Expression of fibronectin-binding protein FbpA modulates adhesion in Streptococcus gordonii

Julie Christie1, Roderick McNaba,2 and Howard F. Jenkinson1

Department of Oral and Dental Science, University of Bristol, Lower Maudlin St, Bristol BS1 2LY, UK1
Department of Microbiology, Eastman Dental Institute, University College London, 256 Gray’s Inn Rd, London WC1X 8LD, UK2

Author for correspondence: Roderick McNab. Tel: +44 1932 822000. Fax: +44 1932 822100. e-mail: scunner_99{at}yahoo.com


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fibronectin binding is considered to be an important virulence factor in streptococcal infections. Adhesion of the oral bacterium Streptococcus gordonii to immobilized forms of fibronectin is mediated, in part, by a high molecular mass wall-anchored protein designated CshA. In this study, a second fibronectin-binding protein of S. gordonii is described that has been designated as FbpA (62·7 kDa). This protein, which is encoded by a gene located immediately downstream of the cshA gene, shows 85 and 81% identity to the fibronectin-binding proteins PavA, of Streptococcus pneumoniae, and FBP54, of Streptococcus pyogenes, respectively. Purified recombinant FbpA bound to immobilized human fibronectin in a dose-dependant manner, and isogenic mutants in which the fbpA gene was inactivated were impaired in their binding to fibronectin. This effect was apparent only for cells in the exponential phase of growth, and was associated with reduced surface hydrophobicity and the surface expression of CshA. Cells in the stationary phase of growth were unaffected in their ability to bind to fibronectin. By utilizing gene promoter fusions with cat (encoding chloramphenicol O-acetyltransferase), it was demonstrated that cshA expression was down-regulated during the exponential phase of growth in the fbpA mutant. Expression of fbpA, but not cshA, was sensitive to atmospheric O2 levels, and was found to be up-regulated in the presence of elevated O2 levels. The results suggest that FbpA plays a regulatory role in the modulation of CshA expression and, thus, affects the adhesion of S. gordonii to fibronectin.

Keywords: oral streptococcus, chloramphenicol O-acetyltransferase reporter, gene expression

Abbreviations: CAT, chloramphenicol O-acetyltransferase

The GenBank accession number for the sequence reported in this paper is X65164.

a Present address: GlaxoSmithKline Consumer Healthcare, St Georges Avenue, Weybridge, Surrey KT13 0DE, UK.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mitis group streptococci [including Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis and Streptococcus sanguis (sanguinis); Kawamura et al., 1995 ] are found at most sites within the human oral cavity and are numerically abundant in dental plaque (Frandsen et al., 1991 ). Colonization and persistence of streptococci in the oral cavity depends, at least in part, on their ability to adhere to oral surfaces, including the salivary pellicle on teeth, to other oral bacteria in plaque and to host epithelial cells (Whittaker et al., 1996 ; Jenkinson & Lamont, 1997 ). Mitis group streptococci are also frequently isolated as the causative agents of infective endocarditis (Douglas et al., 1993 ; Dyson et al., 1999 ). The ability of these bacteria to aggregate platelets and to adhere to endothelial cells, to platelet–fibrin thrombi and to extracellular matrix components (e.g. fibronectin) at sites of native heart-valve damage are thought to contribute to their disease potential (Lowrance et al., 1990 ; Baddour, 1994 ; Herzberg, 1996 ).

Fibronectin is a dimeric glycoprotein (approx. 440 kDa) that is present in a soluble form in plasma and extracellular fluids; it is also present in a fibrillar form on cell surfaces. Both the soluble and cellular forms of fibronectin may be incorporated into the extracellular tissue matrix (reviewed by Romberger, 1997 ). While fibronectin has critical roles in eukaryotic cellular processes, such as adhesion, migration and differentiation, it is also a substrate for the attachment of bacteria. The binding of pathogenic Streptococcus pyogenes and Staphylococcus aureus to epithelial cells via fibronectin facilitates their internalization (Joh et al., 1999 ) and systemic spread within the host.

The best-characterized fibronectin-binding proteins from streptococci and staphylococci share a similar mosaic architecture (Joh et al., 1994 , 1999 ). These cell-surface proteins possess an N-terminal signal peptide for sec-dependent secretion and an LPXTG C-terminal motif for covalent anchorage to cell-wall peptidoglycan (Navarre & Schneewind, 1999 ). A consensus sequence [ED(T/S)(9 or 10 X)GG(3 or 4 X)(I/V)DF, where X is any amino acid] that occurs in multiple copies within the carboxy-terminal amino acid repeat regions of these proteins is important for their fibronectin-binding activities (Joh et al., 1994 , 1999 ). Streptococci also express several atypical fibronectin-binding proteins. These proteins do not contain conventional secretion, anchorage or fibronectin-binding sequences; examples include FBP54 (Courtney et al., 1994 ) and SDH (Pancholi & Fischetti, 1992 ) in Streptococcus pyogenes, and PavA in Streptococcus pneumoniae (Holmes et al., 2001 ).

Fibronectin is present on oral epithelial cells and in submandibular/sublingual and parotid saliva, and is deposited within the acquired pellicle that coats the tooth surface (Zetter et al., 1979 ; Babu et al., 1983 ; Babu & Dabbous, 1986 ; Ericson & Tynelius-Bratthall, 1986 ). A range of oral streptococci have been shown to bind fibronectin (Babu et al., 1983 ; Babu & Dabbous, 1986 ; Ericson & Tynelius-Bratthall, 1986 ; van der Flier et al., 1995 ; Willcox et al., 1995 ) but, in general, little is known about the adhesins involved in this binding. Most molecular studies have been carried out on S. gordonii, which binds to conformationally specific determinants of immobilized human fibronectin that are not presented by soluble plasma fibronectin (Lowrance et al., 1988 ). Fibronectin binding is mediated by a 259 kDa cell-wall-anchored protein, CshA, which also confers cell-surface hydrophobicity and co-aggregation properties with Actinomyces spp., Candida albicans and S. oralis (Holmes et al., 1996 ; McNab et al., 1996 ). CshA is the structural and functional component of the 60 nm long peritrichous fibrils that are present on the S. gordonii cell surface (McNab et al., 1999 ). Isogenic cshA mutants of S. gordonii show approximately 50% reduction in their ability to bind to immobilized human fibronectin when compared to the wild-type (McNab et al., 1996 ), and are unable to colonize the murine oral cavity. There are no significant amino acid sequence similarities between CshA and other wall-anchored staphylococcal and streptococcal fibronectin-binding proteins, perhaps reflecting the binding specificity of CshA for immobilized fibronectin.

