environment is often critical for microbial patho- gens to cause disease (5, 6). Bacteria have developed a variety of mechanisms for uptake and utilization of iron. These mech- anisms include high-affinity siderophore uptake systems (48) and binding protein-dependent transporters that facilitate the acquisition of iron from various host sources, including trans- ferrin, lactoferrin, and heme, which is bound by various host proteins, such as hemoglobin (27, 35, 42). Siderophore trans- port systems are ubiquitous in bacteria, and it has been known for many years that C. diphtheriae secretes a siderophore and contains a siderophore-specific uptake system (10, 30). The structure of the C. diphtheriae siderophore has not been deter- mined; however, a gene required for siderophore biosynthesis and genes encoding an ABC-type siderophore transporter were recently identified in C. diphtheriae and were shown to be regulated by DtxR and iron (15). Hemin is the oxidized form of heme that is found in extra- cellular environments, and it is the form of heme transported by bacterial uptake systems. Hemin iron transport and utiliza- tion systems have been identified in numerous bacterial species and were initially described in gram-negative pathogens, where it was shown that an outer membrane receptor and a periplas- mic binding protein-dependent ABC-type transporter are re- quired for hemin uptake (13, 18, 41). Certain gram-negative species also obtain hemin iron through the use of hemophores, which are low-molecular-weight secreted hemin binding pro- teins that are able to extract hemin from hemoglobin and then transfer the hemin to receptors in the bacterial outer mem- brane (20). Hemin uptake systems have recently been described in gram-positive bacteria, including Staphylococcus aureus (24, 46), Streptococcus pyogenes (3, 19), and Corynebacterium spe- cies (11, 35). S. aureus binds hemin or hemoglobin to its cell envelope through various surface-anchored proteins, termed iron-regulated surface determinants (Isd), which contain sor- tase recognition signals at their C termini that are essential for the covalent linkage of these proteins to the peptidoglycan (24, 46). It is believed that hemin is transported through the cell envelope via a cascade mechanism in which hemin is trans- ferred between various Isd receptors (22, 50). In S. pyogenes, hemin is proposed to bind initially to surface-exposed proteins, designated Shp and Shr, which appear to be anchored to the membrane by a putative transmembrane region in their C termini (21, 49). In both S. aureus and S. pyogenes it is thought that hemin is transferred from the surface-anchored proteins * Corresponding author. Mailing address: DBPAP, CBER, FDA, Bldg. 29, Room 108, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 435-2424. Fax: (301) 402-2776. E-mail: michael.schmitt @fda.hhs.gov. Published ahead of print on 6 February 2009. 2638 on February 14, 2021 by guest http://jb.asm.org/ Downloaded from

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JOURNAL OF BACTERIOLOGY, Apr. 2009, p. 2638–2648 Vol. 191, No. 80021-9193/09/$08.00�0 doi:10.1128/JB.01784-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

HtaA Is an Iron-Regulated Hemin Binding Protein Involved in theUtilization of Heme Iron in Corynebacterium diphtheriae�

Courtni E. Allen and Michael P. Schmitt*Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Center for

Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Received 19 December 2008/Accepted 27 January 2009

Many human pathogens, including Corynebacterium diphtheriae, the causative agent of diphtheria, use hostcompounds such as heme and hemoglobin as essential iron sources. In this study, we examined the Coryne-bacterium hmu hemin transport region, a genetic cluster that contains the hmuTUV genes encoding a previouslydescribed ABC-type hemin transporter and three additional genes, which we have designated htaA, htaB, andhtaC. The hmu gene cluster is composed of three distinct transcriptional units. The htaA gene appears to bepart of an iron- and DtxR-regulated operon that includes hmuTUV, while htaB and htaC are transcribed fromunique DtxR-regulated promoters. Nonpolar deletion of either htaA or the hmuTUV genes resulted in a reducedability to use hemin as an iron source, while deletion of htaB had no effect on hemin iron utilization in C.diphtheriae. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they sharesome sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembraneregions. Protein localization studies with C. diphtheriae showed that HtaA is associated predominantly with thecell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-richbroth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growthmedium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting thatthese proteins may function as cell surface hemin receptors in C. diphtheriae.

Corynebacterium diphtheriae is a gram-positive bacteriumand the etiological agent of diphtheria. Studies of C. diphthe-riae have focused primarily on the structural characterizationand genetic regulation of diphtheria toxin, a secreted exotoxinthat is responsible for much of the morbidity and mortalityassociated with human infection by this pathogen (9). Expres-sion of diphtheria toxin is repressed by iron, and transcriptionof the structural gene, tox, is regulated by the diphtheria toxinrepressor protein, DtxR, in association with iron (4, 36). DtxRis a global iron-dependent repressor in C. diphtheriae thatcontrols the expression of at least 50 genes at more than 20different promoters (7, 14, 15, 29, 34, 38). While iron-depen-dent regulation of bacterial virulence factors has been wellstudied, it is also known that acquisition of iron from theextracellular environment is often critical for microbial patho-gens to cause disease (5, 6). Bacteria have developed a varietyof mechanisms for uptake and utilization of iron. These mech-anisms include high-affinity siderophore uptake systems (48)and binding protein-dependent transporters that facilitate theacquisition of iron from various host sources, including trans-ferrin, lactoferrin, and heme, which is bound by various hostproteins, such as hemoglobin (27, 35, 42). Siderophore trans-port systems are ubiquitous in bacteria, and it has been knownfor many years that C. diphtheriae secretes a siderophore andcontains a siderophore-specific uptake system (10, 30). Thestructure of the C. diphtheriae siderophore has not been deter-mined; however, a gene required for siderophore biosynthesis

and genes encoding an ABC-type siderophore transporterwere recently identified in C. diphtheriae and were shown to beregulated by DtxR and iron (15).

Hemin is the oxidized form of heme that is found in extra-cellular environments, and it is the form of heme transportedby bacterial uptake systems. Hemin iron transport and utiliza-tion systems have been identified in numerous bacterial speciesand were initially described in gram-negative pathogens, whereit was shown that an outer membrane receptor and a periplas-mic binding protein-dependent ABC-type transporter are re-quired for hemin uptake (13, 18, 41). Certain gram-negativespecies also obtain hemin iron through the use of hemophores,which are low-molecular-weight secreted hemin binding pro-teins that are able to extract hemin from hemoglobin and thentransfer the hemin to receptors in the bacterial outer mem-brane (20).

Hemin uptake systems have recently been described ingram-positive bacteria, including Staphylococcus aureus (24,46), Streptococcus pyogenes (3, 19), and Corynebacterium spe-cies (11, 35). S. aureus binds hemin or hemoglobin to its cellenvelope through various surface-anchored proteins, termediron-regulated surface determinants (Isd), which contain sor-tase recognition signals at their C termini that are essential forthe covalent linkage of these proteins to the peptidoglycan (24,46). It is believed that hemin is transported through the cellenvelope via a cascade mechanism in which hemin is trans-ferred between various Isd receptors (22, 50). In S. pyogenes,hemin is proposed to bind initially to surface-exposed proteins,designated Shp and Shr, which appear to be anchored to themembrane by a putative transmembrane region in their Ctermini (21, 49). In both S. aureus and S. pyogenes it is thoughtthat hemin is transferred from the surface-anchored proteins

* Corresponding author. Mailing address: DBPAP, CBER, FDA,Bldg. 29, Room 108, 8800 Rockville Pike, Bethesda, MD 20892.Phone: (301) 435-2424. Fax: (301) 402-2776. E-mail: [email protected].

