Department of Biology, Middlebury College, Middlebury, VT 05753, USA1
Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA2
Author for correspondence: Grace Spatafora. Tel: +1 802 443 5431. Fax: +1 802 443 2072. e-mail: spatafor{at}middlebury.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: oral streptococci, fimA, dtxR, regulation, metals, iron uptake
Abbreviations: IR, inverted repeat
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite a well-documented role for iron in promoting the pathogenesis of some Gram-negative micro-organisms (Bullen et al., 1978 ), the involvement of this micronutrient in disease brought on by Gram-positive bacteria has not been extensively investigated. While early epidemiological and clinical studies suggested that certain trace metals in food and drinking water, including iron, may be associated with the development of dental caries (Adkins & Losee, 1970
; Aranha et al., 1982
), the mechanism(s) by which iron exerts its putative effect(s) on Streptococucs mutans-induced cariogenesis remain unclear. In previous work, the absence of typical hydroxamate- and/or catecholate-like siderophores from S. mutans was suggested by biochemical assays specific for these compounds (Evans et al., 1986
). In our laboratory, dialysis bag experiments (Husson et al., 1993
) conducted in parallel with a universal siderophore detection assay (Schwyn & Neilands, 1987
) have confirmed that S. mutans does not elaborate these small iron-chelating molecules (unpublished observations).
FimA is a 36 kDa fimbrial lipoprotein adhesin on the surface of Streptococcus parasanguis (also known as S. parasanguinis), the primary colonizer of dental plaque and major player in subacute endocarditis (Burnette-Curley et al., 1995 ; Viscount et al., 1997
). Interestingly, reports in the literature implicate a role for S. parasanguis FimA in adherence to fibrin (Burnette-Curley et al., 1995
) but not to saliva-coated hydroxyapatite (Froeliger & Fives-Taylor, 2000
). In the present study, we identified a 34 kDa fimA homologue in S. mutans.
FimA belongs to the lipoprotein receptor antigen I (LraI) family (Fenno et al., 1995 ) of proteins, which is transcribed as part of ABC-transporter-type operons. Reports in the literature indicate that in addition to functioning as adhesins, LraI proteins are also involved in metal ion transport (Dintilhac & Claverys, 1997
; Kolenbrander et al., 1998
). Among the LraI adhesins previously described in the streptococci are ScaA in S. gordonii (Kolenbrander et al., 1994
), PsaA and AdcA in S. pneumoniae (Dintilhac et al., 1997
), ScbA in S. crista (Correia et al., 1996
), SsaB in S. sanguis (also known as S. sanguinis) (Ganeshkumar et al., 1991
), Lmb in S. agalactiae (Spellerberg et al., 1998
) and FimA in S. parasanguis (Burnette-Curley et al., 1995
; Fenno et al., 1995
). Of these adhesins, ScaA and PsaA are known to facilitate the transport of manganese ions (Kolenbrander et al., 1998
; Dintilhac et al., 1997
) while AdcA is reported to be a putative transporter of zinc (Dintilhac et al., 1997
). Other studies describe the translocation of metal ions, including iron, by an ABC transporter lipoprotein in the group A streptococci (Janulcyk et al., 1999
). In the present study, we generated a knockout mutation in the S. mutans fimA gene to define a putative role for the FimA lipoprotein in iron uptake/transport.
The regulation of iron uptake/transport by the fur gene product is well documented in Gram-negative bacteria such as Escherichia coli, Neisseria gonorrhoeae and Vibrio cholerae (Zimmerman et al., 1984 ; Fleming et al., 1983
; Genco & Desai, 1996
; Butteron et al., 1992
), in which FurFe2+ complexes bind to the bacterial chromosome and prevent transcription of iron-dependent genes. Homologues of fur have not been identified in S. mutans. Rather, in the present study we identified a dtxR-like gene (dlg) downstream of the fimA operon on the S. mutans chromosome for which there is a homologue in Corynebacterium diphtheriae. DtxR in C. diphtheriae is an iron-dependent metalloregulatory protein that complexes with Fe2+ to regulate the expression of diphtheria toxin and other virulence-associated genes (Boyd et al., 1990
; Hennecke, 1990
; Schmidt, 1997
; Schiering et al., 1995
). Herein, we present evidence that is consistent with a role for the S. mutans DtxR homologue in iron-dependent regulation of S. mutans fimA expression.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial culture conditions.