Sequencing upstream and downstream of the cshA locus in S. gordonii revealed a gene, downstream of cshA and convergently transcribed, encoding a polypeptide with high sequence similarity to FBP54, a fibronectin-binding protein in S. pyogenes (Courtney et al., 1994 ). This raised the possibility that there was a ‘host-association’ locus on the S. gordonii chromosome that encoded a second adhesin (designated FbpA) mediating attachment of S. gordonii to fibronectin. In this paper, we demonstrate that while FbpA binds to immobilized human fibronectin, the effects observed on the ability of S. gordonii to bind fibronectin upon inactivation of the fbpA gene probably arise through modulation of the expression of CshA.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
The bacterial strains and plasmids used in this study are listed in Table 1. Streptococci were grown at 37 °C on BHY agar containing 37 g brain heart infusion l-1 (Difco), 5 g yeast extract l-1 and 1·5% (w/v) agar. Liquid cultures were grown without shaking in screw-cap tubes or in bottles at 37 °C in BHY medium or in TY-glucose medium (Jenkinson et al., 1993 ). For aeration experiments, cultures (20 ml) were grown in flasks (200 ml) with gyratory shaking (200 r.p.m.). Anaerobic cultures were grown without shaking under 80% N2, 10% CO2 and 10% H2, in an anaerobic cabinet. Escherichia coli strains were grown aerobically at 37 °C in LB (Sambrook et al., 1989 ). The concentrations of the antibiotics used for selection were as follows: 100 µg ampicillin ml-1 (E. coli); 50 µg kanamycin ml-1 (E. coli); 12·5 µg tetracycline ml-1 (E. coli) or 5 µg tetracycline ml-1 (S. gordonii); 50 µg erythromycin ml-1 (E. coli) or 1 µg erythromycin ml-1 (S. gordonii); 100 µg spectinomycin ml-1 (E. coli and S. gordonii); 5 µg chloramphenicol ml-1 (S. gordonii).


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids used in this study

 
DNA and RNA manipulations.
Routine molecular biology techniques were performed as specified by Sambrook et al. (1989) . Plasmid DNA was isolated from E. coli using Concert Minipreps (Gibco Life Technologies) and PCR products were purified by using the Qiaquick Kit (Qiagen). Chromosomal DNA was isolated from S. gordonii as described previously (Jenkinson, 1987 ). S. gordonii cells were transformed with DNA according to the method of Haisman & Jenkinson (1991) . Restriction and modifying enzymes (New England Biolabs) were used under the conditions recommended by the manufacturer. DNA sequencing was performed with cloning-vector-derived primers (M13 forward, M13 reverse, T7 or SP6) or custom-synthesized oligonucleotides (MWG-BIOTECH), using an Applied Biosystems model 373A automated DNA sequencer, or manually using Sequenase (United States Biochemical). Sequence data were analysed with DNAMAN (Lynnon Biosoft), Genejockey or DNA Strider. The BLAST algorithm (Altschul et al., 1990 ) was utilized for database searches.

DNA sequence information 3' to the TGA stop codon of cshA was obtained in two stages, as outlined below. Plasmid pPH1704 contained a 2·2 kb S. gordonii DNA fragment, 1·5 kb of which has been shown to encode the carboxy terminus of CshA (McNab & Jenkinson, 1992 ). The sequence of the remaining 0·7 kb of the S. gordonii DNA fragment was determined and used to design primers for inverse PCR amplification of flanking DNA (McNab et al., 1994 ). Briefly, S. gordonii DL1 genomic DNA was digested with SspI, self-ligated at 3·5 ng µl-1 and then used as a template for PCR (Expand High Fidelity System; Boehringer Mannheim) with the primers CTERM (5'-TTATACAGGACAGAAAACCC-3'; complementary to nucleotides 8298–8317, cshA locus; GenBank accession no. X65164) and IFBPA (5'-GCTCCACCTGATCTTGGTCG-3'; complementary to nucleotides 8907–8926) (30 cycles, 60 °C annealing temperature, 3 min extension at 72 °C). The PCR product (3·63 kb) was purified and subcloned into pUC19 for sequencing.

Total RNA was prepared from S. gordonii cells as described previously (McNab & Jenkinson, 1998 ). RNA samples (10 µg lane-1) were prepared for electrophoresis and separated on 1·2% (w/v) agarose gels. The RNA was then transferred to a nylon membrane, as described by Kroczek & Siebert (1990) . Hybridization of the blots with a 32P-labelled fbpA gene fragment was performed as described by Church & Gilbert (1984) , with solutions containing 8% (w/v) dextran sulphate. The blots were washed three times each for 20 min with 2xSSC containing 0·1% SDS at 65 °C; the radiolabelled bands were detected by autoradiography.

Preparation of antiserum to FbpA.
Antibodies were raised against FbpA that had been produced initially as a glutathione S-transferase fusion protein. Primers HJ30 (5'-ACGGATCCGCAGCAATCGCAAAAGCC-3'; BamHI site shown in bold type; complementary to nucleotides 9765–9794) and HJ31 (5'-TCAATCATGTCGACTTGGACAAG-3'; SalI site shown in bold type; complementary to nucleotides 8366–8388) were used in PCR to amplify an internal fragment of fbpA encoding amino acid residues 39–511. The PCR product was digested with a combination of BamHI and SalI and cloned into pGEX-5X-3 (Amersham Pharmacia Biotech) that had been similarly digested. FbpA from E. coli lysates was purified on a glutathione-Sepharose column, as described by McNab et al. (1996) . Antibodies against recombinant FbpA were raised in New Zealand White rabbits by intramuscular injection of 80 µg of protein; booster injections (30 µg each) were administered after 2 and 3 months.

