Identification of a Locus Involved in the Utilization of Iron by Haemophilus influenzae

INFECrION AND IMMUNITY, OCt. 1994, p. 4515-4525 0019-9567/94/$04.00+0 Copyright 1994, American Society for Microbiology Vol. 62, No. 10 Identification of a Locus Involved in the Utilization of Iron by Haemophilus influenzae JERRY D. SANDERS, LESLIE D. COPE, AND ERIC J. HANSEN* Department of Microbiology, University of Texas Southwestem Medical Center, Dallas, Texas 75235-9048 Received 3 May 1994/Accepted 21 July 1994 Haemophilus influenzae has an absolute requirement for heme for aerobic growth. This organism can satisfy this requirement by synthesizing heme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme. In contrast, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme. A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DLA2 the ability to grow on iron and PPIX. Nucleotide sequence analysis revealed that this NTHI DNA insert contained three genes, designated hita, hitb, and hitc, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmic binding protein-dependent iron transport system in this enteric organism. The NTHI HitA protein also was 69%o identical to the ferric-binding protein ofneisseria gonorrhoeae. Inactivation of the cloned NTHI hitc gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth deficiency of Hib DL42. Construction of an isogenic NTHI TN106 mutant lacking a functional hitc gene revealed that this mutation prevented this strain from growing on a medium containing iron and PPIX in place of heme. This NTHI hitc mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the products encoded by the hitabc genes are essential for the utilization of iron by NTHI. The best-known phenotypic trait of Haemophilus influenzae is its absolute requirement for both heme and NAD (i.e., the classical X and V factors, respectively) for aerobic growth (16). This heme requirement is caused by the inability of H. influenzae to carry out the sequence of biochemical reactions necessary to convert 8-amino-levulinic acid to protoporphyrin IX (PPIX), the immediate biosynthetic precursor of heme (18, 58). H. influenzae does possess the ability to take up PPIX and convert it to heme via the activity of the enzyme ferrochelatase, which inserts a single molecule of iron into the PPIX molecule. Accordingly, PPIX can satisfy the heme requirement of H. influenzae in vitro only if a utilizable form of iron is available in the growth medium (18, 58). H. influenzae possesses the ability to utilize many different sources of iron, including both heme (43) and iron-loaded transferrin (25). In the latter instance, it would appear that H. influenzae, in a manner similar to Neisseria gonorrhoeae and Neisseria meningitidis (7, 50), binds the iron-loaded transferrin molecule to its cell surface (49) and then, by an undefined mechanism(s), extracts the iron from the transferrin and transports it into the cell. It is also known that, in the absence of heme, iron salts and chelates can suffice to provide iron for H. influenzae, provided that PPIX is also available as the source of the porphyrin ring (43, 60). However, there is no information available concerning how iron actually enters the H. influenzae cell after transferrin, heme, or iron chelates are taken up. In this study, we describe the identification of an iron utilization system in H. influenzae that is similar to a periplas- * Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9048. Phone: (214) 648-2284. Fax: (214) 648-2199. 4515 mic binding protein-dependent iron transport system in Serratia marcescens (2, 3, 63). Mutant analysis was used to prove that this system is required for the utilization of iron by H. influenzae when PPIX is substituted for heme in the growth medium. MATERUILS AND METHODS Bacterial strains and culture media. H. influenzae type b (Hib) strain DL42 and nontypeable H. influenzae (NTHI) strain TN106 have both been described in detail (12, 37). The other Hib and NTHI strains used for Western blot (immunoblot) and Southern blot analyses have also been described (11). Mutant and recombinant strains derived from Hib strain DM42 and NTHI strain TN106 are listed in Table 1. H. influenzae strains were routinely grown by using brain heart infusion medium (Difco Laboratories, Detroit, Mich.) supplemented with Levinthal's base (1) (BHIs) at 37 C with aeration (for broth cultures) or at the same temperature in an atmosphere of 5% C02-95% air (for agar plates). The ability of H. influenzae strains to utilize iron and PPIX to satisfy their heme requirement was assessed by using BHI agar plates containing NAD (10,g/ml) and PPIX (20 pug/ml); there is sufficient free iron in this medium for growth of H. influenzae using PPIX in place of heme. Antimicrobial agent supplementation for H. influenzae strains included kanamycin (20,ug/ml), tetracycline (5,ug/ml), or chloramphenicol at either 0.8,ug/ml (for mutants with a cat cartridge insertion in the chromosome) or 2.0,ug/ml (for strains with plasmids containing the cat cartridge). The Escherichia coli cloning host HB101 was grown in LB medium (34) with appropriate antimicrobial agent supplementation. Assessment of utilization of various iron sources by wildtype and mutant NTHI strains. The basal medium was BHI agar containing NAD (10,ug/ml) and deferoxamine mesylate

4516 SANDERS ET AL. INFECT. IMMUN. TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristic(s) Source or reference Strains Hib DL42 Virulent wild-type strain; cannot grow on iron and PPIX 20 DLA2(pJDS150) DL42 containing pjds150; can grow on iron and PPIX This study DL42(pJDS151) DM42 containing pjds151; cannot grow on iron and PPIX This study DL42(pJDS152) DL42 containing pjds152; can grow on iron and PPIX This study DLA2(pJDS153) DM42 containing pjds153; cannot grow on iron and PPIX This study DL42(pJDS154) DM42 containing pjds154; can grow on iron and PPIX This study DL42(pJDS155) DM42 containing pjds155; cannot grow on iron and PPIX This study H. influenzae Rd DB117 Recombination-deficient (rec-1) mutant strain used as a host for library construction 51 and certain subcloning experiments NTHI TN106 Isolate from a transtracheal aspirate from a patient with NTHI pneumonia; wild-type 47 strain which can grow on iron and PPIX TN106.155 TN106 with the hitc gene inactivated, accomplished by transforming TN106 with This study linearized pjds155; cannot grow on iron and PPIX TN106.155.T TN106.155 