©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Characterization of a Haemophilus influenzae Periplasmic Iron Transport Operon (*)

(Received for publication, June 8, 1995)

Pratima Adhikari (1)(§) Shane D. Kirby (2)(¶) Andrew J. Nowalk (1) Kristen L. Veraldi (1) Anthony B. Schryvers (2) Timothy A. Mietzner (1)

From the  (1)Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the (2)Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2`-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.


INTRODUCTION

High affinity acquisition of iron from the host environment is a necessary determinant of virulence for pathogenic bacteria(1, 2, 3, 4, 5, 6, 7, 8, 9) . This acquisition is vital for survival in the human host, where levels of extracellular iron are tightly controlled by the Transferrins (transferrin and lactoferrin), a family of iron-binding proteins that function in the extracellular chelation and transport of host iron(9) . By binding iron with high affinity, Transferrins ensure that all extracellular iron is both efficiently sequestered from pathogenic invaders and mobilized for transport to host tissues. Microorganisms growing in the human host must therefore possess mechanisms for obtaining Transferrin-sequestered iron. For a number of pathogenic members of the Pasteurellaceae (H. influenzae) and Neisseriaceae (Neisseria meningitidis and Neisseria gonorrhoeae), iron acquisition is initiated by cell-surface receptors specific for the Transferrins(10, 11, 12, 13, 14, 15, 16) . Iron is removed from these proteins and transported across the outer membrane, presumably by an energy-dependent TonB-mediated process (17, 18, 19) involving gated-pore properties of the outer membrane receptor (18, 20) . The result is deposition of free iron within the periplasm, where it is separated from the cytosol, its eventual destination, by the cytoplasmic membrane(21) .

Transport of free iron from the periplasmic space into the cytoplasm is proposed to occur by a classic active transport process involving a periplasmic binding protein, a specific cytoplasmic permease, and an energy-supplying nucleotide-binding protein(22) . Much of what is known about the biochemistry of active transport systems has been revealed through the study of model active transport systems for amino acids and sugars in Escherichia coli(22, 23) . Similar systems for siderophore-mediated iron transport have been described for E. coli and related organisms at the genetic level(23) ; however, relatively little is known regarding the basic biochemistry of these iron transport processes.

A genetic locus critical to the transport of iron in H. influenzae has recently been described by Hansen and colleagues (24) . This locus was identified through complementation of a H. influenzae isolate unable to grow on medium containing protoporphyrin IX and free iron. An 11.5-kb (^1)genomic DNA fragment from an isolate proficient for growth on this medium was identified by this analysis(24) . Essential for this phenotype was a 4-kb operon composed of three genes: hitA, hitB, and hitC (hit for Haemophilus Iron Transport) proposed to encode a periplasmic iron-binding protein, a cytoplasmic permease, and a nucleotide-binding protein, respectively. A homologous three-gene operon was originally described for Serratia marcescens and designated sfu for Serratia Ferric-iron Uptake(25) . The sfu operon was isolated based upon its ability to complement an E. coli strain (H-1443) deficient in its ability to produce siderophores for growth on nutrient agar containing 200 µM 2,2`-dipyridyl (dipyridyl), an iron chelator that sequesters free iron in the medium (25, 26) . The open reading frames encoded by the hitABC and sfuABC genes were found to share 38, 37, and 38% identity between respective A, B, and C components at the predicted amino acid level. The similarities between the hit and sfu genetic loci suggest a high level of conservation among two diverse species.

At the protein level, Harkness and colleagues (27) originally observed a quantitatively major, iron-regulated periplasmic protein, subsequently genetically defined as hitA(24) . The predicted open reading frame of hitA is nearly 80% homologous with the ferric iron-binding protein (Fbp) expressed by pathogenic Neisseria(17, 29, 30, 31, 32, 33) . Similarly, the open reading frame of sfuA predicts a protein sequence sharing substantial homology (40% identity) with the Neisserial Fbp(17) . Fbp is a periplasmic iron-binding protein expressed by all pathogenic Neisseria that functions as the binding component of a high affinity active transport system for the assimilation of growth-essential iron from the Transferrins. Purified Fbp binds a single Fe ion with an affinity approaching that of the Transferrins (17, 34, 35) and by a mechanism that is remarkably conserved among this family of proteins, coordinating iron through two tyrosines, a single histidine, and a bicarbonate anion(35) . In our study we will refer to these Fbp homologues as NFbp for Neisseria Fbp derived from the fbp gene locus (33) , HFbp for Haemophilus Fbp derived from the hitA locus(24) , and SFbp for Serratia Fbp derived from the sfuA locus(26) . It is clear is that a common free iron active transport system exists among pathogenic members of the diverse microbial families Enterobacteriaceae (sfu operon), Pasteurellaceae (hit operon), and Neisseriaceae (fbp operon). The existence of this common system may reflect its contribution to the pathogenicity of these organisms.