Purification of the recombinant FbpA polypeptide.
To produce N-terminal His-tagged FbpA for use in protein-binding assays, the entire coding region of FbpA was amplified by PCR from S. gordonii DL1 genomic DNA using the primers HisF1 (5'-TTCGACGGATCCTTTTTACATCAC-3'; BamHI site shown in bold type) and HisR1 (5'-AAGAGAGAGTCGACCCTATTTATA-3'; SalI site shown in bold type). The PCR product was digested with BamHI and SalI and ligated into pQE30 that had been similarly digested. The plasmid construct was then transformed into E. coli M15. Expression of the His-tagged FbpA protein was induced by the addition of 0·01 mM IPTG to cultures in the exponential phase of growth. After a further 4 h incubation at 30 °C, cells were harvested by centrifugation, suspended in lysis buffer [0·05 M NaH2PO4, 250 mM NaCl, pH 8·0, containing 1 mM PMSF and 0·1 % (v/v) protease inhibitor cocktail (Sigma) for use with bacterial cell extracts] and then lysed by sonication. Proteins were extracted from the lysates with 8 M urea, and FbpA was purified on a nickel-NTA resin column, according to the manufacturer’s guidelines. Eluted proteins were dialysed extensively against de-ionized water at 4 °C.

Construction of the fbpA mutant strain.
A portion of the fbpA gene (encoding amino acids 1–175 of FbpA) was amplified by PCR using primers FLPID3 (5'-GTCTTTCTAGAGATTTTTTTTAC-3'; XbaI site shown in bold type) and FLPID4 (5'-CAGTGAAAGGATCCAGACTG-3'; BamHI site shown in bold type). The 525 bp product was purified, digested with a combination of XbaI and BamHI, and then ligated with the integration vector pSF143 (Tao et al., 1992 ), which had been similarly digested. The resultant plasmid, pSFBP1, was transformed into competent S. gordonii DL1 cells with selection for tetracycline resistance to generate recombinant strain UB1060. Southern-blot analysis (using the ECL DNA Labelling and Detection System; Amersham Life Sciences) was performed to confirm that the plasmid had been integrated into the S. gordonii chromosome at the predicted locus.

S. gordonii strain OB507 carries a chromosomal cshA promoter–cat (p-cshAcat) fusion (McNab & Jenkinson, 1998 ). To generate a fbpA knockout mutant in this reporter strain, the PCR product amplified using primers FLPID3 and FLPID4 was digested with XbaI and BamHI, and ligated with integration vector pFW5 (Podbielski et al., 1996 ), which had been similarly digested. The resulting plasmid, pFWFLP1, was inserted onto the S. gordonii OB507 chromosome by transformation and selection for tetracycline and spectinomycin resistance, to generate strain UB1245.

Construction of the strain carrying p-flpAcat.
The fbpA promoter region was amplified by PCR using primers PROMF (5'-TTCTTCTAGATTTATCAGTAAATTACC-3'; XbaI site shown in bold type; complementary to nucleotides 10185–10211, cshA locus) and FUSION2 (5'-GCCTCCGGATTTCGAGCTCCTTGAAAGTTACTATAGAA-3'; complementary to nucleotides 9908–9928). To enable ligation-PCR, FUSION2 carried a 5' extension of 13 bp which was complementary to the 3' end of FUSION1 (see below). A promoterless chloramphenicol O-acetyltransferase (CAT) gene (cat) was amplified from pMH109 (Hudson & Stewart, 1986 ) using primers FUSION1 (5'-TTCTATAGTAACTTTCAAGGAGCTCGAAATCCGGAGGC-3') and Tet/Cat (5'-GGATCCGGGCCACCTCGACC-3'; BamHI site shown in bold type). FUSION1 carried a 5' extension of 19 bp which was complementary to the 3' end of FUSION2. PCR products of the correct size were purified, mixed and used in PCR amplification with primers PROMF and Tet/Cat. The resulting fbpA promoter–cat (p-fbpAcat) fusion product (1·4 kb) was digested with XbaI and BamHI, and then ligated with pFW5 that had been similarly digested, to generate pFWFUSION1. Plasmid pFWFUSION1 was introduced onto the S. gordonii chromosome by transformation and selection for spectinomycin resistance, to generate strain UB1300. The insertion also conferred resistance to S. gordonii against 10 µg chloramphenicol ml-1.

Binding of recombinant FbpA to immobilized fibronectin.
Human fibronectin (Boehringer Mannheim) was applied to microtitre plate wells and additional sites were blocked with BSA, as described previously (McNab et al., 1996 ). Recombinant N-terminal His6-tagged FbpA (1·25 µg) was incubated in fibronectin-coated or BSA-coated (control) wells for 2 h at 37 °C. Wells were washed with TNMC buffer (1 mM Tris/HCl, 150 mM NaCl, 0·1 mM MgCl2, 0·1 mM CaCl2, pH 8·0) and incubated with His5 antibody (Qiagen; 1:1000) for 1 h at room temperature. After rinsing the wells with TNMC, bound antibody was detected with horseradish-peroxidase-conjugated anti-mouse IgG (Dako; 1:1000) and o-phenylenediamine. ELISA (A492) values for protein bound to fibronectin were corrected by subtracting the background value detected for the binding of protein to the BSA-blocked (control) wells. To measure the effects of gelatin on FbpA binding to fibronectin, FbpA was added to fibronectin-coated wells that had been pre-incubated with gelatin (1 mg ml-1) for 1 h at 37 °C.

Hydrophobicity, adhesion and aggregation assays.
Cell-surface hydrophobicity was estimated as the percentage of total cells partitioning with hexadecane (Jenkinson, 1987 ). The adhesion of S. gordonii cells to fibronectin was performed as described previously (McNab et al., 1996 ). Briefly, S. gordonii cells were radiolabelled by growth in TY-glucose medium containing [methyl-3H]thymidine [6 µCi ml-1 (78 Ci mmol-1, 2·89 TBq mmol-1); Amersham]. They were then washed thoroughly and added to microtitre plate wells that had been fibronectin-coated and BSA-blocked as before. Plates were incubated at 37 °C for 2 h with gentle shaking, and the numbers of bound cells were then determined by scintillation counting (Jenkinson et al., 1993 ). The saliva-mediated agglutination of bacterial cells was determined spectrophotometrically using the method described previously by Jenkinson et al. (1993) , with the decrease in the OD600 value being measured over time.