transformed with the 6.5-kb PstI fragment from recombinant plasmid This study pjds152; has a functional hitc gene and can grow on iron and PPIX E. coli HB101 Used as host strain for pbluescript II SK+ 46 Plasmids pls88 Shuttle vector capable of replication in E. coli and H. infiuenzae 61 pgjb103 Shuttle vector capable of replication in E. coli and H. influenzae S pbluescript II SK+ Plasmid vector Stratagene pjds150 pls88 with an 11.5-kb EcoRI fragment of NTHI TN106 DNA containing the intact This study hita, hitb, and hitc genes pjds151 pgjb103 containing a 7.5-kb EcoRI-ClaI fragment derived from the 11.5-kb insert in This study pjds150; contains intact hita and hitb genes pjds152 pgjb103 with the 6.5-kb PstI fragment derived from the 11.5-kb insert in pjds150; This study contains intact hitb and hitc genes pjds153 pgjb103 with the 4.5-kb PvuII-PstI fragment derived from the 6.5-kb PstI insert in This study pjds152; contains an intact hitc gene pjds154 pgjb103 with the 5.8-kb PstI-PvuII fragment derived from the 6.5-kb PstI insert in This study pjds152; contains intact hitb and hitc genes pjds155 pgjb103 with the ClaI sites of the vector removed and the 6.5-kb PstI fragment of This study pjds152 ligated into the PstI site; a cat cartridge was then ligated into the ClaI site within the hitc gene in this insert (Desferal; Ciba-Geigy Ltd., Basel, Switzerland) at a final concentration of either 0.04 or 0.08 mm. A suspension of H. influenzae from a BHIs plate was suspended in BHI broth, centrifuged at 4,000 x g for 10 min at 4 C, and resuspended in BHI broth to yield a Klett-Summerson colorimeter reading (Klett Manufacturing Company, New York, N.Y.) of 35 (equivalent to a final concentration of approximately 2 x 107 CFU/ml). This suspension of organisms was diluted 1:10 in BHI broth, and a 0.1-ml portion of this final suspension was spread on the surface of the test plate. After 30 min, sterile filter paper disks (4-mm diameter) were placed on the surface of the agar, the various iron sources (10,ul) were spotted onto these disks, and the plate was incubated overnight. The iron sources included heme (250,ug/ml), PPIX (250,ug/ml; a negative control), iron-loaded human transferrin (98% iron saturated; Sigma Chemical Co., St. Louis, Mo.) (20 mg/ml), ferric ammonium citrate (5.2 mg/ml), and ferric nitrate (8 mg/ml). Plasmids. The plasmid pbluescript II SK+ was obtained from Stratagene (La Jolla, Calif.). The plasmid shuttle vector pgjb103 (5, 53) was obtained from Gerard J. Barcak, and the plasmid shuttle vector pls88 (61) was provided by William L. Albritton. Recombinant DNA methods. Standard recombinant DNA methods including restriction enzyme digestions, ligation reactions, agarose gel electrophoresis, and plasmid purifications were performed as described previously (34, 46). Restriction enzymes, DNA polymerase Klenow fragment, and BglII, ClaI, and PstI linkers were purchased from New England Biolabs (Beverly, Mass.). The cat gene encoding chloramphenicol acetyltransferase and lacking an internal EcoRI site was derived from the plasmid puc4decat, which was kindly provided by Bruce A. Green. After excision from puc4decat with EcoRI, ClaI linkers were added to the cat cartridge (21). Construction of an NTHI strain TN106 genomic DNA library. Chromosomal DNA purified from NTHI strain TN106 by the method of Marmur (35) was partially digested with EcoRI. DNA fragments (6 to 15 kb) isolated by sucrose density gradient centrifugation were ligated into the EcoRI site of plasmid pls88 (61). This ligation reaction mixture was used to transform H. influenzae Rd strain DB117 as described previously (12, 20), yielding 166,000 recombinants. These recombinants were pooled and grown in BHIs broth, and plasmids representing the NTHI TN106 genomic DNA library were purified from this culture as described previously (12). H. influenzae genetic transformation methods. The glycerol-

VOL. 62, 1994 lactate shock method (52) was used to transform Hib strain DL42 with the shuttle vectors pls88 and pgjb103 and recombinant plasmids derived from these vectors. Transformants were selected on BHIs agar plates containing either kanamycin (20 p.g/ml) (when pls88-based plasmids were used) or tetracycline (5,ug/ml) (for pgjb103-derived plasmids). For transformation of NTHI with linear DNA molecules, either the M1v-based method of Herriott (26) or the BHIs agar plate-based method of Sanders et al. (47) was used. With the latter method, the segregation and outgrowth step was not performed in BHIs broth but instead was accomplished using BHIs agar plates. The resultant bacterial growth was suspended in BHI broth, serially diluted, and plated on appropriate selective media. MAbs. Monoclonal antibody (MAb) 6B8 is directed against the 40-kDa H. influenzae iron-regulated protein previously described as being located in the periplasmic space of this organism (24) and which was designated HitA in this study. To produce a MAb with this antigenic specificity, the HitA protein was purified from Hib strain DL42 for use as an immunogen. Whole cells of this strain, grown overnight on BHI agar plates containing NAD (10,ug/ml), PPIX (0.5,ug/ml), and deferoxamine mesylate (0.8 mm), were disrupted by sonication, and the cell envelopes were pelleted by centrifugation. The resultant supernatant fluid was concentrated by means of a Centriprep concentrator (Amicon, Beverly, Mass.) and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The most abundant protein in this preparation had an apparent molecular mass of 40 kda in SDS-PAGE. N-terminal amino acid sequence analysis of this protein revealed that 13 of the first 14 amino acids were identical to those of the iron-regulated 40-kDa periplasmic protein of H. influenzae (24). This protein was eluted from the gel and used to immunize mice. Spleen cells from these immunized mice were fused with SP2/0-Agl4 plasmacytoma cells, and lymphocyte hybridomas were selected as described previously (44). Culture supernatants from these hybridomas were screened in Western blot analysis using the gel-purified 40-kDa Hib DL42 protein (HitA) as the antigen. MAb 6B8 reacted with a single antigen (approximate molecular mass of 37 to 40 kda) of every Hib and NTHI strain tested to date in Western blot analysis (data not shown). N-terminal amino acid sequence analysis. The HitA protein present in the supernatant from sonicated Hib DL42 cells was subjected to SDS-PAGE by the method