Department of Microbiology and Immunology, Emory School of Medicine, 1510 Clifton Rd, Atlanta, GA 30322, USA
Correspondence
D. Perkins-Balding
dbaldin{at}emory.edu
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ABSTRACT |
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INTRODUCTION |
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Haem/haemoglobin (Hb) receptors have been identified in many bacterial species, both pathogens and commensals (Cope et al., 1994; Henderson & Payne, 1994
; Letoffe et al., 1994
; Stojiljkovic & Hantke, 1992
). These receptors share a large degree of structural homology with the TonB-dependent lactoferrin and transferrin receptors of Gram-negative bacteria (Gray-Owen & Schryvers, 1996
; Schryvers & Stojiljkovic, 1999
; Schryvers et al., 1998
). The common theme of haem/Hb receptors is their ability to bind haem compounds, remove haem from them, and transport haem into the periplasm (Wandersman & Stojiljkovic, 2000
). Haem/Hb receptors can be loosely grouped into three subfamilies based on the level of amino acid homology, overall design and substrate specificity. Haem scavengers' are a subfamily of enterobacterial haem/Hb receptors that share more than 60 % identical amino acid residues and have wide substrate specificity (Bracken et al., 1999
; Wandersman & Stojiljkovic, 2000
). Haem/Hb receptors from Haemophilus, Neisseria and Vibrio spp. share between 25 and 50 % identity and have narrow substrate specificity. The third subfamily of haem utilization systems is found in Serratia, Pseudomonas and Haemophilus spp., where special proteins called haemophores are secreted into the medium. Haemophores bring haem to the outer cell surface, where a TonB-dependent receptor takes haem and transports it into the periplasm (Cope et al., 1994
; Letoffe et al., 1994
, 1998
).
We have identified a high-affinity Hb outer-membrane receptor, HmbR, of Neisseria meningitidis (Stojiljkovic et al., 1995). HmbR enables meningococci to bind and use Hb as a source of iron and porphyrin (Stojiljkovic & Srinivasan, 1997
; Stojiljkovic et al., 1996
, 1995
). HmbR-mediated Hb utilization has been reconstituted in Escherichia coli. As expected for a member of the TonB-dependent family of receptors, HmbR requires expression of the N. meningitidis Ton system to function as a Hb receptor in E. coli (Stojiljkovic & Srinivasan, 1997
). Once HmbR transports haem into the periplasm, it is most likely delivered to the cytoplasm by HemTUV-like proteins, although homologous proteins have not been identified yet in N. meningitidis (Stojiljkovic & Hantke, 1994
). In the cytoplasm, a newly identified haem oxygenase, HemO, is involved in the catabolism of haem in N. meningitidis to iron-biliverdin and CO (Ratliff et al., 2001
; Zhu et al., 2000a
, b
).
Despite a large volume of information on haem/Hb receptors collected in the last decade, important questions about their structure and mechanism of action remain largely unanswered. For example, very little is known about the receptor domains that participate in interactions with haem and haemcarrier complexes. In addition, these receptors must be able to extract haem from different protein carriers prior to the transport step, but the mechanism of haem removal is not understood. In the enterobacterial scavenger subclass of haem/Hb receptors, amino acid residues that participate in the transport of haem through the receptor pore have been identified. Two histidine residues, His128 and His461, are essential for the function of the Yersinia enterocolitica HemR receptor (Bracken et al., 1999).
To date, none of the haem/Hb receptor structures have been solved by crystallography. In order to gain information about the structure of HmbR, our approach followed the analysis of outer-membrane transporters, FepA, FhuA and LamB, using deletions to ascertain which surface-exposed loops of HmbR contribute to specific functions (Boulanger et al., 1996; Braun et al., 1999
; Carmel & Coulton, 1991
; Klebba et al., 1994
; Newton et al., 1999
). This approach exploits the tendency for deletions in loop regions of outer-membrane proteins to have little effect, if any, on proper folding, assembly and localization (Benson et al., 1988
; Klebba et al., 1994
; Newton et al., 1999
). Deletion of transmembrane regions, however, results most often in generating proteins that are either expressed at low levels or proteolytically degraded (Klebba et al., 1994
; Lathrop et al., 1995
; Newton et al., 1999
). This approach is useful in narrowing down the functional regions of an extremely large outer-membrane protein, such as HmbR, so that a more targeted analysis can be done.