Analysis of bacterial proteins.
Bacteria from a late-exponential phase culture (30 ml) of S. gordonii DL1, OB235 cshA3 or UB1060 fbpA were harvested by centrifugation (4000 g, 10 min, 4 °C), washed once in TP buffer [50 mM Tris/HCl, pH 7·8, containing 5% (v/v) protease inhibitor cocktail; 5 ml] and suspended in 0·2 ml spheroplasting buffer [20 mM Tris/HCl, pH 6·8, 10 mM MgCl2, 26% (w/v) raffinose; Jenkinson et al., 1993 ]. Mutanolysin (500 U ml-1; Sigma) and lysozyme (0·2 mg ml-1) were added, and the suspension was incubated at 37 °C for 20 min. TP buffer (0·2 ml) was then added to the suspension, and the cells were disrupted by sonication (4x15 s at 280 W cm-2, with cooling on ice between each pulse) using an Ikasonic U50 hand-held sonicator fitted with a 3-mm-diameter probe (IKA-Werke GMBH, Staufen, Germany). The suspension was fractionated by centrifugation (12000 g, 10 min, 4 °C) and the supernatant (soluble fraction) was carefully removed. The cell-envelope pellet was washed twice by suspension and centrifugation in TP buffer, and the proteins were solubilized from the fractions with 1% (w/v) SDS (final concentration) by heating for 10 min at 90 °C. The solutions were then mixed with loading dye (Jenkinson et al., 1993 ). Proteins were separated by SDS-PAGE on 8% (w/v) acrylamide gels and then transferred to Hybond-C nitrocellulose membranes (Amersham) by electroblotting (McNab & Jenkinson, 1992 ). Antibodies against FbpA were affinity-purified by incubating FbpA antiserum with a nitrocellulose strip containing recombinant FbpA. Bound antibodies were eluted with 0·2 M glycine/HCl, pH 2·5, as described previously (Jenkinson & Easingwood, 1990 ). Antibody binding on immunoblots was detected by using peroxidase-conjugated swine immunoglobulins to rabbit IgG and enhanced chemiluminescence (ECL; Amersham), according to manufacturer’s instructions.

The immunoreactivity of CshA on intact cells was determined by ELISA, as described previously (Holmes et al., 1996 ), using antibodies raised against a recombinant fragment of the CshA polypeptide comprising the N-terminal 844 aa residues of the mature CshA polypeptide (N-CshA; McNab et al., 1996 ).

Preparation of cell-free extracts and CAT assay.
Cell-free extracts were prepared and assayed for CAT activities as described previously (McNab & Jenkinson, 1998 ). Briefly, streptococcal cells in BHY medium (10 ml) were harvested by centrifugation (4000 g, 10 min, 4 °C) and washed once in TPE buffer (100 mM Tris/HCl, pH 7·8, containing 1 mM PMSF and 1 mM EDTA). The bacteria were then resuspended in spheroplasting buffer. Mutanolysin (final concentration 500 U ml-1) was added and the suspension was incubated for 30 min at 37 °C. Cells were disrupted by vortexing with glass beads. The suspensions were then centrifuged (12000 g, 20 min, 4 °C) to remove the glass beads and cell debris; the supernatants were removed for enzyme assays. Protein concentrations were determined by using a Bio-Rad protein assay kit, with bovine {gamma}-globulin as the standard. CAT activity was determined by the spectrophotometric method of Shaw (1975) , utilizing a Shimadzu UV-1201 recording spectrophotometer. The reaction rate was determined from the linear portion of the graph and corrected for the background change in A412. The corrected value was divided by 0·0136 to yield CAT activity expressed as nmol chloramphenicol acetylated min-1 (mg protein)-1 at 37 °C.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence of the fbpA gene
The cshA gene of S. gordonii DL1-Challis encodes a 259 kDa wall-anchored fibronectin adhesin (McNab et al., 1996 ). Analysis of sequence data immediately 3' to cshA, generated during sequencing of the cshA chromosomal locus, revealed the presence of a partial ORF with sequence similarity to FBP54, a fibronectin- and fibrinogen-binding protein of S. pyogenes (Courtney et al., 1994 , 1996 ). To investigate the role of this gene in fibronectin binding, we amplified, by inverse PCR, a 3·63 kb fragment that overlapped with the previously reported cshA sequence. Computer analysis of 2049 bp of sequence downstream of the cshA stop codon (GenBank accession no. X65164, updated to include downstream sequence) revealed a complete ORF, designated FbpA (fibronectin-binding protein A), of 550 aa residues (62693 Da). FbpA demonstrated 81% identity to the FBP54 sequence (550 aa residues) deduced from the S. pyogenes M1 complete genome sequence (GenBank accession no. AAK33911; Ferretti et al., 2001 ) and 85% sequence identity to S. pneumoniae PavA (Holmes et al., 2001 ). The FBP54 polypeptide, as initially discovered by Courtney et al. (1994) , was missing 76 aa residues from the N terminus.

S. gordonii FbpA was encoded on the opposite strand to CshA, with the cshA and fbpA stop codons separated by 75 bp (Fig. 1a). Within this region was a single potential stem–loop structure previously identified as a {rho}-independent transcriptional terminator for cshA (overscored in Fig. 1a). At the 5' end of fbpA there was a potential ribosome-binding site (RBS) (GGAGA) and an extended -10 promoter sequence (underscored in Fig. 1b), comprising a -16 region (TTTG; Voskuil & Chambliss, 1998 ) upstream of a canonical -10 region (TATAAT), positioned appropriately to the fbpA start codon (Fig. 1b). No recognizable -35 sequence was present in the promoter region of fbpA. However, the extended -10 sequence can function naturally in the absence of a -35 site (Sabelnikov et al., 1995 ), and it was predicted that fbpA was expressed from its own promoter. The deduced amino acid sequence of FbpA contained no conventional N-terminal hydrophobic leader or signal peptidase cleavage site to direct export via the general secretory pathway. Also, there was no Gram-positive bacterial cell-wall anchorage motif (LPXTG, where X is any amino acid; Navarre & Schneewind, 1999 ) at the C-terminal end of FbpA. The sequence was relatively leucine rich (61 residues out of 550), a feature previously noted for FBP54 of S. pyogenes (Courtney et al., 1994 ).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Schematic diagram of the S. gordonii DL1 fbpA chromosomal region. (a) Sequence of the fbpAcshA intergenic region (75 bp). The stop codons for fbpA and cshA are in bold type. A stem–loop transcriptional terminator previously identified for cshA is indicated by inverted arrows, and the associated run of T residues (complementary to A residues) is overscored. (b) Comparison of the sequence containing the fbpA promoter region with the corresponding sequence generated by the chromosomal promoter–cat fusion. A potential extended -10 promoter sequence is underscored and RBS sequences are shown in bold type. Changes to the S. gordonii chromosomal sequence resulting from p-fbpA–cat integration are indicated by italics.