of Hunkapiller et al. (29). The gel-resolved HitA protein band was transferred to a polyvinylidene difluoride membrane by the method of Matsudaira (36) and subjected to N-terminal amino acid analysis as described previously (22). Western blot analysis. Detection of the reactivity of the murine MAb 6B8 with the H. influenzae HitA protein in Western blot analysis was accomplished as described previously (30). Nucleotide sequence analysis. H. influenzae DNA inserts contained in pbluescript II SK+ were sequenced by standard techniques, including the use of nested deletions (46). In all cases, both strands were sequenced in their entirety. DNA sequence information was analyzed using the Intelligenetics Suite package and programs from the University of Wisconsin Genetics Computer Group software sequence analysis package (15). Southern blot analysis. Chromosomal DNA purified from wild-type, mutant, and transformant H. influenzae strains was digested to completion with appropriate restriction enzymes and probed in Southern blot analysis (46). The DNA probes used in these experiments included the 1.3-kb cat cartridge H. INFLUENZAE IRON UTILIZATION SYSTEM 4517 Hib DL42 NTHI TN 106 Hib DL42(pLS88) Hib DL42(pJDS1 50) (A) HEME (B) PPIX FIG. 1. Growth of wild-type and recombinant H. influenzae strains on BHI-NAD agar containing either heme (10,ug/ml) (A) or PPIX (20,ug/ml) (B). Mid-logarithmic- hase cultures in BHIs medium were diluted to 1 x 103 to 3 x 103 CFU/ml, and 0.1-ml portions of these suspensions were plated on the two types of solidified media and then incubated overnight as described in Materials and Methods. The strains tested in this experiment included the wild-type Hib strain DL42, the wild-type NTHI strain TN106, Hib DL42 carrying the plasmid shuttle vector pls88, and Hib DL42 carrying the recombinant plasmid pjds150. For the last two strains, the growth medium also contained kanamycin (20,ug/ml). from puc4decat, a 0.89-kb PstI-SspI fragment containing the majority of the hita gene, a 0.64-kb MunI-PvuII fragment containing half of the hitb gene, and a 0.57-kb BsrBI fragment from the hitc gene. RESULTS Growth of H. influenzae strains on iron and PPIX. In preliminary experiments examining the ability of selected Hib and NTHI strains to utilize heme compounds and precursors, it was noted that Hib strain DM42, which grows readily on BHI-NAD medium containing heme (Fig. 1A), could not form single colonies on BHI-NAD agar plates containing iron and PPIX in place of heme (Fig. 1B). Varying the concentration of PPIX in the medium from 0.5 to 100 p,g/ml still did not allow this Hib strain to grow, a finding which indicated that some sort of toxicity associated with relatively high levels of PPIX was not responsible for this phenotype. This finding in itself was not surprising, in that it had been previously reported that 8% of H. influenzae strains could not use iron and PPIX in place of

4518 SANDERS ET AL. INFECT. IMMUN. pjds1 50 4. C1 o L-.0 u C - 4-' 0.~ = QCL~ -=l.r--0 QOCLC(A o9rfi1 I orf 2 hita orf 3 [ ~ m U f) hitb orf 4 hitc orf 5 Iorf 61F orf7 = (a g 4it5 pjds1 51 pjds1 52 pjds1 53 pjds1 54 pjds1 55 1 Kb FIG. 2. Partial restriction enzyme map of the NTHI DNA inserts in pjds150 and related plasmids. All of the DNA extending from the AvaI site located 5' from ORF 1 to the PvuII site beyond ORF 7 in pjds150 was sequenced, and the ORFs detected in this region are depicted together with arrows indicating the predicted direction of transcription of each ORF. The plus and minus signs to the right of each plasmid's DNA insert indicate the ability of that plasmid to transform Hib DL42 to the ability to grow on BHI-NAD plates containing iron and PPIX in place of heme. The triangle beneath the insert from pjds155 indicates the ClaI site where a cat cartridge was inserted into the hitc gene within this plasmid's NTHI DNA insert. heme (25). In contrast, an NTHI strain (TN106) used routinely in this laboratory grew well on the same medium containing iron and PPIX (Fig. 1B). As expected, this same NTHI strain grew well on BHI-NAD agar containing heme (Fig. 1A). Cloning of an NTHI locus that permits growth of Hib DL42 on iron and PPIX. A genetic approach was used to address the interesting difference between Hib DL42 and NTHI TN106 in their relative abilities to grow on iron and PPIX. A genomic library constructed from NTHI strain TN106 in the shuttle vector pls88 was used to transform Hib strain DL42. The transformation reaction mixture was plated on BHI-NAD agar containing PPIX (20,ug/ml) and kanamycin and yielded 32 colonies. Each of these transformants was purified by singlecolony isolation, and their respective recombinant plasmids were isolated by a miniprep technique and used to transform Hib DML2. This second set of transformation reaction mixtures was plated on BHIs agar plates containing kanamycin (to avoid selecting possible spontaneous mutants able to utilize iron and PPIX). One transformant from each reaction mixture was purified and tested for its ability to grow on iron and PPIX. Only 2 of the 32 new transformants grew on iron and PPIX. The recombinant plasmids from these two transformants had very large NTHI DNA inserts of 11.5 and 16 kb. The plasmid with the smaller insert was selected for further study and designated pjds150 (Fig. 2). The recombinant Hib strain DL42(pJDS150) grew readily on iron and PPIX (Fig. 1B), whereas DL42 containing only the shuttle vector pls88 could not form single colonies on the same medium (Fig. 1B). Localization of the relevant genetic locus. Subcloning of restriction fragments from the 11.5-kb NTHI DNA insert in pjds150 involved the use of the shuttle vector pgjb103 which, in the experience of this laboratory, is more amenable for use in transforming both the H. influenzae Rd strain DB117 and Hib strain DL42 (data not shown). Neither the 7.5-kb EcoRI-Clal fragment in pjds151 nor the 4.5-kb PvuII-PstI segment in pjds153 could transform DL42 to grow on iron and PPIX (Fig. 2). However, the 6.5-kb PstI fragment contained in pjds152 was as effective as pjds150 in conferring on Hib DL42 the ability to form single colonies on iron and PPIX (Fig. 2). One additional subcloning experiment which resulted in the construction of pjds154 revealed that the 0.8-kb PvuII-PstI fragment in pjds152 was not involved in converting Hib strain DL42 to this altered phenotype (Fig. 2). Nucleotide sequence analysis. Three fragments of the NTHI insert in pjds150 were subcloned into pbluescript II SK+ for sequencing. These included the 2.7-kb AvaI-PstI, the 2.0-kb + +