� Published ahead of print on 6 February 2009.

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to a substrate binding component that is associated with aheme-dependent ABC transporter that facilitates the passageof hemin into the cytosol.

In Corynebacterium ulcerans, the transport of hemin involvesuse of the ABC-type transporter encoded by the hmuTUVgenes, which share sequence similarity to genes encoding he-min uptake systems in other organisms, including C. diphthe-riae (11, 35). The hmuTUV genes in C. diphtheriae were shownto complement an hmuTUV mutation in C. ulcerans, and it wasfurther demonstrated that the HmuT protein in C. diphtheriaeis a hemin binding lipoprotein that is tethered to the cytoplas-mic membrane by an N-terminal lipid moiety (11). An inser-tion mutation in hmuT had no effect on hemin iron utilizationin C. diphtheriae; however, it is suspected that the mediumconditions used in the study were not optimal for detection ofa hemin uptake deficiency (16, 35). The completion of thegenome sequence of a clinical isolate of C. diphtheriae (8)resulted in identification of htaA, an iron- and DtxR-regulatedgene that is located immediately upstream of hmuT (14). Nopromoter activity was detected in the htaA-hmuT intergenicregion, and it was suspected that htaA and the hmuTUV genesmay constitute a DtxR-regulated operon (14, 35). Two othergenes, designated htaB and htaC, were also identified in thishemin transport gene cluster. The function of the predictedproducts of htaA, htaB, and htaC has not been determined;however, the linkage of these genes to the hmuTUV operonsuggests that they have a possible function in hemin uptake orhemin iron utilization. The use of heme as an iron source in C.diphtheriae also involves the hmuO gene, which encodes aheme oxygenase that catalyzes the degradation of intracellularheme and the subsequent release of the heme-associated iron(16, 31, 47). The hmuO gene in C. diphtheriae is not linked tothe hemin transport gene cluster, but transcription of hmuO isregulated by DtxR and iron, as well as by heme (32, 33).

In this study, we extended our characterization of the hemintransport locus, designated hmu, to examine the expressionand cellular localization of specific hmu gene products. Weshow here that the hmuTUV and htaA genes are involved in theuse of hemin as an iron source in C. diphtheriae. Protein local-ization studies revealed that HtaA, HmuT, and HtaB are as-sociated predominantly with the cytoplasmic membrane whenbacteria are grown in minimal medium, which suggests thatthese proteins may function to transport hemin. We alsodemonstrate that HtaA and HtaB are surface-exposed he-min binding proteins that may function as hemin receptorsin C. diphtheriae.

MATERIALS AND METHODS

Bacterial strains and media. Escherichia coli and C. diphtheriae strains used inthis study are listed in Table 1. Luria-Bertani (LB) medium was used for cultur-ing E. coli, and heart infusion broth (Difco, Detroit, MI) containing 0.2% Tween80 (HIBTW medium) was used for routine growth of C. diphtheriae strains.Bacterial stocks were maintained in 20% glycerol at �80°C. Antibiotics wereadded to LB medium at concentrations of 34 �g/ml for chloramphenicol, 50�g/ml for kanamycin, and 100 �g/ml for ampicillin for E. coli cultures and toHIBTW medium at concentrations of 2 �g/ml for chloramphenicol and 50 �g/mlfor kanamycin for C. diphtheriae cultures. HIBTW medium was made a low-ironmedium by addition of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA)at a concentration of 12 �g/ml (unless indicated otherwise). Modified PGT(mPGT) medium is a semidefined low-iron medium that has been describedpreviously (44). Antibiotics, EDDA, Tween 80, hemin (bovine), and hemoglobin(human) were obtained from Sigma Chemical Co. (St. Louis, MO).

Mutant construction. A previously described allelic replacement technique(45) was used to construct nonpolar deletion mutations in the C. diphtheriae 1737htaA, htaB, and hmuTUV genes, as well as in the complete hmu gene cluster(htaC through htaB). Mutant construction utilized PCR to clone DNA fragmentslocated upstream and downstream of the region targeted for deletion. Thedeleted regions in the htaA, htaB, and hmuTUV mutants are predicted to encodepeptides consisting of approximately 20 amino acids that contain in-frame fu-sions of residues derived from the N and C termini of the original proteins.Primers used for construction of the mutants are listed in Table 2. PCR was usedto confirm the mutations in all of the deletion mutants (not shown). An R47Hpoint mutation was introduced into the C7 dtxR gene using a previously de-scribed procedure (15).

Hemoglobin iron utilization assays. The hemoglobin iron utilization assay hasbeen described previously (16). Briefly, C. diphtheriae strains were grown over-night (20 to 22 h at 37°C) in HIBTW medium and then inoculated to an opticaldensity at 600 nm (OD600) of 0.2 into fresh HIBTW medium that contained 12�g/ml of the iron chelator EDDA. Strains were grown for several hours at 37°Cuntil log phase, at which time bacteria were recovered by centrifugation, resus-pended in mPGT medium, and then inoculated at an OD600 of 0.03 into freshmPGT medium that contained various supplements, as indicated below. After 20to 22 h of growth at 37°C, the OD600s of the cultures were determined.

Experiments that examined the effect of secreted or purified HtaA on hemo-globin iron utilization used HtaA that was either purified from E. coli or obtainedin native form from C. diphtheriae culture supernatants. Culture supernatantswere prepared as follows. C. diphtheriae strain 1737hmu� containing either thecloned htaA gene on pKhtaA or the vector pKN2.6Z was grown to late log phasein low-iron HIBTW medium, and culture supernatants were concentrated byammonium sulfate precipitation. One milliliter of a culture supernatant wasconcentrated to 50 �l and then dialyzed in phosphate-buffered saline (PBS) toremove the ammonium sulfate. The concentrated and dialyzed supernatant (50�l) was added to 1 ml of mPGT medium in the hemoglobin utilization assaydescribed above. Purified glutathione S-transferase (GST)-HtaA and HtaA wereused similarly in the hemoglobin utilization assay, and approximately 1 �g ofpurified protein was added to 1 ml of mPGT culture medium.

