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The evolutionary success of bacterial pathogens may be considered to reside in their extraordinary talent to adapt to the environmental rigors imposed by their human host. This characteristic is exemplified by the bacterial mechanisms used to acquire iron (9, 11, 16, 22). The extracellular secretion of siderophores, low-molecular-weight iron chelators, represents a common means of securing this scarce but critical element (11). It is also becoming apparent that direct binding of host iron-containing proteins to specific receptors located on the bacterial cell surface constitutes another significant bacterial strategy for the capture of iron (9, 16, 22).

The pathogenic Neisseria spp., Neisseria gonorrhoeae and Neisseria meningitidis, produce no siderophores (28, 40). These organisms express an array of outer membrane receptors that specifically interact with human iron-bearing proteins. Genetic studies of the gonococcus and the meningococcus have indicated that iron acquisition from human transferrin and human lactoferrin is initiated by the binding of these glycoproteins to their respective receptors (9, 16). Similarly, the recently described neisserial hemoglobin receptors, HmbR (35, 36) and HpuB (7, 24), mediate the uptake of heme iron from hemoglobin and haptoglobin-hemoglobin complexes.

The events occurring subsequent to receptor binding of the iron ligands are unclear. Iron is released from transferrin and lactoferrin by unknown mechanisms and is deposited in the periplasmic space by a tonB-mediated process (37), presumably by gated pore properties of the lactoferrin and transferrin receptors energized by TonB protein (29). Biochemical data indicates that iron is subsequently transferred to the periplasmic iron-binding protein, FbpA (8). Sequence analysis and genetic complementation studies demonstrate that the gene encoding this protein, fbpA, belongs to an operon termed fbpABC, whose gene products have been proposed to behave as an ATP-binding cassette (ABC) transporter (1). These unidirectional transporter systems are ubiquitous in bacteria and function to deliver scarce nutrients from the periplasmic space into the cytosolic compartment (3, 13, 20). These systems are minimally comprised of three polypeptides, a ligand-specific periplasmic binding protein, one or two proteins embedded in the cytoplasmic membrane, and a membrane-associated nucleotide-binding protein (3, 13, 20). This latter feature is a hallmark of these systems and is responsible for the designation ABC transporter (3, 13, 20).

Similar genetic loci have been described for Haemophilus influenzae (31) and Serratia marcescens (4, 42). Mutation analysis and comparisons of the open reading frames to those of fbpABC indicate that the H. influenzae hitABC and S. marcescens sfuABC operons are functional equivalents of the neisserial fbpABC locus.

Therefore, this investigation was undertaken to address the proposed role of the fbpABC locus as a periplasmic iron transporter by first constructing a meningococcal fbpABC mutant and subsequently determining the phenotype of the mutant with respect to iron acquisition.

Bacterial strains, plasmids, and growth conditions. The bacteria and plasmids used in this study are listed in Table 1. Neisserial strains were grown on chocolate agar at 37°C in an atmosphere of 5% CO₂. Escherichia coli was cultured on Luria-Bertani (LB) agar plates or in LB broth. Transformation of neisserial (35) and E. coli (30) strains was performed as previously described. Antibiotics, where appropriate, were added at the following concentrations: kanamycin, 40 µg/ml; ampicillin, 100 µg/ml; and chloramphenicol, 15 µg/ml (Neisseria spp.) or 25 µg/ml (E. coli).

DNA preparation and manipulation. Gonococcal and meningococcal genomic DNA were prepared by standard methods (30). Isolation and purification of plasmid DNA were performed with Qiagen (Clarita, Canada) columns as described in the manufacturer's specifications. Restriction endonuclease digestions, ligation reactions, and agarose gel electrophoresis were performed as described previously (36). PCR products were recovered from low-melting-point agarose gels and purified by using NuSorb columns. Chromosomal DNA purified from wild-type and mutant N. meningitidis strains was digested to completion with the restriction enzyme XhoI and probed by high-stringency Southern blot analysis (36) employing the digoxigenin chemiluminescent system (Boehringer Mannheim Canada). The DNA probes included the 1.3-kb cat cartridge from TnMax4 and the 1.2-kb fbpA gene.

PCR. PCR amplification of chromosomal DNA from Neisseria spp. was performed in a 25-µl reaction volume containing 1× Taq reaction buffer, 1 U of Taq polymerase (Gibco BRL), 1.5 mM MgCl₂, 0.2 mM deoxynucleoside triphosphates (Gibco BRL), oligonucleotide primers at 0.5 µM each, and 50 ng of genomic DNA as template. Thirty cycles of amplification were performed with a Perkin-Elmer model 480 DNA thermal cycler. The reaction profile consisted of a 30-s denaturation step at 94°C, a 30-s primer annealing step at 52°C, and a 3-min primer extension step at 72°C.

DNA sequencing. DNA sequencing was performed on PCR fragments by the dideoxynucleotide chain termination method (32) using a PRISM Ready Reaction Dye Cycle Sequencing kit (Applied Biosystems) with fluorescence-labelled M13 primers or with synthetic oligonucleotide primers based on known fbpA and cat sequences. All sequence reactions were run and analyzed on an Applied Biosystems 373A automated DNA sequencer.

Western immunoblotting. Whole-cell meningococcal and gonococcal lysates, prepared by using a previously described procedure (5) from cultures grown under iron-limited conditions, were separated on a sodium dodecyl sulfate-10% polyacrylamide gel and electroblotted at 10 V of constant voltage for 12 h at 4°C onto polyvinylidene difluoride membranes (Immobilon-P, 0.45-µm pore size; Millipore Canada) in 25 mM Tris-192 mM glycine-20% (vol/vol) methanol (pH 8.3) by the method of Towbin et al. (39) with a Bio-Rad MiniTransblot apparatus. After saturating for unspecific binding sites with a buffer containing 0.5% (wt/vol) skim milk in 50 mM Tris-HCl (pH 7.5)-1 M NaCl (TBS-M) for 3 h at 25°C, the membrane was probed with a 1:2,000 dilution of an affinity-purified polyclonal antiserum to the gonococcal FbpA (5) (kindly provided by T. Mietzner). After incubating at 35°C for 1 h, the immunoblot was rinsed with three 5-min washes of TBS-M. A 1:2,500 dilution of the second antibody (goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate; Pierce Chemical Co.) was allowed to bind for 1 h at 35°C, and this was followed by three 5-min washes with TBS-M. The membrane was developed with a chloronaphthol-hydrogen peroxide substrate mixture (horseradish peroxidase reagent; Bio-Rad) for 20 min. The paper was washed with water to stop the reaction.

Whole-cell transferrin and lactoferrin binding assays. Whole-cell dot enzyme assays to detect binding to human transferrin and to human lactoferrin were performed as described previously (23).

Construction of the neisserial fbpABC mutant. The wild-type fbpA gene was cloned by PCR amplification from the recombinant plasmid pSBGL, which contains the full-length fbpA gene from gonococcal strain F62 (5). Site-specific primers were designed to encompass the entire coding region of fbpA, including the native Shine-Dalgarno region (primer 403) and 140 bp downstream of the fbpA stop codon (primer 181, modelled on primer F5 [5]). The PCR-amplified fbpA gene was ligated into the TA cloning vector pCR2 (Invitrogen, San Diego, Calif.), creating pCR2NGFA. The fbpA gene in pCR2NGFA was subcloned into the EcoRI site in plasmid pT7-7 (38), taking advantage of the flanking EcoRI sites surrounding the fbpA gene provided by the multiple cloning site of the pCR2 vector. A clone which possessed the fbpA gene in the same orientation as the T7 promoter in pT7-7 was designated pT7-7NGFA. The absence of a NotI restriction site in pT7-7 facilitated the subsequent ligation of the cat gene, encoding chloramphenicol acetyltransferase, into fbpA.

Identification of neisserial fbpABC mutants. Gonococcal strains PID543, F62, and FA19 and meningococcal strain B16B6 were transformed with plasmid pT7-7NGFAC that was linearized with BamHI. After 5 h of phenotypic expression, cells were plated on chocolate agar plates containing chloramphenicol. Three chloramphenicol-resistant colonies derived from meningococcal strain B16B6 were observed within 36 h. These colonies were transferred onto fresh chocolate agar-chloramphenicol plates, and one of these, designated N16FBPA, was selected for further study. No gonococcal transformants were detected despite prolonged incubation of 50 h.

View larger version (51K): [in this window] [in a new window]   FIG. 1.   PCR amplification based analysis of fbpA from the wild-type and mutant strains of N. meningitidis B16B6. (A) Schematic diagram of the neisserial fbpA and fbpB genes displaying the orientations and approximate positions of the oligonucleotide primers used in the PCR amplification assay. (B) Agarose gel demonstrating the PCR products amplified by the oligonucleotide primer pairs. Lanes: b, d, f, h, j, and l, B16B6; c, e, g, i, k, and m, N16FBPA. Primer pairs: 394-395 (lanes b and c), 180-181 (5'fbpA-3'fbpB (lanes f and g), 180-3'fbpB (h and i), 394-3'fbpB (lanes j and k), 395-5'fbpA (lanes l and m). The sizes of standards in kilobases are shown on the left. Figures were imaged with a Hewlett-Packard ScanJet IIp and labelled by using Adobe Photoshop 3.0.

FbpA expression. To confirm the absence of FbpA in the mutant strain, whole-cell lysates were reacted with FbpA-specific polyclonal antiserum in Western immunoblots. The antibody recognized a 37-kDa band corresponding to FbpA in the wild-type meningococcal strain B16B6 (Fig. 2, lane b) and in gonococcal strain PID543 (Fig. 2, lane d). In contrast, no immunoreactive band was observed in the mutant strain N16FBPA (Fig. 2, lane c). These results indicate that FbpA expression was abolished in the mutant.

View larger version (90K): [in this window] [in a new window]   FIG. 2.   Identification of FbpA protein from the wild-type and mutant strains of N. meningitidis B16B6. Whole-cell extracts, prepared from the parental strain B16B6 (lane b), the fbpABC mutant strain N16FBPA (lane c), and N. gonorrhoeae PID543 (lane d) that were grown under iron-restricted conditions, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The blot was probed with a polyclonal antiserum directed against the gonococcal FbpA. Positions of protein standards in kilodaltons are indicated in lane a. Figures were imaged with a Hewlett-Packard ScanJet IIp and labelled by using Adobe Photoshop 3.0.

Growth assays. Growth assays were conducted to determine the ability of the fbpABC mutant to use various iron compounds as the sole exogenous source of iron. All cultures were examined at 22 h of growth. Stationary phase was attained at this time in all cultures (data not shown). The kinetics of growth were identical to those observed at 8 h (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Growth of the wild-type and mutant strains of N. meningitidis B16B6 in iron-limited BHI broth supplemented with various iron compounds. The parental isolate B16B6 and the fbpABC mutant strain N16FBPA were grown in the presence of 8 µM iron-loaded human transferrin (hTf) (A), 8 µM iron-loaded human lactoferrin (hLf) (B), 5 µM ferric chloride (C), 5 µM ferric nitrate (D), 2 µM bovine hemin (bHm) (E), and 0.5 µM human hemoglobin (hHb) (F). Each value represents the mean of three replicate cultures. Standard deviations of the mean are shown by error bars. The absence of error bars indicates that the standard deviation of the three replicate cultures was too small to be represented on the graph.