Division of AIDS, Sexually Transmitted Diseases and Tuberculosis Laboratory Research, National Centers for Infectious Disease, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA1
Author for correspondence: Cheng-Yen Chen. Tel: +1 404 639 3154. Fax: +1 404 639 3976. e-mail: cyc1{at}cdc.gov
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ABSTRACT |
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Keywords: Neisseria gonorrhoeae, iron-storage protein, bacterioferritin
Abbreviations: Bfr, bacterioferritin; DF, Desferal; SSP-PCR, single-specific-primer PCR
The GenBank accession numbers for the sequences reported in this paper are U76633 and U76634.
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INTRODUCTION |
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Two types of iron-storage proteins have been identified in bacteria (Harrison & Arosio, 1996 ): bacterioferritin (Bfr), which contains non-covalently bound haem groups in addition to a non-haem iron core (Stiefel & Watt, 1979
); and ferritin, which does not possess intrinsic haem groups and resembles the iron-containing protein (H-type subunit) of mammalian cells (Ford et al., 1984
). Bfrs are characteristically composed of 24 identical 1822 kDa subunits, which are assembled into a spherical protein shell containing 0·220% (approx. 6002400 iron atoms per molecule) by weight of non-haem iron and 312 non-covalently bound protohaem IX groups (Yariv et al., 1981
; Ford et al., 1984
; Frolow et al., 1994
). Subunits are tightly assembled to form a molecule with fourfold, threefold and twofold symmetry axes (432 symmetry) (Ford et al., 1984
). Results of electron-microscopic studies have indicated that the iron core can be either crystalline (ferrihydrate) or amorphous. Although the mechanism of iron storage in vivo is uncertain, in vitro, iron-core formation involves the oxidation of Fe(II) and hydrolytic polymerization of Fe(III) (Andrews et al., 1993
; Hudson et al., 1993
).
Studies on Neisseria gonorrhoeae have focused primarily on the receptor-mediated mechanisms that this organism uses to obtain iron from host sources; very little is known about the fate of intracellular iron. In this study, we report the identification and purification of a Bfr from N. gonorrhoeae composed of two non-identical subunits; the genes encoding these subunits have been cloned and sequenced. A BfrB-deficient mutant was constructed to examine the biological role of gonococcal Bfr.
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METHODS |
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Purification of gonococcal Bfr.
Overnight growth of N. gonorrhoeae strain F62 was harvested from GC agar plates and resuspended in 20 mM Tris/HCl buffer (pH 7·2). The cell suspension was sonicated (30 s, 6 times) and then centrifuged at 10000 g for 20 min. The supernatant was decanted, heated to 6570 °C for 15 min and centrifuged at 10000 g for 20 min. The heat-treated supernatant was then fractionated by precipitation with (NH4)2SO4. The fraction that precipitated between 30 and 60% saturation was collected by centrifugation (10000 g, 15 min), dissolved in 20 mM Tris/HCl buffer (pH 7·2) and desalted by dialysis against the same buffer overnight at 4 °C. The dialysed material was applied to a Sephacryl S-200-HR (Sigma) gel-filtration column (0·9x60 cm) that had been equilibrated with 20 mM Tris/HCl buffer (pH 7·4) containing 0·15 M NaCl. The molecular mass standards (Bio-Rad) used were thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B12 (1·35 kDa). The iron-stain-positive fractions, corresponding to 300500 kDa molecular mass, were pooled and concentrated by centrifugation (Centriprep-30; Amicon). The retentate was dialysed overnight against 20 mM Tris/HCl buffer (pH 7·2) and then fractionated by anion-exchange chromatography using a DEAE Sepharose CL-6B (Sigma) column (2·8x11 cm). Proteins were eluted with a linear gradient of 0·10·6 M NaCl; Bfr eluted at approximately 0·3 M NaCl. The purity of Bfr was assessed by SDS-PAGE and by iron-specific staining of non-denaturing polyacrylamide gels.
Iron-specific stain.