Plasmid construction. Plasmids used in this study are listed in Table 1. PCR-derived DNA fragments were initially cloned into the pCR-Blunt II-TOPOvector (Invitrogen), and genomic DNA derived from C. diphtheriae strain 1737was used as a template for all PCRs (unless otherwise indicated). The promoterprobe vector pCM502 (32) was used for construction of all lacZ promoterfusions; this vector contains a promoterless lacZ gene and replicates at a low

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant characteristics or use Reference orsource

C. diphtheriae strains1737 Wild type, Gravis biotype, tox� 281737htaA� htaA deletion mutant of 1737 This study1737TUV� hmuTUV deletion mutant of 1737 This study1737htaB� htaB deletion mutant of 1737 This study1737hmu� Deletion mutation in hmu locus of

1737This study

C7(�) Wild type, tox 2C7dtxR dtxR(R47H) mutant of C7(�) This study

E. coli strainsBL21(DE3) Protein expression NovagenDH5� Cloning strain InvitrogenS17-1 RP4 mobilization functions 45TOP10 Cloning of PCR fragments Invitrogen

PlasmidspCR-Blunt-II-

TOPOCloning of PCR fragments, Kanr Invitrogen

pCM502 Promoter probe, Cmr 32phtaB-Z htaB promoter-lacZ fusion in

pCM502This study

pET24a(�) Expression vector (His fusion), Kanr NovagenpET28a(�) Expression vector (His fusion), Kanr NovagenpGEX-6P-1 Expression vector (GST fusion) Ampr GE HealthcarepKN2.6Z C. diphtheriae shuttle vector, Knr 11pKhtaA pKN2.6Z carrying the htaA gene This studypCM2.6 C. diphtheriae shuttle vector, Cmr 36pCD842 pCM2.6 carrying the hmuTUV genes 11pCC1 Copy control vector for htaA cloning Epicentre

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copy number in C. diphtheriae. The htaB promoter-lacZ fusion construct,phtaB-Z, contains a 360-bp PCR-derived DNA product that includes the 3� endof the hmuV gene and the 5� coding region of htaB. To construct plasmidpKhtaA, PCR was used to generate an approximately 2-kb DNA fragment thatcontains the complete coding region and promoter sequence for htaA. Since thecloned htaA gene was unstable at a high copy number in E. coli, the htaA genewas initially cloned into the copy control vector pCC1 (Epicentre, Madison, WI).The insert was subsequently ligated into the EcoRV site in vector pKN2.6Z (11)and transformed into C. ulcerans 712 (40). The DNA sequence of the clonedhtaA gene on plasmid pKhtaA was found to be identical to that reported for thehtaA gene from the C. diphtheriae strain NCTC13129 genome (8).

The vector pET24a� (Novagen, Madison, WI) was used for expression ofsix-histidine (His)-tagged HtaA and HtaB proteins in E. coli. Internal regions ofthe htaA and htaB genes that lack the N-terminal leader sequences (residues 1 to30 for htaA and residues 1 to 24 for htaB) and C-terminal transmembrane andcharged tail regions (residues 565 to 591 for htaA and residues 293 to 325 forhtaB) were amplified by PCR and subsequently ligated into the NdeI-HindIIIsites in pET24a�. The pET28a� vector, which contains a thrombin proteaserecognition site, was also used for expression of htaB. A thrombin cleavage andcapture kit (Novagen) was used to cleave the N-terminal His tag from the HtaBprotein according to the manufacturer’s protocol. The purified HtaB proteinwithout the His tag was used in the hemin binding studies. The pGEX-6P-1vector (GE Healthcare) was used for expression of a GST-HtaA fusion proteinthat contains an N-terminal GST tag. The same region of htaA that was clonedinto the pET24a� vector was ligated into pGEX-6P-1; however, EcoRV andXhoI sites were introduced into the PCR primers used to amplify the htaA gene.

LacZ assays. C. diphtheriae strains containing lacZ fusion constructs weregrown overnight in HIBTW medium and then inoculated at an OD600 of 0.2 intofresh HIBTW medium and grown to log phase. Log-phase cultures were used to

inoculate fresh HIBTW medium to an OD600 of 0.1 and grown overnight in thepresence of various supplements, as indicated below. LacZ activity was deter-mined for overnight cultures as previously described (37).

Protein expression and antibody production. E. coli strain BL21(DE3) carry-ing the cloned htaA or htaB gene on the pET24a� expression vector was grownin 100 ml of LB medium at 37°C to mid-log phase, at which time 1 mMisopropyl-�-D-thiogalactopyranoside (IPTG) was added, and the cultures wereallowed to grow for an additional 2 to 3 h before they were harvested. Thecultures were washed in 10 ml of 20 mM Tris-HCl (pH 7.5), and each pellet wasstored at �20°C overnight. The pellet was thawed and resuspended in buffercontaining 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0) at4°C, and the bacteria were lysed with a French pressure cell, which was followedby removal of cell debris by centrifugation at 10,000 � g at 4°C. The solublefraction containing the HtaB protein was collected and purified using Ni-nitrilo-triacetic acid resin (Qiagen) by a batch affinity method according to the manu-facturer’s instructions. It was observed that the HtaA-His protein formed inclu-sion bodies and could not be purified with the Ni-nitrilotriacetic acid affinityresin. However, we observed that HtaA could be significantly enriched aftersolubilization in 8 M urea, followed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and then excision of the HtaA protein bandfrom the acrylamide gel. The gel-purified HtaA and affinity-purified HtaB pro-teins were used for production of polyclonal antibodies in guinea pigs by stan-dard methods (Cocalico Biologicals Inc.,).

The procedure used for expression of HtaA with the pGEX-6P-1 vector wassimilar to the procedure described above for the pET expression system. TheGST-HtaA protein was found predominantly in the soluble fraction after lysis ofthe bacteria, and the fusion protein was purified using GST resin (GE Health-care) by a batch method according to the manufacturer’s instructions. PurifiedHtaA protein was obtained after removal of the GST tag by cleavage with

TABLE 2. Primers used in this study

Primer Amplified regionApprox

fragmentsize (bp)

Sequencea

Deletion mutantshtaAUP4 Upstream of htaA 1,000 5�-GCGGATATCTCGAGTGCTTTGCCAGG-3�htaAUP2-2 5�-GCGGATCCTTGAATTATCACGGTGC-3�

htaADN1-2 Downstream of htaA 500 5�-GCGGGATCCATTTCCAATCCAGAAATTG-3�htaADN2-2 5�-GCGAAGCTTGATATCACACACCACGGACATAC-3�

UPB1 Upstream of htaB 900 5�-GCGGTCGACGGTTGAGATAAATAACC-3�UPB2 5�-GCGAAGCTTGCAGCCTGAGTCGTTGG-3�

DNB1-1 Downstream of htaB 700 5�-GCGGTCGACTCTAGGTTTAGATTGCG-3�DNB2 5�-GCGGATATCCGAACTCGGTGCAGACG-3�

UPC1 Upstream of htaC 1,100 5�-GCGGTCGACCAAACGATCAAGCCACC-3�UPC2-1 5�-GCGAAGCTTGATATCAGCTGATTTTTGCCATAGCG-3�

hm1-1 Upstream of hmuT 750 5�-GCGATATCCAATGGAACCTTGATGG-3�hm1-2 5�-GCGGATCCACAAACACGAAGCAAAGA-3�

hm1-3 Downstream of hmuV 650 5�-GCGGATCCCAAAACCAAGATGATTTTG-3�hm1-4 5�-GCAAGCTTGATATCCAATTGCGAATAAAAGG-3�

Promoter fusionshtaB-PO1 htaB promoter 360 5�-GCGGTCGACGCGAGGTCGTACTACGC-3�htaB-PO2 5�-GCGGGATCCTCAGCGACAGCTTGAGG-3�

Protein expression24FHtA-His htaA for pET24(a)� without 1,600 5�-GCGCATATGGCCCACCACCACCACCACCACGCTGATAGCAATCAATGC-3�RHtA-TM transmembrane region 5�-GCGAAGCTTCTAGGGATCAATACCGGAATC-3�

GEXFHta-GST htaA for pGEX-6P-1 without 1,600 5�-GCGGATATCAGCTGATAGCAATCAATGC-3�RXhoHta-TM transmembrane region 5�-GCGCTCGAGCTAGGGATCAATACCGGAATC-3�

24FHtB-His htaB for pET24(a)� 5�-GCGCATATGGCCCACCACCACCACCACCACGCTGAAGAACCGGCAGCC-3�

28FHtB htaB for pET28(a)� without 780 5�-GCGCATATGGCTGAAGAACCGGCAGCC-3�RHtB-TM transmembrane region 5�-GCGAAGCTTTTAGTTAAAACCAGAAGTAGA-3�

a Restriction sites are underlined, stop codons are indicated by bold type, and inserted bases are double underlined.

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PreScission protease (GE Healthcare), which was followed by purification stepsperformed according to the manufacturer’s protocol.

SDS-PAGE and Western blot analysis. Proteins were separated by SDS-PAGE (17) and stained with Bio-Safe Coomassie blue (Bio-Rad) by followingthe manufacturer’s instructions. Western blot procedures were performed asdescribed previously (12), and anti-HtaA and anti-HtaB antibodies were used atdilutions of 1:10,000 and 1:20,000, respectively. Antibodies raised against HmuT(35), DtxR (39), and diphtheria toxin (a gift from R. K. Holmes) were used atdilutions of 1:40,000, 1:5,000, and 1:10,000, respectively. Binding of the primaryantibodies to immobilized proteins was detected by using appropriate alkalinephosphatase-labeled secondary antibodies. Alkaline phosphatase activity wasdetected by using established procedures (35).

Protein localization studies. To determine whether proteins were secreted orcell associated, 1-ml cultures were grown to mid-log phase at 37°C in HIBTWmedium containing 12 �g/ml EDDA (low-iron medium). Cultures were centri-fuged briefly to pellet the cells, and the supernatant was recovered and passedthrough a 0.2-�m filter to remove bacteria. Proteins in the filtered supernatantwere precipitated with 10% trichloroacetic acid, washed with ethanol, dried, andthen resuspended in Tris-EDTA buffer (pH 7.4). One half of the sample wasanalyzed after SDS-PAGE and Coomassie blue staining, and the other half of thesample was used for Western analysis. The resulting cell pellet was lysed and thenresuspended in SDS loading buffer, and samples were analyzed as described above.

A previously described procedure was used to determine the cellular locationof cell-associated proteins (11), with the following modifications. Briefly, 200-mlcultures of various C. diphtheriae strains were grown to late log phase in HIBTWmedium at 37°C. Cells were harvested by centrifugation, washed once with PBS(pH 7.4) (Invitrogen), and then resuspended in 10 ml of PBS, which was followedby lysozyme treatment and lysis of the bacteria using a French pressure cell. Celldebris was removed by centrifugation, and the soluble fraction, which containedboth soluble and membrane proteins, was centrifuged at 100,000 � g to pellet thecytoplasmic membrane. The supernatant, which contained the soluble intracel-lular proteins, was recovered and stored at 4°C, while the membrane fraction wassolubilized in 0.1% Triton X-100. C. diphtheriae cultures grown in 50 ml ofmPGT medium were prepared similarly.

Proteinase K experiments. C. diphtheriae 1737htaA�/pKhtaA was grown asdescribed above for the hemoglobin utilization assay, except that 0.5 �M FeSO4

was added to mPGT medium (low-iron conditions) in place of hemoglobin.Cultures were harvested after overnight growth and centrifuged at 7,000 � g for10 min. The cells were resuspended in PBS, which was followed by addition ofproteinase K at a final concentration of 50 �g/ml to the cell suspension. Thereaction mixture was incubated for 30 min at 37°C, at which time the cells werepelleted and then washed two times with PBS. The bacteria were then treatedwith lysozyme followed by 0.75% Sarkosyl to lyse the bacteria. Whole-cell proteinpreparations were boiled under reducing conditions prior to analysis by SDS-PAGE and Western blotting. Control samples were not treated with proteinaseK. Plasmid pKhtaA was used to obtain better detection of the HtaA protein.

Hemin binding analysis. Purified GST-HtaA and HtaB, from which the leaderpeptide and the transmembrane region were deleted (HtaB also lacked thesix-His tag), were analyzed to determine their hemin binding properties byUV-visible spectroscopy using a Beckman DU 640 spectrophotometer. Proteinsat a concentration of 3.5 �M in PBS buffer containing glycerol were assessed todetermine their abilities to bind hemin in the concentration range from 0.5 to 20�M. Proteins were incubated in the presence of hemin for at least 15 min beforespectrophotometric analysis. UV-visible absorption scans of HtaA and HtaBwere done using wavelengths between 280 and 600 nm. Absorbance spectra forall protein-hemin samples were zeroed against a reference cuvette that containedthe same concentration of hemin in PBS buffer in the absence of protein.

Detection of heme-dependent peroxidase activity. The chromogenic com-pound 3,3�,5,5�-tetramethylbenzidine (TMBZ) turns blue in the presence ofheme-dependent peroxidase activity and was used to detect hemin-protein com-plexes as previously described (43). Prior to separation of proteins by SDS-PAGE, samples were incubated for 30 min at room temperature in the presenceor absence of hemin. Hemin was prepared in 0.1 N NaOH and was used at aconcentration of 0.625 �M for HtaA detection or at a concentration of 2.5 �Mfor studies with HtaB. Samples were also incubated in the absence of hemin with0.1 N NaOH. Proteins were not boiled or exposed to reducing agents prior toSDS-PAGE. Lithium dodecyl sulfate (LDS) was used in place of SDS in theHtaB studies as described previously (43). HtaA in culture supernatants wasconcentrated by precipitation with 75% ammonium sulfate followed by dialysis in50 mM Tris (pH 8.0).

Computer analysis. Amino acid sequence similarity searches were done usingthe BLAST program (1) at the National Center for Biotechnology Informationand also using the BLAST server provided at the online site for the Sanger

Institute (http://www.sanger.ac.uk/Projects/C_diphtheriae). The annotated ge-nome sequence of C. diphtheriae strain NCTC13129 (8) is accessible in theEMBL/GenBank database under accession number BX248353.