In this communication, we propose a model for the structure of HmbR, which, like all of the characterized outer-membrane transporters of Gram-negative bacteria, has an extended -sheet that will putatively form an amphiphilic barrel in the outer membrane (Buchanan, 1999
; Koebnik et al., 2000
). Surface-exposed loop regions were predicted from this model and used as a guide for deletion mutagenesis. We identified HmbR mutants that were defective in Hb binding and/or Hb utilization. Further, we demonstrated the importance of the cork-like region, modelled after the plug domains of outer-membrane transporters FepA, FhuA and FecA and the highly conserved central motifs of Hb/haem receptors in the utilization of haemprotein complexes (Bracken et al., 1999
; Wandersman & Stojiljkovic, 2000
).
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METHODS |
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Construction of hmbR deletion and site-directed mutants.
Deletions in hmbR were constructed using restriction sites NotI, PstI and SalI, or by introducing BglII or EcoRI restriction sites at the position of the desired deletion end-points. The majority of inserted restriction sites did not introduce new amino acid residues into the HmbR sequence. By using convenient hmbR restriction sites, mutated receptor genes were reconstituted with the smallest possible PCR-generated hmbR fragment still carrying the desired deletion and cloned into pACYC184 vector (the largest PCR-amplified fragment was 1·2 kb, the smallest 0·1 kb). The nucleotide sequence of all PCR-generated DNA fragments was determined to rule out the existence of secondary mutations.
Construction of an HmbR overexpression plasmid.
Insertion of a His tag into pIRS1173 was done using DNA primers: His-A: (5'-GCATCATCACCATCACCATCTGCA-3') and His-B (5'-GATGGTGATGGTGATGATGCTGCA-3'). The primers were annealed and cloned into PstI-digested plasmid pIRS1173, to produce pSYB1724. The nucleotide sequence was determined to confirm that no unintended mutations had been generated. Hb and haem utilization assays were performed on E. coli strain IR1583 harbouring this plasmid to ensure that HmbR-6His was functional.
Gene replacement of hmbR mutant alleles in N. meningitidis IR1072.
Mutant alleles of hmbR, demonstrating defective Hb binding or Hb utilization phenotypes in E. coli, were used to replace the wild-type copy of hmbR in a serogroup C, clinical isolate of N. meningitidis, IR1072. First, a spontaneous streptomycin-resistant mutant of IR1072, IR4130, was isolated by plating 1x1010 c.f.u. of IR1072 onto GCB agar containing 750 µg streptomycin ml-1. IR4130 was transformed with plasmid pAP1184-FLOB and an hmbR knockout transformant (IR3727) was selected on erythromycin-containing GCB agar plates. Plasmid pAP1184-FLOB is a derivative of pAP1184 (pWKS30 with hmbR[H359R369]), which has the rpsL ermC cassette from pFLOB4300 inserted into the unique BglII site in the hmbR gene (Johnston & Cannon, 1999
). Strain IR3727 containing hmbR : : rpsL ermC mutation (streptomycin sensitive) was subsequently transformed with plasmids carrying hmbR mutant alleles (pAP1183, pAP1184, pAP1185, pAP1192, pAP1203, pAP1325, pAP1383, pAP1384, pAP1402, pAP1420, pAP1492, pAP1561, pAP1562, pAP1565, pAP1725, pAP1751, pAP1752, pAP1753 and pAP1770) and selected on GCB agar containing 750 µg streptomycin ml-1, to generate strains IR3769, IR3770, IR3771, IR3728, IR3793, IR3773, IR3778, IR3729, IR3794, IR3730, IR3795, IR3792, IR3788, IR3780, IR3731, IR3732, IR3796, IR3781 and IR3733, respectively. The genotypes of the mutant hmbR alleles were checked by PCR and a restriction digest with the appropriate restriction endonuclease.
