Cloning and Expression of Two Different Genes from Streptococcus dysgductiue Encoding Fibronectin Receptors*

Binding of bacteria to fibronectin has been impli- cated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcue dysgalactiae to fibronectin. The cells bind to a site in the NHz-terminal domain of the protein via trypsin-sensitive cell surface compo- nents. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding pro- teins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with ‘261-labeled fibronectin, a 140-kDa fibronectin-bind-ing protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibro- nectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction map- ping and cross-hybridization experiments. The two encoded proteins were also immunologically

and bind to specific sites in the central part of the protein. A primary recognition site for the integrin receptors is defined by the amino acid sequence RGD, and synthetic peptides based on this motif inhibit the attachment of many cell types to fibronectin substrates.
Studies during the last decade have shown that also several different genera of bacteria recognize and bind fibronectin and other adhesive matrix proteins (Hook et al., 1989). However, in contrast to eukaryotic cells, little is known about bacterial receptors for fibronectin. A staphylococcal fibronectin receptor protein has been isolated (Mosher and Proctor, 1980;Espersen and Clemmensen, 1982;Froman et al., 1987), and a gene encoding a protein with fibronectin binding activity has been cloned (Flock et al., 1987) and sequenced (Signas et al., 1989). Recently described protein structures "curli," present on the surface of some Escherichia coli, also appear to bind fibronectin (Olsen et al., 1989).
Various strains of group A, C, and G streptococci have been shown to bind fibronectin (Switalski et al., 1982;Myhre and Kuusela, 1983). However, the nature of the bacterial receptor(s) involved in these interactions remains unclear. Previous studies suggest that a protein structure (or structures) located on the surface of most fibronectin-binding streptococcal cells recognizes a site in the NHz-terminal part of the fibronectin molecule (Kuusela et aL, 1984). Streptococcus dysgalactiae cells have also been reported to bind fibronectin at a site located in the COOH-terminal half of the protein. This interaction was mediated by a trypsin-insensitive surface component (Chhatwal and Blobel, 1987;Valentin-Wiegand et aL, 1988). Furthermore, lipoteichoic acid present on the surface of group A streptococci has been proposed to bind fibronectin in a hydrophobic interaction (Beachey and Simpson, 1982).
In the present study we have identified two genes coding for fibronectin-binding proteins in a bovine mastitis isolate of S. dysgalactiue. The two genes were cloned and expressed in E. coli. Recombinant proteins encoded by the cloned genes are shown to bind both intact lz61-fibronectin and its 29-kDa NH2-terminal fragment and to inhibit the binding of "'Ifibronectin to cells of S. dysgalactiae. However, under standard culture conditions only one of the genes seems to be expressed.

MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Culture Media S. dysgaluctiue strain S2 was obtained from W. Mamo (Dept. of Veterinary Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden). E. coli K12 strain TG1 (Carter et al., 1985) was used as a host for transformations with the plasmid vector pUC18 (Norrander et al., 1983). A strain of Streptomyces albus with lytic activity toward streptococci was obtained from Dr. W. Kohler (Insti-tute fur Mikrobiologie und Experimentelle Therapie, Jena, GDR) (Prauser and Kohler, 1965). The Streptococcus and Streptomyces strains were grown in Todd-Hewitt broth (Difco), and E. coli was grown in Luria broth. All cultures were grown at 37 "C. Bacteria were stored at -70 "C in their respective media supplemented with 20% glycerol.
Enzymes and Other Proteins Restriction enzymes and T4 DNA ligase were purchased from International Biotechnologies Inc. (New Haven, CT), New England Biolabs (Beverly, MA), Promega (Madison, WI), or Boehringer Mannheim and used according to the suppliers' recommendations. For exonuclease I11 digestions an Erase-a-Base system kit from Promega was used following a standard protocol from the supplier. Other methods involving DNA techniques were essentially as described (Maniatis et al., 1982).
Collagen type I1 was prepared from bovine nasal cartilage. Fetuin and bovine IgG were purchased from Sigma, and ovalbumin was from Pharmacia LKB Biotechnology Inc.
Purification and Radwlubeling of Fibronectin and Fibronectin Fragments Purification of human fibronectin from plasma was based on affinity chromatography on gelatin-Sepharose (Vuento and Vaheri, 1979;Miekka et al., 1982). The 29-kDa NHp-terminal fibronectin fragment was generated by trypsin digestion of fibronectin and purified as described by Garcia-Pardo et al. (1983). The proteins were labeled with "'iodine (Amersham Corp.) using the chloramine-T method (Hunter, 1978). The specific activity of the labeled native fibronectin used for screening of recombinants was 0.19 MBq/pg; native fibronectin and its 29-kDa NHp-terminal fragment had activities of 0.07 and 0.05 MBq/pg, respectively.