RESULTS

Analysis of the C. diphtheriae hmu hemin transport genecluster. Previous studies of C. diphtheriae identified threegenes, hmuT, hmuU, and hmuV, which encode components ofan ABC-type hemin transporter (11). The HmuT protein wasshown to be a membrane-anchored hemin binding lipoprotein,while HmuU and HmuV were predicted to be the permeaseand ATPase components, respectively. In a subsequent study(14), we showed that the hmuTUV genes were part of a largergene cluster that included the htaA, htaB, and htaC genes (Fig.1A), and we reported that htaA and htaC were transcribedfrom divergent DtxR- and iron-regulated promoters (14). Inthis study, we identified a putative DtxR binding site upstreamof htaB that aligns with 12 of the 19 residues in the consensusDtxR binding sequence and matches 9 of the 11 most highlyconserved bases (Fig. 1A and C). Analysis of an htaB-lacZtranscriptional fusion construct (phtaB-Z) in the wild type andin a dtxR mutant of C. diphtheriae showed that expression fromthe htaB promoter is regulated by iron and DtxR (Fig. 1D).Transcription from the htaB promoter was not affected by thedivalent metals Mn and Zn, and the presence of hemoglobinresulted in a slight decrease in expression, since hemin servesas an iron source in C. diphtheriae (data not shown). Westernanalysis using antibodies raised against HtaA, HtaB, andHmuT demonstrated that the production of each of theseproteins is repressed by iron (Fig. 2A to C), which is consistentwith the study described above and with previous findings (14).Diphtheria toxin, a well-characterized iron-regulated protein,and DtxR, which is constitutively expressed, were included ascontrols in this study (Fig. 2D).

BLAST analysis of the predicted amino sequence of HtaAreveals that it has homology (ranging from 20% to 60% iden-tity) with proteins in several bacterial species that are relatedto C. diphtheriae, including C. ulcerans, Corynebacteriumjeikeium, Corynebacterium glutamicum, Propionibacteriumacnes, and Streptomyces coelicolor. HtaA also contains approx-imately 150 to 200 residues in its N-terminal region that sharealmost 50% sequence similarity to a segment in its C terminus.These two conserved regions are approximately 35% similar toa region in HtaB (Fig. 3A). HtaA and HtaB both containpredicted signal peptides and a putative transmembrane regionin their C termini, which is followed by one (HtaA) or two(HtaB) positively charged residues (Fig. 3A and B). The htaCgene is predicted to encode a putative membrane protein thatshares limited amino acid sequence similarity with proteinswith unknown functions.

Mutations in htaA and hmuTUV result in a reduced abilityto utilize hemoglobin as an iron source in C. diphtheriae. Afunction for HtaA in C. diphtheriae has not been determinedpreviously, although the close proximity of htaA to genes in-volved in hemin transport suggests that HtaA may have a rolein hemin uptake or hemin iron utilization. In previous studies(11, 35), the C. diphtheriae hmuTUV genes were shown tocomplement mutations in the hmuTUV genes in C. ulcerans;however, an insertion mutation in C. diphtheriae hmuT did not

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result in a defect in hemin iron use (35). Since the previousreport, we have developed a more sensitive method for mea-suring hemin iron utilization in Corynebacterium (16). To de-termine the functions of proteins encoded in the hmu genecluster in C. diphtheriae, we constructed various nonpolar de-letions in this region (Fig. 1B). The various mutations in thehmu gene cluster were confirmed by PCR (data not shown)and Western analysis (Fig. 2A to C). C. diphtheriae strain 1737was used to construct the various hmu mutants and to assesshemoglobin iron utilization. The 1737 strain is a clinical isolatefrom the recent Russian diphtheria epidemic (28) and is very

FIG. 2. Western blot analysis of proteins produced by C. diphtheriaewild-type strain 1737 (WT) and various 1737 deletion mutants. Strains weregrown in 1 ml of iron-replete HIBTW medium (�Fe) or iron-depletedmedium (HIBTW medium containing 12 �g/ml EDDA) (�Fe), and proteinspresent either in culture supernatants (HtaA and diphtheria toxin [DT] inpanels A and D, respectively) or in whole-cell extracts (HtaB, HmuT, andDtxR in panels B, C, and D, respectively) were detected by Western blotanalysis using antiserum raised against the proteins indicated on the left.Samples were normalized using OD600 before SDS gels were loaded. htaA�,1737htaA�; TUV�, 1737TUV�; hmu�, 1737hmu�; htaB�, 1737htaB�.

FIG. 1. (A) Genetic map of the hmu locus in C. diphtheriae. The predicted sizes of the various gene products are indicated below the map. P indicates thepresence of a DtxR-regulated promoter, and the arrows indicate the direction of transcription. (B) Regions deleted in the various C. diphtheriae 1737 mutantsconstructed. The deleted regions are aligned with the genetic map shown in panel A. (C) Alignment of the 19-bp putative DtxR binding site upstream of theC. diphtheriae htaB gene with the consensus DtxR binding site. The underlined sequences are the most highly conserved residues (14). (D) Assessment of thepromoter activity (LacZ activity) of an htaB-lacZ transcriptional fusion construct, phtaB-Z, in high-iron HIBTW medium (�Fe) and in iron-depleted conditions(HIBTW medium containing 12 �g/ml EDDA) (�Fe). The values are the means of three experiments. Each result varied less than 15% from the mean. Thedifference between high- and low-iron conditions for the dtxR mutant was statistically significant (P 0.05), and this suggests that the point mutant (R47H)maintains some low-level Fe-dependent repressor activity. See Materials and Methods for experimental details.

FIG. 3. (A) Comparison of various structural characteristics of theHtaA and HtaB proteins. HtaA and HtaB contain a region of sequencesimilarity consisting of approximately 200 amino acids designated theconserved region (CR). SP, signal peptide; TM, transmembrane re-gion; �, positively charged residues. (B) Comparison of the amino acidsequences in the C-terminal tail region for HtaA, HtaB, and thesurface-anchored hemin binding proteins ShR and ShP from S. pyo-genes. The putative membrane-spanning region is underlined, and pos-itively charged residues are indicated by plus signs.

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closely related to the strain used for analysis of the genomesequence (8).

The ability to use hemoglobin as an iron source was analyzedusing low-iron mPGT medium in the presence of 25 �g/mlhemoglobin after 22 to 24 h of growth at 37°C. All strains grewto similar OD600s when 1 �M FeSO4 was added to mPGTmedium (the OD600s were between 3.0 to 3.5), and the growthof all strains was fully inhibited in low-iron mPGT medium inthe absence of an iron source (low-iron mPGT medium con-tains 10 �M EDDA). Deletion of the C. diphtheriae 1737 htaAgene, the hmuTUV genes, or the complete hmu gene clusterresulted in significantly diminished growth compared to thegrowth of the wild-type strain when hemoglobin was suppliedas the sole iron source (Fig. 4A). Deletion of the htaB gene hadno effect on the use of hemoglobin as an iron source inthese assays (Fig. 4A). The presence of the cloned htaA andhmuTUV genes restored the ability to utilize hemoglobin as aniron source in the htaA and hmuTUV mutants, respectively(Fig. 4A). These findings suggest that the products of htaA andhmuTUV are involved in hemin transport or in hemin ironutilization in C. diphtheriae. However, the results also suggestthat additional genes outside the hmu locus encode productsthat have the ability to transport hemin, since hemoglobin wasstill able to stimulate growth of all of the deletion strains. Allof the mutants described with mutations in the C. diphtheriaehmu locus that showed reduced growth when hemoglobin wasadded as the sole iron source exhibited similar reductions ingrowth compared to the wild-type strain when hemin was usedas the only iron source (data not shown).

