Department of Medical Microbiology and Immunology, University of Aarhus, the Bartholin Building, DK-8000 Aarhus C, Denmark1
Division of Oral Health Services Research, Department of Public Health, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan2
Author for correspondence: Mogens Kilian. Tel: +45 89421735. Fax: +45 86176128. e-mail: kilian{at}microbiology.au.dk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: pseudogenes, transferrin-binding protein, haemoglobin-binding protein, haem
The GenBank accession numbers for the tpbA homologue sequences reported in this paper are AY028441 (strain HK1119), AF359437 (HK912), AF359438 (HK989), AF359439 (HK1002), AF359440 (HK988) and AF359441 (HK961); the GenBank accession numbers for the hgpA homologue sequences reported in this paper are AF359442 (HK989), AF359443 (HK988), AF359444 (HK981), AF359445 (HK961), AF359446 (JP2), AF359447 (HK912), AF359448 (HK1605), AF359449 (HK1604), AF359450 (HK1199) and HK359451 (HK1002).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Iron is an essential nutrient to most bacteria. In animal hosts, the vast majority of iron is found intracellularly where it is bound to ferritin or haem compounds, whereas in serum and secretions it is bound to transferrin and lactoferrin. The latter two proteins maintain levels of free extracellular iron at an extremely low level, which is far below that required for the optimal growth of micro-organisms. Consequently, bacteria that successfully colonize humans have developed specific systems to acquire iron (reviewed by Gray-Owen & Schryvers, 1996 ; Guerinot, 1994
; Mietzner & Morse, 1994
; Otto et al., 1992
; Ratledge & Dover, 2000
). Many bacteria rely on siderophores, which are small inorganic chelators with a high affinity for ferric iron. The expression of such siderophores allows bacteria to acquire iron from a variety of environmental and biological sources other than transferrin and lactoferrin. Members of the families Pasteurellaceae and Neisseriaceae do not produce siderophores; instead, they produce surface-exposed proteins that bind haemoglobin, transferrin and, in the case of the Neisseriaceae, lactoferrin, thereby facilitating iron acquisition from these host proteins. Notably, these bacterial receptors are highly specific for iron-binding proteins of the host species (Gray-Owen & Schryvers, 1996
; Ogunnariwo & Schryvers, 1990
; Otto et al., 1992
).
A. actinomycetemcomitans, like other members of the Pasteurellaceae, does not produce siderophores (Winston et al., 1993 ) and little is known about the mechanisms of iron acquisition in this species. Human lactoferrin is bactericidal for the bacterium (Kalmar & Arnold, 1988
). A transferrin-binding protein A gene homologue was detected by PCR in members of the Pasteurellaceae, but its sequence was not characterized further (Ogunnariwo & Schryvers, 1996
). Neither transferrin nor lactoferrin is bound by A. actinomycetemcomitans under iron-restricted conditions (Winston et al., 1993
). However, periplasmic proteins, AfuA and FbpA, with putative iron transport functions have been described, suggesting that an uptake system for iron is actually present on the cell surface of A. actinomycetemcomitans (Graber et al., 1998
; Willemsen et al., 1997
). Finally, Grenier et al. (1997)
reported that A. actinomycetemcomitans can use human haemoglobin as a source of iron. The aim of the present study was to examine further the acquisition of iron by A. actinomycetemcomitans.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Southern blot analysis.