Phenotypic testing of HmbR mutants.
All hmbR constructs were subcloned onto low-copy-number plasmid pWKS30 and transformed into the E. coli IR1583 strain expressing the TonB, ExbB and ExbD proteins of N. meningitidis. The ability of E. coli to use Hb and haem was tested on NBD plates (Nutrient Broth-dipyridyl) as described by Stojiljkovic & Srinivasan (1997). Haemin (bovine), human serum albumin and Hb (human) were obtained from Sigma. To test the ability of N. meningitidis strains to use haem-containing compounds as sole iron sources, a suspension of bacteria was plated onto GCB agar containing 50 mM desferrioxamine mesylate (Desferal, Ciba Geigy). Filter discs (0·25 inches, Schleicher & Schuell) impregnated with test compounds (10 µl of 5 mg ml-1 stock solutions unless otherwise stated) were placed on these plates. Zones of growth around the discs were recorded after overnight incubation at 37 °C in the presence of 5 % CO2 (Stojiljkovic et al., 1995
).
Small-scale purification of HmbR.
E. coli strain IR3519, containing plasmid pSYB1724, was inoculated at a 1 to 100 dilution into 200 ml L-broth containing 30 µg chloramphenicol ml-1. Cells were grown to mid-exponential phase, and HmbR-6His expression was induced from the native promoter using 0·35 mM dipyridyl for 3 h at 37 °C. Outer membranes were prepared from spheroplasts using the method of Hantke (1981). Outer membranes were solubilized in 1 % ZW3-14/TBS (TBS is 50 mM Tris/HCl, 100 mM NaCl, pH 7·5). Solubilized outer membranes were diluted in 25 ml 20 mM phosphate buffer, pH 7·4, 0·5 M NaCl, 0·9 % N-octylglucoside, 0·25 mM PMSF. HmbR-6His was bound to Ni-NTA agarose (Qiagen), washed with 3 vols of the dilution buffer, and eluted in the dilution buffer containing 500 mM imidazole. The yield of purified HmbR was 0·25 mg per ml of culture.
Outer membrane isolation and HmbR quantification.
Preparations of neisserial outer membranes were made as described by Richardson & Stojiljkovic (1999). The total protein content of the outer membranes was determined by solubilization of membrane material followed by its quantification using a modified Lowry assay, the Bio-Rad DC (detergent-compatible) protein assay.
The amount of HmbR and HmbR mutant proteins per outer-membrane sample was determined by Western blotting as follows. Outer-membrane samples containing 140 µg total protein were electrophoresed on a 7·5 % SDS-polyacrylamide gel, and transferred to Hybond-P PVDF membrane (Amersham Pharmacia) using a Schleicher and Schuell electroblotting apparatus. The membrane was blocked in PBST (100 mM phosphate-buffered saline, pH 7·5, 0·5 % Tween 20) containing 5 % dry milk and incubated with rabbit polyclonal antiserum raised against a fusion protein consisting of HmbR and glutathione S-transferase (GST-HmbR). A secondary antibody, anti-rabbit IgG conjugated to alkaline phosphatase (Sigma), and fluorescent (ECF) substrate (Amersham Pharmacia) was used for detection, and visualized on a Molecular Dynamics Storm 860 Phosphorimager. ImageQuant software (Molecular Dynamics) was used to analyse the signal intensity. The amount of HmbR mutant protein per sample was extrapolated from a standard curve that was generated using known quantities of purified HmbR. The amount of the wild-type HmbR in 140 µg of total protein material was 350-fold higher than the lower limit of detection for HmbR by this method; therefore, those outer membranes that did not yield a detectable signal for HmbR were below this limit of detection and were not analysed further. Outer membranes that had detectable levels of HmbR were quantified by this method multiple times. Equal quantities of HmbR and HmbR mutant proteins in outer-membrane samples were then immobilized on nitrocellulose for Hb binding analysis as described below.
Biotinylation of Hb.