 
To detect the expression of FbpA by streptococcal cells, we utilized affinity-purified antibodies raised against recombinant FbpA (see Methods). A major polypeptide band with an approximate molecular mass of 60 kDa was detected on immunoblots of proteins from envelope (containing membrane and wall-associated proteins) and soluble (cytoplasmic) fractions of S. gordonii DL1-Challis cells (Fig. 2, lanes 1 and 2).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Immunoblot analysis of FbpA expression. Proteins were analysed from cytoplasmic (C) or envelope (E) fractions of S. gordonii DL1-Challis (lanes 1 and 2), UB1060 (fbpA) (lanes 3 and 4) or OB235 (cshA) (lanes 5 and 6). Each lane was loaded with 20 µg protein. The positions of molecular mass markers are indicated on the left of the image.

 
Role of FbpA in S. gordonii adhesion to fibronectin
Adhesion of S. gordonii cells to immobilized human fibronectin has been shown to be mediated by CshA (McNab et al., 1996 ). However, since cshA mutants of S. gordonii are only approximately 50% reduced in their binding to fibronectin, at least one other adhesin might be involved (McNab et al., 1996 ). To determine if this additional adhesin was FbpA, the fbpA gene was inactivated as described in Methods. A representative tetracycline-resistant mutant of DL1-Challis, designated UB1060, was isolated and characterized in further studies. Insertion of the antibiotic-resistance determinant at the fbpA locus was confirmed by Southern analysis (data not shown). The FbpA protein was absent in immunoblots of envelope and soluble-protein fractions prepared from mutant strain UB1060 (Fig. 2, lanes 3 and 4). Insertional inactivation of the adjacent cshA gene (strain OB235) did not affect expression of FbpA (Fig. 2, lanes 5 and 6). The growth rates of the mutant strain UB1060 fbpA::tet were similar to those of the wild-type strain at 37 and 42 °C (data not shown).

The ability of wild-type and UB1060 fbpA cells to adhere to immobilized human fibronectin was measured using a microtitre-plate-based assay (see Methods). The adhesion of mid-exponential phase cells of UB1060 to fibronectin was reduced by approximately 25%, when compared with wild-type cells harvested at the same phase of growth (Table 2). However, stationary phase cells of UB1060 and DL1-Challis adhered to fibronectin to approximately the same extent as each other (Table 2). The binding of wild-type or UB1060 cells to fibronectin was inhibited by gelatin to a similar degree (45·2%±3·0%) (data not shown). The adhesion of S. gordonii OB235 cshA3 cells to fibronectin was reduced by approximately 45 %, irrespective of whether the cells were harvested in the exponential or stationary phases of growth (Table 2). Inactivation of fbpA also resulted in significantly reduced surface hydrophobicity of UB1060 cells harvested in the mid-exponential phase of growth, but this reduction was not seen in cells harvested during the stationary phase of growth (Table 2). The hydrophobicity of OB235 (cshA3) was greatly reduced irrespective of the growth phase; this was in accordance with previous results correlating CshA with surface hydrophobicity (McNab & Jenkinson, 1992 ). Saliva-mediated aggregation of exponential or stationary phase cells was unaffected in UB1060, when compared with the wild-type (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 2. Adhesion and surface properties of S. gordonii strains

 
To determine if FbpA bound to human fibronectin, fbpA was cloned into pQE30 and the His6-tagged recombinant protein was expressed in E. coli following IPTG induction. Recombinant FbpA was purified using Ni-NTA affinity chromatography, and it was isolated as a single band by SDS-PAGE (data not shown). Recombinant FbpA bound avidly to immobilized human fibronectin (Fig. 3), but showed no binding to BSA. Conversely, we were unable to detect binding of soluble fibronectin to purified FbpA that had been immobilized on nitrocellulose membranes or in plastic wells (data not shown).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Binding of recombinant FbpA to immobilized human fibronectin. Microtitre plate wells were coated with increasing amounts of fibronectin ({square}) or BSA ({circ}), and FbpA binding was measured by ELISA using a His5 mAb and horseradish-peroxidase-conjugated anti-mouse secondary antibody. All values are means of quadruplicate samples ±SD for three experiments.

 
Inactivation of fbpA affects cshA gene expression
Although recombinant FbpA was shown to bind fibronectin, the effects of fbpA gene inactivation on cell-surface hydrophobicity and adhesion to fibronectin suggested that the expression of CshA might be affected. This was first investigated by ELISA using CshA-reactive antibodies (McNab et al., 1996 ). Mid-exponential phase cells of mutant strain UB1060 fbpA showed a 35% decrease in their surface presentation of CshA when compared with wild-type cells (Table 2), whereas stationary phase cells showed no difference in their surface presentation of this protein. To investigate this further, the fbpA gene was inactivated in a cshA promoter–cat (p-cshAcat) reporter strain (OB507; McNab & Jenkinson, 1998 ) to generate UB1245. Cell-free extracts of OB507 (p-cshAcat) or UB1245 (p-cshAcat fbpA) were prepared from batch-culture samples taken at intervals during growth. CAT-specific activities in cell-free extracts of OB507 were found to increase throughout growth and reached a maximum in the early-stationary phase (Fig. 4), confirming previous results (McNab & Jenkinson, 1998 ). By contrast, CAT-specific activities were significantly lower in cell-free extracts of strain UB1245 cells in the early- and mid-exponential phases of growth. However, by the time UB1245 cultures entered the early-stationary phase their CAT-specific activities approached those of OB507 (Fig. 4). These data suggested that the inactivation of fbpA affected cshA promoter activity during the exponential phase of growth, thus accounting for the reduced surface presentation of CshA on these cells (Table 2).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Specific CAT activity in cells of S. gordonii strain OB507 (p-cshAcat) ({circ}), strain UB1245 (p-cshAcat fbpA) ({bullet}) and strain UB1300 (p-fbpAcat) ({triangleup}) at points during their growth in batch culture ({square}; culture density at OD600 for strain OB507). The results presented (mean±SD for triplicate samples) are from a representative experiment of four repeats. Strains OB507 and UB1245 had similar growth curves.

 
Construction and characterization of the fbpA promoter–cat fusion
To investigate the expression levels of fbpA at various stages during batch-culture growth, a fbpA promoter–cat fusion strain, UB1300, was generated (see Methods). In this construct, the RBS sequence for fbpA (GGAGA) was replaced with the cat RBS sequence (GGAGG), and the RBS–ATG spacing was increased from five nucleotides to eight nucleotides (Fig. 1b), identical to the p-cshAcat construct (McNab & Jenkinson, 1998 ).