Whole-cell DNA was extracted as described previously (Poulsen et al., 1988 ). Briefly, bacterial cells were suspended in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7·5), lysed with 1% SDS under high salt conditions, treated with proteinase K, extracted with phenol/chloroform (1:1) and then precipitated with cold 96% ethanol. After rinsing once with cold 70% ethanol, the DNA was dried and dissolved in TE buffer. Approximately 1 µg whole-cell DNA was digested with EcoRI, electrophoresed overnight through a 1% agarose gel at 1·5 V cm-1 in TAE buffer (0·04 M Tris/acetate, 1 mM EDTA) and visualized by staining with ethidium bromide (Sambrook et al., 1989
). The separated DNA fragments in the gel were transferred onto a Nytran nylon membrane (Protran; Schleicher & Schuell) and fixed by UV irradiation. The transferred DNA was hybridized as described with a final post-hybridization wash in 1xSET (0·15 M NaCl, 0·5 mM EDTA, 20 mM Tris/HCl, pH 7·0), 0·1% SDS and 0·1% sodium pyrophosphate at 60 °C (Poulsen et al., 1994
). The DNA fragments used as probes were prepared by PCR using Ready To Go PCR Beads (Amersham Pharmacia Biotech). Two primer sets were designed, based on the sequence of the transferrin-binding protein A gene (tbpA) and the haemoglobin-binding protein gene (hgpA) homologues identified by searching the preliminary A. actinomycetemcomitans HK1651 genome sequences released by the Advanced Center for Genome Technology (ACGT) at Oklahoma University (http://www.genome.ou.edu/act.html). The primer pair for tbpA was 5'-GGTTATCACTTTTCCGAGCAAC-3' and 5'-CCAGATTAGCCGAAACCTGATG-3', resulting in an amplicon of 531 nt, and the primer pair for hgpA was 5'-CATTGAAAATCACCTATTCCCAAC-3' and 5'-ATAGGACAGATCATGTTCTGTACC-3', which amplified a product of 753 nt. As template in the PCR, we used approximately 1 ng whole-cell DNA from strain HK1651, and the temperature profile for the PCR included an initial denaturation at 94 °C for 5 min followed by 30 cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min, with a final extension at 72 °C for 8 min. The PCR products were purified using Wizard Minicolumns (Promega) and labelled with [
-32P]dATP using the Random Primed DNA labelling Kit (Boehringer Mannheim) according to the manufacturers instructions.
Sequence analysis of the tbpA and hgpA homologues.
For amplification of the tbpA homologue from A. actinomycetemcomitans strains, we used two primer pairs: 5'-CAACACAAATACTGCTTAAGATGC-3' with 5'-ATGCAGGTAATGTCATATCGCG-3', and 5'-GGTTATCACTTTTCCGAGCAAC-3' with 5'-CACTTACGCGTGTGCAACAATC-3'. Amplification using these primer pairs resulted in two overlapping DNA fragments of 1·1 and 1·8 kb, respectively, which covered the putative 2·7 kb tbpA gene. For amplification of hgpA, we used the four pairs of primers: 5'-CTATTCCACCGATGAAGTTTATTTC-3' with 5'-TCGAATGGAATATCAACCGGTGTTCC-3', 5'-CATTGAAAATCCCTATTCCCAAC-3' with 5'-ATACAGATTAGTTACCAGATCATC-3', 5'-CCGGAATATATCCCCGGCGTCACC-3' with 5'-TTGTACCAAATGGGCGGATTGATC-3', and 5'-CACATATATTAAGCTGTGATGC-3' with 5'-CCGCTTCGGTATTGAACCTGTC-3'. Amplification using these primer pairs resulted in amplicons of 1·7, 1·1, 1·0 and 1·9 kb, respectively, which covered the 3·2 kb hgpA gene. PCR and purification of the subsequent products were carried out as described above. For DNA sequencing, we used the same primers as for the PCR and additional primers designed on the basis of the preliminary genome sequence of strain HK1651. Sequencing of both strands of the amplified fragments was achieved by using the Thermo Sequenase Dye Terminator Cycle Sequencing Kit (Amersham Life Science), and the resulting products were analysed on an automated DNA sequencer (model 377; Applied Biosystems). Sequence data were analysed with programs included in the GCG package (Genetics Computer Group, University of Wisconsin, Madison, WI, USA).
Insertional mutagenesis of hgpA.
