Genetic characterization of the spaA1 determinant of the surface protein antigen gene from Streptococcus sobrinus 6715

Inr. J. Biochem. Vol. 21, No. II, Printed in Great Britain

pp.

1245-1254,

0020-71 IX/89 $3.00 + 0.00 Pergamon Press plc

1989

GENETIC CHARACTERIZATION OF THE spaA 1 DETERMINANT OF THE SURFACE PROTEIN ANTIGEN GENE FROM STREPTOCOCCUS SOBRINUS 6715 SHIGENO

SAITO,* YOSHIMITSUABIKO, TEEUYOSHIKOYANAGI, YORIMITSUYAZAWA and HISASHI TAKIGUCHI

Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan [Tel. 0473(68) 61 II] (Received 17 April 1989) Abstract-l. The surface protein antigen-encoding gene spaA 1, previously cloned from Streptococcus sobrinus 6715 (serotype g) into the plasmid pACYC184, was examined. 2. The gene product of pYA726 was identified both by the minicell method and by in vitro transcription-translation system as a protein whose mol. wt was estimated to be 140,000. 3. The spaA 1 gene was localized on the 3.75 kb AuaI-BumHI fragment of the cloned DNA. 4. An internal spaA 1 promoter located close to one end of the 1.25 kb AuaI fragment within the 3.75 kb AuaI-BumHI fragment initiated the transcription for the insert DNA of S. sobrinus autonomously.

INTRODUCTION

Mutans Streptococci are believed to be the principal etiological agent of dental caries in humans and in experimental animals (Hamada and Slade, 1980). Streptococcus sobrinus (serotype g) produces a number of enzymes and other extracellular proteins. These proteins are dextranase, glucosyltransferase, fructosyltransferase, glucan-binding protein and surface protein antigens. Lehner et al. (1981) reported the extracellular protein antigen of S. mutans (serotype c), designated antigen I/II, which was found to possess antigenic determinants important in the induction of immunity to S. mutans-induced dental caries in rhesus monkeys. A few years ago we purified to homogeneity the surface protein antigen A (spaA protein) from S. sobrinus 6715 (serotype g) and found it to be immunologically cross reactive with antigen I/II (Holt et al., 1982). The spaA product is a major protein, comprising ca 35% of the protein on the cell surface of S. sobrinus (Curtiss et al., 1986). On the other hand, we previously described the cloning of the gene for the spaA protein from S. sobrinus 6715, using cosmid cloning and immunological screening (Holt et a[., 1982). In immunological analysis extracts from Escherichia coli cells harboring the recombinant plasmid pYA726 reacted with both antiserum to the antigen I/II protein and antiserum to the spaA protein purified from S. sobrinus 6715 (anti-spaA) (Holt et al., 1982). More recently, Holt and Ogundipe (1987) reported that S. sobrinus spaA protein contained two antigenic determinants, and E. coli strains with plasmid pYA726 specified a protein termed spaA 1 which was found to possess one of these determinants.

*To whom all correspondence

should be addressed.

Curtiss et al. (1983) and Curtiss (1985) suggested that the spaA gene of S. sobrinus was involved in sucrose-independent adherence to saliva-coated hydroxyapatite, served as a glucan-binding protein, was involved in sucrose-dependent aggregation, and was probably translationally modified to dextranase. The cloned spaA 1 determinant of the spaA protein should provide a new approach to the analysis of the structure and function of this important surface protein. The present study was thus undertaken to localize the spaA 1 determinant on the 8.46 kb BumHI insert from the S. sobrinus DNA, to determine the promoter region for the SpaA 1 determinant and to identify the antigenically reactive regions of the determinant. Construction of subclones of pYA726, measurement of protein synthesis by an in vitro transcription-translation system and in E. coli minicells, immunoprecipitation of plasmid gene products, and E. coli RNA polymerase binding were used for these purposes. MATERIALS AND

METHODS

Bacierial strains and plusmids E. coli K-12 strains HBlOl (Boyer and Roulland-Dussoix, 1969) and xl274 (F- minA1 tsx-63 purE41 I- pdxC3 minB2 recA1 meK65 rpsL97 T3’ ~~1-14 h-277 cycA 1 cycB2) (provided by Roy Curtiss III, Washington University, St Louis, MO., U.S.A.) were used as the recipient strains for transformation. The construction of pYA726 has been described previously (Holt et al., 1982). Plasmids pACYC184 (Chang and Cohen, 1978), pBR322 (Bolivar er al., 1977) and pBR328 (Soberon er al., 1980) were used as vectors for subcloning experiments.

