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Journal of Bacteriology, May 2004, p. 3266-3269, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3266-3269.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of a Nucleotide-Binding Domain Associated with Neisserial Iron Transport

Gloria H. Y. Lau,¹ Ross T. A. MacGillivray,¹ and Michael E. P. Murphy^1,2*

Department of Biochemistry and Molecular Biology,¹ Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada²

Received 22 October 2003/ Accepted 3 February 2004

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The fbpABC operon in Neisseria gonorrhoeae encodes an ATP-binding cassette transporter required for iron uptake from the host ferric binding proteins. The gene for the nucleotide-binding domain (fbpC) expressed in Escherichia coli has intrinsic ATPase activity (0.5 mmol/min/mg) uncoupled from the iron transport process. The FbpC E164D mutant is found to have a 10-fold reduction in specific activity. FbpC is covalently modified by 8-azido-[³²P]ATP, indicating that FbpC is a functional ATPase that likely combines with FbpB to form a ferric iron transporter.

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The ability of the pathogen Neisseria gonorrhoeae to sequester its iron requirement from the human host plays an important role in establishing infection and causing disease (4). This bacterium possesses surface receptors that interact specifically with such host iron-binding proteins as transferrin (11) and lactoferrin (12). Iron is removed from these host iron-binding proteins and transferred to the periplasmic iron-binding protein FbpA (ferric iron-binding protein), which initiates the periplasm-to-cytosol transport of iron.

Adhikari et al. (1996) determined the nucleotide sequence of the fbpABC operon in N. gonorrhoeae (1) and proposed that these three genes encode a periplasmic binding protein, cytoplasmic permease, and a nucleotide-binding domain, respectively. The addition of a plasmid containing the fbpABC operon enables siderophore-deficient Escherichia coli strains to grow on nutrient agar containing an inhibitory concentration of 2,2'-dipyridyl, an iron chelator, showing that this operon functions as an iron transport system at the periplasm-to-cytosol level (1). Initial attempts to detect the expression of FbpB (511 amino acids) and FbpC (352 amino acids) in iron-stressed N. gonorrhoeae or in E. coli constructs containing the fbpABC operon have been unsuccessful (1, 21). However, Khun and coworkers recently showed the presence of fbpAB and fbpBC transcripts in Neisseria meningitidis by reverse transcription-PCR, suggesting that the fbpABC locus is transcribed as a single contiguous message in that organism (9). The similarity between N. gonorrhoeae and N. meningitidis genomes suggests that a complete fbpABC operon exists and is transcribed as a contiguous message in N. gonorrhoeae.

The amino acid sequence of FbpC, the nucleotide-binding domain, is conserved in other ATP-binding cassette (ABC) transporters and is proposed to provide the energy for the periplasm-to-cytosol transport of iron. To show that FbpC is a functional nucleotide-binding domain, we used an E. coli-based recombinant system to purify and characterize recombinant FbpC.

Cloning, expression, and purification of recombinant FbpC_His6. The fbpC gene was PCR amplified from the genomic DNA of a clinical isolate (Q3,21) of N. gonorrhoeae (British Columbia Center for Disease Control, Vancouver, Canada) by using oligonucleotides that contain HindIII, NdeI, and XhoI restriction sites as well as a stop codon (Table 1). Cloning of the N. gonorrhoeae fbpC gene into the pET28a expression vector produced a stable genetic construct, pEfbpC3. DNA sequence analysis confirmed that the sequence of the amplified fbpC fragment was identical to the available N. gonorrhoeae genome sequence (strain FA 1090; http://www.genome.ou.edu/gono.html) except at nucleotide position 567, which represents a silent mutation. (The GenBank accession number for the completed N. gonorrhoeae genome is AE004969.)

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TABLE 1. Bacterial strains, plasmids, and primers used in this study

FbpC_His6 was successfully overexpressed in E. coli HMS174(DE3) cells containing the pEfbpC3 plasmid. Purification of FbpC_His6 involves nickel-chelate chromatography (elution at 250 mM imidazole) and dialysis overnight followed by ion-exchange chromatography using Q-Sepharose. This procedure resulted in FbpC protein that was >95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1A). N-terminal sequence analysis and the apparent molecular mass of approximately 40 kDa determined by SDS-PAGE confirmed the identity of the expressed FbpC.

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FIG. 1. Purification and ATP binding by FbpCHis6. (A) SDS-PAGE of purified FbpCHis6 after metal chelate (lane 1) and high-Q (lane 2) chromatography. Molecular mass markers are in the leftmost lane and are labeled in kilodaltons. (B) Autoradiogram showing binding of 8-azido-[-32P]ATP to FbpCHis6. Purified FbpCHis6 was preincubated in photoaffinity labeling reaction mixtures (40 mM Tris-HCl [pH 8], 5 mM MgCl2, 50 mM NaCl, 5 mM CaCl2, 7.5% glycerol) with different concentrations of unlabeled ATP prior to UV irradiation. Lane 1, 0 mM; lane 2, 1 mM; lane 3, 2 mM; lane 4, 5 mM; lane 5, 10 mM. Approximately 1.5 µCi of labeled azido-ATP was used in each reaction.