By contrast to cshA expression, which increases in the late-exponential phase of growth, fbpA promoter activity remained at a relatively constant level throughout growth and into the stationary phase (Fig. 4). However, fbpA promoter activity was consistently lower (five- to eightfold) than that of the cshA promoter. An fbpA mRNA transcript of approximately 1850 nt was visualized in Northern-blot analysis of RNA isolated from cells of S. gordonii DL1. In support of the CAT reporter data, the levels of fbpA mRNA with respect to total RNA did not alter significantly throughout growth and into the stationary phase (Fig. 5).



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 5. Northern-blot analysis of fbpA transcription. Samples containing 10 µg total RNA extracted from cells harvested at the early-exponential (lane 1), mid-exponential (lane 2), late-exponential (lane 3) and stationary (lane 4) phases of growth were separated by agarose gel electrophoresis, transferred to a nylon membrane and probed with a radiolabelled fbpA gene fragment. The positions of molecular mass markers (nucleotides, x103) are indicated at the left of the image. The fbpA transcript (approx. 1850 nt) is indicated by an arrow.

 
Effects of environmental O2 levels on flpA gene expression
Since the expression of the fibronectin-binding proteins PrtF1 (SfbI) and SOF of group A streptococci is regulated by O2 or CO2 (Okada et al., 1993 ; VanHeyningen et al., 1993 ; McLandsborough & Cleary, 1995 ), we determined whether environmental O2 levels affected cshA or fbpA transcription. The activity of the cshA promoter was apparently unaffected by cultivation of cells in static broth, under reduced O2 (anaerobic cabinet) or under aeration (Table 3). By contrast, in the p-fbpAcat fusion strain, the specific CAT activity was elevated nearly threefold in aerated cultures compared with the activity seen in anaerobic cultures (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. Effect of O2 on fbpA or cshA gene expression in cells in the mid-exponential phase of growth

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fibronectin binding by bacteria is an important property that may promote colonization of the host and contribute to disease progression. The list of fibronectin-binding proteins expressed by streptococci is increasing, and at least eight different fibronectin-binding proteins have now been identified in group A streptococci alone (Joh et al., 1999 ; Terao et al., 2001 ). In this study, we have identified FbpA as a second fibronectin-binding protein in oral S. gordonii. The fbpA gene lies immediately downstream of the cshA gene that encodes a major fibronectin-binding protein of S. gordonii (McNab et al., 1994 , 1996 ).

FbpA of S. gordonii exhibits several features that distinguish it from the other fibronectin-binding proteins of Gram-positive bacteria that have been characterized to date. FbpA does not contain sequences that closely match recognized motifs involved in binding fibronectin, suggesting that binding may be mediated by a novel mechanism. In support of this, streptococcal and staphylococcal proteins that contain consensus fibronectin-binding motifs are able to bind soluble and immobilized fibronectin, generally interacting with the N-terminal fibrin- and heparin-binding domain of fibronectin (Joh et al., 1999 ). Like CshA, FbpA binds to only the immobilized form of fibronectin and, since these two proteins show no amino acid similarity, their mechanisms of fibronectin binding may be different or may involve protein configurations that are not easily defined through linear sequence motifs.

The inactivation of fbpA was associated with a significant reduction in the fibronectin binding and cell-surface hydrophobicity of S. gordonii cells harvested at the exponential phase of growth. However, our results suggest that these phenotypic effects resulted from a reduction in the expression of CshA. Although we cannot rule out that, under some growth conditions, FbpA may mediate adhesion of S. gordonii to fibronectin, it would appear that the CshA-independent mechanism of binding of stationary phase cells is not due to FbpA. In support of this, we have not been able to conclusively demonstrate cell-surface localization of FbpA by immunogold electron microscopy using affinity-purified FbpA antibodies (data not shown). By contrast, the orthologous PavA protein of S. pneumoniae, which partly mediates pneumococcal cell adhesion to fibronectin (Holmes et al., 2001 ), has been shown to be cell-surface exposed. PavA and FbpA both appear to partition between the soluble (cytoplasmic) and envelope (containing membrane and wall-associated proteins) fractions of cells. Neither of these proteins carries conventional secretory (signal peptide) nor cell-surface anchorage sequences, so the mechanism by which PavA is secreted and becomes cell-surface-associated is currently unknown.

The ability of bacteria to sense and respond to environmental influences is integral to their survival and adaptation within the host (Finlay & Falkow, 1997 ). The level of O2 varies at host sites and bacteria respond to this. Fibronectin-binding protein PrtF1 of S. pyogenes is maximally expressed under aerobic conditions (VanHeyningen et al., 1993 ), and this is thought to enhance colonization at superficial body sites. We have also shown that O2 levels may directly regulate the expression of FbpA, but that this does not significantly impact on CshA production. The cshA promoter is apparently under complex regulatory control, since it has been demonstrated that maximal cshA promoter activity in the early-stationary phase of growth requires a functional Hpp oligopeptide transport system (McNab & Jenkinson, 1998 ) as well as the co-production of CshB (McNab & Jenkinson, 2000 ). HppA, which is an oligopeptide-binding protein (Jenkinson et al., 1996 ), is thought to be involved in sensing an extracellular signal that modulates the expression of cshA in a cell-density-dependent manner. The data presented here suggest that FbpA provides another layer of control on CshA expression during the exponential phase of growth. It is unclear how this operates at present, and we have been unable to show by gel-shift assay that purified FbpA binds to the cshA promoter (data not shown). It is possible that FbpA may act as an accessory factor involved in HppA-dependent peptide signal detection, or as an independent regulator which would allow the integration of multiple sensory signals at the level of adhesin gene transcription. In S. pyogenes, Mga positively regulates the expression of several virulence factors, including SOF and Fba, at the transcriptional level (McLandsborough & Cleary, 1995 ; Terao et al., 2001 ). The expression and activity of Mga is, in turn, affected by various environmental stimuli (Okada et al., 1993 ; Podbielski et al., 1992 ), and an additional level of control occurs through Nra, a negative regulator of Mga transcription whose expression is cell-density-dependent (Podbielski et al., 1999 ). Nra also directly regulates the expression of the fibronectin-binding protein PrtF2 (Podbielski et al., 1999 ). PrtF, another fibronectin-binding protein of group A streptococci, is positively regulated by the transcriptional factor RofA, whose expression is elevated under anaerobic conditions (Fogg et al., 1994 , 1997 ). These multiple layers of regulation controlling the expression of fibronectin-binding proteins in streptococci highlight the importance of detecting and integrating spatial and temporal information for colonization and survival within the host.