Human Hb (Sigma) was biotinylated using sulfo-NHS-LC-biotin (Pierce Endogen) and purified over a 10 ml desalting column. Biotin incorporation was determined by the HABA [2-(4'-hydroxyazobenzene)benzoic acid] assay (Pierce Endogen), with 1·9 mol biotin incorporated per mol dissociated (1
1) Hb dimers.
Binding of biotinylated Hb to outer membranes immobilized on nitrocellulose.
Outer-membrane samples from N. meningitidis, each containing 200 ng HmbR (quantified as described above), were resuspended in 200 µl TBS and serially diluted twofold. The outer membranes were transferred onto Hybond-P (PVDF) membrane (Amersham Pharmacia) using a microsample filtration manifold (Minifold I, Schleicher & Schuell). The PVDF membrane was blocked with 5 % skim milk in TBST (TBS+0·1 % Tween 20) for 2 h at room temperature. The membrane was incubated with 50 nM biotinylated Hb (16 µl of 2 mg ml-1 stock) in 10 ml 5 % skim milk/TBST for 1 h at room temperature. The membrane was rinsed three times with TBST for 15 min. Hb binding was detected by incubation of the membrane with 5 µCi [35S]streptavidin (Amersham Pharmacia) diluted in 10 ml TBST solution for 1 h at room temperature. After rinsing the membrane with TBST three times for 15 min, Hb binding was quantified using a Molecular Dynamics Storm 860 Phosphorimager and by radiocounting in a Beckman LS6500 scintillation counter. Measurements using both methods were compared for consistency. Error in Hb binding values due to non-specific binding of Hb to outer membranes was corrected by subtracting the radiocounts (phosphorcounts) of equivalent quantities of HmbR- outer membranes.
Construction of hmbR alleles expressed from an araC-regulated promoter.
The 3·3 kb BamHIHindIII DNA fragment carrying the hmbR gene was first cloned into pBAD24 from pIRS626 (Guzman et al., 1995; Stojiljkovic et al., 1995
). The DNA fragment carrying the 5' end of the hmbR gene was PCR-amplified using the HmbREcoRI and internal HmbR primers. Amplified fragment was digested with EcoRI and NotI restriction endonucleases and cloned into hmbR-containing pBAD24. The resulting plasmid pBAD1352 contains the hmbR start codon positioned immediately downstream of the ribosome-binding site.
hmbR mutant alleles were subcloned into pBAD1352 from pACYC184-derivative plasmids, pIRS1402, pIRS1725, pIRS1192, pIRS1770, pIRS1185 and pIRS1492. The hmbR-internal NotISalI DNA fragment within each of these plasmids was gel purified and cloned into the NotISalI region of pBAD1352, replacing the wild-type sequence. Restriction analysis and sequencing was used to confirm the presence of mutant alleles. Outer membranes were prepared from 2·5x109 cells of each strain and Western blotting using rabbit polyclonal antiserum against HmbR was performed on these samples to demonstrate outer-membrane localization of mutant HmbRs and equal protein expression.
In vivo binding of biotinylated Hb to whole cells and flow cytometry.
Hb binding to whole cells was measured using flow cytometry on a FACS Calibur flow cytometer (Becton Dickinson). Bacteria were inoculated at a 1 : 100 dilution from an overnight culture into fresh Luria broth containing ampicillin (100 µg ml-1) and -aminolaevulinic acid (50 mg ml-1). Cells were grown at 37 °C with shaking to an OD600 of 0·30·4, and then HmbR expression was induced for 1·5 h with 0·04 % arabinose (unless otherwise indicated). Approximately 0·6x108 cells were harvested and washed with PBS-BSA (20 mM sodium phosphate buffer, pH 7·4, 150 mM NaCl, 10 mM MgCl2, 1 % BSA). Cells were resuspended in PBS-BSA with 1 µM biotinylated Hb and incubated on ice for 10 min. Cells were then diluted tenfold, fixed in 1 % formaldehyde PBS-BSA buffer, and washed. PE-conjugated streptavidin (Caltag Laboratories) was used to label cells with bound Hb. Data were collected for 2x104 cells per sample as determined by forward light scatter and side scatter intensity, and were analysed using the FlowJo software package (Tree Star).