Wild-type S. mutans UA130 (serotype c) was grown overnight at 37 °C and 5% CO2 in 14 ml ToddHewitt broth (THB). S. mutans GMS700 and GMS800 were grown overnight as described in THB supplemented with kanamycin (250 µg ml-1), and GMS850 was grown in THB supplemented with erythromycin (10 µg ml-1). Cultures were stored in THB containing 20% (v/v) sterile glycerol at -80 °C.
For growth-curve determinations and Northern hybridization experiments, S. mutans was grown at 37 °C with 5% CO2 as described, and the cells harvested by centrifugation at 7000 r.p.m. for 10 min in an SS34 rotor. The cell pellets were washed three times in iron-depleted FMC and resuspended in 1 ml of the same. Fourteen millilitres of FMC containing various concentrations of iron were then inoculated with 150 µl of this cell concentrate and grown as described for up to 24 h.
Total RNA for spot blotting was derived from overnight cultures of S. mutans UA130 and GMS800 grown in THB or THB supplemented with kanamycin, respectively. These cultures were used to inoculate 45 ml THB and grown to early-, mid- and late-exponential and stationary phases as described.
Protein isolation and SDS-PAGE.
For the preparation of cell lysates, 14 ml volumes of FMC containing 0·0110 µM ferric citrate were inoculated, and the cells grown and harvested as described. The cell pellets were resuspended in cold phosphate-buffered saline containing 0·5 mM PMSF and the cell suspensions transferred to microcentrifuge tubes containing 1/3 vol. zirconium beads (0·1 mm). The cells were disrupted in a Mini-Bead Beater (Biospec) at 4 °C for 3 min and cellular debris removed by low-speed centrifugation for 1 min in a refrigerated microcentrifuge. Total protein determinations were performed for each sample using a BCA protein assay kit (Pierce) and bovine serum albumin as a standard. The cell lysates were combined with an equal volume of 2xsample buffer (Laemmli, 1970 ) and incubated at room temperature for 2 h prior to loading onto a 515% SDS-PAGE gradient gel. Electrophoresis proceeded at 90 V for 16 h, followed by staining with Coomassie brilliant blue R-250 (Sigma) and/or silver stain (AMRESCO).
Western blotting.
S. mutans proteins (50 µg per lane) were resolved on each of two SDS-PAGE gels, one of which was stained with Coomassie blue. Proteins from the other gel were transferred electrophoretically to nitrocellulose membranes in a Hoeffer Western transfer apparatus at 0·2 A and 4 °C overnight. The membrane was blocked in 100 ml 5% (w/v) powdered skim milk for 1 h with gentle agitation, washed twice in 100 ml Tris-buffered saline/Tween for 10 min each, and then allowed to react with a 1:5000 dilution of a polyclonal rabbit antiserum directed against FimA from S. parasanguis (provided by Dr Paula Fives-Taylor, University of Vermont). Following three additional washes as described, a 1:10000 dilution of a horseradish-peroxidase-conjugated goat anti-rabbit antiserum (Sigma or Pierce) was applied. After several more washes, the blots were prepared for visualization by enhanced chemiluminescence (ECL) according to the manufacturers recommendations (Amersham Life Science).
Cloning of the S. mutans fimA gene and construction of a fimA knockout mutation.