Studies on the HFbp, NFbp, and SFbp homologues and their respective operons predict that they should function similarly. This report describes the ability of purified HFbp to efficiently compete for dipyridyl-bound iron in vitro. Like the sfu operon, hitABC can complement the siderophore-deficient E. coli strain H-1443 to growth on dipyridyl-containing medium. We further demonstrate that labeled iron from this medium is initially bound to periplasmic HFbp and can only be transported into the cell if a functional permease and nucleotide-binding protein are present. These studies explain why the siderophore-deficient E. coli expressing the hit operon can be complemented to growth on dipyridyl-containing media. Furthermore, they represent the first comprehensive biochemical analysis of a periplasmic iron transport system.


EXPERIMENTAL PROCEDURES

Strains and General Reagents

Type b H. influenzae strain DL63 was obtained from E. Hansen, University of Texas Southwestern Medical Center, Dallas, Texas. E. coli strains DH5alpha and JM109 were purchased from Promega (Madison, WI). E. coli strain H-1443 was kindly provided by Dr. Volkmar Braun, Universität Tübingen, Tübingen, Germany.

The plasmid pJDS150 was a generous gift of Dr. Eric Hansen, University of Texas Southwestern. Plasmid pBR322 was purchased from Promega (Madison, WI). Oligonucleotides were prepared using an Applied Biosystems International model 391 DNA Synthesizer (Foster City, CA) and were deprotected and purified as per the manufacturer's instructions. Taq polymerase used was purchased from either Life Technologies, Inc. or Boehringer Mannheim. Random hexamers used in generating labeled PCR probes, T4 DNA ligase, and the restriction enzymes EcoRI, EcoRV, BamHI, and SmaI were purchased from Boehringer Mannheim. Nutrient broth, trypticase soy broth, components for Luria-Bertani Broth (LB), NZY agar, Difco agar, and other media components were purchased from Difco (Detroit, MI). Cetyltrimethylammonium bromide, 2,2`-dipyridyl, CM-Sepharose, DEAE-Sepharose, MgS0(4), tetracycline, and ampicillin were purchased from Sigma. Low molecular weight protein standards for SDS-PAGE analysis were purchased from Pharmacia Biotech Inc. The radioisotopes [alpha-P] and [Fe](NO(3))(3) were purchased from DuPont NEN. Eco-Lite scintillation mixture was purchased from ICN Biomedicals Inc. (Irvine, CA), and samples were counted using a Packard 1600TR Tri-Carb liquid scintillation analyzer (Packard Instrument Company, Meridian, CT). Whatman no. 4 filter paper was purchased from Whatman (Maidstone, United Kingdom). The Amicon concentration cell and Diaflo ultrafiltration membranes were from Amicon (Lexington, MA).

Cloning of the hitA Gene Region and the Minimal hitABC Operon

A 700-bp hitA gene fragment was PCR amplified from H. influenzae DL63 chromosomal DNA by designing a primer based on the N-terminal amino acid sequence of a 40-kDa iron-regulated periplasmic protein suspected to be the H. influenzae Fbp analog (27) and by designing a second primer to a conserved region shared by the closely related sfuA(26) and gonococcal fbp genes(33) . PCR reactions were performed in 50 µl volumes by methods previously described(28) . The PCR profile consisted of 25 cycles of 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 2.5 min which was followed by a final extension of 72 °C for 10 min. Cloning of an intact hitA containing construct was achieved by screening a previously prepared Zap II H. influenzae DL63 chromosomal DNA library using the alpha-P 700-bp random hexamer-labeled hitA PCR product as a probe. The Zap II gene library was prepared by random mechanical shearing of DL63 chromosomal DNA followed by blunt ending the sheared products using S(1) nuclease. The blunted products were methylated using EcoRI methylase to protect genomic EcoRI sites and then treated with Klenow to ensure blunt ending of the genomic DNA fragments. The blunted, methylated fragments were ligated to EcoRI linkers using T4 DNA ligase and then digested with EcoRI restriction endonuclease. The DNA was then size-fractionated on a sucrose density gradient to obtain 5-10-kb fragments which were subsequently cloned into the unique EcoRI site within the lacZ gene pBluescript portion of the Zap II vector. Plating of the library was done in accordance with the manufacturer's directions except for the following modifications: LB broth containing 0.7% Difco agar and 10 mM MgS0(4) was used in the place of NZY agar; similarly, LB top agar was used to replace NZY top agar. Plaque lifts were performed on the plated library by applying nitrocellulose filters to plates prechilled to 4 °C and incubating the plates with the applied filters at this temperature for 30 min. Subsequently, filters were marked for orientation, removed from the plates, and screened according to the Stratagene protocol. After two rounds of screening, a number of positive clones were identified, and the pBluescript SKII phagemids were excised as per the manufacturer's instructions. One of these clones was designated pBSJ1 and was shown to contain an 3.5-kb genomic DNA fragment which included the intact hitA gene, approximately two-thirds of the coding region for hitB, and 1.3 kb of noncoding sequence upstream of hitA (Fig. 1).