Preparation of DNA
Streptococcal cells (final concentration 2.5 X lo9 colony-forming units/ml) were incubated for 3 h in a mixture of equal volumes of 10 mM Tris-HC1 buffer (pH 7.9) containing 150 mM NaC1, 100 mM EDTA, and 1 mg/ml lysozyme, and streptolytic culture medium from S . albus. Chromosomal DNA was then prepared according to Marmur (1961). Plasmid DNA from E. coli clones was prepared using a boiling method and LiCl extraction (Monstein and Geijer, 1986).
Construction of a Genomic Library from S. dysgalactiue, Strain S2, in E. coli TG1 and Detection of Clones Expressing Fibronectin Binding Activity Chromosomal DNA from S. dysgalactiue S2 wau partially digested with Sau3Al and fractionated on a 1% agarose gel. Fractions containing DNA of 3-9 kilobase pairs (kb)' were mixed with pUC18 after the plasmid had been digested with BamHI and alkaline phosphatase. The mixture was ligated with T4 DNA ligase and used for transformation of E. coli TG1 to ampicillin resistance. Seven hundred white colonies collected from LB agar plates supplemented with 50 pg/ml ampicillin, 0.004% 5-bromo-4-chloro-3-indolyl~-D-galactopyranoside (Sigma), and 0.1 mM isopropyl 8-D-thiogalactopyranoside (Sigma) were replica plated to nitrocellulose filters. The bacteria bound to the nitrocellulose filters were lysed in chloroform vapor for 5 min and then washed 3 X 10 min in 100 mM Tris-HC1 (pH 7.5), 150 mM NaCl, and 0.05% Tween 80 (buffer A). Nonspecific protein binding sites on the filters were blocked by incubation of the filters for 2 h at 37 "C in 100 mM Tris-HC1 (pH 7.5), 150 mM NaCI, and 1% fat-free milk powder. Subsequently, '=I-labeled fibronectin was added, and the incubation was continued overnight at 22 "C. The filters were washed 3 X 10 min in buffer A. Positive clones were detected on an x-ray film (0-max, Amersham Corp.) after a 2-day exposure.
Dot-Blot Hybridization Plasmid DNA was denatured, diluted, and bound to a nitrocellulose filter. The probes were prepared from the plasmids containing streptococcal DNA by digestion with appropriate restriction enzymes (see legend to Fig. 5), gel purified, and labeled with [a-32P]dATP by a random-priming method (Multiprime DNA labeling system, Amersham Corp.). The labeled probes were allowed to hybridize with the 'The abbreviations used are: kb, kilobase pair(s); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; bp, base pairs. immobilized DNA at 42 "C overnight. Subsequently, the filters were washed with 0.1 X SSC (1 X SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) supplemented with 0.1% SDS at 65 "C and analyzed by autoradiography.
Assays for Fibronectin Binding Direct Binding Assay S. dysgalactiue S2 was grown overnight in Todd-Hewitt broth. Bacteria were collected by centrifugation and suspended in phosphate buffered saline (PBS; 140 mM NaCl, 10 mM phosphate, pH 7.4) to a density of 1 X 10" cells m1-I. 5 X 10' bacteria were incubated with 5 X 10' cpm of '251-labeled fibronectin in 0.5 ml of PBS containing 0.1% bovine serum albumin and 0.1% Tween 80 to block nonspecific binding to cells and tubes. The mixture was rotated in an end-overend mixer for 1 h at 22 "C. Incubation was stopped by the addition of 2.5 ml of ice-cold 0.1% Tween 80 in PBS, and the tubes were centrifuged at 1,400 X g for 15 min. After removal of the supernatant, the radioactivity associated with the cells was quantified in a counter (LKB Wallac, Turku, Finland). Bacteria incubated with 5 X lo' cpm of '251-labeled intact fibronectin or the 29-kDa NHp-terminal fragment bound 6,000-6,500 and 12,000-12,500 cpm, respectively. Radioactivity recovered from incubation mixtures lacking bacteria (background) was 300-500 cpm and subtracted in each test.