VOL. 62, 1994 PstI-PvuII, and the 3.8-kb PvuII-PvuII fragments (Fig. 2). Upon evaluation of the nucleotide sequences of these three fragments, it appeared that a small DNA fragment located between the AvaI-PstI and PstI-PvuII fragments was missing, and therefore, double-stranded DNA sequencing of this region in pjds151 was performed. This analysis revealed the presence of a small (76-bp) PstI fragment located between these two fragments (Fig. 2). Seven open reading frames (ORFs) were readily identifiable within the 8.5 kb of DNA sequence (Fig. 2). Five of these ORFs were arranged sequentially on one strand, with the other two in tandem on the opposite strand. The first ORF (ORF 1 in Fig. 2) encoded a predicted protein with a calculated molecular weight of 26,598 which was 52% identical to the 24.5-kDa hypothetical protein from Pseudomonas aeruginosa suggested to be involved in proline biosynthesis (57). The protein encoded by the second ORF (ORF 2 in Fig. 2) had no significant homology with any proteins in the available data bases. On the opposite DNA strand, ORF 6 encoded a protein with a molecular weight of 30,470 which was 24% identical to the D-alanine-D-alanine ligase B of E. coli (45), while the protein encoded by ORF 7 on the same DNA strand had a calculated molecular weight of 41,470 and was 61% identical to the succinyl-diaminopimelate desuccinylase of E. coli (8, 62). Identification ofh. influenzae proteins putatively involved in iron utilization. The three ORFs in the middle of the sequenced DNA (ORFs 3 to 5 in Fig. 2) encoded proteins with significant similarity to a set of proteins (SfuA, SfuB, and SfuC) from S. marcescens that are involved in a periplasmic binding protein-dependent iron transport system (2, 3, 63). The protein encoded by ORF 3 had a calculated molecular weight of 36,218 and was 38% identical to the SfuA protein of S. marcescens (see Fig. 4) (2). This S. marcescens protein is the periplasmic binding component of this iron transport system (2, 3). The H. influenzae protein encoded by ORF 3 was also 69% identical to the major iron-regulated protein of N. gonorrhoeae, designated as ferric-binding protein (Fbp) (6). Because of the similarity between this H. influenzae protein and both the SfuA and Fbp molecules and because the last two proteins are involved in iron transport in their respective organisms (2, 3, 10), we tentatively designated the protein encoded by ORF 3 as H. influenzae iron transport protein A (HitA). A MAb (6B8) raised against the 40-kDa iron-regulated periplasmic protein from Hib strain DL42 reacted with the recombinant NTHI HitA protein expressed in E. coli (data not shown). Hydrophobicity analysis of the N-terminal region of the NTHI HitA protein indicated that the first 23 amino acids formed a hydrophobic domain similar to that of leader peptides (54). N-terminal amino acid sequence analysis of the 40-kDa periplasmic, iron-regulated protein expressed by Hib strain DL42 revealed that the N-terminal sequence of this mature Hib protein was identical to that of residues 24 to 37 of the NTHI HitA protein (Fig. 3) and nearly identical to the N terminus of the periplasmic iron-regulated protein from another H. influenzae strain (24), suggesting that the NTHI HitA protein was synthesized with a leader peptide. It was also noted that the hita gene was preceded by a 19-nucleotide sequence that weakly resembled (11 of 19 matches) the E. coli Fur consensus binding sequence (33). Whether this sequence plays a role in the expression of the HitA protein is not known. The fourth ORF detected in the sequenced DNA started approximately 120 nucleotides downstream from the stop codon in hita. The protein encoded by ORF 4 had a calculated molecular weight of 51,232 and was 37% identical to the S. marcescens SfuB protein (Fig. 4), which functions as an integral H. INFLUENZAE IRON UTILIZATION SYSTEM 4519 cytoplasmic membrane protein in the S. marcescens iron transport system (2). This protein encoded by ORF 4 was designated HitB. The putative start codon for this ORF was TTG (Fig. 3), based on the observation that the deduced amino acid sequence following this codon was similar to the N-terminal region of the SfuB protein (Fig. 4). ORF 5, the last of these three ORFs, overlapped the extreme 3' end of ORF 4 and encoded a protein with a molecular weight of 40,410. This protein exhibited 38% identity with the SfuC protein of S. marcescens (Fig. 4), which functions as an ATP-binding protein in the S. marcescens iron transport system (2), and was designated HitC. Similarly to SfuC, the HitC protein possessed the consensus A and B regions common to nucleotide-binding proteins of ABC transporters (17, 27) (Fig. 4). In addition, it should be noted that the start codon for the hitc gene overlaps the end of the hitb gene (Fig. 3). In S. marcescens, the start codon for the sfuc gene similarly overlaps the stop codon of the sfub gene (2). Use of mutant analysis to demonstrate involvement of the hitabc genes in utilization of iron and PPIX. The subcloning experiments described above had localized the genetic locus in pjds150 which allowed Hib strain DM42 to utilize iron and PPIX to the 5.8 kb PstI-PvuII fragment in pjds154. The possible involvement of the NTHI hitabc genes in this complementation activity was investigated directly by mutant analysis. A cat cartridge was inserted into the ClaI site within the hitc gene in pjds152, resulting in the mutated plasmid pjds155 (Fig. 2). When pjds155 was transformed into Hib strain DML2, the resultant recombinant could not grow on BHI-NAD agar plates containing iron and PPIX (Fig. 2). Construction of an isogenic NTHI hitc mutant. The inabilities of pjds155 and pjds151 to allow growth of Hib strain DM42 on medium containing iron and PPIX raised the possibility that the hitc gene product was directly involved in the growth of H. influenzae on this medium. To investigate this, pjds155 with the mutated hitc gene was linearized by digestion with BglII and used to transform the wild-type NTHI strain TN106. Transformants were selected on BHIs medium containing chloramphenicol. From a total of 52 chloramphenicol-resistant transformants, eight were tested for growth on BHI-NAD agar containing iron and PPIX in place of heme and found to be incapable of forming single