Figure 1: Plasmid map of the HFbp-expressing plasmid pBSJ1. As described in the text, an 3.5-kb fragment encoding 1.3 kb upstream and 1.2 kb downstream of the HFbp coding sequence was excised from a positive Zap II clone and inserted into the EcoRI site of the plasmid pBS SK. Ori = origin of replication, AmpR = beta-lactamase gene, lacZ = beta-galactosidase gene.



The fragment containing the minimal hitABC from H. influenzae was prepared by PCR amplification as described in Fig. 2. PCR reactions were performed in 100 µl volumes using standard conditions previously described (33) and 10 units of Taq polymerase and 10 units of Taq extender. Amplification was achieved by 27 cycles of denaturation (95 °C for 1.5 min), annealing (60 °C for 2 min), and extension (72 °C for 3 min). At cycle 17, the reactions were replenished with an additional 5 units of both Taq polymerase and Taq extender. Specifically, primers were designed to the extreme ends of the hitABC sequence (24) that included 250 bp upstream and 230 bp downstream of this operon. For the upstream primer, hitO-5`, there was an engineered 5` SmaI site; the downstream primer hitO-3` included a 3` BamHI site (Table 1). Using these primers and the plasmid pJDS150 as template, a PCR fragment of approximately 4.2 kb was generated. Following PCR, the amplified fragment was gel purified and digested simultaneously with BamHI and SmaI for about 4 h at 37 °C. The PCR fragment was combined with the 4.2-kb EcoRV-BamHI fragment of pBR322 (gel-purified) at a 3:1 ratio of insert to vector. Ligation was achieved using standard conditions(33) . This ligation was used to transform competent E. coli strain H-1443 and the transformants selected on LB agar containing 100 µg/ml ampicillin. Transformants were screened for tetracycline sensitivity on LB plates containing 25 µg/ml of this antibiotic. Tetracycline sensitive clones were screened for plasmid DNA and the presence of hitABC insert verified by PCR amplification as described above.


Figure 2: SDS-PAGE comparison of HFbp and NFbp. 5 µg of each protein were run on a 12% acrylamide gel as specified under ``Experimental Procedures.'' Numbers on the left refer to molecular weights estimated from a reference curve of standard protein relative mobilities.





A hitC deletion mutant was constructed in order to demonstrate the essential nature of this gene to the complementation of H-1443 to growth on nutrient agar containing 200 µM dipyridyl (NA/Dip). A 1.3-kb fragment of DNA was deleted from the ClaI site (400 bp from the stop codon in hitC) to the NarI site (at position 1205 in pBR322). This was achieved by complete digestion of pAHIO with NarI followed by a partial ClaI digest. From this partial digest the approximately 7-kb partial product that contains the deleted hitC gene was gel-purified. Subsequently, this fragment was ligated under standard conditions and used to transform E. coli H-1443 cells to ampicillin resistance. Positive clones were confirmed by restriction digest analysis and the plasmid expressing this mutation designated pAHIODeltahitC.