Inhibition Assay
Solubilized proteins were analyzed for fibronectin binding activity by measuring their ability to compete with streptococcal cells for binding of 1261-labeled fibronectin. The assay was performed in the same way as described above for the direct binding assay, with the exception that aliquots of soluble protein were added to the assay mixture.
Solubilization of Fibronectin-binding Proteins S. dysgalactiae $32 was grown at 37 "C for 18 h. The cells were collected by centrifugation and suspended in 50 mM Tris-HC1 (pH 7.6) containing 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (Merck), and 1 mM N-ethylmaleimide (Sigma) to a density of 5 X 10" cells/ml. Cell densities were estimated by comparing the absorbance of the sample with a previously prepared standard curve relating Am to the cell number determined by counting cells in a Petroff Hausser chamber. The suspension was sonicated (3 X 1 rnin), and bacterial debris was removed by centrifugation (30 min, 10,000 X g). The supernatant containing the solubilized proteins was stored at -70 "C until used.
Lysates of E. coli clones were prepared by sonication as described above or by incubating the bacteria (2.5 X lo9 cells/ml) in 50 mM Tris-HC1 buffer (pH 7.9) containing lysozyme (1 mg/ml) and 100 mM EDTA at 37 "C for 30 min. The lysates were clarified by centrifugation and stored at -70 "C until used.
Isolation and Purification of Fibronectin-binding Proteins from E. coli Clones E. coli clones containing the plasmids pSDFlOO or pSDF2OO were grown in LB medium at 37 "C for 18 h. Bacteria were collected by centrifugation and lysed by sonication as described. A four-step procedure was developed for the purification of fibronectin-binding proteins.
Step 1. Ion-exchange Chromatography After the addition of solid urea to a final concentration of 2.0 M, the lysate was applied to a column of Q-Sepharose Fast Flow (Pharmacia), equilibrated with 50 mM Tris-HC1 (pH 7.6) containing 2.0 M urea, 1 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide. The column was washed with the same buffer and eluted with an NaCl gradient (0.0-0.6 M) in the same buffer. Fractions showing fibronectin binding activity as indicated in the inhibition assay were pooled and dialyzed against water.
Step 2. Ammonium Sulfate Precipitation Fibronectin-binding components in the pooled fractions were precipitated by adding solid ammonium sulfate to 60% saturation. The precipitate was dialyzed against water before further fractionation. Step

Fast Protein Liquid Chromatography
Further purification of the fibronectin-binding proteins was ob-Fibronectin Receptors in Streptococcus dysgalactiae tained by ion-exchange chromatography using a Mono Q column, fitted to a fast protein liquid chromatography system (Pharmacia), and equilibrated with 25 mM Tris-HC1 (pH 7.6). Bound material was eluted from the column with an NaCl gradient (0.0-0.6 M). Samples containing fibronectin-binding proteins were dialyzed against water and lyophilized.

Step 4. Affinity Chromatography
Fibronectin-binding material from the Mono Q column was dissolved in PBS and applied to a column of Sepharose 4B, which was previously substituted with the 29-kDa fibronectin fragment and equilibrated with PBS. The column was washed with PBS containing 1.0 M NaCl, and proteins adsorbed to the affinity matrix were eluted with 2.0 M guanidine hydrochloride in PBS, dialyzed against water, and lyophilized.
This purification procedure resulted in a 174-and 42-fold increase in the specific activity of fibronectin-binding proteins in lysates of E. coli harboring pSDFlOO and pSDF200, respectively. However, when these proteins were examined by SDS-PAGE they appeared to have undergone extensive degradation, and the preparations consisted of several polypeptides with M, values of 15,000-140,000. Essentially all of these peptides bound '261-labeled fibronectin when analyzed in a Western blot type assay.

Production and Purification of Antibodies
The purified pSDFlOO and pSDF2OO gene products were used as antigens for the immunization of male New Zealand White rabbits. After preimmune bleeding, rabbits were immunized intramuscularly with 50 pg of either pSDF100or pSDF2OO-encoded purified protein emulsified in Freund's incomplete adjuvant. Additional injections of 50 pg were given after 10 and 20 days, with immune serum being collected on day 30.
Affinity matrices for isolation of monospecific antibody were prepared by directly passing E. coli pSDFlOO and pSDF2OO cell lysates through a column of Sepharose 4B substituted with the 29-kDa fibronectin fragment, as described in the protein purification protocol. Protein retained on this matrix was eluted and after dialysis was coupled to 2 ml of cyanogen bromide-activated Sepharose CL-4B (Pharmacia) according to the method recommended by the manufacturer. The amounts of protein used for coupling were 4 mg for pSDF2OO and 0.7 mg for pSDF100. Immune serum (10 ml) was passed twice through a column packed with the affinity matrix, previously equilibrated in 10 mM Tris-HC1 (pH 7.5). After washing with this buffer, retained antibodies were eluted by consecutively applying 100 mM glycine (pH 2.5) and 100 mM triethylamine (pH 11.5). An intermediate wash with 10 mM Tris-HC1 (pH 8.8) was included between the elutions. The eluted fractions were immediately neutralized by adding %o of the fraction volume of 1 M Tris-HC1 (pH 8.0). Prior to use for Western blotting, affinity-purified anti-pSDF100 antibody was passed through the pSDF2OO protein affinity column to adsorb antibodies recognizing common epitopes. Likewise, anti-pSDF2OO was passed through the pSDFlOO protein matrix.

SDS-PAGE, Western Blotting, and Autoradiography
For SDS-PAGE, the gel and electrophoresis buffers were those of Laemmli (1970). Resolving gels were cast with a 3-15% gradient of acrylamide. The Bio-Rad Trans-Blot apparatus was used for transfer of proteins to Immobilon-P membranes (Millipore; Bedford, MA) using the transfer buffer of Towbin et al. (1979). After transfer, the membranes were incubated in 5% (w/v) non-fat milk powder to block additional protein binding sites.
For immunoblots, the membranes were incubated overnight at 4 "C with affinity-purified antibody diluted 1,000-fold in Tris-buffered saline (20 mM Tris-HCl,500 mM NaC1, pH 7.5) containing 3% bovine serum albumin. The secondary antibody was Bio-Rad goat anti-rabbit alkaline phosphatase conjugate, used as recommended by the supplier with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as the color development substrate.
For detection of fibronectin binding activity, the blocked membranes were incubated overnight at 4 "C, under gentle agitation, in 30 ml of Tris-buffered saline containing 5 X 10' cpm of IZ6I-labeled fibronectin or 29-kDa NH2-terminal fibronectin fragment. After thorough washing, the membranes were dried at 37 "C and placed into a film cassette with Fuji RXlOO x-ray film. The film was developed after overnight exposure at 4 "C.

Demonstration of Cell Surface Localization of
Fibronectin-binding Proteins One hundred mg (wet weight) of S. dysgaluctiae 52 was suspended in 0.5 ml of PBS and digested with trypsin (Sigma, type XI11 75 pg/ ml) for 1 h at 37 "C. Trypsin was inactivated with soybean trypsin inhibitor (300 pglml), and the cells were pelleted by centrifugation. The supernatant was taken as the trypsin-released fraction while the cell pellet was suspended in SDS-PAGE reducing buffer and boiled to release remaining cell-associated proteins. Cells that had not been treated with trypsin were also boiled in reducing buffer for use as a control.
Protein solubilized from the control cells, trypsin-treated cells, and protein released from the cell surface by trypsin digestion was subjected to SDS-PAGE, transferred to Immobilon-P, and probed with affinity-purified antibody to pSDF2OO antigen. The blot was developed using the Bio-Rad amplified Immune-Blot assay kit. In aparallel experiment, trypsin-treated cells were assayed for fibronectin binding relative to control cells with 'Z61-labeled 29-kDa fibronectin fragment as ligand. When 5 x lo4 cpm of '251-labeled fibronectin are added to 5 X lo8 cells 6,000-6,500 cpm of ligand typically bind to the cells whereas only 300-500 cpm were recovered from incubation mixtures not containing bacteria. A binding site for S. dysgalactiae is located in the NH2-terminal domain of fibronectin since a 1261-labeled fragment encompassing this region also bound to the bacterial cells. Binding of 12'I-labeled fibronectin of its NH2-terminal fragment to bacterial cells was a time-dependent process. Furthermore, the bacteria could be saturated with labeled ligand, and digestion of cells with trypsin resulted in the reduction of the ability to bind fibronectin (data not shown). The bacteria were also incubated with '261-labeled intact fibronectin or the 29-kDa NHz-terminal tryptic fragment of fibronectin in the presence of increasing amounts of the corresponding cold protein or peptide. The cold 29-kDa fragment inhibited the binding of both ' "1labeled molecules to the same degree as cold intact fibronectin ( Fig. 1). A number of unrelated proteins were tested for their ability to interfere with the binding of '261-labeled fibronectin to bacteria. At the concentrations tested, these proteins, which included collagen, ovalbumin, fetuin, and nonimmune IgG, did not interfere with the bacterial binding of lZ5I-labeled intact fibronectin or its NH2-terminal fragment (Table I). Taken together these results indicate that a protein (or proteins) on the surface of s. dysgalactiae acts as a specific fibronectin receptor(s) recognizing the NH2-terminal domain in the protein.

Characterization of '25Z-Labeled Fibronectin
Identification of Fibronectin-binding Protein($ in S. dysgalactim Strain S2-A lysate of S. dysgalactiae S2 prepared by sonicating the bacterial cells in a buffer containing protease inhibitors was found t o inhibit the binding of '251-labeled intact fibronectin or the 29-kDa fibronectin fragment in a concentration-dependent manner (Fig. 2). When the lysate was passed over a column of fibronectin-Sepharose, the inhibitory activity was removed. On the other hand, when the lysate was passed through a column of albumin-Sepharose, the activity was only marginally reduced (data not shown). These results suggest that the inhibitory activity of the lysate was caused by the presence of fibronectin-binding proteins.
The lysate was fractionated by SDS-PAGE, transferred to an Immobilon-P filter, and probed with '261-labeled intact fibronectin or the 29-kDa NH2-terminal fragment. Autoradiographic analyses revealed that a major fibronectin-binding component reacting with the 12'I-labeled 29-kDa fragment had migrated in the electrophoresis as a protein of 140 kDa The inhibition is expressed as percentage of binding. Labeled ligand bound in the absence of unlabeled fibronectin was set to 100% (equivalent to 0% inhibition). Radioactivity recovered in the absence of bacterial cells was considered background. (Fig. 3). This component reacted weakly or not at all when the filter was incubated with intact lZ5I-fibronectin, which might reflect a difference in affinity for the two ligand forms. Additional fibronectin-binding polypeptides of smaller size were also present. These might represent degradation products or different gene products.
The growth medium of strain S2 was tested for the presence of extracellular fibronectin-binding proteins by measuring the inhibitory activity of the culture medium on the binding of '261-labeled fibronectin to cells of S2. No inhibitory activity was detected.

Construction of a Genomic Library from S. dysgalactiae Strain S2 in E. coli TGl and Detection of Clones Expressing
Fibronectin Binding Activity-A genomic library from S. dysgalactiae S2 in E. coli TG1 was prepared as described under "Materials and Methods." Three positive clones, with recombinant plasmids called pSDF100, pSDF200, and pSDF300, were identified. Since the restriction cleavage patterns of the inserts in two of the recombinant plasmids, pSDFlOO and pSDF300, were partially overlapping, only pSDFlOO (with an insert of 4.9 kb), encoding the highest fibronectin binding activity of the two, and pSDF2OO (with an insert of 6.9 kb) were studied further. Fig. 4, a and b, shows the completely different restriction maps of the inserts in pSDFlOO and pSDF2OO.

TABLE I Inhibition of the binding of '251-lubeled fibronectin or the 29-kDa
NHz-terminal fibronectin fragment to S. dysgalactiae S2 by different amounts of unlabeled proteins Bacteria (5 X 10' ) were incubated with 5 X 10' cpm of 1251-labeled fibronectin or 1251-labeled 29-kDa fibronectin fragment in the absence or presence of unlabeled proteins. Values represent means & standard errors of three seDarate exDeriments. To localize the domains in the inserts of pSDFlOO and pSDFZOO which encode the fibronectin binding activity, subcloning was performed in pUC18 using restriction sites of the inserts (Fig. 4, a and b). Cleavage of the insert in pSDFlOO with XbaI generated a 1.4-kb fragment, which, after ligation into pUC18 (pSDFlOZ), coded for a protein with fibronectin binding activity. Digestion of the insert in pSDF1OZ with exonuclease I11 for different time periods resulted in a number of subclones with inserts of decreasing size. The corresponding proteins retained the ability to bind fibronectin until only approximately 500 bp remained of the pSDF1OZ insert. A subjected to SDS-polyacrylamide gradient gel electrophoresis (3-15%), transferred to Immobilon-P, and probed with '"I-fibronectin ( l a n e 1 ) or '"1-29 kDa fragment of fibronectin ( l a n e 2). The migration position of prestained molecular mass standards (Bethesda Research Laboratories) is indicated. The molecular masses of the protein standards, in daltons are: myosin heavy chain, 200,000; phosphorylase 6 , 97,400; bovine serum albumin, 68,000; ovalbumin, 43,000; chymotrypsinogen, 25,700; @-lactoglobulin, 18,400; and lysozyme 14,300. The ratio of the different reactive proteins varied from one experiment to another, which possibly was the consequence of varying cleavage of a native protein by proteolytic enzymes present in the lysate.
further shortening of the insert resulted in a loss of fibronectin binding by the encoded protein. The negative test results for subclones pSDNlO2 and pSDN103 concerning the ability to bind fibronectin are not conclusive since one cannot exclude the possibility that the inserts are in the wrong reading frame or are inserted in the wrong orientation. However, the results locate at least one domain encoding fibronectin binding activity to the insert in pSDF102.
By using a similar strategy the region in pSDF2OO coding for fibronectin binding activity was localized to a 1-kb XhoI-HindIII fragment (pSDF203) in the 3'-end of the insert. The HindIII site is within the multilinker. Digestion of the insert in pSDF203 with exonuclease I11 gave a series of subclones with inserts of decreasing size. Subclone pSDF203c3 with an insert of approximately 750 bp coded for a protein with fibronectin binding activity. Subclones pSDF203c6 and pSDF203c8 with inserts of 600 and 500 bp, respectively, coded for proteins with reduced fibronectin binding activities. Subclones with shorter inserts coded for inactive proteins.
To investigate possible relationship between the inserts in pSDFlOO and pSDF200, DNA fragments from the domains in the respective inserts, which code for fibronectin binding activity, were selected as probes to be used in cross-hybridization experiments. A 0.6-kb XbaI-BstXI fragment, originating from pSDF100, present in pSDFlO2 and the XhoI-Hind111 fragment, subcloned in pSDF203, were used as probes (marked as bold lines in Fig. 4, a and b) in hybridizations to decreasing amounts of plasmid DNA from pSDF100, pSDF200, pUC18, and chromosomal DNA from S. dysgaluctiae S2 on a nitrocellulose filter (Fig. 5). The probe from pSDFlO2 gave strong signals when hybridized to pSDF100. A very weak signal was recorded when the probe from pSDF102 was hybridized to pSDF200, and no signal was obtained in hybridizations to pUC18. Analyses using the probe from pSDF202 gave a strong signal with pSDF200 and no signals with pSDFlOO and pUC18. These data support the results obtained by restriction analyses (Fig. 4, a and b), indicating that the streptococcal DNA inserts in pSDFlOO and pSDF2OO originate from two different genes. Both probes also hybridized to chromosomal DNA from strain S2 verifying that the two genes were derived from this strain.
Characterization of the Proteins Encoded by pSDFl00 and pSDF200"Lysates of E. coli TG1 clones containing pSDFlOO and pSDF200, respectively, were found to inhibit the binding of '2SI-labeled intact fibronectin or the NH2-terminal29-kDa fibronectin fragment to S2 cells in a concentration-dependent  (Fig. 5). The fibronectin binding activities of lysates from the different subclones, expressed as percentage inhibition of binding of labeled fibronectin to S. dysgalactiae S2, are indicated. More than 85% inhibition, +; less than 15% inhibition, -. C, deletions made from the 3' end of the insert in pSDFlO2. Panel b, restriction map and subclones of the insert in pSDF2OO. A, restriction map of the insert in pSDF2OO containing a 6.9-kb insert from S. dysgalactiae S2 in pUC18. B, various subclones constructed in order to determine the regions of the genes coding for the fibronectin binding activity. The bold line in pSDF203 indicates the probe used in dot-blot hybridization (Fig. 5). The fibronectin binding activities of lysates from the different subclones, expressed as percentage inhibition of binding of labeled fibronectin to S. dysgalactiae S2, are indicated. More than 85% inhibition, +; between 40 and 30% inhibition (+); less than 15% inhibition, -. C, deletions made from the 3' end of the insert in pSDF203.  a pSDFlO2 (lanes A , B, C, and D) fragment or XhoI-Hind111 (present within the multilinker of the vector) fragment, 1,000 bp, from pSDF203 (lanes E, F, G, and H).
The probes were selected to cover the domains coding for fibronectin binding activity (see legend to Fig. 4). The amounts of plasmid DNA bound to the filter are indicated. or heat-killed (filled symbols) E. coli TG1 clones with pSDFlOO (circles) or pSDF2OO (sqwres). As a negative control, lysate from cells of E. coli TG1 with pUC18 vector (triangles) was used. Data are expressed as percentage of the control, where binding to bacteria incubated in the absence of potential inhibitors was set as 0%. fashion (Fig. 6). Heat treatment of the E. coli clones before preparation of the lysates had no effect on the inhibitory capacity. Both lysates could completely inhibit binding of the ligands to streptococcal cells although a somewhat higher concentration of pSDFlOO lysate protein was required to obtain a level of inhibition comparable to pSDFZOO. This result suggests that the lysate from the pSDF100-containing clone has a lower concentration of active components, caused either by less efficient expression or by a greater sensitivity to proteolysis. Alternatively, the difference in specific activity of the two lysate preparations could reflect a difference in affinity by the corresponding receptor proteins for fibronectin. However, since complete inhibition was obtained by either lysate, the two gene products appear to bind to similar domain(s) in the fibronectin molecule. A lysate prepared from E. coli TG1 containing pUC18 had no inhibitory activity on the binding of '251-fibronectin to the S2 cells (Fig. 6).
The presence of fibronectin-binding components was demonstrated further in experiments in which proteins in the two lysates were fractionated by SDS-PAGE, transferred to Immobilon-P, and probed with '2sI-labeled intact fibronectin or the 29-kDa NH2-terminal fragment (Fig. 7). For both pSDF100-and pSDF2OO-encoded proteins there was considerable heterogeneity in the size of the polypeptides which bound the two ligands. However, all bands that bound '251-fibronectin also bound 1251-labeled 29-kDa fragments.
In the E. coli pSDF2OO cell lysate, the largest polypeptide with binding activity was 140 kDa, and this was true also for the S. dysgalactiue S2 cell lysate. For E. coli pSDF100, a fibronectin-binding polypeptide with an apparent M, of 140,000, was also observed. However, a strong signal was also obtained from a polypeptide of slightly higher molecular weight. When a lysate from E. coli containing pUC18 was analyzed in a similar fashion, no fibronectin-binding components were detected (Fig. 7).
Similar profiles were revealed on Western blots probed with monospecific antibodies raised against the isolated recombinant fibronectin-binding proteins from clones containing pSDF2OO and pSDF100, respectively (Fig. 8). Antibodies against the pSDF2OO gene product identified a protein of 140 kDa, and several smaller, presumably degradation products, in lysates of both E. coli clone pSDF2OO and S. dysgaluctiue S2 but did not react with polypeptides from clones containing pSDFlOO or pUC18 (Fig. 8). Antibodies to fibronectin-binding proteins encoded by pSDFlOO only gave a detectable signal with material from the pSDF100-containing clone revealing a polypeptide of molecular mass somewhat larger than 140 kDa. The lysate from S. dysgaluctiue S2 did not contain any protein that could be detected with the antibody against the pSDF100-encoded fibronectin-binding protein, suggesting that this protein was not expressed in the cells in appreciable amounts. Furthermore, components in lysates of E. coli clones containing pSDF2OO or pUC18 did not react with the pSDF100-encoded protein.  Evidence for surface localization of the pSDF2OO-encoded protein of S. dysgalactiae was obtained from an experiment in which cells were treated with trypsin followed by Western immunoblotting. Control extracts of boiled cells showed the presence of 140-kDa pSDF2OO antigen as in Fig. 8B, and there was no evidence of this intact antigen in extracts from cells pretreated with trypsin. In the supernatant from the trypsintreated cells three antigenic polypeptides with molecular masses of 76,58, and 39 kDa were apparent (data not shown). This is in agreement with data from binding assays in which the trypsin-treated cells showed a 60% reduction in fibronectin binding ability relative to control cells.

Fibronectin Receptors in Streptococcus dysgalactiae
Our results indicate that S. dysgalactiae S2 contains at least two separate genes encoding immunologically distinct fibronectin-binding proteins, both of which bind the 29-kDa NH2terminal domain of fibronectin. However, it appears that under the culture conditions employed, only the gene cloned in pSDF2OO is expressed on the cell surface in detectable quantities by strain S2.

DISCUSSION
S. dysgalactiae belongs to group C streptococci and is an important pathogen for cattle. The strain S2 examined in this study was isolated from a case of bovine mastitis. A previous study (Mamo et al., 1987) has indicated that this strain expresses fibronectin-binding cell surface receptors which are at least partly of proteinaceous nature since they are susceptible to treatment of cells with proteolytic enzymes. In the present paper we report on the identification of two separate genes in S. dysgalactiae S2 coding for fibronectin-binding proteins. The genes were characterized by restriction mapping (Fig. 4, a and b), and regions of the genes encoding fibronectin binding activities were localized by subcloning in pUC18. From these regions it was possible to select restriction fragments to be used as DNA probes, which in hybridization experiments specifically recognized the respective gene (Fig.  5).
Our data clearly establish the presence of cell surface protein components that mediate receptor-ligand interactions. An antibody raised against one of the expressed proteins (encoded by pSDF2OO) reacted with several polypeptides when a lysate of S. dysgalactiae S2 was analyzed by Western blot (Fig. 8). Polypeptides of similar molecular weight also bound fibronectin when the transferred peptides were probed with 12sI-labeled ligand (Fig. 7). Furthermore, when whole cells were incubated with trypsin there was a 60% reduction in fibronectin binding, which could be correlated with the loss of a 140-kDa antigen in Western immunoblots and the concomitant appearance of discrete antigenic polypeptides in the cell-free supernatant. Additional data in support of a cell surface localization come from preliminary nucleotide sequence analysis. The pSDF100-and pSDF2OO-encoded proteins were found to possess typical signal sequences for protein secretion and COOH-terminal sequences similar to those observed in several other Gram-positive cell wall proteins. This may account for the failure to obtain total loss of fibronectin binding by treatment of cells with trypsin. The fibronectin binding domain may be close to the COOH terminus, adjacent to the membrane anchor and cell wall spanning sequences as with Staphylococcus aureus fibronectinbinding protein (Signas et al., 1989). Polypeptides retaining binding activity could therefore remain firmly associated to the cell wall. It is likely that the many fibronectin-binding and immunoreactive polypeptides in S. dysgalactiae S2 (Figs. 3 and 8) and E. coli pSDF2OO (Figs. 7 and 8) represent degradation products of a high molecular weight native receptor, possibly with an M , of 140,000, as this is the size of the largest polypeptide identified both in immunoblots and blots probed with '251-labeled ligand. The second fibronectinbinding protein, encoded by pSDF100, is not produced at detectable levels in S. dysgalactiae S2 under the conditions of our experiments (Fig. 8). This observation raises the possibility that external factors may regulate the expression of the two fibronectin receptor genes in S. dysgalactiae which will be examined in future studies.
Previous studies on the binding site in fibronectin for S. dysgalactiae have resulted in apparently different conclusions. Mamo et al. (1987) found that strain S2 bound to a site in the NHz-terminal region of fibronectin whereas Chhatwal et al. (1987) reported that a different S. dysgalactiae strain, also isolated from bovine mastitis, interacts with an internal site close to the RGD-containing sequence recognized by eukaryotic receptors of the integrin family. Our data confirm the previous results that strain S2 primarily binds to the NHZterminal domain of fibronectin. Purified unlabeled 29-kDa fragment effectively inhibits the binding of '251-labeled fibronectin to streptococcal cells (Fig. 1). The fibronectin receptor encoded by pSDF2OO binds '261-labeled 29-kDa fragment of fibronectin, as does the released material from strain S2 (Figs. 7 and 3). Furthermore, it inhibits the binding of the 29-kDa fragment and intact fibronectin to streptococcal cells (Fig. 6). In view of these considerations, it is tempting to suggest that the protein encoded by the recombinant plasmid pSDF2OO represents the major fibronectin receptor of S. dysgalactiae S2. It is possible that the strain studied by Chhatwal et al. (1987) expresses a different receptor with different binding specificity. However, this hypothetical receptor is not the pSDFlOO product, which also recognizes the NHz-terminal domain of fibronectin. Hence, the streptococcal fibronectinbinding proteins encoded by pSDFlOO and pSDF200 have a ligand specificity similar to that of the staphylococcal fibronectin receptor characterized previously (Flock et al., 1987;Froman et al., 1987). Studies are currently in progress to characterize and compare further the streptococcal and staphylococcal fibronectin-receptor proteins, their active sites, and the binding sites in fibronectin.