The primers and plasmids used to clone and subsequently disrupt the S. mutans fimA gene are shown in Tables 1 and 2
. The 5' end of the S. mutans fimA gene was amplified with primers SmfimA-F and SmfimA-R using sequence information derived from the GenBank database (accession number AF232688) and cloned into pGEMT-EZ (Promega) according to the recommendations of the supplier. The resulting recombinant, pMM1, includes a unique HindIII site 13 bp downstream of the fimA initiation codon which we exploited in subsequent knockout mutagenesis experiments. Specifically, a 2·0 kb
Km-2 kanamycin-resistance cassette (aphA3
) from plasmid pBR322Km
was cloned into the HindIII site within the fimA coding sequence on pMM1, and E. coli transformants harbouring the fimA knockout mutation were selected on L-agar supplemented with kanamycin (50 µg ml-1). The knockout mutation was confirmed by sequence analysis of plasmid DNA isolated and purified on mini-prep spin columns (Qiagen). We disrupted the ampicillin-resistance gene resident on the resulting pMM2 construct with PvuI, and recircularized the 5·5 kb fragment with T4 DNA ligase (Promega) following gel extraction (Qiagen). Plasmid DNA (pMM3) was isolated from E. coli transformants demonstrating resistance to kanamycin, and confirmed by restriction enzyme mapping. To confirm ampicillin sensitivity, transformants were also replica-plated onto L-agar supplemented with 50 µg ampicillin ml-1. Finally, pMM3 was moved into S. mutans by electroporation (Spatafora et al., 1995
), and the double crossover event selected for on ToddHewitt agar (TH agar) plates supplemented with kanamycin (250 µg ml-1). The resulting S. mutans knockout mutant (GMS700) was confirmed by Southern blot analysis of restricted chromosomal DNA isolated as described by Sambrook et al. (1989)
. A DNA fragment internal to the fimA coding sequence was generated using primers fimA.pb-F and fimA.pb-R and radiolabelled for hybridization experiments.
|
|
Construction of GMS850, a S. mutans fimA/dlg double knockout mutant.
Primers ermAMHin-F and ermAMHin-R (Table 2) were used to amplify an erythromycin-resistance determinant (ermAM) from plasmid pSG236 (Goodman & Gao, 2000
). The resulting 0·94 kb amplicon was digested with HindIII and ligated into the HindIII site located within the fimA coding sequence cloned on plasmid pMM1. E. coli transformants were selected on L-agar supplemented with 300 µg erythromycin ml-1 from which putative recombinants (pfimEm1) were isolated and purified on Qiagen spin columns. The pfimEm1 construct was confirmed by restriction mapping, and digested with PvuI to disrupt the ampicillin-resistance gene on the plasmid. The resulting construct, pfimEm2, was subsequently used to transform the S. mutans dlg knockout mutant (GMS800) as described. Streptococcal transformants demonstrating resistance to both erythromycin (10 µg ml-1) and kanamycin (250 µg ml-1) on TH agar plates were selected for Southern blot analysis to confirm knockout mutations in the fimA and dlg genes on the S. mutans chromosome (data not shown).
DNA isolation.
Chromosomal DNA was isolated from S. mutans UA130, GMS700, GMS800 and GMS850 using a modification of the method of Marmur (1961) . The DNA was purified by caesium chloride/ethidium bromide equilibrium density-gradient centrifugation and digested with restriction enzymes (Promega) according to the recommendations of the supplier. Plasmid DNA was extracted and purified from E. coli transformants using Qiagen spin columns (Qiagen).
PCR.
Primers used to generate amplicons for cloning and/or nick translation are shown in Table 2. Amplification with Red Taq polymerase (Sigma) was performed in a Hybaid PCR Express thermocycler with the following cycling conditions: 94 °C for 1 min, 50 °C for 2 min and 72 °C for 2 min repeated for 35 cycles, followed by a 72 °C extension for 10 min.
Southern blotting.
Restricted chromosomal DNAs from S. mutans UA130, GMS700 and GMS850, or from PCR products generated as described above, were resolved on 0·8% agarose gels and transferred to nitrocellulose membranes according to the method of Southern (1975) . The DNAs were cross-linked to the membranes in a FisherBrand cross-linker (FB-UVXL-1000) and probed with a 770 bp fimA-specific or a 500 bp dlg-specific amplicon that had been previously radiolabelled with [32P]dATP by nick translation (Rigby et al., 1977
). Primers fimA.pb-F and fimA.pb-R or Sm-dlg-pb-F and Sm-dlg-pb-R (Table 2
) were used to amplify the fimA- or dlg-specific probes, respectively. Filters were hybridized in 1% BSA, 300 mM sodium phosphate, 7% SDS and 100 mM EDTA, pH 8, at 60 °C for 16 h in a FisherBrand hybridization oven with gentle agitation. The membranes were then washed once for 10 min at 60 °C in 0·5% BSA, 40 mM sodium phosphate, 5% SDS and 1 mM EDTA, and twice for 10 min each at 60 °C in 40 mM sodium phosphate, 1% SDS and 1 mM EDTA. Autoradiography (Kodak BIOMAX ML film) proceeded for up to 24 h at -80 °C in the presence of an intensifying screen.