Isolation of HFbp

Similar to what was observed for the overexpression of NFbp in an E. coli background(32) , overnight growth of JM109(pBSJ1) resulted in a distinctly red pellet upon centrifugation of a 1.5 ml suspension, suggestive of the overexpression of HFbp. This was confirmed by SDS-PAGE analysis of crude CTAB lysates from E. coli JM109 (pBSJ1), prepared as described previously(32) . This analysis indicated a major protein with a molecular mass of 40 kDa (data not shown). Purification of HFbp was achieved by a modification of the method of Berish et al.(32) using 2-liter cultures of JM109(pBSJ1) in LB supplemented with 200 µg/ml ampicillin, grown with aeration at 37 °C for 22-24 h. The cells were harvested by pelleting at 5000 times g for 15 min at 4 °C and washed once in phosphate-buffered saline followed by suspension in 25 ml of 1 M Tris, pH 8.0. Lysis of the cells was performed by treating this cell suspension with 25 ml of a 4% (w/v) CTAB solution accompanied by shaking for 1 h at 37 °C. The solution was then diluted to 400 ml with dH(2)O, the particulates removed by centrifugation (7000 times g for 15 min at 4 °C), and the solubilized material reserved. The pelleted debris were suspended in 25 ml of 1 M Tris, pH 8.0, and subjected to a second round of CTAB lysis and pelleting of particulate material as described above. The lysates from the two CTAB lysis steps were then combined and diluted to a final volume of 1 liter with dH(2)O and clarified by filtration through Whatman no. 4 filter paper. The clarified lysate was applied to a CM-Sepharose CL-6B column connected in series to a DEAE-Sephacel column (6 cm diameter times 15 cm length and 6 cm diameter times 7 cm length, respectively). Equilibration of the CM-Sepharose column was accomplished by washing with five volumes of 1 M NaOH followed by five volumes of 10 mM Tris base, pH 8.0, containing 1 M NaCl (high salt buffer) and five volumes of 10 mM Tris base, pH 8.0 (low salt buffer). The DEAE column was equilibrated by washing with five volumes of high salt buffer followed by five volumes of low salt buffer. The soluble CTAB extract was applied to the equilibrated system at a flow rate of 1.5 ml/min and the HFbp-containing eluant collected. This eluant, which contained >95% HFbp, was concentrated 10-fold in an Amicon cell using a 10-kDa cutoff Diaflo ultrafiltration membrane. Alternatively, the protein was precipitated by bringing the eluant to a final concentration of 80% ethanol (v/v) to yield a pure HFbp precipitate.

Biochemical Characterization of HFbp

The purification of NFbp was performed as described previously(32) . Protein determinations were obtained using a modified Lowry method(32) . HFbp and NFbp preparations (5 µg each) were analyzed on a 12% acrylamide gel using reducing SDS-PAGE conditions as described previously(32) . Molecular mass estimates were obtained from this analysis using least-squares method from molecular weight standards on the same gel. Predicted molecular masses of HFbp and NFbp were obtained from their previously published DNA sequences(24, 33) . Isoelectric focusing was performed as described previously for the gonococcal Fbp(32) . Visible absorbance spectra were determined from a 2-mg/ml sample of purified HFbp in 20 mM Tris, pH 8.0, containing 200 mM NaCl, as described previously(35) . HFbp iron affinity was estimated from citrate competition assays performed using a method nearly identical to that described for NFbp by Chen et al.(17) . For this analysis, aliquots of HFbp were incubated with increasing concentrations of citrate, pH 8.0, and deferration of HFbp was monitored by decrease in absorbance at 483 nm.

Partitioning of Labeled Iron between the Periplasm and Non-periplasmic Compartments of E. coli Expressing hit Constructs

Bacterial cultures for these assays were prepared by inoculation with a single colony in 5 ml of nutrient broth containing 100 µg/ml ampicillin (if required) and grown for 8 h at 37 °C. A 100-µl aliquot of the culture was plated onto nutrient agar containing 75 µM dipyridyl, 100 µg/ml ampicillin (if required), and 10^7 counts/min [Fe](NO(3))(3) (0.1 µmol). Plates were grown at 37 °C for 12 h and the bacteria harvested. The bacteria were then washed three times in phosphate-buffered saline, suspended to an OD of 0.68 in phosphate-buffered saline, and 1.5 ml of each culture was pelleted. The periplasmic fraction was isolated using a modification of the method of Ames(22) . Briefly, pellets were resuspended in 20 µl of chloroform, vortexed, and incubated at 25 °C for 15 min. 100 µl of 10 mM Tris, pH 8.0, was added to each sample, followed by vortexing. Samples were pelleted by centrifugation for 5 min at 7000 times g and the aqueous phase containing the periplasmic fraction was removed. The remaining chloroform suspension represents the non-periplasmic fraction of the bacteria. Samples were counted after dissolving them in 3 ml of scintillation mixture.

Complementation of aroB E. coli to Growth on Dipyridyl-containing Agar by the hit Operon

Plating data were obtained by growing strains to mid-log phase in LB supplemented with ampicillin (where necessary) and diluted to a concentration that would allow 100-200 colony forming units/plate. Under these conditions, the aroBE. coli strain H-1443 grows on nutrient agar containing 100 µM dipyridyl but not on NA/Dip. The hitA expressing E. coli H-1443(pBSJ1), hitABC expressing H-1443(pAHIO), and the deletion mutant H-1443 (pAHIODeltahitC) were investigated for single-colony growth on nutrient agar containing 100 µM and 200 µM dipyridyl. DH5alpha was used as a positive control (data not shown), whereas H-1443 and H-1443(pBR322) were used as negative controls. Growth was scored as positive when pinpoint single colonies occurred after 20 h at 37 °C and negative if no isolated colonies were observed after 20 h.