Cellular localization of the C. diphtheriae hmu gene prod-ucts. To better understand the function of the hmu gene prod-ucts in hemin iron utilization, studies were done to identify thecellular locations of the various proteins encoded in this genecluster. Studies were initially done to determine whether var-ious proteins are cell associated or secreted into the culturemedium. As shown in Fig. 5A, HtaA was predominantly cellassociated when C. diphtheriae was grown in low-iron mPGTminimal medium, although low levels of HtaA were detectedin the culture supernatant. Surprisingly, HtaA was found al-most exclusively in the supernatant fraction when bacteriawere grown in low-iron HIBTW medium, and HtaA exhibiteda secretion profile almost identical to that of diphtheria toxin,a well-known secreted protein in C. diphtheriae (Fig. 5A). Ex-amination of four additional C. diphtheriae strains, strains1716, 1718, 1897, and 1751 (28), showed that they also secretedHtaA during growth in HIBTW medium (Fig. 5B). The HtaBand HmuT proteins were both found to be predominantly cellassociated, although smaller quantities were observed in thesupernatant fraction (Fig. 5A). DtxR, an intracellular proteinused as a control to monitor cell lysis, was found exclusively inthe cellular fraction, which indicates that the presence of HtaAin the supernatant was not the result of leakage or lysis of thebacteria during growth in HIBTW medium. The localization ofdiphtheria toxin, HtaB, and HmuT after growth in mPGTmedium was similar to that observed after growth in HIBTWmedium (data not shown).

The results of cellular fractionation studies and Westernanalysis indicate that HtaA is associated primarily with thecytoplasmic membrane when C. diphtheriae 1737 is grown inmPGT medium (Fig. 5C). HtaA and diphtheria toxin were not

detected in any cellular fraction when bacteria were grown inHIBTW medium, since these proteins are secreted into thesupernatant in this medium (Fig. 5A). HtaB and HmuT arelocalized primarily to the cytoplasmic membrane, while thetranscriptional regulatory protein, DtxR, is found exclusively inthe soluble cytosolic fraction (Fig. 5C).

Secreted HtaA is not involved in hemin iron utilization.While it is not clear what function, if any, the secreted HtaA

FIG. 4. Utilization of hemoglobin as an iron source by C. diphthe-riae wild-type strain 1737 and mutant strains. (A) OD600s of culturesgrown for 20 to 22 h in mPGT medium containing 10 �M EDDA and25 �g/ml hemoglobin (human). The plasmids carried by the variousstrains included pKhtaA containing the cloned htaA gene (pA), vectorpKN2.6Z (pK), vector pCM2.6 (pCM), and pCD842 containing thecloned hmuTUV genes (p842). wt, wild-type strain 1737; A�,1737htaA�; TUV�, 1737TUV�; hmu�, 1737hmu�; htaB�, 1737htaB�. (B) Results of experiments performed like the experimentsdescribed for panel A, except that cultures of 1737htaA� were sup-plemented with 50 �l of concentrated culture supernatant from1737hmu� that contained either the vector pKN2.6Z (SupK) or thecloned htaA gene on pKhtaA (SupA). The presence of HtaA in culturesupernatants in 1737hmu�/pKhtaA was confirmed by SDS-PAGEanalysis (not shown). The results are the averages and standard devi-ations of three independent experiments. The values for 1737htaA�/pKN2.6Z are significantly different from the values for 1737/pKN2.6Zand 1737htaA�/pKhtaA (�, P 0.05), and the values for 1737TUV�/pCM2.6 are significantly different from the values for 1737/pKN2.6Zand 1737TUV�/pCD842 (��, P 0.05).

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protein has in hemin iron utilization in C. diphtheriae, additionof HtaA (either purified or concentrated from culture super-natants) to cultures of 1737htaA� did not enhance the growthof this htaA mutant in the presence of hemoglobin (Fig. 4B).These results suggest that the secreted HtaA protein is notinvolved in the use of hemin iron by C. diphtheriae but that thedominant form of HtaA involved in the uptake of hemin ismost likely bound to the cytoplasmic membrane.

Surface exposure of hmu gene products. To determinewhether HtaA, HtaB, or HmuT is exposed on the cell surfaceand therefore may be accessible to bind hemin or possiblyhemoglobin, C. diphtheriae was grown in low-iron mPGT me-dium, which was followed by treatment of the intact bacteriawith proteinase K. This protease is predicted to cleave proteins

that are exposed outside the cell wall. As shown in Fig. 5D,HtaA and HtaB were fully digested by proteinase K, suggestingthat these proteins are exposed on the surface of the bacteria.The levels of HmuT were reduced after protease treatment,but this protein was not fully digested like HtaA and HtaB.This finding suggests that HmuT is not as accessible to pro-teinase K as the other two proteins and may be partially pro-tected by the cell wall; a similar observation was reportedpreviously for the lipoprotein component of the ABC-typehemin transporter in S. aureus (24). DtxR, which is an intra-cellular protein, was not digested by proteinase K (Fig. 5D).

HtaA and HtaB are hemin binding proteins. UV-visiblespectroscopy was used to analyze the hemin binding propertiesof the purified GST-HtaA and HtaB proteins. The GST-HtaAand HtaB proteins at a concentration of 3.5 �M were incu-bated at room temperature for at least 15 min with hemin atconcentrations ranging from 0.5 �M to 10 �M. UV-visibleabsorption scans of the protein-hemin samples indicated thatboth GST-HtaA and HtaB showed strong absorption peaks at406 nm (Fig. 6A and B), which is an absorption maximumconsistent with the binding of hemin (24). Absorption mea-surements for HtaA in the absence of the GST tag could not beobtained due to proteolytic degradation of HtaA, which oc-curred after cleavage of the GST tag and the subsequent dial-ysis that was needed to remove the GST-protease cleavagebuffer. The GST-protease cleavage buffer was found to inter-fere with absorbance measurements for the HtaA-hemin com-plex. When the GST protein alone was incubated with 5 �Mhemin, it showed no absorption peak in the 400-nm region(Fig. 6C), indicating that it is does not bind hemin, as previ-ously reported (24). Measurements of absorption at 406 nm forGST-HtaA and HtaB with increasing hemin concentrationswere used to determine dissociation constants of 1.9 0.4 �Mfor GST-HtaA and 4.9 0.7 �M for HtaB (Fig. 6D and E).