RNA isolation.
For Northern hybridization experiments, total intact RNA was isolated from S. mutans UA130 cultures grown as described in FMC supplemented with 0·0110 µM ferric citrate. The cultures were centrifuged at 4 °C and 6000 r.p.m. in an SS34 rotor for 5 min and the cell pellets resuspended in 4 ml ice-cold sterile Tris/EDTA (TE ) buffer, pH 8·0. To the cell suspensions, 3·5 g sterile, chilled, acid-washed glass beads (Sigma, 150220 µm diameter) were added and immediately mixed with 3 ml phenol/chloroform/isoamyl alcohol (PCI), pH 4·3 (Sigma). After the addition of 0·25 ml 10% SDS, the mixture was vortexed for 4 min with intermittent cooling on ice. The cell mixture was then centrifuged as described above and the aqueous phase extracted three more times with PCI, pH 4·3. Nucleic acid was precipitated overnight at -20 °C in the presence of 0·1 vol. 10 M LiCl and 2 vols ethanol. The RNA was pelleted by centrifugation at 8000 r.p.m. and 4 °C in an SS34 rotor for 15 min and subsequently washed in ice-cold 70% ethanol. Finally, the RNA pellets were air-dried, resuspended in 50 µl sterile diethyl pyrocarbonate-treated water, and stored frozen at -80 °C. For spot blot hybridization studies, total RNA was isolated from S. mutans by disruption in a reciprocating shaking device (FastPrep, Bio101) according to the Bio101 Fast RNA Blue protocol.
Northern and spot blots.
Total RNA isolated from UA130 and GMS700 was resolved on a 0·8% formaldehyde agarose gel, transferred to a nitrocellulose membrane, and cross-linked in a FisherBrand cross-linker. Spot blots of total RNA isolated from GMS800 were prepared using a Schleicher and Schuell Minifold I apparatus according to the recommendations of the supplier. RNAs were probed with a radiolabelled 770 bp amplicon that is internal to the fimA coding sequence, or (as an internal control) with a 500 bp rpsL amplicon derived from the S. mutans chromosome using primers rpsL-F and rpsL-R (Table 2). Hybridization and wash conditions were as described for Southern blotting.
Metal ion uptake.
S. mutans UA130, GMS700, GMS800 and GMS850 were grown overnight in THB with 250 µg kanamycin ml-1 or 10 µg erythromycin ml-1 when appropriate. The precultures (250 µl) were used to inoculate 50 ml prewarmed THB and grown to early exponential phase. Then 7·4x104 Bq 55FeCl3 (3 µM) or 54MnCl2 (0·03 µM) was added to 1 ml cells and the cultures were grown overnight as described. The bacteria were pelleted and washed three times in fresh THB, and radioactivity was measured in a scintillation counter calibrated for 55Fe or 54Mn. Control cultures grown in parallel and in the absence of radioisotope were serially diluted and plated on TH agar plates for bacterial enumeration.
Cariogenic potential of S. mutans GMS700 and GMS800 in germ-free rats.
The cariogenic potential of S. mutans UA130, GMS700 and GMS800 was determined in young gnotobiotic Fischer rats. Nineteen-day-old weanling rats were challenged orally with approximately 108 c.f.u. ml-1 of the appropriate test strain. Animals were maintained on a sterile caries-promoting diet containing 5% sucrose (Michalek et al., 1975 ) provided ad libitum. Colonization was assessed 2 d post-challenge and then weekly for the duration of the experiment by collecting faecal swab samples and culturing them on Mitis Salivarius (MS) agar (Difco) with or without kanamycin. Rats killed 35 d post-challenge were scored for caries (Keyes, 1958
), and plaque microbiology was assessed on MS agar with appropriate selection to confirm the presence of UA130, GMS700 or GMS800.
Statistical analysis.