Orthologues of FbpA are present in a wide range of streptococci, in Lactococcus lactis, in Bacillus subtilis and in some Gram-negative species of bacteria. The PavA protein of S. pneumoniae is essential for pneumococcal virulence (Holmes et al., 2001 ; Lau et al., 2001 ), while a promoter trap screening system has demonstrated that an FbpA-like protein in Streptococcus suis is expressed during experimental infection of piglets (Smith et al., 2001 ). It has also been shown that mice immunized with recombinant FBP54 were protected against S. pyogenes infection (Kawabata et al., 2001 ). Thus, while the role of these FbpA-like proteins in streptococcal infections are not yet understood, they are important for bacterial growth and survival within the host and they may provide novel targets for disease intervention.


   ACKNOWLEDGEMENTS
 
We are grateful to L. Tao (College of Dentistry, University of Illinois at Chicago, USA) and A. Podbielski (Institute of Microbiology, Virology and Hygiene, University Hospital Rostock, Germany) for the provision of plasmids, and N. J. Mordan for skilled electron microscopy assistance. We also thank A. R. Holmes for helpful discussions. J.C. was in receipt of a University of Bristol Postgraduate Scholarship.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.[Medline]

Babu, J. P. & Dabbous, M. K. (1986). Interaction of salivary fibronectin with oral streptococci. J Dent Res 65, 1094-1100.[Abstract]

Babu, J. P., Simpson, W. A., Courtney, H. S. & Beachey, E. H. (1983). Interaction of human plasma fibronectin with cariogenic and non-cariogenic oral streptococci. Infect Immun 41, 162-168.[Medline]

Baddour, L. M. (1994). Virulence factors among gram-positive bacteria in experimental endocarditis. Infect Immun 62, 2143-2148.[Medline]

Church, G. M. & Gilbert, W. (1984). Genomic sequencing. Proc Natl Acad Sci USA 81, 1991-1995.[Abstract]

Courtney, H. S., Li, Y., Dale, J. B. & Hasty, D. L. (1994). Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect Immun 62, 3937-3946.[Abstract]

Courtney, H. S., Dale, J. B. & Hasty, D. L. (1996). Differential effects of the streptococcal fibronectin-binding protein, FNB54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells. Infect Immun 64, 2415-2419.[Abstract]

Douglas, C. W. I., Heath, J., Hampton, K. K. & Preston, F. E. (1993). Identity of viridans streptococci isolated from cases of infective endocarditis. J Med Microbiol 39, 179-182.[Abstract]

Dyson, C., Barnes, R. A. & Harrison, G. A. (1999). Infective endocarditis: an epidemiological review of 128 episodes. J Infect 38, 87-93.[Medline]

Ericson, D. & Tynelius-Bratthall, G. (1986). Absorption of fibronectin from human saliva by strains of oral streptococci. Scand J Dent Res 94, 377-379.[Medline]

Ferretti, J. J., McShan, W. M., Adjic, D. & 20 other authors (2001). Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98, 4658–4663.[Abstract/Free Full Text]

Finlay, B. B. & Falkow, S. (1997). Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61, 136-169.[Abstract]

Fogg, G. C. & Caparon, M. G. (1997). Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA. J Bacteriol 179, 6172-6180.[Abstract]

Fogg, G. C., Gibson, C. M. & Caparon, M. G. (1994). The identification of rofA, a positive-acting regulatory component of prtF expression: use of an m{gamma}{delta}-based shuttle mutagenesis strategy in Streptococcus pyogenes. Mol Microbiol 11, 671-684.[Medline]

Frandsen, E. V. G., Pedrazzolli, V. & Kilian, M. (1991). Ecology of viridans streptococci in the oral cavity and pharynx. Oral Microbiol Immunol 6, 129-133.[Medline]

Haisman, R. J. & Jenkinson, H. F. (1991). Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase. J Gen Microbiol 137, 483-489.[Medline]

Hanahan, D. (1985). Techniques for transformation of E. coli. In DNA Cloning , pp. 109-135. Edited by D. M. Glover. Oxford:IRL Press.

Herzberg, M. C. (1996). Platelet–streptococcal interactions in endocarditis. Crit Rev Oral Biol Med 7, 222-236.[Abstract]

Holmes, A. R., McNab, R. & Jenkinson, H. F. (1996). Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin–receptor interactions. Infect Immun 64, 4680-4685.[Abstract]

Holmes, A. R., McNab, R., Millsap, K. W., Rohde, M., Hammerschmidt, S., Mawdsley, J. L. & Jenkinson, H. F. (2001). The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol 41, 1395-1408.[Medline]

Hudson, M. C. & Stewart, G. C. (1986). Differential utilization of Staphylococcus aureus promoter sequences by Escherichia coli and Bacillus subtilis. Gene 48, 93-100.[Medline]

Jenkinson, H. F. (1987). Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation. J Gen Microbiol 133, 1909-1918.[Medline]

Jenkinson, H. F. & Easingwood, R. A. (1990). Insertional inactivation of the gene encoding a 76-kilodalton cell surface polypeptide in Streptococcus gordonii Challis has a pleiotropic effect on cell surface composition and properties. Infect Immun 58, 3689-3697.[Medline]

Jenkinson, H. F. & Lamont, R. J. (1997). Streptococcal adhesion and colonization. Crit Rev Oral Biol Med 8, 175-200.[Abstract]

Jenkinson, H. F., Terry, S. D., McNab, R. & Tannock, G. W. (1993). Inactivation of the gene encoding surface protein SspA in Streptococcus gordonii DL1 affects cell interactions with human salivary agglutinin and oral actinomyces. Infect Immun 61, 3199-3208.[Abstract]

Jenkinson, H. F., Baker, R. A. & Tannock, G. W. (1996). A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J Bacteriol 178, 68-77.[Abstract]

Joh, H. J., House-Pompeo, K., Patti, J. M., Gurusiddappa, S. & Höök, M. (1994). Fibronectin receptors from gram-positive bacteria: comparison of active sites. Biochemistry 33, 6086-6092.[Medline]

Joh, D., Wann, E. R., Kreikemeyer, B., Speziale, P. & Höök, M. (1999). Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol 18, 211-223.[Medline]

Kawabata, S., Kunitomo, E., Terao, Y., Nakagawa, I., Kikuchi, K., Totsuka, K. & Hamada, S. (2001). Systemic and mucosal immunizations with fibronectin-binding protein FBP54 induce protective immune responses against Streptococcus pyogenes challenge in mice. Infect Immun 69, 924-930.[Abstract/Free Full Text]