The chromogenic substrate TMBZ, which has been usedpreviously to detect heme-dependent peroxidase activity that isassociated with hemin binding proteins (43), was used to ana-lyze the ability of the native form of the HtaA protein to bindhemin. TMBZ staining of secreted proteins from C. diphtheriae1737hmu� carrying plasmid pKhtaA grown in low-ironHIBTW medium showed that heme-dependent peroxidase ac-tivity was uniquely associated with the HtaA protein (Fig. 6F, �Hemin gel, lane 5). Peroxidase activity was also associated withHtaA that was present in culture supernatants of wild-typestrain 1737 in the absence of any plasmid (not shown). Noproteins were stained with TMBZ in supernatants from1737hmu� carrying only the plasmid vector (Fig. 6F, � Hemingel, lane 4). It was noted that HtaA exhibited hemin bindingeven in the absence of prebinding with hemin (Fig. 6F, �Hemin gel), which suggests that HtaA produced in C. diphthe-riae is bound to hemin (Fig. 6F, � Hemin gel, lane 5). PurifiedHtaA (without the GST tag) and the GST-HtaA fusion protein(lacking the signal peptide and the putative C-terminal mem-brane-spanning region) exhibited heme-dependent peroxidaseactivity in the presence of TMBZ (Fig. 6F, � Hemin gel, lanes2 and 3). Compared to HtaA, the purified HtaB proteinshowed very weak, nonspecific, heme-dependent peroxidaseactivity in the presence of TMBZ (data not shown). This ob-servation suggests that the HtaB-hemin complex is not stable

FIG. 5. (A) Western blot analysis to measure secretion or cell as-sociation of proteins expressed from C. diphtheriae wild-type strain1737. One-milliliter cultures were grown for 20 to 22 h at 37°C iniron-depleted HIBTW medium or in mPGT medium (only results forHtaA are shown for growth in mPGT medium). Polyclonal antiserum(specific to the proteins indicated on the left) was used for detection ofproteins associated with the cell pellet (Cell) and the supernatantfraction (Sup). Doublet bands for HtaA and HtaB indicate breakdownproducts, which exhibited some variability between protein prepara-tions. See Materials and Methods for a description of the method usedfor sample preparation. DT, diphtheria toxin. (B) Western blot detec-tion of HtaA in culture medium from various C. diphtheriae clinicalisolates after growth in iron-depleted HIBTW medium. (C) Westernblot analysis to identify the localization of proteins after cell fraction-ation of C. diphtheriae wild-type strain 1737. Cultures were grown inHIBTW medium or in mPGT medium (only results for HtaA areshown for mPGT medium), and polyclonal antiserum (specific to pro-teins indicated on the left) was used for detection of total cellularproteins. T, membrane and cytosolic fractions; S, soluble cytosolicproteins; CM, cytoplasmic membrane proteins. The total cellular frac-tion for HtaA was collected from the whole-cell lysate when the cellswere harvested. Samples were loaded using equivalent protein levels.(D) Western blotting to detect protein levels after proteinase K treat-ment of C. diphtheriae 1737hta�/pKhtaA grown in low-iron mPGTmedium. The antisera used for detection are indicated on the left(�HtaA, anti-HtaA; �HtaB, anti-HtaB; �HmuT, anti-HmuT; �DtxR,anti-DtxR), and the presence (�) or absence (�) of proteinase K (50�g/ml) is indicated at the top. See Materials and Methods for a de-scription of experimental details.

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under the conditions used to detect TMBZ-dependent perox-idase activity.

DISCUSSION

While hemin transport systems were first described in gram-negative bacteria (13, 41), the Corynebacterium HmuTUV sys-tem was the first hemin transporter to be identified in a gram-positive species (11). Over the last several years, hemintransport systems have been described in several additionalgram-positive organisms, including the important humanpathogens S. aureus (24) and S. pyogenes (3, 19). While bothgram-negative and gram-positive bacteria employ ABC-typetransporters and an associated substrate binding protein tomove hemin through the cytoplasmic membrane, these organ-isms utilize distinctly different components to bind hemin orhemoglobin to the cell surface. While gram-negative bacteria

bind hemin or hemoglobin to large outer membrane proteins,gram-positive bacteria, which do not have distinct outer mem-branes, bind hemin or hemoglobin to proteins anchored to thecell envelope. These surface receptors either are covalentlylinked to the cell wall peptidoglycan via sortases, as observedwith the Isd proteins in S. aureus (24, 46, 50), or are tetheredto the cytoplasmic membrane through a transmembrane C-terminal tail region, as proposed for the Shp and Shr proteinsin S. pyogenes (3, 19, 21, 49) and for HtaA and HtaB in C.diphtheriae.

Both HtaA and HtaB contain N-terminal signal sequencesand have putative transmembrane regions in their C termini,structural features that are also present in the S. pyogenes hemebinding proteins Shr and Shp (3, 19). Although HtaA andHtaB share structural and possibly functional similarities withthese S. pyogenes proteins, BLAST analysis shows that HtaAand HtaB have no significant sequence similarity to either Shp

FIG. 6. HtaA and HtaB are hemin binding proteins. (A and B) UV-visible spectroscopy was used to examine GST-HtaA (A) or HtaB (B) ata concentration of 3.5 �M in the presence of various hemin concentrations. Absorption scans were done at 300 to 600 nm. (C) Absorption scansfor GST-HtaA, HtaB, and GST in the presence of 5 �M hemin. (D and E) Absorption at 406 nm for GST-HtaA (D) and HtaB (E) at various heminconcentrations. Different concentrations of hemin (0 to 20 �M) were incubated with protein (3.5 �M) for at least 15 min at 20 to 25°C prior tomeasurement of absorbance. The values are the means and standard errors of three independent experiments. (F) Protein preparations wereincubated with 0.625 �M hemin (� Hemin) or with control buffer (� Hemin) prior to separation by SDS-PAGE and staining either withCoomassie blue (left panel) or with TMBZ to detect heme-dependent peroxidase activity as described in Materials and Methods (right panel).Lane 1, purified GST protein; lane 2, purified GST-HtaA fusion; lane 3, purified HtaA; lanes 4 and 5, ammonium sulfate-precipitated supernatantof 1737hmu� (hmu�) carrying either the vector (lane 4) or the cloned htaA gene on plasmid pKhtaA (lane 5); lane M, molecular weight marker.The � Hemin gel in the right panel (TMBZ stained) is a duplicate of the left panel (Coomassie blue stained).

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or Shr. A comparison of the C-terminal regions of these fourproteins reveals that they all contain a putative membrane-spanning region that is followed by a positively charged tailsequence, which in S. pyogenes is proposed to be involved inanchoring the proteins to the cytoplasmic membrane (Fig. 3B)(3, 19). HtaA contains only a single charge in this tail region,and membrane prediction models suggest that the HtaA trans-membrane segment is relatively weak compared to the mem-brane-spanning regions in HtaB, Shp, and Shr (http://www.ch.embnet.org/software/TMPRED_form.html).