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RESULTS |
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Mutations.
Nineteen deletion and site-directed mutations in HmbR were created to test the predictions of our topological model. Mutations were primarily generated to delete or partially delete putative surface loops; eight (of eleven) of the largest loops were targeted in this analysis. In addition, several deletions were made in the putative cork domain. Allelic exchange was used to replace the wild-type hmbR allele with these mutant alleles in the chromosome of N. meningitidis IR4130. Western blots indicated that HmbR mutant proteins were expressed in the outer membranes of N. meningitidis, which included deletions in the cork domain, L2, L3, L5, L6, L7, L8 and L10 (Fig. 2a). Most of the HmbR mutant proteins that were defective in outer-membrane export or assembly had deletions in predicted transmembrane regions, as expected from deletions of related ferric siderophore transporters (Klebba et al., 1994
; Lathrop et al., 1995
; Newton et al., 1999
). Two independent deletions of L11 were attempted, but neither produced a mutant protein that could localize to the outer membrane. Ten additional mutants were tested in E. coli and were not transformed into N. meningitidis because they failed to localize to the outer membrane.
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Phenotypic characterization of hmbR deletion derivatives in N. meningitidis
Six of the nineteen strains containing hmbR mutant alleles in N. meningitidis (hpuAB) strain IR1072 were functionally defective in either Hb binding or haem utilization and are shown in Fig. 1 and Table 2
.
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Hb utilization.
The HmbR mutants 1402 (cork), 1725 (L2), 1192 (L3), 1185 (L6), 1492 (L6) and 1770 (L7) were unable to support growth of meningococci on Hb as a sole source of iron (Fig. 3b). However, several other HmbR mutants with lowered expression, on the basis of antibody binding, were fully functional in this assay.
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Hb binding to HmbR mutant proteins in vivo
In the previous in vitro binding assay, N. meningitidis outer membranes were isolated by detergent extraction and were subject to drying on nitrocellulose. To confirm these Hb binding phenotypes seen in N. meningitidis and to address the possibility that the assay conditions may have affected the binding activity of HmbR mutants, an in vivo Hb binding analysis was performed using whole cells of E. coli expressing HmbR variants at high levels from an araC-regulated promoter. The amount of Hb binding by whole cells was assayed by measuring association to fluorochrome PE-conjugated streptavidin using flow cytometry. The greatest sensitivity for the flow cytometry analysis of Hb binding was achieved in E. coli; when using N. meningitidis in this assay the difference between the fluorescence values was small (roughly tenfold) with the wild-type HmbR-expressing cells poorly labelled. Increasing HmbR expression using iron induction and IPTG induction, as well as using a non-encapsulated strain, did not improve labelling enough that differences in mutants could be evaluated with any level of confidence (data not shown).
SDS-PAGE analysis and Western blotting confirmed that HmbR expression could be induced from the araC-regulated promoter, and nutrition assays demonstrated the ability of E. coli IR1583 expressing HmbR to grow with Hb as a sole source of iron (data not shown). Different concentrations of the arabinose inducer were tested to determine the concentration required to achieve the maximum level of HmbR expression (data not shown). Outer membranes were prepared from E. coli strains expressing HmbR mutant proteins and examined using SDS-PAGE, stained with Coomassie blue (Fig. 4a), and immunoblotting using rabbit polyclonal antiserum against HmbR (Fig. 4b
). HmbR mutant proteins overexpressed in E. coli IR1583 localized to the outer membrane and were expressed at high levels, according to a standard curve using purified HmbR. Relative to each other, only mutant 1402 (cork) seemed to be expressed at lowered levels.