Kawamura, Y., Hou, X.-G., Sultana, F., Miura, H. & Ezaki, T. (1995). Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45, 406-408.[Abstract]

Kroczek, R. A. & Siebert, E. (1990). Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal Biochem 184, 90-95.[Medline]

Lau, G. W., Haataja, S., Lonetto, M., Kensit, S. E., Marra, A., Bryant, A. P., McDevitt, D., Morrison, D. A. & Holden, D. W. (2001). A functional genomic analysis of type 3 Streptococcus pneumoniae virulence. Mol Microbiol 40, 555-571.[Medline]

Lowrance, J. H., Hasty, D. L. & Simpson, W. A. (1988). Adherence of Streptococcus sanguis to conformationally specific determinants in fibronectin. Infect Immun 56, 2279-2285.[Medline]

Lowrance, J. H., Baddour, L. M. & Simpson, W. A. (1990). The role of fibronectin binding in the rat model of experimental endocarditis caused by Streptococcus sanguis. J Clin Invest 86, 7-13.[Medline]

McLandsborough, L. A. & Cleary, P. P. (1995). Insertional inactivation of virR in streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein. FEMS Microbiol Lett 128, 45-51.[Medline]

McNab, R. & Jenkinson, H. F. (1992). Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii. Mol Microbiol 6, 2939-2949.[Medline]

McNab, R. & Jenkinson, H. F. (1998). Altered adherence properties of a Streptococcus gordonii hppA (oligopeptide permease) mutant result from transcriptional effects on cshA adhesin gene expression. Microbiology 144, 127-136.[Abstract]

McNab, R. & Jenkinson, H. F. (2000). Regulation of cell surface adhesin expression in Streptococcus gordonii. In Streptococci and Streptococcal Diseases Entering the New Millennium , pp. 337-340. Edited by D. R. Martin & J. R. Tagg. Wellington, New Zealand:Securacopy.

McNab, R., Jenkinson, H. F., Loach, D. M. & Tannock, G. W. (1994). Cell-surface-associated polypeptides CshA and CshB of high molecular mass are colonization determinants in the oral bacterium Streptococcus gordonii. Mol Microbiol 14, 743-754.[Medline]

McNab, R., Holmes, A. R., Clarke, J. M., Tannock, G. W. & Jenkinson, H. F. (1996). Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin. Infect Immun 64, 4204-4210.[Abstract]

McNab, R., Forbes, H., Handley, P. S., Loach, D. M., Tannock, G. W. & Jenkinson, H. F. (1999). Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties. J Bacteriol 181, 3087-3095.[Abstract/Free Full Text]

Navarre, W. W. & Schneewind, O. (1999). Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 63, 174-229.[Abstract/Free Full Text]

Okada, N., Geist, R. T. & Caparon, M. G. (1993). Positive transcriptional control of mry regulates virulence in the group A streptococcus. Mol Microbiol 7, 893-903.[Medline]

Pakula, R. & Walczak, W. (1963). On the nature of the competence of transformable streptococci. J Gen Microbiol 31, 125-133.[Medline]

Pancholi, V. & Fischetti, V. A. (1992). A major surface protein of group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J Exp Med 176, 415-426.[Abstract]

Podbielski, A., Peterson, J. A. & Cleary, P. (1992). Surface protein–CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus pyogenes. Mol Microbiol 6, 2253-2265.[Medline]

Podbielski, A., Spellerberg, B., Woischnik, M., Pohl, B. & Lutticken, R. (1996). Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS). Gene 177, 137-147.[Medline]

Podbielski, A., Woischnik, M., Leonard, B. A. B. & Schmidt, K.-H. (1999). Characterization of nra, a global negative regulator gene in group A streptococci. Mol Microbiol 31, 1051-1064.[Medline]

Romberger, D. J. (1997). Fibronectin. Int J Biochem Cell Biol 29, 939-943.[Medline]

Sabelnikov, A. G., Greenberg, B. & Lacks, S. A. (1995). An extended -10 promoter alone directs transcription of the DpnII operon of Streptococcus pneumoniae. J Mol Biol 250, 144-155.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Shaw, W. V. (1975). Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43, 737-755.[Medline]

Smith, H. E., Buijs, H., de Vries, R., Wisselink, H. J., Stockhofe-Zurwieden, N. & Smits, M. A. (2001). Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets. Microbiology 147, 271-280.[Abstract/Free Full Text]

Tao, L., LeBlanc, D. J. & Ferretti, J. J. (1992). Novel streptococcal–integration shuttle vectors for gene cloning and inactivation. Gene 120, 105-110.[Medline]

Terao, Y., Kawabata, S., Kunitomo, E., Murakami, J., Nakagawa, I. & Hamada, S. (2001). Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator. Mol Microbiol 42, 75-86.[Medline]

van der Flier, M., Chhun, N., Wizeman, T. M., Min, J., McCarthy, J. B. & Tuomanen, E. I. (1995). Adherence of Streptococcus pneumoniae to immobilized fibronectin. Infect Immun 63, 4317-4322.[Abstract]

VanHeyningen, T., Fogg, G., Yates, D., Hanski, E. & Caparon, M. (1993). Adherence and fibronectin binding are environmentally regulated in the group A streptococci. Mol Microbiol 9, 1213-1222.[Medline]

Voskuil, M. I. & Chambliss, G. H. (1998). The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Res 26, 3584-3590.[Abstract/Free Full Text]

Whittaker, C. J., Klier, C. M. & Kolenbrander, P. E. (1996). Mechanisms of adhesion by oral bacteria. Annu Rev Microbiol 50, 513-552.[Medline]

Willcox, M. D. P., Loo, C. Y., Harty, D. W. S. & Knox, K. W. (1995). Fibronectin binding by Streptococcus milleri group strains and partial characterisation of the fibronectin receptor of Streptococcus anginosus F4. Microb Pathog 19, 129-137.[Medline]

Zetter, B. R., Daniels, T. E., Quadra-White, C. & Greenspan, J. S. (1979). LETS protein in normal and pathological human oral epithelium. J Dent Res 58, 484-488.[Abstract]

Received 25 October 2001; revised 14 February 2002; accepted 19 February 2002.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Christie, J.
Articles by Jenkinson, H. F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Christie, J.
Articles by Jenkinson, H. F.
Agricola
Articles by Christie, J.
Articles by Jenkinson, H. F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2002 Society for General Microbiology.