RESULTS

Cloning and Overexpression of Recombinant HFbp

Cloning and sequencing of the hitABC operon were recently reported by Sanders et al.(24) . Independently, we have cloned the hitA region gene. As described under ``Experimental Procedures,'' the gene encoding HFbp was cloned based upon amino acid sequence homology between NFbp (33) and SFbp(26) . To accomplish this, PCR was used to amplify a 700-bp fragment using a primer based on known N-terminal amino acid sequence of Hfbp (27) (F3, Table 1) and a second primer (F6, Table 1) which is based on conserved sequences between NFbp (33) and SFbp(26) . After sequencing to confirm that it encoded an Fbp homologue, this fragment was labeled and used to screen a ZapII library of H. influenzae DL63 DNA as described under ``Experimental Procedures.'' One positive recombinant phage was subjected to the in vivo excision protocol, allowing for isolation of the recombinant plasmid designated pBSJ1. This plasmid (Fig. 1) was used for the overexpression of HFbp (``Experimental Procedures''). Sequence comparison of the pBSJ1 insert with the published hitABC operon indicates that this construct contains the entire coding region for hitA and two-thirds of the hitB open reading frame in addition to 1.3 kb of DNA upstream of hitA (data not shown).

Purification and Biochemical Characterization of HFbp

The isolation of HFbp was performed using a modification of the NFbp purification procedure of Berish et al.(33) . Cetyltrimethylammonium bromide-solubilized JM109(pBSJ1) cell extracts were applied to tandemly arranged CM-Sepharose and DEAE-Sephacel columns. In contrast to the purification of NFbp, in which NFbp binds tightly to CM-Sepharose, HFbp binds to neither the cation nor the anion exchanger. While most solubilized proteins were retained on the columns, HFbp remained in the eluant (data not shown). HFbp in the eluant was concentrated by ultrafiltration, using an Amicon PM10 filter with a molecular mass cutoff of 10 kDa. Yields of recombinant HFbp by this procedure were consistently between 60-90 mg/liter of cell culture harvested and were of greater than 95% purity as judged by SDS-PAGE analysis (Fig. 2).

Biochemical analyses of purified HFbp were performed to compare its physical and functional properties with those of NFbp. The biochemical attributes of NFbp have been extensively reported (17, 32, 33, 36) and are listed as part of Table 2. Physical comparison of HFbp with NFbp reveals that both proteins share similar predicted molecular masses, although their migration in SDS-PAGE is noticeably different (Table 2, Fig. 2). The isoelectric points of HFbp and NFbp differ by more than a full pH unit. This difference in charge may explain the disparity in SDS-PAGE mobility and affinity for ion exchange resins. However, two other functional indices highlight the similarity that the proteins share in their coordination of iron. The visible absorbance maximum of the ferrated protein is nearly identical for the two, indicating that iron is bound within a very similar ligand field in both HFbp and NFbp. Secondly, the affinities for Fe are identical, again emphasizing the functional homology between the two proteins. These and other (37) observations provide compelling evidence for the functional homology of NFbp and HFbp.



Periplasmic Acquisition of Iron from Ferric-2,2`-dipyridyl

As the objective of this study was to demonstrate that NFbp and HFbp function similarly, the ability of these deferrated purified protein preparations to obtain iron from Fe-dipyridyl was investigated. Dipyridyl is a well-characterized organic iron chelator(25) . Fig. 3illustrates that increasing concentrations of Fe-dipyridyl, when added to apoHFbp, cause increasing saturation of HFbp with iron. At high concentrations of Fe-dipyridyl, HFbp was completely saturated. These data demonstrate the ability of HFbp to compete for iron bound to dipyridyl in vitro and suggest that it should be possible for HFbp to compete for iron bound to dipyridyl in the periplasm.


Figure 3: Iron saturation of HFbp by Fe(dipyridyl)(2). Increasing amounts of Fe(dipyridyl)(2) were added to 60 µM apo-HFbp as described under ``Experimental Procedures.'' Binding of iron by HFbp was monitored by the increase in absorbance at 483 nm, the visible maximum of the ferrated protein. This data demonstrates that apoHFbp can efficiently compete for dipyridyl-bound iron in vitro.