colonies on this medium. One of these, designated TN106.155, was selected for further study. To confirm that proper allelic exchange had occurred in this mutant strain, chromosomal DNA preparations from the wildtype NTHI strain TN106 and the mutant strain TN106.155 were digested with PstI and probed in Southern blot analysis with probes for both the hitc gene and the cat cartridge. When the wild-type chromosomal DNA was probed with a 0.57-kb BsrBI fragment from the hitc gene, a 6.5-kb PstI fragment bound this probe, as expected (Fig. 5, panel 1, lane A). In contrast, the chromosomal DNA from the mutant strain TN106.155 had a 7.8-kb PstI fragment that hybridized with this hitc probe (Fig. 5, panel 1, lane B), consistent with the presence of the 1.3-kb cat cartridge within the hitc gene in the chromosome of this mutant. The presence of the cat cartridge within the hitc gene of the mutant strain was confirmed by the use of the cat cartridge probe (Fig. 5, panel 2, lane B). As expected, the DNA of the wild-type strain did not bind this probe (Fig. 5, panel 2, lane A). Utilization of iron sources by the wild-type and mutant NTHI strains. To determine directly whether the hitc gene product was involved in iron utilization, the mutant and wild-type NTHI strains were plated on BHI-NAD agar containing PPIX (as the porphyrin source) and deferoxamine

4520 SANDERS ET AL. INFECT. IMMUN. 1 127 CAATGATAAATTGATTGATGAAACTTATGATTGCAGTGAGTTATTTAATGCGCTTTGTGATGTATTAACAAATTTAGGTTATATCCAGCCTGCAAACCTATAATCTGTGGACGGTGCGTGTTATGTA 128-35 -35-10 -10 SD 254 CCGTTTTGTTTTTCATCTAAATATCAAT TTAAAAAATT6,""''...'MAGGCATAG AATAG-CTGC AATTCAG GCTTAACTGAATATTTGCACTCAACATAAGGAGCTTTTCA 255 350 ATG CAA TTT AAA CAT TTC AAA CTT GCT ACC CTT GCT GCA GCA CTT GCT TTT TCT GCT MT AGT TTT GCT GAT ATT ACC GTT TAT MT GGT CAG CAC M Q F K H F K L A T L A A A L A F S A N S F A D I T V Y N G Q 351 H 446 AAA GM GCC GCT GCT GCA GTG GCA AAA GCC TTT GM CAG GM ACA GGC ATT AAA GTT ACG CTA MT AGC GGG AM AGT GCG CM CTT GCA GGT CM K E A A A A V A K A F E Q E T G I K V T L N S G K S A Q L A G Q 447 542 TTA AAA GM GM GGC GAT AAA ACA CCC GCC GAT GTT TTC TAT ACT GM CM ACA GCG ACT TTT GCT GAT CTT TCT GM GCA GGG CTT TTA GCA CCA L K E E G D K T P A D V F Y T E Q T A T F A D L S E A G L L A P 543 638 ATT TCA GM CM ACA ATT AAA CM ACT GCA CM AAA GGC GTA CCA CTT GCA CCG AAA AAA GAC TGG GTT GCA TTA AGT GGC CGT TCT CGC GTA GTG I S E Q T I K Q T A Q K G V P L A P K K D W V A L S G R S R V V 639 734 GTT TAC GAT CAC ACT AAA TTA TCT GM AAA GAT ATG GM AAA TCA GTG CTT GAT TAT GCA ACA CCA AAA TGG AAA GGC AAA ATC GGT TAT GTA TCA V Y D H T K L S E K D M E K S V L D Y A T P K W K G K I G Y V S 735 830 ACT TCT GGT GCG TTC TTA GAG CM GTT GTT GCT TTA AGC AAA ATG AAA GGG GAC AAA GTT GCG CTT MT TGG TTA AAA GGC TTA AAA GAG MC GGT T S G A F L E Q V V A L S K M K G D K V A L N W L K G L K E N G 831 926 AAA CTT TAT GCT AAA MT AGT GTG GCA TTA CM GCG GTC GM MT GGT GM GTG CCT GCT GCG TTA ATC MT MC TAT TAT TGG TAT MC CTT GCA K L Y A K N S V A L Q A V E N G E V P A A L I N N Y Y W Y N L A 927 1022 AAA GAA AAA GGC GTG GM MT CTA AAA AGC CGT CTT TAT TTT GTT CGC CAC CM GAT CCA GGT GCG TTA GTT AGC TAT TCA GGT GCA GCA GTA TTA K E K G V E N L K S R L Y F V R H Q D P G A L V S Y S G A A V L 1023 1118 AAA GCC TCT AAA MT CM GCT GM GCA CM AAA TTC GTT GAT TTC TTA GCA AGT AAA AAA GGT CM GM GCA TTA GTG GCA GTG CGT GCA GM TAT K A S K N Q A E A Q K F V D F L A S K K G Q E A L V A V R A E Y 1119 1214 CCA TTA CGC GCT GAT GTG GTT TCG CCA TTT MT CTT GM CCT TAT GM AAA TTA GM GCA CCA GTG GTA TCC GCA ACA ACA GCT CM GAT AAA GAG P L R A D V V S P F N L E P Y E K L E A P V V S A T T A Q D K E 1215-35 -35-10 -10 1328 CAT GCG ACC AAA TTA ATT GM GM GCT GGA TTG AAA TM TCCAAATATTGCGTGTGTGGATTTGAAAAMTTCACGCACGCGTTTACMTTCTCTACCAAAATTMCCGCTTGC H A T K L I E E A G L K 1329 SD 1433 GTGCGTGMCTGAAATGTTCACATTCCTAAAAGGATTTAACT TTG CCT CGC AGA CCG CCA TTC TGG CTT ACT TTA CTT ATC ATC TTA ATC GGA CTT CCG TTA TGT M P R R P P F W L T L L I I L I G L P L C 1434 1529 TTG CCG TTT CTG TAT GTC ATT TTG CGT GCG ACA GM GTG GGA TTA ACG CGA AGT GTT GAG CTA TTG TTT CGC CCT CGA ATG GCT GM TTA TTA AGC L P F L Y V I L R A T E V G L T R S V E L L F R P R M A E L L S 1530 1625 MT ACA ATG CTT TTA ATG GTT TGT GTA ACT ATT GGT GCT ATT TCA CTT GGT ACG CTT TGT GCT TTT TTA CTT GM CGT TAT CGC TTT TTC GGT AAA N T M L L M V C V T I G A I S L G T L C A F L L E R Y R F F G K 1626 1721 GCT TTT TTT GM GTG GCG ATG ACG TTA CCC CTT TGT ATT CCC GCC TTT GTA AGT TGT TTT ACT TGG ATC AGC CTC ACA TTC CGC GTT GAG GGT TTT A F F E V A M T L P L C I P A F V S C F T W I S L T F R V E G F 1722 1817 TGG GGA ACA ATT GGT ATT ATG ACA TTA AGT TCA TTT CCT TTG GCT TAT TTG CCC GTT TCC GCC ATA CTA AAA AGA CTT GAT CGT TCT CTT GM GAA W G T I G I M T L S S F P L A Y L P V S A I L K R L D R S L E E 1818 1913 GTA AGC CTT TCG CTT GGT AAA AGC CCT GTG TAT ACC TTT TGG TAT GCT ATT TTT CCG CAA CTT AAA CCC GCC ATT GGT AGT AGT ATC TTA CTG ATT V S L S L G K S P V Y T F W Y A I F P Q L K P A I G S S I L L I 1914 2009 GCT TTA CAT ATG TTG GTC GM TTT GGT GCA GTG TCA ATT TTA AAT TAT CM ACC TTT ACC ACT GCC ATT TTC CM GM TAT GM ATG TCT TTT MC A L H M L V E F G A V S I L N Y Q T F T T A I F Q E Y E M S F N 2010 2105 MC AGC ACT GCT GCA TTA TTA TCT GCC GTT TTA ATG GCG ATT TGT ATA CTT ATT GTT TTT GGG GM ATT TTC TTT CGT GGA AAA CM ACA CTT TAC N S T A A L L S A V L M A I C I L I V F G E I F F R G K Q T L Y 2106 2201 CAC AGT GGA AAA GGT GTT ATG CGT CCT TAT CTC GTA AAA ACA CTT TCC TTT GGA AAA CM TGT TTA ACC TTC GGA TTT TTC TCT AGC ATA TTC ATT H S G K G V M R P Y L V K T L S F G K Q C L T F G F F S S I F I 2202 2297 TTA AGC ATT GGC GTG CCT GTG ATT ATG TTA ATT TAC TGG CTT ATT GTG GGA ACT TCA CTC GM AGT GCG GGT GAT TTT TCA GM TTT TTA TCG GCA L S I G V P V I M L I Y W L I V G T S L E S A G D F S E F L S A Nucleotide sequence of a 4.2-kb segment of NTHI TN106 DNA from pjds150 containing the hitabc genes; the deduced amino acid FIG. 3. sequence is listed below the nucleotide sequence. The ORF for hita extends from nucleotides 255 to 1253, the ORF for hitb extends from nucleotides 1371 to 2891, and the ORF for hitc extends from nucleotides 2878 to 3948. Putative -35 regions, -10 regions, and Shine-Dalgarno sites are indicated in front of each ORF. A possible Fur box is indicated upstream from hita by shading. An inverted repeat immediately downstream from the hitc gene is indicated by opposing arrows. The putative leader peptide for the HitA protein is indicated by underlining.