A fragment of hgpA (positions 2852647 in the ORF of the hgpA gene from strain HK912) was amplified by PCR from 1 ng whole-cell DNA from A. actinomycetemcomitans HK912 using the primers 5'-GGCTTTGCTGTGCGTGGTGTGGA-3' and 5'-ATCGGAATATCGCCATCCATTC-3'. The amplicons were electrophoresed through a 1% agarose gel, excised, purified using the GeneClean II kit (BIO 101) and cloned into the pGEM-T Easy vector (Promega) using Escherichia coli XL-1 Blue for propagation. The resulting plasmid was termed pGEM-hgpA. The kanamycin-resistance gene from the 1·7 kb transprimer transposon in pGPS1.1 was inserted into the hgpA gene fragment in pGEM-hgpA using TnsABC Transposase as recommended by the manufacturer (New England Biolabs). The integration was shown by sequencing to be at position 1814 with a five nucleotide duplication of the target sequence. DNA from the resulting plasmid was linearized by digestion with SphI, extracted with phenol and introduced into A. actinomycetemcomitans by electroporation as described (Sreenivasan et al., 1991 ; Suzuki et al., 2000
). The cells were plated onto TSBYE agar containing 25 mg kanamycin l-1 to select for transformants (Sreenivasan et al., 1991
). Insertion of the kanamycin-resistance gene into the genomic hgpA gene was confirmed by PCR using primers 5'-CATTGAAAATCACCTATTCCCAAC-3' and 5'-ATACAGATTAGTTACCAGATCAT-3', which flanked the insertion and yielded a 1088 nt amplicon from the original hgpA gene.
Accession numbers.
The tbpA homologues have been deposited in GenBank under accession numbers AY028441 (strain HK1199), AF359437 (HK912), AF359438 (HK989), AF359439 (HK1002), AF359440 (HK988) and AF359441 (HK961). The hgpA homologues have been deposited in GenBank under accession numbers AF359442 (HK989), AF359443 (HK988), AF359444 (HK981), AF359445 (HK961), AF359446 (JP2), AF359447 (HK912), AF359448 (HK1605), AF359449 (HK1604), AF359450 (HK1199) and AF359451(HK1002).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Screening of the preliminary genome sequence of A. actinomycetemcomitans HK1651, which is a representative of the JP2 clone, revealed that it harbours a stretch of 3·2 kb with 70·9% identity to the Haemophilus influenzae hgpA gene encoding HgpA. Using a 0·75 kb fragment of this hgpA homologue in strain HK1651 as a probe in Southern blot analysis of EcoRI-restricted whole-cell DNA, we found that the remaining 10 strains of A. actinomycetemcomitans also harboured a single sequence similar to this probe (Fig. 2). The complete hgpA homologue was amplified by PCR and sequenced for nine of the 10 strains. In the last strain, HK961, the primers used in the PCR failed to amplify the start of the gene; consequently, the sequence of the first 400 nt of the 3·2 kb hgpA homologue was not determined for this strain.
|
|
|
An alignment of the deduced HgpA amino acid sequences from the seven strains that showed an intact ORF revealed >99% mutual identity. The sequences were 76% similar, including 12 gaps, to the H. influenzae haemoglobin-binding protein A. A search in the GenBank database revealed no other sequences with a higher degree of homology to the proposed A. actinomycetemcomitans HgpA protein. The 25 residues in the N terminus were predicted by the computer program SIGNALP (Nielsen et al., 1997 ) to constitute a signal peptide resulting in a secreted protein of 113·8 kDa with an isoelectric point of 9·74. As for most outer-membrane proteins, the C-terminal residue is a phenylalanine (Struyve et al., 1991
). The sequence NEIVVSGSSEG near the N terminus of the protein is homologous to the TonB box found in other TonB-dependent receptors (Elkins et al., 1995
; Lundrigan & Kadner, 1986
). None of the A. actinomycetemcomitans hgpA genes showed an equivalent to the CCAA repeats in the N-terminal part of the H. influenzae hgpA gene, which are supposed to be subject to slipped-strand mispairing resulting in the deletion/addition of repeats, thereby potentiating phase variation of the HgpA protein (Ren et al., 1998
).