After electrophoresis of cell-free extracts in non-denaturing polyacrylamide gels, iron-containing proteins were visualized by staining with a solution of 0·75 mM 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine, sodium salt (Ferrozine; Sigma) and 15 mM thioglycolic acid in 2% (v/v) acetic acid (Chung, 1985 ). To locate column fractions containing iron, an aliquot of each column fraction (200 µl) was added to the Ferrozine reagent (5 µl) and the absorbance at 562 nm was measured after 15 min. Iron-containing protein bands or column fractions exhibited a visible red colour within 20 min of staining. The iron-stained gel was subsequently stained with Coomassie blue and destained with 10% (v/v) acetic acid in order to visualize the presence of any other cellular proteins.
N-terminal sequence analysis.
Purified Bfr was subjected to SDS-PAGE and electro-transferred onto a PVDF membrane (Schleicher & Schuell). Protein bands were excised from the transblot and sequenced by standard Edman chemistry on a Beckman PI2090 Integrated Micro-sequencing System.
PCR and DNA sequence analysis.
bfrB was PCR-amplified using a forward degenerate primer (964FP, Table 1), deduced from the N-terminal amino acid sequence of gonococcal BfrB, and a reverse degenerate primer (966RP, Table 1
), based on a conserved region in the C-terminal of the E. coli Bfr (amino acids 128134) (Andrews et al., 1989
; Denoel et al., 1995
). PCR conditions consisted of 30 cycles of denaturation at 94 °C for 1·5 min, annealing at 62 °C for 2 min and extension at 72 °C for 2 min. The flanking regions and the remaining sequences of gonococcal bfrA and bfrB were completed by using single-specific-primer PCR (SSP-PCR) (Shyamala & Ames, 1989
). The Applied Biosystems model 373 DNA sequencing system (Perkin-Elmer) was used for sequencing the PCR products according to the manufacturers cycle sequencing protocol by using dye-terminator chemistry (Sanger et al., 1977
).
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Expression of gonococcal Bfr.
Whole-cell lysates prepared from E. coli DH5, DH5
with pUC18, or DH5
with pCYC-1 were fractionated by SDS-PAGE and electro-transferred onto a nitrocellulose membrane (Schleicher & Schuell). The blot was probed with anti-E. coli Bfr polyclonal antiserum (1:1000) and bound antibody detected with horseradish-peroxidase-labelled goat anti-rabbit IgG conjugate (Bio-Rad).
Construction of a BfrB-deficient mutant of N. gonorrhoeae.
The sequence encompassing bfrA and bfrB and their corresponding flanking regions were amplified by PCR using synthetic oligonucleotide primers (FPEco2 and RPBam2, Table 1) designed to introduce an EcoRI restriction site 1·1 kb upstream of the start codon of bfrA and a BamHI site 163 bp downstream of the stop codon of bfrB. The resulting 2·4 kb PCR fragment was double-digested with BamHI and EcoRI and ligated into pUC18. The single HindIII restriction site on pUC18 had been previously eliminated by digesting with the enzyme, end-filled using the Klenow fragment and religated. Competent E. coli DH5
cells were subsequently used for transformation. Ampicillin-resistant transformants were obtained on LB medium containing 50 µg ampicillin ml-1. Plasmid minipreps were prepared to confirm the presence of the 2·4 kb DNA insert. The recombinant plasmid, designated pCYC-2, was digested with HindIII, which cuts only once in the coding region of bfrB, and ligated with the purified
fragment prepared with HindIII ends. Competent E. coli DH5
cells were transformed as previously described and transformants selected on LB medium containing 30 µg ampicillin ml-1and 30 µg spectinomycin ml-1. The pCYC-2 containing the insertionally inactivated bfrB, designated pCYC-3, was purified from several transformants to confirm the insertion of the
fragment and then linearized with EcoRI prior to transforming piliated cells of N. gonorrhoeae strain F62. Gonococcal transformants were selected on GC agar plates containing 30 µg streptomycin ml-1 and 30 µg spectinomycin ml-1. The presence of the
fragment in bfrB was confirmed by PCR amplification of the BfrB structural gene. One of the transformants, designated F62BfrB
, was selected for further studies.
Sensitivity to H2O2 and paraquat.