Means and standard errors for caries scores and bacterial metal ion uptake were evaluated by analysis of variance using the Duncan and KruskalWallis tests, respectively. Differences were considered to be significant at P0·05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Cloning of the S. mutans fimA gene and confirmation of a fimA knockout mutation in S. mutans GMS700
The strategy used to clone and interrupt the S. mutans fimA coding sequence in E. coli is illustrated in Fig. 3(a). The
Km-2 cassette (aphA3
) resident on pMM3 was then used to disrupt the wild-type fimA gene on the S. mutans UA130 chromosome by allelic exchange. The resulting knockout mutation in GMS700 was confirmed by Southern blot using a 770 bp fimA-specific probe. Insertion of the 1·8 kb
Km-2 cassette into the fimA coding sequence on the GMS700 chromosome is supported by a shift in the fimA-containing DNA fragment from 2·6 kb in UA130 to 4·4 kb in GMS700 (Fig. 3b
).
|
|
Spot blot analysis
Total RNA isolated from S. mutans UA130 and GMS800 grown in THB was spotted onto a nitrocellulose membrane and hybridized with a fimA-specific probe. The data revealed increased fimA expression in the dlg mutant relative to the UA130 progenitor during the mid-exponential, late-exponential and stationary phases of growth (Fig. 5).
|
|
S. mutans fimA and dlg knockout mutations do not significantly affect caries formation in vivo
The cariogenic potential of S. mutans GMS700 and GMS800 was examined in germ-free rats. The results of these experiments are summarized in Table 4. The mean caries scores for rats infected with either knockout mutant were not significantly different from those infected with the wild-type UA130 progenitor.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work in our laboratory applied inductively coupled argon plasma (ICAP) analysis (Coish & Sinton, 1992 ) to reveal the iron content of human saliva. We defined salivary iron concentrations as ranging from 0·1 to 1·0 µM (unpublished observations), and therefore included these concentrations in our in vitro studies. The growth of S. mutans was restricted in a medium containing 0·4 µM iron, indicating that S. mutans is likely to experience iron starvation in the oral cavity, especially during non-mealtimes. The results of dialysis bag and chrome azural assays performed in our laboratory support a receptor-mediated mechanism for S. mutans iron uptake/transport (unpublished observations), which is consistent with previous reports in the literature indicating that S. mutans does not elaborate siderophores (Evans et al., 1986
). Moreover, ferric chloride, ferric/ferrous citrate and transferrin all proved to be suitable iron sources for S. mutans growth in iron-starved cultures (data not shown). This is supported by reports that describe a membrane-associated flavin reductase in S. mutans that reduces ferric (III) iron into its more soluble ferrous (II) form prior to internalization (Evans et al., 1986
). A transferrin receptor has not yet been identified in S. mutans, however, nor has one been described in other Gram-positive pathogens known to utilize transferrin as an iron source (Williams & Griffiths, 1992
).
The FimA lipoprotein is encoded by the last of three genes which comprise a tricistronic ABC transporter operon on the S. mutans chromosome (Kitten et al., 2000 ). The first ORF shares up to 62% amino acid identity with other streptococcal ATP-binding proteins, including ORF1 in S. gordonii (Kolenbrander et al., 1994
) and PsaB in S. pneumoniae (Novak et al., 1998
). The second shares up to 81% amino acid identity with other streptococcal hydrophobic transmembrane proteins within ABC transporter operons, including PsaA in S. gordonii (Kolenbrander et al., 1994
) and FimB in S. parasanguis (Froeliger & Fives-Taylor, 2000
). The S. mutans fimA gene product shares up to 76% amino acid identity with other streptococcal LraI proteins, including ScbA in S. cristae (Correia et al., 1996
), Adc in S. pneumoniae (Dintilhac & Claverys, 1997
), ScaA in S. gordonii (Kolenbrander et al., 1994
), and FimA in S. parasanguis (Fenno et al., 1995
), all of which encode high-affinity metal ion transport proteins. That a fimA null mutation was not lethal in S. mutans indicates that other mechanisms for iron uptake/transport are functional in this oral pathogen. The presence of other iron transporters in S. mutans is also supported by the equivalent amounts of 55Fe uptake we observed for the fimA knockout mutant and its wild-type progenitor.