An experimental approach based upon that described for defining the sfuABC operon by Zimmerman et al.(25) was used to investigate iron transport from the periplasmic space to the cytoplasm. This approach employs E. coli H-1443, an aroB strain which is deficient in the synthesis of aromatic compounds (38) including amino acids and the siderophore enterochelin(39) . The growth of this strain is inhibited by 200 µM dipyridyl in nutrient agar; however, at concentrations of 100 µM dipyridyl H-1443 will grow, presumably due to low affinity iron uptake systems (Table 3). In order to examine the in vivo competition for dipyridyl-bound iron by HFbp, H-1443 was grown under conditions in which trace concentrations of Fe-dipyridyl were incorporated into 75 µM dipyridyl-containing nutrient agar. This strain and an HFbp-expressing isogenic variant containing the plasmid pBSJ1 were propagated for 12 h. At this time organisms were harvested and washed, and periplasmic fractions were extracted from cells as described under ``Experimental Procedures.'' The concentrations of labeled iron associated with the periplasm and the non-periplasmic components were compared for both strains (Fig. 4). The results demonstrate that both strains had equivalent levels of radioactivity associated with the non-periplasmic fraction. This is consistent with the observation that these bacteria share common low affinity systems for iron uptake. In contrast, the strain expressing HFbp contained 25-fold more Fe in the periplasm than did the plasmid-free H-1443. This is consistent with the prediction that the presence of HFbp in the periplasm would allow accumulation of free iron from Fe-dipyridyl at this site. This demonstrates that, similar to the in vitro ability of apoHFbp to mobilize iron bound to dipyridyl, periplasmic Hfbp can effectively liberate Fe from dipyridyl.




Figure 4: Competition for dipyridyl-bound labeled iron by HFbp in the periplasm. E. coli strain H-1443, with and without the HFbp-producing plasmid pBSJ1, was grown overnight on nutrient agar containing 75 µM dipyridyl and Fe, as specified under ``Experimental Procedures.'' Bacteria were scraped from plates, washed, and separated into periplasmic and non-periplasmic fractions. The amount of iron in either fraction was determined by scintillation counting. The results demonstrate the ability of the strain expressing HFbp to efficiently concentrate iron in the periplasm.



Complementation of a Siderophore-deficient Strain of E. coli for Growth on Medium containing Ferric-dipyridyl

Production of a functional siderophore by aroBE. coli strain DH5alpha permits its growth on nutrient agar containing 200 µM dipyridyl (NA/Dip) (Table 3). In contrast, the aroBE. coli strain H-1443 and the HFbp-expressing pBSJ1 transformant could not be propagated on NA/Dip but could be grown on nutrient agar containing 100 µM dipyridyl (Table 3). Thus, the presence of the HFbp is not sufficient for the removal and transport of iron into the cytoplasm. This implies that a functional operon is required for complementation, as can be inferred from the original studies of the sfuABC operon demonstrating that the entire operon was required for complementation to growth on this medium (25, 39) .

Based upon the above observations, we investigated the ability of the hitABC operon to confer growth upon aroB H-1443 on NA/Dip. The plasmid pJDS150 derived by Hansen and colleagues (24) contains an H. influenzae DNA fragment encoding the 4.0-kb hitABC operon and an additional 7.5 kb outside of this operon (Table 1, ``Experimental Procedures''). This plasmid conferred upon E. coli strain H-1443 the ability to grow as small colonies on NA/Dip (data not shown). To demonstrate that only the hitABC genes were required for this functional complementation, a 4.2-kb PCR fragment containing only hitABC was amplified from pJDS150 and cloned into pBR322. The resulting plasmid pAHIO (Fig. 5) allowed H-1443 to grow as single colonies on NA/Dip (Fig. 6, Table 3). Isolated colonies of H-1443(pJDS150) (data not shown) or H-1443 (pAHIO) were approximately 3-fold smaller than those obtained from plating the aroB DH5alpha (data not shown). The negative controls, untransformed H-1443 (data not shown) and H-1443(pBR322) (Fig. 6), were incapable of growth on NA/Dip.


Figure 5: Outline of the construction of the hitABC-containing plasmid pAHIO. As described under ``Experimental Procedures,'' a PCR fragment containing the hitABC operon with minimal flanking sequence was amplified from pJDS150. Using SmaI and BamHI ends, the fragment was cloned into the EcoRV and BamHI sites of pBR322.




Figure 6: Complementation of aroBE. coli for growth on NA/Dip. E. coli strain H-1443 containing either pAHIO or pBR322 were grown to mid-log and plated to obtain 100-200 colony-forming units/plate, on either NA or NA/Dip. Plates A and C represent the growth of H-1443(pBR322) and H-1443(pAHIO), respectively, on NA/Dip, while plates B and D represent their growth on NA. Pinpoint colonies observed in plate C demonstrate the ability of the hitABC operon to complement H-1443 to growth on dipyridyl-containing media.