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DISCUSSION |
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Our mutagenesis-based approach to identify the functional domains of HmbR followed the precedents of other outer-membrane protein studies, including LamB and the ferric siderophore receptors (Klebba et al., 1994; Newton et al., 1999
). A growing body of evidence indicates that surface-exposed peptide loops of outer-membrane transporters confer specificity for a substrate (Barnard et al., 2001
; Newton et al., 1999
; Scott et al., 2001
). Recently, the specificity of TonB-gated porins, FepA and FhuA, and their high affinity for ferric siderophores was attributed to their surface loops by equilibrium binding analyses (Scott et al., 2001
). Both loop deletions and PCR mutagenesis of FepA point to substrate specificity function associated with loop domains (Barnard et al., 2001
; Newton et al., 1999
). Co-crystal structures of FhuAferrichrome and FecAdiferricdicitrate demonstrate binding of ligand to external loops and cork domain (Ferguson et al., 2002
; Lambert et al., 1999
). Our purpose for targeting surface-exposed loops of HmbR was to reduce the formidable task of studying individual amino acids in such a large protein, and to identify the contributions of individual loops in Hb binding, haem stripping and transport. Previously, mutagenesis of FepA and FhuA has shown that, with some exceptions, deletions and insertions in surface-exposed loops are usually tolerated while deletions of transmembrane regions lead to defects in receptor assembly and/or export (Braun et al., 1999
; Buchanan et al., 1999
; Ferguson et al., 1998
; Killmann et al., 1996
; Koebnik & Braun, 1993
; Lathrop et al., 1995
; Liu et al., 1993
; Locher et al., 1998
; Newton et al., 1999
; Postle, 1999
). In addition, HmbR variant proteins described in this study were shown to retain TonB-dependent haem transport function. Productive interactions with the TonB/ExbBD complex and a stable pore for haem to pass through are good indicators that gross changes in conformation have not resulted from loop deletions.
This study has produced several important insights into HmbR structurefunction relationships despite potential pitfalls of mutagenesis-based approaches. First, it separated the functions of haem from Hb utilization (in E. coli) since all HmbR mutants expressed in the outer membrane were efficient in haem use irrespective of their Hb utilization phenotypes. Haem usage was also shown to be a TonB-dependent activity in E. coli, suggesting that haem is passing through the transporter in a physiologically relevant manner and not simply leaking through a disordered outer membrane. The lack of appropriate mutants in TonB-independent haem uptake in N. meningitidis prevented us from determining whether this also holds for HmbR expressed in meningococci. We assessed haem utilization as an indicator of proper conformation and outer-membrane localization, because any mutations, especially deletions and insertions, can produce abnormalities in protein structure that may affect protein function in an irrelevant way. Since haem transport was shown to be a TonB-dependent process in E. coli, mutant receptors were also interacting with TonB in an appropriate manner. Only mutants that were proficient in haem utilization and present in the bacterial outer membrane by Western blot analysis were selected for further study. Loss of haem utilization was consistent with a change in tertiary structure of functional importance.
Second, the study revealed two types of Hb utilization-negative mutant receptors, those deficient in the Hb binding step and those deficient in Hb utilization steps that follow Hb binding. Deletion mutations which allowed HmbR to bind Hb well, but did not allow the use of Hb as an iron source, localized to the carboxyl-terminal region of HmbR in L6, L7 and the cork domain. These HmbR mutants were proficient in use of free haem, which suggests that the defect is limited to the removal of haem from Hb. The Hb binding of these variant proteins was not at the wild-type level, but they were not dramatically impaired. Since haem removal may be a complex process, it is possible that there are multiple sites of haem contact on several surface loops and the loss of any of these sites may compromise haem removal. This may explain how deletions in L6 exhibit slightly different Hb-binding activities. It is important to emphasize that the outer-membrane localization properties of these HmbR mutants were indistinguishable from those of the wild-type HmbR; again, however, the possibility of secondary effects of deletions on the HmbR structure cannot entirely be ruled out. Deletion of proximal loops, however, L5 and L8, had no effect on Hb binding or haem utilization, suggesting that they are not involved in either process.
