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Streptococcus gordonii is a prominent colonizer of dental plaque, a microbial biofilm that is strongly associated with the development of caries and periodontal diseases. S. gordonii exhibits a wide range of adherence properties that are well characterized at the molecular level (8). Ssp cell surface proteins are multifunctional adhesins that participate in binding reactions with salivary agglutinins and with other plaque bacteria such as Porphyromonas gingivalis, an aggressive periodontal pathogen (1, 2, 7). Such interbacterial adhesive interactions are important in the development of plaque and in its transition from a benign accumulation to a potentially pathogenic entity.

In strains of S. gordonii thus far examined, tandem genes encode SspA and SspB polypeptides that are highly similar with respect to structure and adhesive function (2, 6). However, the sspA and sspB genes possess individual promoter regions and are differentially regulated in response to environmental conditions (4). Further, as measured by promoter-cat reporter constructs, transcriptional activity of the sspA promoter is about threefold higher than that of the sspB promoter over a range of growth conditions (4). SspA and SspB may thus have distinct roles to play for the organism. In this study we investigated regulation of the sspB gene by the sspA gene product.

SspA binds to the sspB promoter region. To determine whether the SspA polypeptide can bind to the sspB upstream region, a gel mobility shift assay was performed using the BandShift kit (Amersham Pharmacia Biotech). Recombinant SspA protein was expressed from the sspA gene and regulatory sequences (2) in Escherichia coli DH5 cultured in Luria-Bertani broth. Crude periplasmic preparations were generated by osmotic shock (5). The SspA polypeptide was further purified by Sepharose 6B (Pharmacia) and DEAE-Sephadex (Sigma) chromatography. Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a single band of SspA protein was detected following silver staining. The region 347 to 17 bp from the sspB translational start site (3) was amplified by PCR and cloned into pCRII (TOPO vector; Invitrogen). A 364-bp double-stranded EcoRI fragment containing the sspB promoter region was used for gel shift analysis. DNA (5 ng) was 5' labeled with -³²P and purified on an Atlas Nucleospin column (Clontech). Binding reactions of SspA (4 µg of protein) and target DNA were carried out in binding buffer (40 mM Tris-HCl [pH 7.5], 200 mM NaCl, 2 mM dithiothreitol, 50% glycerol, 1% Nonidet P-40) and mixed with a synthetic competitor poly(dI-dC) in a total of 20 µl and then incubated at room temperature for 20 min. Samples were analyzed on a 5% polyacrylamide gel (run in low-ionic-strength gel buffer: 70 mM Tris-HCl [pH 7.5], 30 mM sodium acetate, 10 mM EDTA) for 90 min at 10 V/cm at 4°C. As shown in Fig. 1, purified SspA protein was capable of binding to and retarding the mobility of the sspB upstream region. SspA-DNA complex formation was not affected by E. coli DNA (0.1 mg/ml), indicating specificity of binding (Fig. 1, lane 4). In contrast, the presence of excess unlabeled target DNA abolished the SspA-mediated gel shift (Fig. 1, lane 3). Additional evidence of specificity was provided by the failure of an E. coli extract containing an irrelevant protein (EBNA-1) to shift the target DNA (Fig. 1, lane 5).

View larger version (105K): [in this window] [in a new window]   FIG. 1.   Electrophoretic mobility shift analysis of binding of SspA to the sspB upstream promoter-containing region. A 364-bp 32P-labeled sspB upstream fragment was used as the probe. Reaction mixtures contained probe alone (lane 1), probe with SspA protein (lane 2), probe with SspA and excess unlabeled probe (lane 3), probe with SspA protein and excess E. coli DNA (lane 4), or probe with sonicated E. coli extract containing cloned DNA binding domain from EBNA-1 (lane 5).

SspA binding protects a 32-bp region of the sspB promoter. To identify the SspA binding site upstream of the sspB gene, DNase I footprinting was performed. As preliminary results indicated that the protected region was at the 5' end of the region used for gel shift analysis, a 5' extended target was used for footprinting. A 279-bp upstream EcoRI-XmaI sspB DNA fragment (418 to 139 from the translational start site) was amplified by PCR and mixed with purified SspA protein in binding buffer (described above) containing 10 mM MgCl₂. Digestion was initiated by the addition of 2 U of DNase I (Amersham Pharmacia Biotech). The reactions were stopped by the addition of diluted DNase stop solution (768 mM sodium acetate, 128 mM EDTA, 0.56% sodium dodecyl sulfate, 256 µg of yeast RNA/ml) (Amersham Pharmacia Biotech), and the mixtures were extracted with an equal volume of phenol-chloroform (1:1). The nucleic acids were then precipitated, dried, resuspended in loading buffer (deionized formamide containing 10 mM EDTA, 0.3% bromophenol blue, and 0.3% xylene cyanol), and electrophoresed in 8% polyacrylamide gels containing 7 M urea. The Maxam and Gilbert A+G sequencing ladder (9) was used as a size standard for the DNase I protection experiments. The results revealed that a 32-bp region of the sspB upstream sequence is fully protected from DNase I digestion in the presence of SspA (Fig. 2). This protected sequence spans 342 to 311 bp from the translational start site of the sspB promoter and is 233 to 264 bp upstream of the predicted 35 promoter element.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   (A) DNase I footprint of protection by SspA of a 279-bp sspB upstream region. Lane 1, Maxam and Gilbert A+G ladder; lanes 2 to 5, DNase I treatment in the presence of 0, 1, 2, and 4 µg of SspA, respectively. The 32-bp protected sequence is indicated by a bracket. (B) DNA sequence upstream of sspB. The 32-bp region protected from DNase I digestion is underlined.

Disruption of the sspA gene affects sspB expression. To determine whether the binding of SspA to the sspB upstream region influences transcription of sspB in S. gordonii, an sspB promoter-cat86 reporter fusion was introduced into the genome of an SspA-deficient strain of S. gordonii. S. gordonii strains were cultured aerobically in Trypticase peptone broth supplemented with yeast extract (5 mg/ml) and 0.5% glucose at 37°C. S. gordonii OB220 (kindly provided by Howard Jenkinson, University of Bristol) is derived from strain DL1 and contains an insertional inactivation of the sspA gene (2). Recombinant strain HA00 was described previously (4) and is a derivative of DL1 containing a chromosomal sspB promoter-cat reporter fusion. Strain HA02 was constructed by a strategy similar to that for HA00 and is a derivative of OB220 (SspA deficient) containing a chromosomal sspB promoter-cat reporter fusion. Briefly, suicide plasmid pHA145 (4), which encodes tetracycline resistance and contains a 1.1-kb BamHI fragment with the S. gordonii sspB promoter-cat fusion, was isolated by Wizard Miniprep (Promega) and integrated into the chromosome of OB220. Transformants resulting from a single (Campbell) crossover were selected on brain heart infusion agar containing tetracycline (10 µg/ml).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Growth phase and expression of sspB in an SspA+ (strain HA00) or SspA (strain HA02) background. Optical densities (O.D.) and CAT activities were measured at different incubation times. The data are from a representative of three experiments.