Insertional inactivation of the hgpA homologue
We used insertional inactivation of the proposed hgpA gene to verify that it was responsible for the ability of the strains to utilize human haemoglobin as a source of iron. A kanamycin-resistance gene was inserted into a 2·4 kb fragment of the hgpA gene from strain HK912; this DNA construct was introduced by electroporation into four different strains of A. actinomycetemcomitans, each of which was able to grow on haemoglobin and had an apparently functional hgpA gene. After plating onto selective media, colonies with possible integrations were analysed by PCR using hgpA-specific primers that flanked the inserted kanamycin-resistance gene. In this assay, a 1·1 kb fragment of the parental hgpA gene was amplified, whereas the mutated hgpA gene resulted in a PCR product of 2·8 kb. For strains HK912, HK988 and HK1002 we did not obtain mutants with the resistance-marker gene integrated into the chromosomal hgpA gene, whereas for strain HK989 several such mutants were recovered. Differences in transformability among A. actinomycetemcomitans strains have been reported previously (Galli et al., 2002 ). Notably, in contrast to the parental strain HK989, the mutated version of strain HK989 was unable to grow on human haemoglobin (Fig. 4
), thus confirming that in strain HK989 the hgpA gene is functional in the acquisition of iron from human haemoglobin.
Homologues of the transferrin-binding-protein gene tbpA
A search in the preliminary genome sequence of A. actinomycetemcomitans HK1651 revealed a sequence with significant homology to the H. influenzae tbpA gene (Gray-Owen et al., 1995 ), which encodes the conserved A subunit of the heterodimeric TbpATbpB complex that constitutes the transferrin-binding receptor in this species (Gray-Owen & Schryvers, 1996
). However, this 2·7 kb sequence represented a non-functional pseudogene, as a deletion of a single nucleotide at position 279 interrupted the ORF and a C to T transition at position 1288 created a stop codon (positions are relative to the ORF of the H. influenzae tbpA gene, GenBank accession no. U10882). We confirmed this deletion and transition by PCR amplification of the pseudogene and subsequently sequenced the tbpA homologue of strain HK1651. In addition, the genomic sequence upstream of this tbpA homologue in strain HK1651 did not have the potential of encoding a TbpB protein. For the remaining 10 strains included in this study, we used hybridization analysis to assay for the presence of genomic sequences with homology to the tbpA pseudogene. Strain HK1651 was also included in this analysis to detect potential functional duplications of tbpA. A Southern blot of EcoRI-restricted whole-cell DNA was hybridized with a 0·53 kb fragment of the HK1651 tbpA pseudogene. All except one of the 11 strains examined showed a single EcoRI fragment hybridizing with the probe, indicating that a single copy of a tbpA homologue was present in each of these strains (Fig. 5
). Thus, strain HK1651 does not appear to harbour a functional tbpA gene in addition to the pseudogene. Genomic DNA of the exceptional serotype c strain HK981 did not hybridize with the probe, indicating that the tbpA gene sequences were deleted in this strain.
To further characterize the genomic sequences with homology to tbpA, we amplified by PCR and sequenced these tbpA homologues from an additional six strains of A. actinomycetemcomitans, HK912, HK961, HK988, HK989, HK1002 and HK1199, which represented the different serotypes of this organism. The ORF in all six of the resulting sequences was interrupted by mutations and, therefore, presumably represents a pseudogene. The sequence of the tbpA homologue in strains HK1199 and HK1651, both representatives of the JP2 clone, was identical. The tbpA sequences of the serotype a, b and d strains HK989, HK988 and HK1002, respectively, were very similar (>99·5% similar) to that of HK1651 and included the single nucleotide deletion at position 279, whereas the mutation at position 1288 that created the stop codon in strain HK1651 was absent in these three strains. In the serotype b strain HK912, the tbpA homologue was also very similar to the HK1651 tbpA sequence, except for a large out-of-frame deletion of 860 nt at positions 11502010. This is in agreement with the relatively weak hybridization observed for strain HK912 in the Southern blot analysis, since a major part of the DNA fragment used as a probe covered this deletion. The serotype e strain HK961 tbpA sequence was the most distinct (94·9% similar to that of strain HK1651). Compared to the H. influenzae tbpA gene it showed two out-of-frame mutations, a deletion of 40 nt at positions 617657 and an insertion of a single nucleotide at position 2421. The deleterious mutations in the different tbpA pseudogenes are summarized in Fig. 6.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several species in the family Pasteurellaceae, including H. influenzae and Haemophilus ducreyi, express related surface-exposed haemoglobin-binding proteins that facilitate the acquisition of iron from human haemoglobin (Elkins et al., 1995 ; Jin et al., 1996
; Ren et al., 1998
). In H. influenzae there is even a redundancy in the function of haemoglobin binding as a single strain may express three distinct haemoglobin-binding proteins, termed HgpA, HgpB and HgpC, each of which is subject to phase variation (Morton et al., 1999
). A. actinomycetemcomitans has been reported to use human haemoglobin as a source of iron for growth, and interactions with lipopolysaccharides have been suggested to mediate the binding of haemoglobin in this organism (Grenier et al., 1997
).