VOL. 62, 1994 H. INFLUENZAE IRON UTILIZATION SYSTEM 4521 2298 2393 TTC AGC MT TCA TTT ATC ATT TCA GGC TTA GGC GCA TTG CTT ACC GTA ATG TGC GCC TTG CCA TTA GTT TGG GCA GCT GTT CGT TAC CGT AGT TAT F S N S F I I S G L G A L L T V M C A L P L V W A A V R Y R S Y 2394 2489 TTA ACG ATT TGG ATC GAC CGC TTG CCA TAT CTA TTG CAC GCT GTG CCA GGC TTA GTC ATT GCC TTA TCA TTA GTC TAT TTT TCC ATC CAT TAC GCT L T I W I D R L P Y L L H A V P G L V I A L S L V Y F S I H Y A 2490 2585 MC GAC CTA TAT CAA ACC TTT TTT GTG ATT ATC ATT GCC TAC TTT ATG CTC TAT TTG CCA ATG GCA CM ACT ACA TTA AGA GCG TCT TTA GAG CM N D L Y Q T F F V I I I A Y F M L Y L P M A Q T T L R A S L E Q 2586 2681 CTT TCC GAT CAA ATT GM AAA GTC GGA CM AGT CTT GGG CGA MC CCT TTC TAT ATT TTT CGC ACC TTG ACG CTA CCA GCC ATA TTA CCC GGT GTG L S D Q I E K V G Q S L G R N P F Y I F R T L T L P A I L P G V 2682 2777 GCA GCC GCA TTT GCT TTG GTT TTT TTG MT TTA ATG AAA GAG CTT ACC GCG ACG CTT TTG CTC ACA TCA MT GAT ATT MA ACC TTA TCC ATT GCT A A A F A L V F L N L M K E L T A T L L L T S N D I K T L S I A 2778 2873 GTG TGG GM CAT ACC AGC GAT GCA CAA TAT GCT GCC GCC ACC CCT TAT GCG TTA ATG CTC GTT CTT TTT TCG GGT ATT CCT GTA TTT TTA TTG AAA V W E H T S D A Q Y A A A T P Y A L M L V L F S G I P V F L L K 2874 2970 AAA TAT GCG TTT AAA TAAA ATG ATA MT MT CCG TTA CTA ACC GTT AAA MT CTC MT MA TTT TTT MT GAA CM CM GTT CTG CAC GAT ATT TCA K Y A F K M R L N K M I N N P L L T V K N L N K F F N E Q Q V L H D I S 2971 3066 TTC AGC TTA CM CGC GGA GM ATC CTC TTT TTA CTT GGT GCT TCA GGC TGT GGC AAA ACT ACA TTA TTA CGT GCC ATT GCA GGT TTT GM CM CCC F S L Q R G E I L F L L G A S G C G K T T L L R A I A G F E Q P 3067 3162 TCT MT GGC GM ATT TGG CTA AAA GAG CGG TTA ATT TTT GGC GAG MT TTT MT CTT CCA ACG CM CM CGC CAT CTC GGT TAT GTG GTG CM GAG S N G E I W L K E R L I F G E N F N L P T Q Q R H L G Y V V Q E 3163 3258 GGC ATA CTT TTT CCT CAC TTA MT GTC TAT CGC MC ATT GCT TAC GGA TTA GGC MC GGC AAA GGA AAA MT AGC GM GM AAA ACG CGG ATT GM G I L F P H L N V Y R N I A Y G L G N G K G K N S E E K T R I E 3259 3354 CM ATA ATG CAA CTA ACG GGT ATT TTT GM CTG GCG GAT CGC TTT CCA CAT CM CTT TCA GGC GGA CM CM CM CGC GTA GCA TTG GCG CGT GCT Q I M Q L T G I F E L A D R F P H Q L S G G Q Q Q R V A L A R A 3355 3450 TTA GCC CCT MT CCA GM CTG ATT TTA TTA GAC GAG CCT TTC AGT GCC TTA GAC GAG CAT CTT CGC CM CM ATT CGC CM GAG ATG CTC CM GCA L A P N P E L I L L D E P F S A L D E H L R Q Q I R Q E M L Q A 3451 3546 CTT CGC CM AGC GGT GCT TCC GCA ATT TTC GTT ACT CAC GAC CGT GAT GM GCC TTA CGC TAC GCT GAT AAA ATC GCC ATT ATT CAG CM GGT AAA L R Q S G A S A I F V T H D R D E A L R Y A D K I A I I Q Q G K 3547 3642 ATT TTA CM ATC GAT ACG CCT CGC ACC CTT TAT TGG TCG CCT MT CAT CTT GM ACA GCA AAA TTT ATG GGG GM AGT ATT GTT TTG CCT GCA MT I L Q I D T P R T L Y W S P N H L E T A K F M G E S I V L P A N 3643 3738 CTA CTC GAT GM MT ACC GCT CM TGC CM TTA GGC MT ATT CCT ATA AAA MT AAA TCA ATC TCA CAA MT CM GGT AGG ATT TTG CTT CGT CCA L L D E N T A Q C Q L G N I P I K N K S I S Q N Q G R I L L R P 3739 3834 GM CM TTT AGC CTG TTT AAA ACA TCA GAA MT CCA ACC GCA CTT TTT MT GGA CM ATA AAA CM ATT GM TTT AAA GGA AAA ATT ACC TCC ATT E Q F S L F K T S E N P T A L F N G Q I K Q I E F K G K I T S I 3835 3930 CM ATT GM ATT MT GGC TAC GCA ATA TGG ATT GAG MC GTT ATT TCT CCC GAT TTA TCT ATT GGC GAT MT TTG CCT GTT TAT TTA CAT AAA AAA Q I E I N G Y A I W I E N V I S P D L S I G D N L P V Y L H K K 3931 4051 GGG CTT TTT TAC GCT TM CMTAAAGCCTTATCTTCACGATMGGCTTATTTTTAMCMTATATTCGGMAAAATTTGTTCMGATGTGATATTAAACAGTCTGTGCCTAAAATATTTTC G L F Y A -- > <--------- 4052 4178 CCCTTTCCACGCCTGTTGCAAAATCTCTGGAGAAAATTGCTGGCTTGCTAATTCAGCTAACGGCAAATAACTGATATGCCAAGGCTCCCGCCCCACTTTCTTATTGGACTGCATATTCATAAAAGGC FIG. 3.-Continued. mesylate (to chelate free iron in the medium). Filter paper BHI-NAD agar plates containing iron and PPIX. All 30 disks containing various iron sources were then placed on the transformants that were obtained in this experiment were surface of these agar plates. The wild-type TN106 strain was chloramphenicol sensitive, a finding which suggested that able to use all of the iron sources tested, including heme, allelic exchange had replaced the mutated hitc gene with the transferrin, ferric ammonium citrate, and ferric nitrate, and wild-type allele in each of these strains. This was confirmed by exhibited a readily detectable zone of growth around the disks Southern blot analysis of one of these transformants, desigcontaining these compounds (Fig. 6). In contrast, the mutant nated TN106.155.T (Fig. 5, panels 1 and 2, lanes C). As strain TN106.155, while it could use heme, was unable to expected, this transformant strain was able to utilize all of the utilize the other three iron sources described above for growth iron sources that could not be utilized by the hitc mutant (Fig. on this medium (Fig. 6). 6). Elimination of the hitc mutation in the mutant strain Detection of hitabc genes in other H. influenzae strains. TN106.155. To reconstitute the wild-type genotype, mutant Chromosomal DNA preparations from two NTHI strains TN106.155 was transformed with the 6.5-kb PstI fragment of (TN106 and AAR200) and three Hib strains (DM42, DL63, NTHI chromosomal DNA from pjds152 and the desired and 760705) were digested with PstI and used in Southern blot transformants were selected by virtue of their ability to grow on analysis together with probes for the hitabc genes. A single