The results of Western and Northern blot analyses reveal an inverse relationship between S. mutans fimA expression and iron availability. This is consistent with a role for iron in the regulation of fimA expression. In fact, located downstream of the S. mutans fimA operon is a 654 bp dtxR-like gene (dlg) which shares up to 54% similarity at the amino acid level with other iron-dependent repressor proteins including SirR in Staphylococcus epidermidis (Cockayne et al., 1998 ), TroR in Treponema pallidum (Hardham et al., 1997
) and DtxR in Corynebacterium diphtheriae (Tao & Murphy, 1994
). Upstream of the dlg start codon are putative ShineDalgarno, and -10 and -35 consensus sequences, indicating that dlg expression may be driven by an independent promoter. Within the promoter region that precedes the S. mutans fimA operon is a 38 bp inverted repeat (IR) sequence to which DtxR-like metalloregulatory proteins typically bind (Kitten et al., 2000
). This suggests that expression of dlg may also be controlled by the promoter that drives expression of the ABC transporter operon, resulting in transcriptional readthrough of fimA. Taken collectively, the structural organization of the S. mutans fimA locus is consistent with a role for the dlg gene product in regulating iron-responsive fimA expression in S. mutans.
Gram-negative pathogens typically use Fur, a 17 kDa cytosolic metalloregulatory protein that is also regulated by iron, to modulate the expression of genes important for virulence (Bullen et al., 1978 ; Finkelstein et al., 1983
). Western blots performed in our laboratory revealed no evidence of a Fur-like homologue in S. mutans but confirmed the presence of a DtxR-like metalloregulator (data not shown). In Corynebacterium diphtheriae, DtxR utilizes iron to regulate virulence genes whose products are necessary for adherence and toxin production (Tao & Murphy, 1994
). MntR and IdeR are DtxR-like proteins in Bacillus subtilis (Que & Helmann, 2000
) and Mycobacterium tuberculosis (Pohl et al., 1999
), respectively, which regulate manganese or iron uptake/transport functions. In the present study, we propose a model for Dlg metalloregulation in S. mutans that may involve the formation of DlgFe2+ complexes. Specifically, we propose that when iron is plentiful, DlgFe2+ complexes form and associate with the IR sequence upstream of the S. mutans fimA operon, thereby decreasing iron uptake by down-regulating expression of the fimA iron transporter (Fig. 7a
). In contrast, when iron is limiting, as it is in the human host, we propose that Fe2+ is not available as a co-repressor to complex with Dlg. Thus, Dlg cannot bind at the IR sequence and fimA expression is likely to become derepressed, thereby promoting iron scavenging (Fig. 7b
). Evidence supporting this model includes the increase in fimA expression noted for the GMS800 dlg knockout mutant on Western and Northern blots, and the increase in 55Fe uptake by GMS800 which is compromised in the fimA/dlg double knockout mutant, GMS850. Taken collectively, these findings indicate that Dlg is a repressor of S. mutans fimA expression, and that the fimA gene product is involved in iron transport in this oral pathogen. Gel mobility shift assays are currently under way in our laboratory to confirm the S. mutans IR sequences as putative Dlg-binding sites.
|
Finally, the disruption of dlg did not affect S. mutans-induced cariogenesis in germ-free rats. This is not surprising since the expression of virulence factors is paramount for bacterial survival in the host environment. Indeed, the expression of genes whose products promote S. mutans caries development is likely to be subject to multiple mechanisms of control, not all of which belong to the proposed Dlg regulon. We also noted that the cariogenic potential of S. mutans was not significantly affected by a fimA knockout mutation in a rat caries model. This is consistent with a recent report by Froeliger & Fives-Taylor (2000) suggesting that FimA is not significantly involved in S. parasanguis adhesion to saliva-coated hydroxyapatite. However, recent in vivo studies support a role for S. mutans FimA in an endocarditis model (Burnette-Curley et al., 1995
; Kitten et al., 2000
). We propose that fimbrial adhesins, rather than the glucan products of sucrose-dependent glucosyltransferases, mediate adherence when S. mutans translocates into the bloodstream. This is supported by previous work in our laboratory which revealed induction of fimA expression upon exposure of S. parasanguis cultures to horse or bovine serum (unpublished observations). How iron might be involved in mediating the expression of fimA under these host conditions has not yet been determined.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aranha, H., Strachan, R. C., Arceneaux, J. & Byers, B. (1982). Effect of trace metals on growth of Streptococcus mutans in a teflon chemostat. Infect Immun 35, 456-460.[Medline]
Archibald, F. (1983). Lactobacillus plantarum, an organism not requiring iron. FEMS Microbiol Lett 19, 29-32.