The present study demonstrates that sequences homologous to the H. influenzae hgpA gene are present in A. actinomycetemcomitans. The function of the hgpA homologue was confirmed by the demonstration that human haemoglobin could be utilized as a source of iron by A. actinomycetemcomitans, combined with the observation that mutational inactivation of the hgpA gene resulted in a loss of this property (Fig. 4). However, in contrast to all other strains, representatives of the JP2 clone and the single representative of serotype e were unable to grow on human haemoglobin as a source of iron and harboured a hgpA pseudogene. Whether this difference is associated with the increased pathogenic potential of the JP2 clone remains to be examined.
We have previously demonstrated that the population structure of A. actinomycetemcomitans is basically clonal and that the genetically distinct subpopulations within the species generally correlate with the serotypes a through e (Hayashida et al., 2000 ; Poulsen et al., 1994
). The finding that the proportional similarities among the hgpA sequences of the 11 strains of A. actinomycetemcomitans studied here correlated with the previously observed genetic relationships within the species is in full agreement with these conclusions (Fig. 3
). Thus, the hgpA sequences in all seven serotype b strains were very similar, confirming that the high-toxic serotype b strains are evolutionarily closely related to the low-toxic serotype b strains. In addition, the identical hgpA gene sequences in the serotype a and d strains are in accordance with the close genetic relatedness of these serotypes. Likewise, the very different hgpA sequence of the serotype e strain is in agreement with its distant genetic relationship to the other serotypes.
In the tbpA pseudogenes, the out-of-frame mutation at position 279 apparently occurred before serotype diversification and before the 530 nt deletion in the ltx promoter that led to the high toxicity of the JP2 clone (Fig. 6). Conversely, the high degree of similarity between hgpA sequences of the high- and low-toxic serotype b strains suggests that the mutations rendering hgpA inactive in the highly toxic JP2 clone are relatively recent in evolution. The sequence similarities do not provide information on the evolutionary origin of the deleterious mutations in the tbpA and hgpA genes in the single serotype e strain, except to infer that they have occurred independently of the evolution of the other tbpA and hgpA pseudogenes. The presence of evolutionarily unrelated hgpA pseudogenes in A. actinomycetemcomitans strains suggests that there has been no selection for a functional hgpA gene in these strains.
It has been shown that A. actinomycetemcomitans Y4 (a low-toxic serotype b strain) binds to and can utilize haem as a source of iron (Graber et al., 1998 ; Grenier et al., 1997
). In agreement with this observation, all strains examined in our study were capable of growing in the presence of haemin as the only source of iron. It is conceivable that free haem may be accessible in the natural environment of A. actinomycetemcomitans, the periodontal pocket, as a result of proteolysis of haem-containing proteins by proteases released by other micro-organisms. In this context, it is worth noting that Porphyromonas gingivalis, another suspected periodontal pathogen, produces a lysine-specific cysteine proteinase, Kgp, which degrades and releases haem from haemoglobin, haemopexin, haptoglobin and transferrin (Genco & Dixon, 2001
). An analogous synergistic cooperation in iron acquisition from haemoglobin has been demonstrated during abscess formation between E. coli, which releases a haemoglobin protease, and Bacteroides fragilis (Otto et al., 2002
).