4522 SANDERS ET AL. INFECT. IMMUN. 10 20 30 40 50 60 HitA HItA MQFKHFKL---ATLA--AALAFSA-NS FAD-- ITVYNGQHKEAAAAVAKAFEQETG IKVTLNSGKSAQLAGQ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 HM SfuA MKLRISSLGPVALLASSMMLAFGAQAASADQGIVIYNAQHENLVKSWVDGFTKDTGIKVTLRNGGDSELGNQ 10 20 30 40 50 60 70 HitB SfuB 70 80 90 100 110 120 130 LKEEGDKTPADVFYTEQTATFADLSEAGLLAPISEQTIKQTAQKGVPLAPKKDWVALSGRSRVVVYDHTKLS if, 011 H ll, 111 11, Ill LVQEGSASPADVFLTENSPAMVLVDNAKLFAPLDAATLAQVEPQYRP--SSHGRWIGIAARSTVFVYNPAKLS 80 90 100 110 120 130 140 140 150 160 170 180 190 200 EKDMEKSVLDYATPKWKGKIGYVSTSGA-FLEQVVALSKMKGDKVALNWLKGLKENGKLYAKNSVALQAVEN DAQLPKSLLDLAKPEWKG-RWAASPSGADFQAIVSALLELKGEKATLAWLKAMKTNFTAYKGNSTVMKAVNA 150 160 170 180 190 200 210 210 220 230 240 250 260 270 GEVPAALINNYYWYNLAKEKGVENLKSRLYFVRHQDPGALVSYSGAAVLKASKNQAEAQKFVDFLASKKGQE I' 10 H, I'll Ill 11 I 'l 11 I'l1 111! GQVDSGVIYHYYPFVDGAKTGENSNNIKLYYFKHQDPGAFVSISGGGVLASSKHQQQAQAFIKWITGKQGQE 220 230 240 250 260 270 280 290 300 310 320 330 x ALVAVPA-EYPLRADVVSPFNLEPYEKLEAPVVSATTAQDKEHATKLIEEAGLK I ILIE O OIILV 'I L V T ILRTNNAFEYAVGVGAASNPKLVPLKDLDAPKVDAAQLNSK-KVVELMTEAGLL 10 20 30 40 50 60 70 MPR--RPPFWLTLLIILIGLPLCLPFLYVILRATEVGLTRSVELLFRPRMAELLSNTMLLMVCVTIGAISLGTLC VPRHPRPGAIVVVSAVLLSLLALLPLGFVIGVAFETGWQTVKALVFRPRVAELLLNTLLLVVLTLPICAVLGVAL 20 30 40 50 60 70 80 90 80 90 100 110 120 130 140 AFLLERYRFFGKAFFEVAMTLPLCIPAFVSCFTWISLTFRVEGFWGTIGIMTLSSFPLAYLPVSAILKRLDRSLE AWLTERTTLPGRRLWAVLATAPLAVPAFVQSYAWIISLVPSMHGLGAGVFISVLAYFPFIYLPAAAVLRRLDPGIE 100 110 120 130 140 150 160 160 170 180 190 200 210 220 EVSLSLGKSPVYTFWYAIFPQLKPAIGSSILLIALHMLVEFGAVSILNYQTFTTAIFQEYEMSFNNSTAALLSAV DVATSLGSRPPAVFFRVVLPQLKLAVWGGSLLIALHLLAEYGLYAMIRFDTFTTAIFDQFQSTFNGPAANMLAGV 170 180 190 200 210 220 230 240 230 240 250 260 270 280 290 LMAICILIVFGEIFFRGKQTLYHSGKGVMRPYLVKTLSFGKQCLTFGFFSSIFILSIGVPVIMLLIYWLIVG---- LVLCCLGLLLLEAISRGRARYARVGSGSARSQTPRRLSPPLAALALLLPIALTALALGVPFITLARWLWLGGFEV 250 260 270 280 290 300 310 300 310 320 330 340 350 360 ---TSLESAGDFSEFLSAFSNSFI ISGLGALLTVMCALPLVWAAVRYRSYLTIWIDRLPYLLHAVPGLVIALSLV WRNAELWPALWQTLSLSA--------AGALLITLCAIPMAWLSVRYPARLYRVLEGCNYVTSSLPGIVVALALV 320 330 340 350 360 370 380 370 380 390 400 410 420 430 440 YFSIHYANDLYQTFFVIIIAYFMLYLPMAQTTLRASLEQLSDQIEKVGQSLGRNPFYIFRTLTLPAILPGVAAAF TITIHSFRPIYQTEITLLLAYLLMFMPRALINLRAGIAQAPVELENVARSLGKSPAQALWSTTLRLAAPGVAAGA 390 400 410 420 430 440 450 450 460 470 480 490 500 x ALVFLNLMKELTATLLLTSNDIKTLSIAVWEHTSDAQYAAATPYALMLVLFSGIPVFLL----KKYAFK llll 'l ALVFLAIANELTATLLLAPNGTRSTLATGFWALTSEIDYVAAAPYALIMVALSLPLTWLLYSQSKRTAGL 470 480 490 500 510 520 x 20 30 40 50 60 70 80 HitC NPLLTVKNLNKFFNEQQVLHDISFSLQRGEILFLLGASGCGKTTLLRAIAGFEQPSNGEIWLKERLIFGENFN SfuC MSTLELHGIGKSYNAIRVLEHIDLQVAA.SRTAIV9P8GS0R4 LLRIIAGFEIPDCGQILLQGQAMGNGSGW 10 20 30 40 50 60 70 90 100 110 120 130 140 150 LPTQQRHLGYVVQEGILFPHLNVYRNIAYGLGNGKGKNSEEKTRIEQIM-QLTGIFELADRFPHQLSGGQQQR VPAHLRGIGFVPQDGALFPHFTVAGNIGFGL---KGGKREKQRRIEALMEMVALDRRLAALWPHELSGGQQQR 80 90 100 110 120 130 140 160 170 180 190 200 210 220 VALARALAPNPELILLDEPFSALDEHLRQQIRQEHLQALRQSGASAIFVTHDRDEALRYADKIAIIQQGKILQ VAAASQRt''t)ZFSALDTGLRAATRKAVAELLTEAKVAS ILVTHDQSEALSFADQVAVMRSGRLAQ 150 160 170 180 190 200 210 240 250 260 270 280 290 IDTPRTLYWSPNHLETAKFMGESIVLPANLLDENTAQCQLGNIPIKNKSISQNQGRILLRPEQFSLFKTSENP VGAPQDLYLRPVDEPTASFLGETLVLTA-ELAHGWADCALGRIAVDDRQRS-GPARIMLRPEQ--IQIGLSDP 220 230 240 250 260 270 280 310 320 330 340 350 x TALFNGQIKQIEFKG--KITSIQIEINGYAIWIENVISPDLSIGDNLPVYLHKKGLFYA AQRGQAVITGIDFAGFVSTLNLQMAATGAQLEIKTVSREGLRPG--AQVTLNVMGQAHIFAG 290 300 310 320 330 340 x 8kb 6 kb ABC _ ABC 1 2 FIG. 5. Southern blot analysis of PstI-digested chromosomal DNA from the wild-type, mutant, and transformant strains of NTHI TN106. DNA preparations from the wild-type strain (lanes A), the hitc mutant strain TN106.155 (lanes B), and the transformant strain TN106.155.T (lanes C) were probed either with a 0.57-kb BsrBI fragment from the hitc gene (panel 1) or with the cat cartridge (panel 2). Size position markers (in kilobases) are indicated on the left. possessed a PstI fragment of approximately 9.5 kb that hybridized these three probes (Fig. 7). DISCUSSION The paucity of free iron in the human body (9, 55, 56) requires that colonizing or invading microorganisms possess a mechanism(s) to obtain iron from host factors that sequester 2 4 ABCA FIG. 4. Comparison of the deduced amino acid sequences of the H. influenzae HitA, HitB, and HitC proteins with those of the SfuA, SfuB, and SfuC proteins of S. marcescens, respectively. The deduced amino acid sequences of the three S. marcescens proteins were published previously (2). The shaded regions in the SfuC protein indicate the FIG. 6. Utilization of iron sources by wild-type NTHI strain consensus A and B regions common to nucleotide-binding proteins of TN106, hitc mutant strain TN106.155, and transformant strain ABC transporters (17, 27). TN1O6.155.T. Suspensions of 2 x 105 CFU of the wild-type (A), mutant (B), and transformant (C) strains were spread onto BHI-NAD agar plates containing PPIX (as the source of porphyrin) and deferoxamine mesylate (to chelate free iron in the medium). Filter paper disks were placed on these plates and spotted with the following compounds: DNA fragment from each strain bound all three probes. row Wlth 1, heme; row 2, PPIX (a negative control); row 3, ferric nitrate; NTHI strain TN1O6, this fragment was 6.5 kb in size, as would row 4, iron-loaded human transferrin; row 5, ferric ammonium citrate. be expected from the restriction map of pjds150 (Fig. 2). In The plates were incubated overnight as described in Materials and contrast, the other NTHI strain and the three Hib strains all Methods.