In this study, the hmu hemin transport system in C. diphthe-riae was analyzed and shown to include genes whose productsare involved in the utilization of hemin and hemoglobin as ironsources. Deletion of htaA, hmuTUV, and the complete hmulocus resulted in a reduction in hemin iron use; however, eachof the mutants maintained the ability to use hemin iron forgrowth, which indicates that additional uptake and/or utiliza-tion systems for hemin iron acquisition are active in C. diph-theriae. The presence of more than one hemin iron utilizationand uptake system has been described or proposed for severalbacteria, including the gram-negative organisms Vibrio chol-erae (26) and Yersinia enterocolitica (41) and the gram-positiveorganisms S. pyogenes (3) and S. aureus (24). For many of theseorganisms, it was reported that mutations in the hemin-specificABC transporters did not eliminate the ability of these speciesto use hemin as an iron source, which suggested that alterna-tive transporters were active. Analysis of the genome of C.diphtheriae has revealed several ABC-type iron or siderophoretransporters (8), all of which show some sequence similarity topreviously identified hemin transporters. However, the prod-ucts encoded by the hmuTUV genes show the highest levels ofsequence similarity to previously described hemin uptake sys-tems (11). Genes that are predicted to encode proteins withsequence similarity to HtaA (DIP0522 and ChtA) and HtaB(ChtB) are present in the C. diphtheriae genome, and all ofthese factors contain putative N-terminal secretion signals, aswell as C-terminal transmembrane regions (8, 14). It is possiblethat DIP0522 and/or ChtA may contribute to the hemin ironuptake activity observed in an htaA mutant, and similarly, theproduct of chtB may complement an htaB mutant. Furtherstudies are needed to determine whether DIP0522, ChtA,ChtB, HtaB, or any of the other related iron uptake systemsencoded in the C. diphtheriae genome is involved in hemin ironutilization.

BLAST analysis (1; http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) has revealed that bacterial species related to C. diph-theriae, including C. ulcerans, C. glutamicum, C. jeikeium, andStreptomyces coelicolor, all contain genes whose predictedproducts have significant sequence similarity to HtaA, and allof these putative proteins contain transmembrane regions intheir C termini followed by positively charged residues, anobservation that indicates that there is a common anchoringmechanism in this group of related proteins. However, somedifferences in the anchoring of these proteins to the cell enve-lope have been observed. A recent study of Propionibacteriumacnes, an organism related to Corynebacterium, identified aputative hemin iron transport protein (PA-21693) that hassequence similarity to HtaA but contains a sortase anchoringmotif in its C terminus, suggesting that PA-21693 is covalentlyanchored to the cell wall through the action of a sortase (23).

It is not clear why certain hemin or hemoglobin binding pro-teins in gram-positive bacteria are covalently anchored to thecell wall by sortases, while others, such as HtaA and Shp, aretethered to the surface by a single membrane-spanning region.A covalent attachment to the cell wall appears to be moresecure than binding to the cell by a single transmembraneregion. All of the gram-positive species that are known tocontain surface-anchored hemin binding proteins produce sor-tases and numerous sortase substrates, indicating that a lack ofsortases is not the reason for the differences in the anchoringof these proteins. It is possible that differences in how theseproteins are attached to the cell envelope may be attributed tothe different environments that these organisms inhabit and/orto additional functions that may be associated with these sur-face proteins, such as adhesins.

An unexpected finding in this study was the observation thatHtaA is secreted during growth in nutrient-rich HIBTW me-dium but is predominantly cell associated during growth inmPGT medium, a semidefined minimal medium that is com-monly used to culture C. diphtheriae strains (44). The presenceof a C-terminal transmembrane region in HtaA predicts thatthe protein is associated with the cytoplasmic membrane,which is observed for HtaB when C. diphtheriae is grown ineither HIBTW or mPGT medium. However, analysis of theC-terminal amino acid sequence of HtaA indicates that HtaAhas a relatively weak transmembrane region compared to themembrane-spanning region in HtaB (http://www.ch.embnet.org/software/TMPRED_form.html). Although a less-than-optimal membrane-spanning sequence may indicate a weakerassociation with the membrane, it would not account for thedifferences in localization observed between organisms grownin HIBTW and mPGT media. The reason for the difference inlocalization of HtaA between organisms grown in the twomedia is not known, although differences in membrane com-position, permeability, cellular metabolism, or the presence ofproteases may contribute to the observed differences in thelocation of HtaA. While it is not known if HtaA is secreted ormembrane bound in vivo, studies reported here showed thatwhen the HtaA protein was added to mPGT culture medium,there was no stimulation of growth of the C. diphtheriae 1737htaA deletion mutant when hemoglobin was the sole ironsource (Fig. 4B). This finding suggests that the secreted formof HtaA may not be involved in hemin iron uptake.

In a previous report (35), we were unable to demonstrate ahemin iron utilization deficiency in a C. diphtheriae hmuT vec-tor integration mutant (RT5), but we observed that a similarlyconstructed hmuT mutant of C. ulcerans was defective in he-min iron use. This previous study was performed using nutri-ent-rich HIBTW medium, whereas mPGT medium was used asthe growth medium in the studies described in this report. TheC. diphtheriae RT5 mutant does exhibit a hemin iron utilizationdefect similar to that observed with the hmuTUV deletionmutant when cells are grown in mPGT medium (M. P. Schmitt,unpublished observation). The reason for the lack of a pheno-type for the C. diphtheriae hmuT mutant in HIBTW medium isnot known; however, as noted above, C. diphtheriae hmu mu-tants are able to use hemin iron even in mPGT medium, whichsuggests that there are alternate hemin uptake or utilizationsystems. It is possible that these systems (or others) may havemore robust activity during growth in HIBTW medium than

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during growth in mPGT medium and can fully substitute forthe hmu system during growth in HIBTW medium. Differencesin metabolism or in heme or iron requirements may also con-tribute to the observed difference in hemin iron use betweenthe two media.

Work in our laboratory has demonstrated that hemin andhemoglobin are equally capable of supplying iron for growth ofC. diphtheriae strains (16, 31, 35). While it is not known how C.diphtheriae acquires the hemin moiety from hemoglobin, arecent study has suggested that one mechanism by which he-min iron is obtained for use by bacteria is through the spon-taneous release of hemin from hemoglobin (25). It is alsopossible that secreted or surface-exposed proteases in C. diph-theriae contribute to the breakdown of hemoglobin and subse-quent release of hemin. In the model shown in Fig. 7, wepropose that hemin initially binds to HtaA at one or both ofthe conserved domain regions. Hemin binding sites have notbeen identified for HtaA, and future studies should determineif these conserved regions are involved in hemin binding ortransport. It is predicted that hemin bound to HtaA is trans-ferred to HtaB, which may function in an intermediate step inthe movement of hemin from HtaA to HmuT. This mechanismof action for HtaB would be similar to the function proposedfor some of the Isd proteins from S. aureus, which are involvedin the movement of hemin through the cell wall (22, 46, 50).HmuT is proposed to deliver hemin to the HmuU permease, acomponent of the ABC transporter (HmuU and HmuV),which facilitates the uptake of hemin into the cytosol, where itis thought to be degraded by the heme oxygenase enzymeHmuO, releasing iron for cellular metabolism.

ACKNOWLEDGMENTS

We thank Lori Keating and Karen Meysick for helpful comments onthe manuscript.

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