The hitABC iron acquisition operon appears to behave as a classical active transport system dependent upon a periplasmic binding protein, a cytoplasmic permease, and a nucleotide-binding protein. Therefore, disruption of any of these three components should eliminate the function of the system. In order to demonstrate the requirement for a functional hitABC operon for growth, a 400-bp C-terminal deletion of the hitC gene, designated pAHIODeltahitC, was constructed (``Experimental Procedures''). Consistent with the above prediction, H-1443(pAHIODeltahitC) was unable to grow on NA/Dip (Table 3).

Complementation for growth is a gross measure of the molecular events contributed by the hitABC operon. By examining the distribution of labeled iron in the cell, the efficiency of iron transport at the molecular level can be measured. We investigated the movement of iron from the periplasm into the cytosol using Fe to correlate transport with the presence or absence of the hitABC components. Using isogenic variants expressing all or part of the operon, the distribution of Fe into the periplasmic or the non-periplasmic cell-associated fractions was analyzed. As described above, bacteria were grown on Fe-dipyridyl/nutrient agar media, harvested, and separated into the periplasmic and the non-periplasmic cell-associated fractions. Fig. 7shows the distribution of Fe for each of the isogenic variants. The HFbp-expressing H-1443(pBSJ1) showed a level of radioactivity in the periplasmic fraction similar to that observed for the hitABC operon-expressing H-1443(pAHIO). This is consistent with the previous assertion that HFbp is effectively mobilizing Fe from dipyridyl and concentrating it in the periplasmic space of these bacteria. However, the greatly increased amount of non-periplasmic cell-associated radioactivity in H-1443(pAHIO) compared with H-1443(pBSJ1) indicates that the effective transport of periplasmic Fe is dependent on the complete hitABC operon encoded by pAHIO. The profile of the H-1443(pAHIODeltahitC) bacteria is nearly identical to that of the H-1443 (pBSJ1) strain. This is consistent with the fact that pAHIODeltahitC should produce comparable amounts of HFbp but be unable to mediate transport of iron due to the nucleotide-binding protein deletion.


Figure 7: Comparison of labeled iron uptake by E. coli strain H-1443 isogenic variants containing one of three plasmids: pBSJ1 (HFbp producing), pAHIO (complete hit operon), or pAHIODeltahitC (complete hit operon with deletion in the hitC gene). These analogs were grown overnight on NA containing 75 µM dipyridyl and Fe as specified under ``Experimental Procedures.'' The isogenic bacterial cultures were scraped from plates, washed, and separated into periplasmic and cell pellet fractions. The amount of iron in either fraction was determined by scintillation counting. These results demonstrate that an intact hitABC operon is required for the transport of iron from the periplasm to the cytosol, forming the basis for the functional complementation observed in Fig. 6.




DISCUSSION

This study represents the characterization of a periplasmic free iron transport system at the genetic and biochemical levels. By cloning, overexpressing, and purifying the hitA gene product, we demonstrated that HFbp competes for iron bound to dipyridyl in vitro. Furthermore, HFbp can compete in vivo for dipyridyl-bound iron in the E. coli periplasm. Finally, growth on dipyridyl-containing agar can be conferred upon the E. coli host by expression of a functional operon. The model proposed in Fig. 8illustrates the mechanism by which dipyridyl-bound iron at sufficient concentrations diffuses across a permeable outer membrane into the periplasm, where HFbp is able to compete for this iron source. Subsequent transport of iron across the cytoplasmic membrane can only be accomplished in the presence of a functional cytoplasmic membrane permease and a nucleotide-binding protein, as evidenced by growth as pin-point colonies on NA/Dip (Fig. 6) and the increased non-periplasmic cell-associated concentration of labeled iron (Fig. 7). The observation that these colonies were small relative to those produced by aroBE. coli indicates that dipyridyl-associated iron is limiting the growth of the transformed E. coli under these conditions. The implication of this observation is that studying the hit operon in this E. coli background may provide an insightful model for detecting subtle alterations in iron-transport properties of this operon. In toto, these studies elucidate the component steps in the active transport of free iron from the periplasm to the cytosol at the biochemical level.


Figure 8: Model for hitABC-mediated iron acquisition from dipyridyl in aroBE. coli. The E. coli strain H-1443 is unable to access dipyridyl-bound iron since it lacks the production of a periplasmic transport system. Expression of HFbp (pBSJ1) allows concentration of iron from dipyridyl in the periplasm, without further transport to the cytosol (Fig. 7). Supplying the intact hitABC operon (pAHIO) allows both the concentration of iron in the periplasm and subsequent transport into the cytosol. As such, this transport system represents the molecular basis for the growth of pAHIO-containing strains on media containing the iron chelator dipyridyl.