The region of HmbR deleted in L7 (mutant 1770, V489G502) is part of a highly conserved amino acid sequence in all haem/Hb receptors (Bracken et al., 1999
). Deletion of this conserved domain resulted in a Hb-binding-proficient, but Hb-utilization-deficient phenotype. A portion of this conserved motif including the NPNL box has homology to the more distantly related FepA transporter (Bracken et al., 1999
). Much like HmbR, deletions in L7 (and L8) of FepA resulted in no binding or transport of ferric enterobactin, and resulted in what was described as an open channel based on increased susceptibility to large antibiotics (Newton et al., 1999
). Structural studies of FecA and FecAdiferricdicitrate complex have indicated substantial changes in the position of L7 and L8 of FecA upon ligand binding (11 Å and 15 Å, respectively) (Van Der Helm et al., 2002
). L7 in FecA does not appear to be involved in ligand binding, although L8 shows several interactions (Van Der Helm et al., 2002
). This region is also homologous with the region of HemR that contains the functionally important His461 residue. Like the L7 deletion in HmbR, HemR His461 mutants were unable to use any haemprotein complex including Hb while retaining the ability to use free haem as an iron source (Bracken et al., 1999
). Histidine residues often serve as axial ligands for haem binding; therefore, we targeted several histidine residues in L7 (and region) for mutagenesis (H448, H501 and H520). Replacement of these residues with alanine, however, resulted in Hb utilization phenotypes in N. meningitidis that were indistinguishable from the wild phenotype (data not shown). Ligandreceptor interactions may be multideterminant in HmbR, or the histidines in L7 may not directly interact with haem.
Mutant 1402 contains a deletion within the putative cork domain of HmbR (H64N76) and also has a Hb-binding-proficient, but Hb-utilization-deficient phenotype. This amino-terminal deletion of HmbR does not remove the putative TonB box (residues E7A13) which is conserved among the TonB-dependent transporters. The phenotype of this HmbR mutant suggests that the cork domain plays an active role in the use of haem associated with Hb. Indeed, a smaller deletion (three amino acids) in the same region (1183,
H64K66) was also partially affected in usage of Hb when expressed in meningococci. Both mutants were fully capable of supporting growth on haem, when expressed in E. coli, suggesting that transport functions of HmbR are not grossly affected. The involvement of the putative cork domain of HmbR in Hb usage is in contrast to the many recent studies of the cork domains of ferric siderophore receptors which report no significant role of the cork in substrate binding or transport (Braun et al., 1999
; Howard et al., 2001
; Killmann et al., 2001
; Scott et al., 2001
; Usher et al., 2001
). In fact without any contributions of the cork domain, FhuA has been reported to function as a TonB-dependent transporter, transporting at a rate of 45 % of wild-type FhuA (Braun et al., 1999
). Recently, some controversy has arisen as to whether in vivo reconstitution of active transporters has occurred in cork-deletion studies of FhuA and FepA (Vakharia & Postle, 2002
). Nonetheless, one difference between the ferric siderophore receptors and HmbR is that the whole siderophore traverses the outer membrane via the siderophore receptor, as free haem does with HmbR, but a second function, the removal of haem from Hb, must precede haem transport in HmbR. We propose an explanation where the cork domain of HmbR might be essential in the removal of haem from Hb, and that it is not necessarily involved in the binding or transport of free haem.
A third important finding of our study is that deletions of L2 (1725) and L3 (1192) dramatically affected the ability of HmbR to bind and use Hb while preserving the use of haem as an iron source. Both in vitro analysis using purified outer membranes from N. meningitidis and in vivo measurements of Hb binding to E. coli cells expressing HmbR mutant proteins support the involvement of this region of HmbR in Hb binding. We recognize that indirect effects of deletion mutations in loops may perturb the tertiary structure of surface domains other than the deleted loop. Clearly, additional mutagenic studies focusing on smaller regions or on individual residues, which are not as likely to have an impact on structure, will be required. However, the initial target region for analysis has been narrowed by this study. Biochemical studies will also be designed to corroborate the validity of these predictions and explain the mechanism by which HmbR allows usage of Hb and free haem as sources of iron.
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ACKNOWLEDGEMENTS |
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Received 29 April 2003;
revised 9 July 2003;
accepted 9 September 2003.