VOL. 62, 1994 H. INFLUENZAE IRON UTILIZATION SYSTEM 4523 gkb - 6kb AB C DE A B C D E A B C D E hydrophilic protein with nucleotide-binding domains (SfuC) (2, 3). The exact function of the SfuA molecule has been VW -t- inferred to be the binding of ferric iron (3), which enters the S. marcescens cell as a complex with citrate (2, 3, 63). Similarly, Sue the gonococcal Fbp protein, which is 37% identical to the SfuA molecule, has been shown to at least transiently bind iron (10). It should be noted that Fbp and HitA are even more similar to each other (69% identity) than they are to SfuA. This fact and the localization of the HitA protein to the periplasmic compartment of H. influenzae (24) reinforce the likely role of HitA 2 3 as the ligand-binding component of an iron transport system FIG. 7. Reactivity of the N' THI TN106 hita, hitb, and hitc gene that appears to be a member of the ABC transporter family probes with chromosomal DNA from wild-type NTHI and Hib strains. (17, 27). Chromosomal DNA preparatioins from NTHI strain TN106 (lanes A), The successful construction of an NTHI hitc mutant per- NTHI strain AAR200 (lanes B) H,ib strain DL42 (lanes C), Hib strain mitted in vitro testing that demonstrated directly the functional DL63 (lanes D), and Hib strair 760705 (lanes E) were digested with involvement of the protein products of the hitabc gene cluster PstI and probed in Southern blc t analysis with the following probes: a in iron utilization by H. influenzae. In contrast, several attempts 0.89-kb PstI-SspI fragment contaming the majority of the hita gene to use allelic exchange to introduce a mutated hita gene (panel 1), a 0.64-kb MunI-PvuII fragment from the hitb gene (panel 2), and a 0.57-kb BsrBI fragment fr om the hitc gene (panel 3). Molecular containing a cat cartridge into H. influenzae have met with size markers (in kilobases) are indicated on the left. failure (data not shown), suggesting that a functional HitA protein may be essential for the growth of H. influenzae. Similarly, mutant analysis of the function of Fbp, a HitA homolog, in the gonococcus has not been achieved to date this essential element. Graam-negative enteric bacteria and because attempts to construct anfbp mutant have been unsucccomplish this task by means of cessful (10). Whether N. gonorrhoeae also contains HitB and pseudomonads generally a( siderophore production andithe TonB-dependent transport of HitC homologs is not known with certainty, but the 136 bp of the siderophore-iron compil[ex into the bacterial cell (14, 19, published nucleotide sequence immediately downstream from 41). The mechanisms by wrhich siderophore-bound iron are the stop codon for the gonococcal fbp gene (6) encodes an transported across the out( er membrane, periplasmic space, incomplete polypeptide that has 57% identity with the N- and cytoplasmic membrane have been the subject of many terminal region of HitB (data not shown). elegant studies (4, 19, 41). The fact that the presence of the NTHI TN106 hitabc genes Other bacterial pathogenss, including both H. influenzae and in trans allowed Hib strain DML2 to grow on BHI-NAD agar pathogenic Neisseria specie, s, possess a siderophore-indepen- plates containing iron and PPIX indicates that this Hib strain dent mechanism(s) for iron, acquisition that involves the direct likely is deficient in its ability to transport iron. However, binding of iron-carrying sernim glycoproteins (e.g., transferrin) Southern blot analysis (Fig. 7) revealed that Hib strain DM42 to the bacterial cell (42). This binding of transferrin to the cell possessed chromosomal DNA regions that hybridized to the surface involves two proteinis, designated Tbpl and Thp2, and NTHI hita, hitb, and hitc gene probes. Moreover, Western the former protein from both N. gonorrhoeae (13) and N. blot analysis using the HitA-specific MAb 6B8 indicated that meningitidis (32) has been shown to possess similarity to Hib strain DLM2 does express a 40-kDa protein reactive with TonB-dependent outer meimbrane proteins. A recent study this MAb (data not shown); whether this Hib HitA protein is suggests that, in these path ogenic Neisseria species, once the functional has not yet been determined. However, the fact that iron has been removed from transferrin and gains access to the the mutation in the hitc gene in the recombinant plasmid periplasmic space, it is bounid by Fbp as part of the process of pjds155 eliminated the ability of this plasmid to complement transporting the iron molecaule across the cytoplasmic mem- the growth deficiency of Hib strain DM42 suggests that this Hib brane (10). It should also be noted that Fbp has been shown to strain may simply lack a functional HitC protein. be the major iron-repressib] le protein of both N. gonorrhoeae The results of the present study indicate that the H. influ- enzae HitA, HitB, and HitC proteins are likely involved in the and N. meningitidis (6, 38, 3i9). While studies involving eitther the regulation of H. influenzae utilization of iron once this element has gained access to the outer membrane protein expression by environmental iron periplasmic space. Elimination, by mutation, of a functional levels (31, 40, 59) or the identification of H. influenzae outer HitC protein resulted in an NTHI strain that could not utilize membrane proteins that bin d iron-containing compounds (23, iron chelates or iron bound to transferrin while still retaining 28, 31, 48, 49) have beer1 published, there has been no the ability to use heme as a source of iron (Fig. 6). Whether the identification to date of an JH. influenzae gene product(s) that HitA and HitB proteins are essential for iron utilization was is essential for the utilization of iron in any form. In the present not addressed directly in this study, and the inability to study, it was shown that an NITHI hitc mutant could not utilize construct a viable hita mutant (data not shown) may preclude either free or protein-bound iron (Fig. 6). Moreover, the ability genetic analysis of the role of HitA in this process. Nonetheof H. influenzae to utilize Fi'PIX as a source of porphyrin in less, it is clear that, at least in vitro, utilization of transferrinplace of heme appeared to b)e dependent on the presence of a bound iron by H. influenzae is dependent on the presence of a functional hitabc gene cluster. functional HitC protein. The H. influenzae hitabc lgene products proved to be similar to the proteins encoded by tile sfuabc operon in S. marcescens (2, 3). These three proteins ( of this enteric organism have been ACKNOWLEDGMENTS implicated in a periplasmic binding protein-dependent iron transport system (2, 3). Thlis system involves a periplasmic ligand-binding protein (SfuLA), a very hydrophobic protein localized to the cytoplasmic membrane (SfuB), and a rather This study was supported by Public Health Service grants A117621 and A123366 to E.J.H. L.D.C. was supported by NCI training grant CA09082-19. We are grateful to Cindy Patterson for her assistance in the

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