Common themes in the transport of molecules across membranes have begun to emerge from diverse and detailed studies of prokaryotic and eukaryotic systems. For example, we have recently demonstrated that NFbp functions in the transport of iron across the periplasm in a manner analogous to the transport of iron in serum by the vertebrate Transferrins(35) . This analogy holds not only at the level of function, but also at the level of structure: an identical set of iron-binding ligands is used by NFbp and by transferrin(35) . As a closely related protein homologue, HFbp can be reasonably predicted to function in a similar capacity. Another common theme is the utilization of an ABC (for ATP Binding Cassette) transporter-exporter protein complex(40, 43) . ABC transporter-exporters require energy for transport. This energy is supplied by hydrolysis of a nucleotide triphosphate, which is facilitated by the nucleotide-binding protein. In eukaryotic systems (e.g. the mammalian P-glycoprotein drug exporter, MDR), ATP hydrolysis and membrane permease activities are contained on a single polypeptide. In contrast, the cytoplasmic permease and nucleotide binding activities exist on two separate polypeptides in the bacterial ABC importers(40, 41, 42) . This is consistent with the data presented above for the transport of growth-essential iron by the hitABC operon.

An important aspect of these studies is the correlation of this operon family with bacterial pathogens. The ability to optimally compete for iron from the host environment correlates with pathogenicity of Neisseria species(44, 45) . The microbial pathogens N. gonorrhoeae and N. meningitidis both express antigenically detectable levels of NFbp when propagated under conditions of iron stress, whereas the closely related commensal Neisseria species (e.g. N. sicca and N. perflava) do not(32, 36) . Since we have shown that HFbp requires a functional permease and nucleotide-binding protein to function, it is presumed that these are also present in pathogenic Neisseria but not in commensal Neisseria. Furthermore, the studies of Sanders et al.(24) demonstrated that a functional hitABC operon was associated with a non-typable H. influenzae isolate. This functional operon was used to complement a type b H. influenzae to growth on iron-limited medium. H. influenzae causes a spectrum of disease, ranging from asymptomatic colonization to invasive bacterial meningitis(46) . It is possible that the presence of a functional hit operon may contribute to the differences in pathogenicity of individual H. influenzae isolates.

In addition to the periplasm-to-cytoplasm transport of iron investigated in this report, it has been demonstrated that delivering iron to the periplasmic space is a critical step in obtaining iron from the host environment. We have recently defined a human transferrin-binding protein complex oriented at the surface of H. influenzae(16) . This complex is required as the first step in the process of assimilation of iron from human transferrin. In the current study we circumvent the delivery of iron from transferrin into the periplasm by substituting dipyridyl as an iron source to a E. coli host lacking a functional transferrin receptor complex. Two molecules of dipyridyl bind a single molecule of iron at physiologic pH, giving the complex a molecular mass of less than 400 Da, a size that is freely diffusible through the E. coli porin. Alternatively, since dipyridyl is a hydrophobic compound it may diffuse through the outer membrane by partitioning into the lipid bilayer in a manner similar to what is observed for erythromycin(47) . In either case iron is delivered to the periplasm and the requirement for a functional outer membrane receptor complex is subverted. These studies demonstrate that the hitABC operon can function independent of the transferrin receptor. This correlates with the fact that the sfuABC system from S. marcescens has been demonstrated to be TonB independent, a characteristic which would preclude the involvement of an energy-dependent outer membrane receptor complex. The significance of this mechanism of iron uptake in such a diverse range of microorganisms and its role in pathogenicity is unknown.

HFbp and NFbp are homologous in their biochemical attributes. Because of this relatedness, it is likely that both proteins serve similar functions in these two bacterial species. It has been previously demonstrated that NFbp transiently associates with labeled iron mobilized from transferrin. This evidence has implicated NFbp in the transport of iron from the transferrin-binding complex to the cytoplasm (17) . This study illustrates that, similar to the sfuABC system, the hitABC system is capable of functioning in the absence of Tbp1/2 by acquiring iron directly from sources which diffuse into the periplasm. As a result, the hitABC transporter and the Neisserial equivalent may function in a broader role as a high affinity, periplasmic iron scavenging system. Further characterization of this system will enhance our understanding of this mechanism of iron acquisition and allow a more detailed molecular understanding of how the Fbp family of proteins participates in the process of periplasmic iron transport.


FOOTNOTES

*
This investigation was supported by Grant 1R29AI32226-01 from the National Institutes of Allergy and Infectious Disease (to T. A. M.) and Grant MT-10350 from the Medical Research Council of Canada (to A. B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 412-648-9245; Fax: 412-624-1401.

Supported by a Studentship from the Alberta Heritage Foundation for Medical Research.

(^1)
The abbreviations used are: kb, kilobase(s); Fbp, ferric iron-binding protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); NA/Dip, nutrient agar containing 200 µM dipyridyl.


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