The high content of hydrophobic residues in FbpC (1) and the hypothesis that ABC nucleotide-binding domains are anchored to the cytoplasmic membrane via interaction with the integral subunits (8) may explain the low solubility of the overproduced FbpC fusion proteins, which is also consistent with the previous findings that both MalK and HisP are prone to aggregation (17, 23). The addition of ATP to a concentration of at least 2 mM throughout the purification procedure was found to be essential in maintaining FbpC_His6 solubility, as the final yield of FbpC was reduced by more than 50% if ATP was omitted. Soluble HisP has also shown a requirement for 5 mM ATP and 20% glycerol throughout the purification procedure to obtain sufficient quantities for characterization (17). Increasing the glycerol concentration to 50% significantly improved solubility of pure FbpC_His6 but rendered the purified protein harder to work with. Therefore, final, pure FbpC_His6 was immediately aliquoted and stored at –80°C in 20% glycerol and 2 mM ATP. EDTA was also added to a concentration of 1 mM to avoid hydrolysis of ATP.

Binding of [-³²P]N₃ATP to purified FbpC_His6. A photoaffinity ATP analog, 8-azido-[-³²P]ATP (ICN Biomedical Inc.), was used to label specifically the purified FbpC_His6 protein. Purified FbpC_His6 was found to bind [-³²P]N₃ATP (Fig. 1B), indicating an active conformation with a nucleotide-binding site. The protein band was absent in the control experiment in which UV irradiation was omitted (data not shown). Preincubation with 5 mM unlabeled ATP led to reduced intensity in the FbpC band, suggesting the specific binding of ATP to FbpC. Photoaffinity labeling of FbpC was reduced as the concentration of unlabeled ATP was increased (Fig. 1B).

Purified FbpC_His6 displays ATPase activity. Purified FbpC_His6 was first characterized by its ability to hydrolyze ATP into ADP and inorganic phosphate (3, 17). Figure 2A shows linear ATPase activity of FbpC_His6 with respect to time in the presence of Mg²⁺. This activity was linear up to at least 10 min and was dependent on the Mg²⁺ cation. The maximal activity was displayed when the concentration of Mg²⁺ was between 1 and 2 mM (Fig. 2B). The specific activity of FbpC_His6 was 0.5 ± 0.1 µmol/min/mg (100 mM Tris-HCl [pH 8.0], 40 mM NaCl, 4 mM ATP, 20% [vol/vol] glycerol, 2 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol), comparable to that determined for MalK of the maltose transporter (0.7 to 1.3 µmol/min/mg) (14, 20) and HisP of the histidine permease in Salmonella enterica serovar Typhimurium (0.5 µmol/min/mg) (17).

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FIG. 2. ATPase activity of FbpCHis6. (A) Linearity of ATP-hydrolyzing activity of FbpCHis6 (60 µg/ml) with respect to time in the presence () or absence () of MgCl2. Pi, inorganic phosphate. (B) Mg2+ dependence of FbpCHis6 activity. (C) pH dependence of FbpCHis6 activity. Buffers used were MES (morpholineethanesulfonic acid)-Na, pH 6.0 to 6.5; MOPS (morpholinepropanesulfonic acid)-Na, pH 7.0; Tris-Cl, pH 7.5 to 8.8; CHES [(N-cyclohexylamino)ethanesulfonic acid], pH 9.3; and CAPS [3-(cyclohexylamino)-1-propanesulfonic acid], pH 10.2. (D) Glycerol dependence of FbpCHis6 activity. The ATPase specific activity is expressed in nanomoles of inorganic phosphate liberated per minute per milligram of protein.

The optimum pH for FbpC_His6 activity was found to be slightly alkaline, from pH 7.5 to 8.0 (Fig. 2C). The presence of glycerol at concentrations of up to 20% was found to stimulate the rate of ATP hydrolysis (Fig. 2D), with the activity dropping off at higher glycerol concentrations, possibly due to disruption of hydrophobic interactions (17). This finding is different from that observed for HisP, where glycerol concentrations of greater than 10% (vol/vol) resulted in a decrease in ATP hydrolysis. In general, the characteristics of the enzyme, such as its Mg²⁺ and pH dependence, are in agreement with previous findings for isolated MalK (14) and HisP (17).