Boyd, J., Oza, M. N. & Murphy, J. (1990). Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent element (dtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci USA 87, 5968-5972.[Abstract]
Bullen, J. J., Rogers, H. J. & Griffiths, E. (1978). Role of iron in bacterial infections. Curr Top Microbiol Immunol 80, 1-35.[Medline]
Burnette-Curley, D., Wells, V., Viscount, H., Munro, C. L., Fenno, J. C., Fives-Taylor, P. & Macrina, F. L. (1995). FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis. Infect Immun 63, 4669-4674.[Abstract]
Butteron, J., Stoebner, J., Payne, S. & Calderwood, S. (1992). Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor in Vibrio cholerae. J Bacteriol 174, 3729-3738.[Abstract]
Cockayne, A., Hill, P. J., Powell, N. B., Bishop, K., Sims, C. & Williams, P. (1998). Molecular cloning of a 32 kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC tansporter. Infect Immun 66, 3767-3774.
Coish, R. A. & Sinton, C. W. (1992). Geochemistry of mafic dikes in the Adirondack mountains: implications for late Proterozoic continental rifting. Contrib Mineral Petrol 110, 500-514.
Correia, F. F., DiRienzo, J. M., McKay, T. L. & Rosan, B. (1996). scbA from Streptococcus crista CC5A: an atypical member of the lraI gene family. Infect Immun 64, 2114-2121.[Abstract]
Dintilhac, A. & Claverys, J. P. (1997). The adc locus, which affects competence for genetic transformation in Streptococcus pneumoniae, encodes an ABC transporter with a putative lipoprotein homologous to a family of streptococcal adhesins. Res Microbiol 148, 119-131.[Medline]
Dintilhac, A., Alloing, G., Granadel, C. & Claverys, J. P. (1997). Competence and virulence of Streptococcus pneumoniae: AdcA and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol Microbiol 25, 727-739.[Medline]
Evans, S., Arceneaux, J., Byers, B., Martin, M. & Aranha, H. (1986). Ferrous iron transport in Streptococcus mutans. J Bacteriol 168, 1096-1099.[Medline]
Federle, M. J., McIver, K. S. & Scott, J. R. (1999). A response regulator that represses transcription of several virulence operons in the group A streptococcus. J Bacteriol 181, 3649-3657.
Fenno, J. C., Shaikh, A., Spatafora, G. & Fives-Taylor, P. (1995). The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system. Mol Microbiol 15, 849-863.[Medline]
Finkelstein, R., Sciortino, C. & McIntosh, M. (1983). Role of iron in microbehost interactions. Rev Infect Dis 5, S759-S777.[Medline]
Fleming, T., Nahlika, M. & McIntosh, M. (1983). Regulation of enterobacteria iron transport in Escherichia coli: characterization of ent::Mud(Aprlac) operon fusions. J Bacteriol 156, 1171-1177.[Medline]
Froeliger, E. H. & Fives-Taylor, P. (2000). Streptococcus parasanguis FimA does not contribute to adherence to SHA (abstract). J Dent Res 79, 337.
Ganeshkumar, N., Hannam, P. M., Kolenbrander, P. E. & McBride, B. C. (1991). Nucleotide sequence of a gene coding for a saliva-binding protein (SsaB) from Streptococcus sanguis 12 and possible role of the protein in coaggregation with Actinomyces. Infect Immun 59, 1093-1099.[Medline]
Goodman, S. D. & Gao, Q. (2000). Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5. Plasmid 4, 85-98.
Genco, C. & Desai, P. (1996). Iron acquisition in the pathogenic Neisseria. Trends Microbiol 4, 185-191.[Medline]
Hardham, J. M., Stamm, L. V., Porcella, S. F. & 7 other authors (1997). Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog. Gene 197, 4764.[Medline]
Hennecke, H. (1990). Regulation of bacterial gene expression by metalprotein complexes. Mol Microbiol 4, 1621-1628.[Medline]
Husson, M., Legrand, D., Spik, G. & Leclerc, H. (1993). Iron acquisition by Helicobacter pylori: importance of human lactoferrin. Infect Immun 61, 2694-2697.[Abstract]
Janulcyk, R., Pallon, J. & Björck, L. (1999). Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations. Mol Microbiol 344, 596-606.