In conclusion, A. actinomycetemcomitans is undergoing evolutionary changes relative to its closest taxonomic relatives with regard to its strategies for acquiring iron. The lack of a functional tbpA gene adequately explains the observation that this species cannot utilize human transferrin as a source of iron. The inability to use human haemoglobin as an iron source discriminates strains of the high-toxic JP2 clone from low-toxic serotype b strains and from most other strains of A. actinomycetemcomitans that express a functional haemoglobin-binding protein like several other members of the family Pasteurellaceae.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Contreras, A., Rusitanonta, T., Chen, C., Wagner, W. G., Michalowicz, B. S. & Slots, J. (2000). Frequency of 530-bp deletion in Actinobacillus actinomycetemcomitans leukotoxin promoter region. Oral Microbiol Immunol 15, 338-340.[Medline]
Elkins, C., Chen, C.-J. & Thomas, C. E. (1995). Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi. Infect Immun 63, 2194-2200.[Abstract]
Escolar, L., Pérez-Martín, J. & de Lorenzo, V. (1999). Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181, 6223-6229.
Galli, D. M., Kerr, M. S., Fair, A. D., Permpanich, P. & LeBlanc, D. J. (2002). Parameters associated with cloning in Actinobacillus actinomycetemcomitans. Plasmid 47, 138-147.[Medline]
Genco, C. A. & Dixon, D. W. (2001). Emerging strategies in microbial haem capture. Mol Microbiol 39, 1-11.[Medline]
Graber, K. R., Smoot, L. M. & Actis, L. A. (1998). Expression of iron binding proteins and hemin binding activity in the dental pathogen Actinobacillus actinomycetemcomitans. FEMS Microbiol Lett 163, 135-142.[Medline]
Gray-Owen, S. D. & Schryvers, A. B. (1996). Bacterial transferrin and lactoferrin receptors. Trends Microbiol 4, 185-191.[Medline]
Gray-Owen, S. D., Loosmore, S. & Schryvers, A. B. (1995). Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae. Infect Immun 63, 1201-1210.[Abstract]
Grenier, D., Leduc, A. & Mayrand, D. (1997). Interaction between Actinobacillus actinomycetemcomitans lipopolysaccharides and human hemoglobin. FEMS Microbiol Lett 151, 77-81.[Medline]
Guerinot, M. L. (1994). Microbial iron transport. Annu Rev Microbiol 48, 743-772.[Medline]
Haraszthy, V. I., Lally, E. T., Haraszthy, G. G. & Zambon, J. J. (2002). Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans. Infect Immun 70, 3170-3179.
Haubek, D., Poulsen, K., Westergaard, J., Dahlén, G. & Kilian, M. (1996). Highly toxic clone of Actinobacillus actinomycetemcomitans in geographically widespread cases of juvenile periodontitis in adolescents of Africa origin. J Clin Microbiol 34, 1576-1578.[Abstract]
Haubek, D., Dirienzo, J. M., Tinoco, E. M. B., Westergaard, J., Lopéz, N. J., Chung, C. P., Poulsen, K. & Kilian, M. (1997). Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis. J Clin Microbiol 35, 3037-3042.[Abstract]
Haubek, D., Ennibi, O.-K., Poulsen, K., Poulsen, S., Benzarti, N. & Kilian, M. (2001). Early-onset periodontitis in Morocco is associated with the highly leukotoxic clone of Actinobacillus actinomycetemcomitans. J Dent Res 80, 1580-1583.[Abstract]
Hawley, D. K. & McClure, W. R. (1983). Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11, 2237-2255.[Abstract]
Hayashida, H., Poulsen, K., Takagi, O. & Kilian, M. (2000). Phylogenetic associations of ISAa1 and IS150-like insertion sequences in Actinobacillus actinomycetemcomitans. Microbiology 146, 1977-1985.