Site-directed mutagenesis. To ensure that the ATPase activity displayed by the purified FbpC_His6 preparation was not due to contaminating ATPases and to establish the enzymatic importance of Glu164, PCR-based mutagenesis was performed (15) to create the FbpC mutant E164D. The FbpC mutant was hypothesized to be defective in hydrolysis and to have little effect on ATP binding based on previous structural and mutational analyses of HisP in the Salmonella histidine transport system (22). The HisP crystal structure shows that residue Glu179 in HisP is responsible for forming a hydrogen bond with a water molecule that interacts with the -phosphate of the bound ATP at the active site (6). The E179D mutant of HisP eliminates transport activity but allows ATP binding (22), suggesting that this residue is necessary only for ATP hydrolysis but does not greatly affect nucleotide binding.

The E164D mutant construct was cloned, used to transform the E. coli strain HMS174(DE3), and overexpressed, and the mutant protein was purified using a protocol identical to that used for the wild type (Table 1). The E164D mutant has a specific activity of approximately 0.047 µmol/min/mg, representing a greater than 10-fold reduction in specific activity compared to that of the wild-type FbpC, suggesting that Glu164 is an important residue in ATP hydrolysis and that the activity observed from the wild-type can be attributed to the purified FbpC_His6 protein and not some impurities from the preparation.

All bacterial ABC importers share conserved nucleotide-binding domains that provide energy for the transport of many growth-essential nutrients through the hydrolysis of ATP (5). The FbpC protein of the periplasmic iron transport complex corresponding to the N. gonorrhoeae fbpABC operon shares 51% amino acid identity with HitC corresponding to the hitABC operon in Haemophilus influenzae and 40% identity with SfuC corresponding to the sfuABC operon in Serratia marcescens; both are nucleotide-binding subunits of closely related Fe(III) periplasmic transporters (13). Unlike the more distantly related HisP of histidine permease (6, 10, 16, 17) and MalK of maltose transporter in Salmonella serovar Typhimurium (7, 14, 19, 23), the bacterial iron transport systems are not well characterized. However, siderophore-deficient E. coli strains unable to utilize many iron sources can be complemented by the addition of a plasmid containing the hitABC (2), sfuABC (24), or fbpABC operon (1), indicating the importance of these operons in iron transport.

An elegant experiment by Sanders et al. showed that insertional inactivation of the hitC gene produced an isogenic nontypeable H. influenzae (NTHI) strain unable to utilize iron bound to transferrin or iron chelators. Interestingly, reconstitution of the wild-type genotype by replacing the mutated hitC gene with the wild-type allele by allelic exchange created a new strain that was able to utilize all of these iron sources (18), indicating the critical role of HitC in the iron transport process. Moreover, reverse transcription-PCR of the fbpABC locus in N. meningitidis detected an fbpC transcript (9); in contrast, no fbpC transcripts were detected in RNA samples from N. gonorrhoeae cultures (1, 21). Detection of fbpBC transcripts implies a functional role for FbpB and FbpC in neisserial periplasmic iron transport. Genetic conservation of the fbpABC operon also suggests that this observation applies to N. gonorrhoeae. However, an N. gonorrhoeae fbpC mutant (constructed by insertional inactivation) was previously shown to be capable of growth with transferrin as the sole exogenous iron source (21), conflicting with the evidence obtained in the H. influenzae iron transport model.

In this study, we have purified recombinant FbpC_His6 and showed that the isolated protein, like many traffic ATPases, has intrinsic ATPase activity uncoupled from the iron transport process. Given the diversity of pathogenic bacteria that express ABC transporter operons that take up iron at the periplasm-to-cytosol level and their amino acid sequence similarity, FbpC is likely to function as an ATP-binding subunit in neisserial iron transport.

 
This work was supported by CIHR grants MT-14767 and MOP-49597 to M.E.P.M. and R.T.A.M. M.E.P.M. is a CIHR scholar, and G.H.Y.L. was supported by an NSERC studentship.

We thank members of Michael Gold's laboratory for help with the photoaffinity labeling experiment. We also thank David Chan and the British Columbia Center for Disease Control. We thank Elena Bekker, Martin Boulanger, and Elitza Tocheva for many helpful comments and careful reading of the manuscript. We acknowledge the Gonococcal Genome Sequencing Project, supported by USPHS/NIH grant #AI38399, and B. A. Roe, L. Song, S. P. Lin, X. Yuan, S. Clifton, Tom Ducey, Lisa Lewis, and D. W. Dyer at the University of Oklahoma.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of British Columbia, #300 6174 University Blvd., Vancouver, BC V6T 1Z3, Canada. Phone: (604) 822-8022. Fax: (604) 822-6041. E-mail: memurphy{at}interchange.ubc.ca.

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Journal of Bacteriology, May 2004, p. 3266-3269, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3266-3269.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lau, G. H. Y. Articles by Murphy, M. E. P. Search for Related Content PubMed PubMed Citation Articles by Lau, G. H. Y. Articles by Murphy, M. E. P.