Keyes, P. (1958). Dental caries in the molar teeth of rats. II. A method for diagnosing and scoring several types of lesions simultaneously. J Dent Res 37, 1088-1099.
Kitten, T., Munro, C. L., Michalek, S. M. & Macrina, F. L. (2000). Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence. Infect Immun 68, 4441-4451.
Kolenbrander, P. E., Andersen, R. N. & Ganeshkumar, N. (1994). Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette. Infect Immun 62, 4469-4480.[Abstract]
Kolenbrander, P. E., Andersen, R. N., Baker, R. A. & Jenkinson, H. F. (1998). The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake. J Bacteriol 180, 290-295.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Litwin, C. M. & Calderwood, S. B. (1993). Role of iron in regulation of virulence genes. Clin Microbiol Rev 6, 137-149.[Abstract]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208-218.
Michalek, S., McGhee, J. & Navia, J. (1975). Virulence of Streptococcus mutans: a sensitive method for evaluating cariogenicity in young gnotobiotic rats. Infect Immun 12, 69-75.[Medline]
Nakayama, K. (1992). Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants. J Bacteriol 174, 4928-4934.[Abstract]
Novak, R., Braun, J. S., Charpentier, E. & Tuomanen, E. (1998). Penicillin tolerance genes of Streptococcus pneuomoniae: the ABC-type manganese permease complex, Psa. Mol Microbiol 29, 1285-1296.[Medline]
Pohl, E., Holmes, R. K. & Hol, W. G. J. (1999). Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal binding sites fully occupied. J Mol Biol 285, 1145-1156.[Medline]
Posey, J. E. & Gherardini, F. C. (2000). Lack of a role for iron in the Lyme disease pathogen. Science 288, 1651-1653.
Que, Q. & Helmann, J. D. (2000). Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheriae toxin repressor family of proteins. Mol Microbiol 35, 1454-1468.[Medline]
Rigby, P. W. J., Dieckman, M., Phodes, C. & Berg, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. I. J Mol Biol 113, 237-251.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schiering, N., Tao, X., Zeng, H., Murphy, J., Petsko, G. & Ringe, D. (1995). Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae. Proc Natl Acad Sci USA 92, 9843-9850.[Abstract]
Schmidt, M. (1997). Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme. Infect Immun 65, 4634-4641.[Abstract]
Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47-56.[Medline]
Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503-517.[Medline]
Spatafora, G. & Moore, M. (1998). Growth of Streptococcus mutans in an iron-limiting medium. Methods Cell Sci 20, 217-221.
Spatafora, G., Rohrer, K., Barnard, D. & Michalek, S. (1995). A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo. Infect Immun 63, 2556-2563.[Abstract]
Spellerberg, B., Rozdzinski, E., Martin, S., Weber-Heynemann, J., Schnitzler, N., Lütticken, R. & Podbielski, A. (1998). Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin. Infect Immun 67, 871-878.
Tao, X. & Murphy, J. R. (1994). Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol 14, 191-197.[Medline]
Terleckyj, B., Willett, N. P. & Shockman, G. D. (1975). Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect Immun 11, 649-655.[Medline]
Viscount, H. B., Munro, C. L., Burnette-Curley, D., Peterson, D. L. & Macrina, F. L. (1997). Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats. Infect Immun 65, 994-1002.[Abstract]
Weinberg, E. D. (1978). Iron and infection. Microbiol Rev 42, 45-66.
Williams, P. & Griffiths, E. (1992). Bacterial transferrin receptors structure, function, and contribution to virulence. Med Microbiol Immunol 181, 301-322.[Medline]
Wooldridge, K. G. & Williams, P. H. (1993). Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol Rev 12, 325-348.[Medline]
Zimmerman, L., Hantke, K. & Braun, V. (1984). Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12. J Bacteriol 159, 271-277.[Medline]
Received 23 January 2001;
accepted 2 March 2001.