The sensitivity of N. gonorrhoeae strain F62 and its isogenic BfrB-deficient mutant to H2O2 and paraquat was determined by a disc-diffusion assay. Gonococci were grown overnight on GC agar plates and resuspended in GC broth to a concentration of 108 c.f.u ml-1 and 100 µl of the cell suspension was spread onto GC agar plates. Filter paper discs (1/4 in. in diameter, Schleicher & Schuell), containing various amounts of H2O2 (05 µmol) or paraquat (03 µmol), were placed on the surface of the plate. After 24 h incubation at 35 °C with 5% CO2, plates were examined for zones of growth inhibition. The zone of growth inhibition was determined by measuring the diameter (cm) of the clear zone surrounding the disc minus the diameter of the disc. The mean zone of inhibition±standard deviation was calculated from the results of three separate experiments.
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RESULTS |
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PCR and DNA sequence analysis
Degenerate primers deduced from the N-terminal amino acid sequence of the gonococcal BfrB and from a region of E. coli Bfr (amino acids 128134) were used to PCR-amplify the majority of the BfrB gene. The flanking regions and the remaining sequence of the BfrB gene were obtained by SSP-PCR. Results of this additional sequencing indicated that the DNA sequence encoding bfrA was upstream of bfrB. The complete nucleotide and deduced amino acid sequences of the gonococcal Bfrs indicated two ORFs of 462 bp and 471 bp, corresponding to positions 148609 and 6401110, respectively. The two Brf genes were located in tandem with an intervening gap of 27 bases. Potential ShineDalgarno ribosome-binding sites (GAGAG for bfrA and AGGAG for bfrB) were located 610 bp upstream from the ATG start codon of bfrA and bfrB, respectively. Putative -10 and -35 promoter regions were also identified and a GC-rich region of dyad symmetry located 43 bp downstream from the stop codon in bfrB could function as a rho-independent transcription terminator. There was a potential Fur-binding sequence (12 of 19 bases were identical to the consensus neisserial fur sequences; Genco & Desai, 1996 ) in front of the -35 hexamer. There was 55·7% homology between the DNA sequences of bfrA and bfrB; and 39·7% identity for the amino acid sequences of BfrA and BfrB. The deduced amino acid sequence of BfrA consisted of 154 amino acids with a predicted molecular mass of 17961 Da and an isoelectric point (pI) of 4·57; BfrB consisted of 157 amino acids, and had a molecular mass of 18014 Da and a pI of 4·58. The deduced amino acid sequences of BfrA and BfrB showed 41·3% and 56·1% identity to E. coli Bfr, respectively.
Cloning and expression of Bfr
The entire coding sequence encompassing bfrA and bfrB was amplified by PCR and ligated into pUC18 and in-frame with lacZ. Competent E. coli DH5 cells were transformed, and transformants containing pCYC-1 were analysed for expression of gonococcal Bfr on Western blots probed with anti-E. coli Bfr polyclonal antiserum. The results in Fig. 2
show that E. coli DH5
or E. coli DH5
containing pUC18 exhibited a single Bfr subunit of 20 kDa. The transformants expressed two additional proteins corresponding in size to the gonococcal BfrA and B (Fig. 2a
, lane 3), and both proteins reacted with the polyclonal antiserum to E. coli Bfr (Fig. 2b
, lane 3). These additional proteins were not observed in E. coli DH5
or E. coli DH5
containing pUC18.
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Effect of iron on the growth of BfrB-deficient mutant
N. gonorrhoeae strains F62 and F62BfrB were grown in liquid GC medium with or without 30 µM DF. Growth of both strains was markedly reduced under iron-limited conditions (Fig. 3
); however, growth of strain F62BfrB
was inhibited to a greater degree than was the growth of strain F62 under identical growth conditions.