Jin, H., Ren, Z., Pozsgay, J. M., Elkins, C., Whitby, P. W., Morton, D. J. & Stull, T. L. (1996). Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae. Infect Immun 64, 3134-3141.[Abstract]
Kalmar, J. R. & Arnold, R. R. (1988). Killing of Actinobacillus actinomycetemcomitans by human lactoferrin. Infect Immun 56, 2552-2557.[Medline]
Lundrigan, M. D. & Kadner, R. J. (1986). Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. J Biol Chem 261, 10797-10801.
Mietzner, T. A. & Morse, S. A. (1994). The role of iron-binding proteins in the survival of pathogenic bacteria. Annu Rev Nutr 14, 471-493.[Medline]
Morton, D. J., Whitby, P. W., Jin, H., Ren, Z. & Stull, T. L. (1999). Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b. Infect Immun 67, 2729-2739.
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6.[Abstract]
Ogunnariwo, J. A. & Schryvers, A. B. (1990). Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor. Infect Immun 58, 2091-2097.[Medline]
Ogunnariwo, J. A. & Schryvers, A. B. (1996). Rapid identification and cloning of bacterial transferrin and lactoferrin receptor protein genes. J Bacteriol 178, 7326-7328.[Abstract]
Otto, B. R., Verweij-van Vught, A. M. & MacLaren, D. M. (1992). Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit Rev Microbiol 18, 217-233.[Medline]
Otto, B. R., van Dooren, S. J., Dozois, C. M., Luirink, J. & Oudega, B. (2002). Escherichia coli hemoglobin protease autotransporter contributes to synergistic abscess formation and heme-dependent growth of Bacteroides fragilis. Infect Immun 70, 5-10.
Poulsen, K., Hjorth, J. P. & Kilian, M. (1988). Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenza serotype b strains. Infect Immun 56, 987-992.[Medline]
Poulsen, K., Theilade, E., Lally, E. T., Demuth, D. R. & Kilian, M. (1994). Population structure of Actinobacillus actinomycetemcomitans: a framework for studies of disease-associated properties. Microbiology 140, 2049-2060.[Abstract]
Ratledge, C. & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881-941.[Medline]
Ren, Z., Jin, H., Morton, D. J. & Stull, T. L. (1998). hgbB, a gene encoding a second Haemophilus influenzae hemoglobin- and hemoglobin-haptoglobin-binding protein. Infect Immun 66, 4733-4741.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Slots, J., Reynolds, H. S. & Genco, R. J. (1980). Actinobacillus actinomycetemcomitans in human periodontal disease: a cross-sectional microbiological investigation. Infect Immun 29, 1013-1020.[Medline]
Sreenivasan, P. K., LeBlanc, D. J., Lee, L. N. & Fives-Taylor, P. (1991). Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids. Infect Immun 59, 4621-4627.[Medline]
Stormo, G. D., Schneider, T. D. & Gold, L. M. (1982). Characterization of translational initiation sites in E. coli. Nucleic Acids Res 10, 2971-2996.[Abstract]
Struyve, M., Moons, M. & Tommassen, J. (1991). Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol 218, 141-148.[Medline]
Suzuki, N., Nakano, Y., Yoshida, Y., Nakao, H., Yamashita, Y. & Koga, T. (2000). Genetic analysis of the gene cluster for the synthesis of serotype a-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans. B B A 1517, 135-138.
Willemsen, P. T., Vulto, I., Boxem, M. & de Graaff, J. (1997). Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans. J Bacteriol 179, 4949-4952.[Abstract]
Winston, J. L., Chen, C.-K., Neiders, M. E. & Dyer, D. W. (1993). Membrane protein expression by Actinobacillus actinomycetemcomitans in response to iron availability. J Dent Res 72, 1366-1373.[Abstract]
Zambon, J. J. (1985). Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 12, 1-20.[Medline]
Received 26 July 2002;
accepted 9 September 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |