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Microorganisms have developed diverse and elaborate systems to obtain iron, which is present in limited quantities in the human host. Some pathogenic bacteria produce siderophores, which efficiently scavenge ferric iron from the environment and facilitate the growth of the organism (27). The pathogenic Neisseria species do not produce siderophores (17, 26) but instead produce an array of distinct iron-regulated outer membrane receptors which specifically interact with different iron binding proteins. These include the transferrin (TF) binding proteins, Tbp1 and Tbp2, the lactoferrin (LF) binding proteins, LbpA and LbpB (2, 5, 14, 24), and the hemoglobin receptors, HmbR (22, 23) and HpuB (6, 7, 15), which facilitate the uptake of heme iron from hemoglobin and hemoglobin-haptoglobin complexes, respectively. Although the binding of iron-containing ligands to these receptors is well characterized, the subsequent steps of iron removal and transport into the bacterial cytoplasm are not well defined. Previous studies have determined that iron is removed from TF and LF in an energy-dependent manner but that neither TF nor LF is internalized. Once the iron is removed from TF, it is bound by the ferric binding protein (FbpA). FbpA functions within the periplasm, shuttling iron from TF through the periplasm and to the cytoplasmic membrane. Our studies indicate that iron from heme interacts with FbpA (8); however, the specificity of this interaction and the fate of iron following its interaction with FbpA are not well defined. Neisseria meningitidis and N. gonorrhoeae fbpA mutants were recently demonstrated to be deficient in the ability to use iron from human TF and LF; however, the use of iron from heme and hemoglobin was unimpaired (12, 25). These results, together with our biochemical data obtained with N. gonorrhoeae, suggest that more than one periplasmic binding protein may be used for the transport of iron from hemin.

We have previously reported on the cloning and sequencing of the gonococcal fbpA gene (4). DNase footprinting studies indicate that E. coli Fur binds to a 42-bp site within the fbpA promoter (9). We have also established that the fbpA promoter is regulated by Fur and iron in E. coli and that the level of transcriptional regulation of fbpA in the gonococcus is directly related to the degree of iron restriction. Two open reading frames downstream of the gonococcal fbpA, designated fbpB and fbpC, have been identified (1). The proteins encoded by these loci are homologous to the products of portions of previously described operons expressed by Haemophilus influenzae (hitABC), Serratia marcescens (sfuABC), and Yersinia enterocolitica (yfuABC) (3, 19, 20). The fbpABC locus has been proposed to encode for a periplasmic protein-dependent transport system, with FbpA functioning as the periplasmic binding protein, FbpB (511 amino acids) functioning as the hydrophobic membrane protein, and FbpC (352 amino acids) functioning as the cytoplasmic membrane-associated nucleotide binding protein. However, attempts to detect proteins corresponding to FbpB and FbpC in iron-stressed gonococcal membranes or in Escherichia coli constructs expressing the fbpABC operon have failed. Likewise, in previous studies we were unable to detect fbpB- or fbpC-specific transcripts by Northern blot analysis (10), and thus the exact role of these proteins in iron transport remains to be defined. In this study, we have constructed and characterized a gonococcal fbpC mutant and have further defined the transcription of the N. gonorrhoeae fbpABC locus.

Bacterial strains and growth conditions. N. gonorrhoeae F62 (obtained from R. P. Williams, Baylor College of Medicine, Houston, Tex.) was maintained on complex medium (gonococcal base; Difco Laboratories, Detroit, Mich.) agar containing 1% IsoVitaleX (BBL, Baltimore, Md.) and grown aerobically under 5% CO₂ at 37°C. Broth cultures were grown in chemically defined medium (CDM) (16) supplemented with 4.2% NaHCO₃. All glassware was washed with 10% nitric acid and thoroughly rinsed in deionized water to remove residual iron. Cold hemin (Na plus K salt) was purchased from Porphyrin Products Inc. (Salt Lake City, Utah), and apotransferrin and ferric nitrate were purchased from Sigma Chemical Co. (St. Louis, Mo.). Cold hemin was dissolved in distilled H₂O and prepared fresh for each experiment. N. gonorrhoeae F62 was grown in CDM plus 25 µM Desferal (Ciba-Giegy) (CDM/25D) for 3 h aerobically at 37°C, washed, and resuspended in CDM/25D. This culture served as the inoculum into fresh CDM/25D with a starting absorbance at 660 nm (A₆₆₀) of 0.06. To this was added either iron-loaded human TF (5 µM, 30% iron saturated), ferric nitrate (10 µM), or hemin (4 µM). Growth was monitored by measuring the A₆₆₀ hourly for 5 h.

Construction and characterization of a gonococcal fbpC mutant. Plasmid pAFbpO, which contains a 3.6-kb DNA fragment corresponding to the fbpABC locus ligated into the EcoRV and BamHI sites of pBSKS (1) was used to construct an fbpC mutant. To disrupt the fbpC gene, a 600-bp BstEII-AccI internal fragment of the fbpC gene was deleted and the linearized plasmid was blunt ended with T4 DNA polymerase and subsequently dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim) to reduce the religation background. A 1.3-kb EcoRI fragment encompassing the Tn903 kanamycin cassette was excised from pUC4K-KSAC (18) and blunt ended with T4 DNA polymerase. The kanamycin cassette was ligated with the linearized pAFbpOC vector in the presence of T4 DNA ligase overnight at 14°C, and the ligation mix was used to transform high-efficiency JM109 cells (Promega Corp., Madison, Wis.). Transformants were selected on Luria-Bertani agar plates containing kanamycin (50 µg/ml).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of the Neisseria fbpABC operon. (A) Positions of the oligonucleotide primers used in RT-PCR. (B) Positions of the oligonucleotide primers used to confirm the presence of the kanamycin cassette (indicated by shading) in the recombinant plasmid construct and the fbpC mutant N. gonorrhoeae F62C.1.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Growth of N. gonorrhoeae F62 (A) and N. gonorrhoeae F62C.1 (B). N. gonorrhoeae was grown in CDM/25D for 3 h, washed, and resuspended in CDM/25D. This culture served as the inoculum into fresh CDM/25D with a starting A660 of 0.06. To this was added either iron-loaded human TF (5 µM, 30% iron saturated), ferric nitrate (Fe, 10 µM), or hemin (Hm, 4 µM). Cultures without added iron served as negative controls (-Fe). Growth was monitored hourly by A660 for 5 h. Results are from one experiment and are representative of three separate experiments. Mean values are plotted, and the standard deviations are included for each datum point. OD660, optical density at 660 nm (equivalent to A660).

Analysis of fbpABC transcription. In previous studies we demonstrated by Northern blot analysis that the gonococcal fbpA gene is transcribed as a monocistronic mRNA independently of fbpB and fbpC (10). In this same study we could not detect fbpB- or fbpC-specific polycistronic or monocistronic transcripts by Northern blot analysis. To precisely define the mechanism of regulation of fbpABC, we examined transcription of the fbpABC locus by reverse transcription-PCR (RT-PCR) analysis. RT-PCR amplification was performed with the Titian one-tube RT-PCR system (Boehringer Mannheim) as specified by the manufacturer. To eliminate DNA contamination, RNA samples were treated with RNase-free DNase (Promega Corp., Madison, Wis.) and an RT-PCR negative control experiment with DNase-treated RNA samples was performed in parallel with the RT-PCR (Fig. 3, lane 9). RT-PCR analysis was performed with several sets of primers (Table 1 and Fig. 1) and with RNA isolated from N. gonorrhoeae F62 grown under iron-replete (CDM with 50 µM ferric nitrate) and iron-depleted (CDM with 20 µM Desferal) conditions. Constitutive expression of the gonococcal rmp gene (11) was confirmed by RT-PCR and used as a positive control (Fig. 3, lane 8).

View larger version (35K): [in this window] [in a new window]   FIG. 3.   RT-PCR analysis for detection of fbpA, fbpAB, and fbpC transcripts in N. gonorrhoeae grown under iron-depleted and -replete conditions. Lanes: 1, DNA molecular size standards; 2, 4, 6, and 8, RNA obtained from N. gonorrhoeae under iron-depleted conditions; 3, 5, 7, and 9, RNA grown under iron-replete conditions; 2 and 3 RT-PCR with primer pairs SB3 and FBP(S) specific for fbpA; 4 and 5, RT-PCR with primer pairs FbpA.PR1 and FbpB.PR2 to amplify fbpA and the 5' end of fbpB; 6 and 7, RT-PCR with primer pairs Fbp.C1 and Fbp.C2 to amplify fbpC; 8, RT-PCR with primer pairs RMP F1 and RMP R1 to amplify the rmp gene (positive control); 9, DNA control.

Concluding remarks. The gonococcal fbpC gene has been predicted to encode a cytoplasmic membrane-associated nucleotide binding protein which functions together with FbpA in the transport of iron into the cell (1). However, the exact role of the gonococcal FbpC in iron transport has not been previously defined. Characterization of the gonococcal fbpC mutant described here indicates that the gonococcal FbpC is not essential for iron transport from TF, heme, or inorganic iron (ferric nitrate). Our studies also indicate that the gonococcal fbpABC locus is not a functional equivalent of the ABC transport system described in H. influenzae (hitABC) (19). Previous studies with H. influenzae hitA and hitC mutants have demonstrated that the absence of either the hitA or hitC gene abolishes the function of the ABC transport system (13, 19). N. meningitidis fbpA and N. gonorrhoeae fbpA mutants were recently shown to be unable to use iron supplied from human TF, LF, or inorganic iron, supporting the pivotal role of FbpA in iron transport (12, 25). The observed phenotype of the meningococcal fbpA mutant could not be attributed to a specific gene of the operon, since a polar effect on downstream fbpB and fbpC genes was not ruled out by complementation analysis studies. However, characterization of a specific N. gonorrhoeae fbpC mutant, as described in the present study, indicates that FbpC is not essential for transport of iron in the gonococcus.