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DISCUSSION |
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In this study, we have identified and purified a Bfr from N. gonorrhoeae, and subsequently cloned and sequenced the corresponding genes. The identification of gonococcal Bfr was facilitated by an iron-specific stain, which is based on the interaction between the iron atoms stored in the Bfr and a sensitive chromogenic ligand, Ferrozine, which results in the formation of a reddish-coloured complex. Bfr was the only protein in non-denaturing PAGE of supernatants of sonicated cell suspensions that could be readily stained with Ferrozine, indicating the high content of iron molecules within this protein. Additional data indicating that this protein is Bfr include heat stability (6570 °C for 15 min) (Smith et al., 1988 ; Andrews et al., 1993
), molecular mass between 300 and 500 kDa for the holoprotein and between 18 and 22 kDa for its subunits, N-terminal sequence homology to Bfr subunits from other bacteria, and immunological cross-reactivity with anti-E. coli Bfr antiserum.
Unlike most Bfrs, which are homopolymers composed of identical subunits, gonococcal Bfr comprises two similar, but not identical, subunits. Harker & Wullstein (1985) reported that the Bfr from A. vinelandii comprised two non-identical subunits; however, Grossman et al. (1992)
were able to clone only one gene for this protein. More convincing data for structural heterogeneity of Bfr were obtained from Pseudomonas aeruginosa, in which the first 69 and 55 residues of the N-terminal amino acid sequence of the
and ß subunits differed considerably (Moore et al., 1994
). The corresponding genes for the
and ß subunits of P. aeruginosa Bfr have not been cloned to further substantiate these findings. The results of our study are the first to locate genes encoding different Bfr subunits and verify the existence of structural heterogeneity in this iron-storage protein. Structural heterogeneity of Bfr may be less complicated than in mammalian ferritins, where the structural complexity is due to combinations of various ratios of two subunits, heavy (H) and light (L), which differ in size, amino acid composition, surface charge and immunoreactivity (Theil, 1987
; Harrison & Arosio, 1996
).
How the biosynthesis of Bfr or prokaryotic ferritin is regulated by iron awaits elucidation. In E. coli, the induction of bfr expression by iron was found to be dependent on the ferric uptake regulator protein (Fur), although not to the direct interaction between Fur and the bfr gene (Harrison & Arosio, 1996 ). It is not clear how Fur acts as an intermediate and what other cellular components are involved in this regulation. The genes encoding the two gonococcal Bfr subunits were located in tandem with an intervening gap of only 27 bp, suggesting that they might be co-transcribed and co-regulated. The presence of a potential iron box within the 5' region of bfrA in front of a putative -35 hexamer suggests that gonococcal Bfr might be negatively regulated by iron. However, this contradicts results obtained with other micro-organisms, suggesting that in spite of the presence of potential iron boxes the production of Bfr was either up-regulated by, or indifferent to, the iron concentration present in the growth medium (Grossman et al., 1992
; Cristina et al., 1994
; Evans et al., 1995
). The genetic regulation of gonococcal Bfr is currently under investigation.
Despite the structural similarity between ferritins and Bfrs, they share little homology in their amino acid sequences and exhibit no immunological cross-reactivity (Wai et al., 1995 ), suggesting that they have different origins. Most studies suggest micro-organisms possess either a ferritin or a Bfr homologue (Stiefel & Watt, 1979
; Moore et al., 1986
; Kurokwa et al., 1989
; Brooks et al., 1991
; Rocha et al., 1992
; Laulhère et al., 1993
; Cristina et al., 1994
; Inglis et al., 1994
; Denoel et al., 1995
; Evans et al., 1995
; Penfold et al., 1996
). However, the presence of both ferritin and Bfr has been reported in E. coli (Yariv et al., 1981
; Izuhara et al., 1991
) and Helicobacter pylori (Doig et al., 1993
; Evans et al., 1995
). It is not clear why relatively few bacteria possess both types of iron-storage proteins and what advantage this apparent redundancy has. In vitro data indicate that the rate at which iron is incorporated into apoferritin is approximately fivefold faster than into apoBfr, suggesting that ferritin might compete for iron in vivo more efficiently than Bfr (Andrews et al., 1993
). We cannot rule out the possibility that the two biochemically and genetically different iron-storage proteins might have other specific or essential functions. For example, E. coli Bfr is also known as cytochrome b1 and may additionally function as an electron-storage molecule (Smith et al., 1988
). We have been unable to identify a potential ferritin homologue in N. gonorrhoeae by homology searches comparing the sequences of the E. coli and Haemophilus influenzae ferritin against the gonococcal (University of Oklahoma, N. gonorrhoeae strain FA1090) and meningococcal (The Sanger Center, Neisseria meningitidis serogroup A strain Z2491) genome databases. Alternatively, the gonococcus may have a ferritin gene that has little homology to those of E. coli or H. influenzae.
The homology of the amino acid sequences of gonococcal BfrA and BfrB to those of other Bfrs (Andrews et al., 1989 ; Kurokwa et al., 1989
; Brooks et al., 1991
; Grossman et al., 1992
; Laulhère et al., 1993
; Inglis et al., 1994
; Denoel et al., 1995
) for which full-length or partial sequences are available is shown in Fig. 5
. Gonococcal BfrA and BfrB shared 39·7% identity. The percentage identity compared to other bacterial Bfrs ranged from 39·5% to 59·6% for BfrA, and from 41·6% to 62·2 % for BfrB. A seven amino acid motif consisting of Glu-18, -51, -94, -127, His-54, -130 and Tyr-25, which constitutes the ferroxidase centre (Andrews et al., 1991
; Brun et al., 1995
) and is associated with the binuclear metal-binding site, was fully conserved in BfrA (Fig. 5
); however, only four of these seven residues were conserved in BfrB. Conversely, the methionine residue (Met-52), which provides the axial ligand for haem binding (Grossman et al., 1992
; Cheesman et al., 1993
), was conserved only in BfrB. These data demonstrate that the structure and function of Bfr are conserved among prokaryotes and suggest that both subunits are required to form a functional Bfr, with BfrA providing the ferroxidase centre and BfrB the haem-binding ligand. Interestingly, the seven amino acids which constitute the ferroxidase centre were conserved in E. coli ferritin but only five were retained in E. coli Bfr (Andrews et al., 1991
, 1992
). It has been suggested that the ferritin and Bfr of E. coli are the bacterial counterparts of the mammalian H-rich and L-rich ferritins with respective roles in short-term iron flux and long-term iron storage (Andrews et al., 1993
). It is not clear whether gonococcal Bfr comprises equal molar ratios of the A and B subunits, or whether different Bfr subunits associate to form a Bfr that is either BfrA-rich or BfrB-rich. However, the absence of iron-specific staining of Bfr by Ferrozine in the F62BfrB
mutant (data not shown) suggests that BfrA subunit alone can not form a functional iron-storage protein.
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Eukaryotic ferritins function by buffering the free-iron concentration in the cells, thus protecting cells from harmful iron-catalysed oxidative damage (Harrison & Arosio, 1996 ). Similarly, a ferritin homologue from Campylobacter jejuni was recently reported to contribute to protection against oxidative stress (Wai et al., 1996
). We investigated the role of the gonococcal Bfr in protecting against oxidative stress by insertionally inactivating BfrB and examining the sensitivity of a BfrB-deficient mutant to hydrogen peroxide and paraquat. The growth of the BfrB-deficient mutant of N. gonorrhoeae was less than that of the parent strain under both iron-sufficient and iron-limiting conditions, suggesting that Bfr plays a role in iron storage. In addition, the BfrB-deficient mutant was more sensitive to H2O2 and paraquat than the parent strain, suggesting that the accumulation of intracellular free iron caused by the absence of BfrB may lead to hypersensitivity to H2O2. Furthermore, BfrA alone was not capable of protecting the cell from oxidative injury.
A ferritin- and Bfr-deficient double mutant of E. coli grew poorly under iron-limited conditions (Simon Andrews, personal communication), suggesting the possibility of another internal iron pool. In our study, insertional inactivation of BfrB was not lethal to the growth of the gonococcus and provides further support for an internal iron pool. However, questions remain as to how this iron pool interacts with iron-storage or iron-requiring proteins. Although ferritins and Bfrs are involved in iron storage and in protection against oxidative stress, the exact role of ferritin and Bfr in iron metabolism has yet to be elucidated since the intracellular levels of these proteins present are considered too low to serve as major iron-storage compounds (Braun, 1997 ).
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ACKNOWLEDGEMENTS |
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Received 1 March 1999;
revised 18 May 1999;
accepted 7 June 1999.