Media Unless otherwise stated, the complex L broth medium (Lennox, 1955) and L agar plates containing 1.35% agar were used for growth of E. coli strains. M9 salts contained @r liter of distilled water): NalHPO,, 6 g; KH,PD,, 3 g; NaCl, 0.5 g; NH,Cl, 1 g; 1 M CaCl,, 0.1 ml, 1 M MgSOl,

1245

1246

SH~OEN~

!hmO

1 ml. Bacteria carrying recombinant plasmids were grown on media containing ampicillin (25 pg/ml), tetracycline (12.5 pg/ml) or chloramphenicol (25 pg/ml). Prepararion of plasmid DNA

Amplification of plasmid DNA was by chloramphenicol (170pg/ml) or by spectinomycin (3OOpg/ml) (Chang and Cohen, 1978). Plasmid DNA was isolated from 1 1. amplified cultures and purified from cleared lysates by cesium chloride-ethidium bromide equilibrium density gradient centrifugation as described previously (Holt et al., 1982). The method of Kado and Liu (1981) was used to screen the plasmid contents of large numbers of transformants. The rapid alkaline extraction procedure of Birnboim and Doly (1979) was used to isolate small amounts of DNA from 1 ml overnight cultures for restriction endonuclease digestion or transformation. Restriction endonuclease digestion of plasmid DNAs

Restriction endonculeases were obtained from commercial sources. The conditions specified by the suppliers were used for the digestion of DNA by each enzyme. Subcloning experiments

DNAs were digested with the restriction endonucleases described in Results. In some cases the vector was pretreated with calf intestinal alkaline phosphatase to remove 5’-terminal phosphates and to prevent recircularization of olasmid. The endonuclease-digested DNAs were ligated with T4 DNA ligase, and used for transformation (Maniatis et al., 1982). Gel electrophoresis of DNA For screening recombinant plasmid DNA, we routinely used 0.8% horizontal agarose gel electrophoresis using TBE buffer (Maniatis et al., 1982). For mapping restriction endonuclease sites, DNA fragments generated by restriction endonuclease digestion were analyzed on 3, 1.8, 1.2 or 0.8% agarose horizontal gels or 4% polyacrylamide vertical gels. Molecular weight standards for supercoiled plasmid DNA were pACYC184, pBR322 and the plasmids from strain V517 (Macrina er al., 1978). Molecular weight standards for linear DNA fragments generated by restriction endonuclease digestion were HindIII-digested 1~1857 DNA and HaeIII-digested (pXl74 RF1 DNA. In vitro transcription-lranslation

The coupled DNA expression system was purchased from New England Nuclear and used as described in their instructions, with 2.5 pg of DNA template for each reaction. Labeled polypeptides were resolved by SDS-PAGE followed by fluorography. Minicell method

Minicells of E. coli x 1274 or plasmid-containing x 1274 derivatives were purified by a slight modification of the procedures of Frazer and Curtiss (1975) and Clark-Curtiss and Curtiss (1983). A 11. vol of L broth with or without antibiotics was inoculated with lOpI of a cell suspension previously stored in 30% glycerol-L broth at -20°C. After incubation for 15 hr at 37”C, the minicells were purified by one differential centrifugation and three or four velocity centrifugations in discontinuous sucrose gradients. The purified minicells (1 vegetative cell/10s-106 minicells) were suspended in M9 salts supplemented with 0.5% glucose, adenine (40 g/ml), pyridoxine (2 g/ml) and 10% methionine assay medium (Difco) and incubated for 5 min at 37°C. The cells were then incubated in the presence of [“S]methionine (New England Nuclear, 1083 Ci/mmol) for 60 min. Aliquots of the labeled minicells were harvested, washed three times with cold buffered saline with gelatin (Curtiss, 1965) and then lysed by boiling for 5 min in 50 pl

et al.

of Laemmli’s sample buffer (2.3% SDS; 5% 2-mercaptoethanol; 10% glycerol; 0. I % Bromophenol Blue; 62.5 mM Tris-HCl, pH 6.8). A portion (104cpm) of each lysate was subjected to SDS-PAGE and fluorography. Immunological procedures

Anti-spaA antiserum was prepared as described previously (Holt er al., 1982). Protein, synthesized in minicells, that reacted with anti-spud antiserum was identified by a slight modification of the procedure of Oliver and Beckwith (1982). Minicells, which were radioactively labeled as described above, were harvested, washed three times with buffer A (10 mM Tris-HCl, pH 8.0), suspended in 100 ~1 of buffer B (10 mM Tri-HCl, pH 8.0; -1% SDS; 1 mM EDTA). and incubated for 60min at 37°C. The SDSsolubilized suspension was diluted with 200 ~1 of buffer C (2% Triton X-100; 50mM Tris-HCl, pH 8.0; 150mM NaCl: 1 mM EDTA). and nreabsorbed bv incubation for 10 min with 10 mg of Zysoibin (fixed and-killed Sraphylococcus aureus cells bearing protein A, Zymed Laboratories) followed by centrifugation. The resultant supematant was incubated overnight at 4°C with 10 pl of antiserum. Then, 3 mg of Zysorbin was added to the mixture, which was then incubated for 60 min at 4°C. After centrifugation, the immune complexes were washed three times by vigorous Vortex mixing in 0.6 ml of buffer D (1% Triton X-100; 50 mM Tris-HCl, pH 7.6; 1 M NaCl) and three times in buffer A. The immunoprecipitates were suspended in 50 pl of Laemmli’s sample buffer. The suspension was heated at 100°C for 5min and cooled to room temperature. After centrifugation, aliquots of the supernatant were used for SDS-PAGE and fluorography. SDS-PAGE

SDS-polyacrylamide gels were prepared essentially according to the method of Laemmli (1970). The radiolabeled samples were electrophoresed on linear 4 to 20% SDSpolyacrylamide gradient gels. After staining and destaining, the gels were soaked with a scintillator (Er?hance, New England Nuclear) and dried for fluorography. Standard proteins with molecular weights ranging from 3 1,000 to 200,000 (Bio-Rad Laboratories) were run on the same gel. RNA polymerase binding assay

This experiment was performed as described by Wishart er al. (1983) except that an S & S membrane filter (0.45 pm, Schleicher & Schiill) was used instead of a Millipore nitrocellulose filter. RESULTS Construction p YA 726

of

subclone

plasmids

derived

from

Plasmid pYA726 has been reproted to specify 1 determinant of S. sobrinus spaA protein in an 8.46 kb BamHI-generated fragment of S. sobrinus 6715 DNA inserted into the tetracycline resistance gene of pACYC184 (Holt and Ogundipe, 1987). To localize the spaA 1 determinant on the cloned fragment of S. sobrinus DNA, we attempted to reduce the size of S. sobrinus DNA cloned into pACYC184 (Fig. 1). To subclone the BamHI,-HincII, fragment of pYA726 into the vector pACYC 184, pYA726 was digested with BamHI and HincII (for the nomenclature of fragments, see the legend to Fig. 1). pACYC184 is 4.04 kb, codes for resistance to chloramphenicol and tetracycline and contains a single site for BamHI and two sites for HincII (Chang and Cohen, 1978). The BamHI site and one of the HincII

spaA

Characterization Plasmids

WV................

Inserted

of the spaA 1 gene DNA from S. sobrinus . . . . . . . . . . . . . . . . . . . . . 4

& pYA726

1 : i.

pYA74 1

:. I

1247

82

8.48

135

Pst I ; :_

1 2.23

El., +k$

2.82

i

4.27

“y;*%o I:

ii **

: :

% ;:

i

; :

pYA745

:.

2.82

;. i.

:

pYA746 pMD756 pMD759 pMD749 pMD750 pYA743

pYA740

Fig. 1. Simplified restriction map of the DNA inserts in plasmids. The construction of the plasmids is described in the text. Vector DNAs used pACYC184 for pYA726, pYA741, pYA746 and pYA743, pBR322 for pYA745, pMD749, pMD750 and pYA740, and pBR328 for pMD756 and pMD759. Restriction endonucleases: BarnHI( &SRI(E), HincII(Hc), Psr I(Pst), pVuII(Pv) and Aua I(Av). The arrow indicates the direction of transcription of gene spuA 1 (deduced from deletion analysis). Distance between sites are given in kb.

sites are in the gene coding for tetracycline, and the loss of the 1.33 kb ~~~~II-~~~HI vector fragment and the subsequent insertion of the B~~HI,-~~~~II~ fragment of S. sob&w DNA into this site inactivates the gene for tetracycline resistance. Therefore, after digestion, ligation and transformation of E. coli HBlOl, chloramphenicol-resistant transfor-

mants were primarily selected and then screened for tetracycline sensitivity. Chloramphenicol-resistant, tetracycline-sensitive transformants were screened by a quick lysis procedure for plasmid isolation by Kado and Liu as described in Materials and Methods to detect plasmids having the expected molecular size of about 6.98 kb (the 4.27 kb BarnHI,--HineIl, S. sobrims fragment of pYA726 plus the 2.71 kb long Born HI-HticII vector fragment of pACYC 184). One of these plasmids, designated pYA741, contained the desired fragment. Larger deletions were made by removing DNA fragments generated by the restriction endonucleases BarnHI, EcoRI or HincII. pYA740 was constructed by digesting pYA726 with BamHI and EcoRI and inserting the Barn HI,-Eco RI* fragment into pBR322. Thus, pYA740 includes the other end of the 8.46 kb BamHI fragment of S. sobrinus DNA in pYA726. The restriction endonuclease HincII cleaved pYA726 into five fragments with molecular sizes of 4.56, 2.89, 2.82, 1.19 and 1.04 kb (data not shown). After cleavage, ligation to HincII-cut pACYCl84 and ~~sfo~ation of HBlOl, chloramphe~col-resis~nt, tetracycline-sensitive clones were selected. The resulting plasmids pYA743 and pYA746 included the

1.19 kb H&I&-H&II, and the 2.82 kb HincII,Hi&I, S. sobrims DNA fragments, respectively. We next attempted to subclone the 5.93 kb PsrI-EcoRI, S. sobrinus DNA fragment obtained by complete digestion of pYA726 DNA with PstI and partial digestion with EcoRI into pBR322 DNA which had been completely digested with PstI and EcoRI. The recombinant plasmid pYA745 included the 2.82 kb HincII,-HincII, fragment of pYA746 and parts of the S. sobrinus cloned fragments in pYA741 and pYA743 within the 5.93 kb PstI-EcoRI, fragment. Expression of the plasmids in vitro and in minicells indicated that the 2.82 kb HincII fragment of pYA726 is useful in that it apparently carries the regulatory region of the spaA I dete~inant (see below). To localize the regulatory region and the amino terminus of the spaA 1 determinant, we attempted to construct subclones derived for the 2.82 kb H&II,-HincII, fragment of pYA726. The 1.42 kb PuuII,-PuuII, fragment of pYA726 was inserted into the PuuII site of the cloning vector pBR328, yielding plasmid pMD756. Subcloning of the individual 0.44 kb HincII,-AuaI, and 1.13 kb Aua I,-HincII, restriction fragments was achieved from pYA746 by digestion with HincII and AuaI, conversion of AuaI-generated cohesive ends to blunt ends with Sl-nuclease, followed by ligation into the PuuII site of pBR322. The resulting plasmids were designated pMD749 and pMD750, respectively. pMD759 was constructed from pMD756 by digestion with AYuI, religation, selecting for ampicillin resistance, and screening for tetracycline sensitivity.

1248

SHIGENOSAITOef al.

PolypepGdes synthesized by p YA 726 and its derivatives

To determine what polypeptides are actually encoded by the cloned spaA 1 coding DNA sequence, the expression of pYA726 and its derivatives in vitro and in minicells was studied. As shown in Fig. 2 (lane 1) and Fig. 3A (lane I), the radioactively labeled polypeptides synthesized by pYA726 in the in vitro transcription-translation and minicell systms migrated at positions in SDSpolyacrylamide gels corresponding to 140,000 and 26,000 Da. The 26,000 Da polypeptide comigrated with and is presumed to be the pACYC184-encoded chloramphenicol acetyltransferase (Fig. 2, lane 9; and Pratt et al., 1981). The effect of deletions of portions of the 8.46 kb S. sobrinus DNA in pYA726 on the sizes of polypeptides synthesized was examined next. The subclone plasmids indicated in Fig. 1 are presumably missing a part of the spaA I determinant. We examined the polypeptides specified by these subclone plasmids because we anticipated that plasmids carrying the spaA I promoter and part of the DNA sequence coding for the amino-terminus of the spaA 1 protein would make a polypeptide smaller than the intact spaA 1 protein, whereas plasmids carrying only that part of the gene coding for the carboxyl-terminus of the spaA 1 protein would not make any polypeptides. Both in vitro and in minicells (Fig. 2, lane 3; and Fig. 3A, lane 3), pYA746, which contains the 2.82 kb HincII,-HincII, S. sobrinus DNA fragment of pYA726, synthesized a polypeptide with an apparent mol. wt of 92,000 in addition to the 26,000 Da chloramphenicol acetyltransferase presumably encoded by pACYC184. The 2.82 kb HincII fragment contained in pYA746 has a coding capacity for a 103,000 Da polypeptide. The 92,000 Da polypeptide is probably derived from the 140,000 Da polypeptide specified by the S. sobrinus DNA insertion in pYA726. If so, it would contain the aminoterminal amino acids of the spaA 1 protein. Thus its size was useful for identifying the likely starting point of the spaA 1 structural gene. Subclone plasmid pYA745, which includes the 2.82 kb HincII,-HincII, fragment within the 5.93 kb Pst I-&o RI, fragment in the same orientation as in pYA726, synthesized a ca 95,000 Da polypeptide both in vitro and in minicells (Fig. 2, lane 2; and data not shown in minicells). Since the ampicillin resistance gene of the vector pBR322 had been inactivated in pYA745 by inserting the 5.93 kb PsrI-EcoRI, fragment at the PstI site, p-lactamase was not observed. The large 5.93 kb PstI-EcoRI, fragment gave only a 3000 Da larger polypeptide than that synthesized by the 2.82 kb HincII fragment of pYA746. On the other hand, no spaA l-determined protein was found in vitro for pYA741 or in minicells harboring pYA741 (Fig. 2, lane 8; and data not shown in minicells). These results show that pYA741 probably does not contain sequences to code for protein(s). For subclone plasmids pYA740 and pYA743 also, no spaA l-determined protein was found either in vitro or in minicells (data not shown). pMD756, harboring the 1.42 kb PvuII fragment

from the 2.82 kb HincII fragment of pYA726, in addition to two proteins of mol. wt of 28,000 and 31,000 coded by the ampicillin resistance gene (Sutcliffe, 1978; Sancar et al., 1979) and a 37,000 Da protein coded by the tetracycline resistance gene of pBR328 (Sancar et al., 1979), exhibited a major unique protein migrating at an approximate mol. wt of 48,000 seemed to be specified by spaA 1 determinant (Fig. 2, lane 4; and Fig. 3A, lane 5). pMD759, which had the 1.25 kb AvaI fragment from the 1.42 kb Pvu II fragment of pYA746, synthesized a polypeptide of a size similar to that of the pMD756 gene product (Fig. 2, lane 5; and Fig. 3B, lane 1). From these obserations, we inferred that the spaA 1 determinant is located between the AvaI, and BarnHI, sites, the spaA 1 promoter is apparently located close to the AvaI, site, and the gene is transcribed from this promoter towards BamHI, on the map (Fig. 1). Reaction between the spaA spaA antiserum

gene products and anti-

Synthesis of the spaA 1 protein by cloned and subcloned DNA fragments was examined by an immunoprecipitation assay using anti-spud antiserum. The 140,000 Da polypeptide synthesized by the E. coli minicells harboring plasmid pYA726 was immunoprecipitated by anti-spaA antiserum, whereas the 26,000 Da polypeptide was not immunoreactive with the antiserum (Fig. 3A, lane 2), thus demonstrating that the 140,000 Da polypeptide synthesized by pYA726 is the spaA 1 gene product. The results that the polypeptides synthesized by the subclones pYA746, pMD756 and pMD759 also were immunoprecipitated by anti-spud antiserum indicate that the 1.25 kb Ava I fragment harbored by pMD759 encodes the amino-terminal amino acids and parts of the antigenic determinants of the spaA 1 protein (Fig. 3A, lanes 4 and 6; and Fig. 3B, lane 2). RNA polymerase binding assay

To localize the spaA 1 promoter more precisely, pYA746 DNA was digested with restriction endonuclease, AvaI, and mixed with E. coli RNA polymerase, and mixture was filtered through membrane filters. The fragments bound to the RNA polymerase were eluted from filters and were electrophoresed on 1.2% agarose gels. The RNA polymerase-bound 1.25 kb AvaI fragment of pYA746 were retained on the filter (Fig. 4, lane 4). This result indicates that the 1.25 kb AvaI fragment contains an E. coli RNA polymerase binding site. DISCUSSION

The initial purpose of this study was to localize the gene for the spaA 1 determinant on cloned fragment of the S. sobrinus DNA, to establish the promoter region for the gene and to identify the antigenically reactive regions of the protein coded by the gene. The 8.46 kb BamHI fragment of S. sobrinus 6715 DNA contained in pYA726 was found to express a polypeptide with mol. wt ca 140,000 (Fig. 2, lane 1; and Fig. 3A, lane 1) which was recognized by antispaA antiserum (Fig. 3A, lane 2). Other products with

200

116 97

43

Cat

12

3

4

5

6

7

8

9

10

Fig. 2. In vitro transcription-translation of plasmid-encoded proteins. Proteins were labeled with [35S]methionine, separated on a linear 420% SDSpolyacrylamide gradient gel and fluorographed: 1, pYAl26; 2, pYAl45; 3, pYA746; 4, pMD756; 5, pMD759; 6, pMD749; 7, pMD750; 8, pYA741; 9, pACYC184; 10, no DNA. Ap and Cat indicated the migration of gene products encoded by vectors (blu and cur). The apparent masses of the various polypeptides are indicated in kDa. Arrows indicate the gene products specified by spa_4 1 gene.

1249

200

-

116

-

97

-

66

-

43

-

Tc Ap

-

Cat

-

1

1

2 Fig. 3(A)+or

3

4

legend see opposite.

1250

5

6

Tc AP

1

2

3

4

5

6

Fig. 3(B) Fig. 3. Immunoprecipitation of [35S]methionine-labeled proteins expressed in plasmid-containing minicells. Immunoprecipitation of radiolabeled minicell extracts with anti-s@ antiserum and S. aureus protein A were performed as described in the text. The precipitates obtained were analyzed by SDS-polyacrylamide gradient gel and fluorography. (A) 1, pYA726 whole minicell lysates; 2, pYA726 immunoprecipitates of minicell lysates; 3, pYA746 whole minicell lysates; 4, pYA746 immunoprecipitates of minicell lysates; 5, pYA756 whole minicell lysates; 6, pYA756 immunoprecipitates of minicell lysates. (B) 1, pYA759 whole minicell lysates; 2, pYA759 immunoprecipitates of minicell lysates; 3, pYA749 whole minicell lysates; 4, pYA749 immunoprecipitates of minicell lysates; 5, pYA750 whole minicell lysates; 6, pYA750 immunoprecipitates of minicell lysates. Arrows indicate the gene products specified by spaA I gene.

1251

1

2

3

4

Fig. 4. Binding of restriction fragments to E. coli RNA polymerase. pYA746 DNA was digested with AvaI restriction endonuclease and mixed with E. co/i RNA polymerase, and mixture was filtered through membrane filters. The fragments bound to the RNA polymerase were eluted from filters and were electrophoresed on 1.2% agarose gels. 1, complete digest of pYA746 DNA; 2, DNA fragments which passed through into the filtrate after incubation with RNA polymerase; 3, DNA fragments which were retained on membrane after incubation without RNA polymerase; 4, DNA fragments which were retained on membrane after incubation with RNA polymerase.

1252

Characterization

slightly lower molecular weights were also detected by the antiserum (Fig. 4, lane 2) and might be degradation products of the spaA 1 protein. Thus, this observation indicated that the 140,000 Da polypeptide was the major gene product of the gene for the spaA 1 determinant. Expression of the plasmids in vitro and in minicells indicated that the 2.82 kb HincII fragment of pYA726 was useful in that it apparently carried the regulatory region of the spaA 1 determinant and one end seemed to be very close to the amino terminus of the gene for the spaA 1 determinant. To learn the orientation of the 2.82 kb WincII fragment of pYA746 with respect to the vector, restriction map ping of this and adjacent fragments was examined. pYA746 DNA was digested with the AvaI, BgZI and PvuII restriction endonucleases used singly or in pairs (Fig. 1 for AvuI and PvuII sites; data not shown for BglI sites). In comparison with pYA746 and pYA726 the Hi&I fragment was found to be reversed in orientation (data not shown). pYA746 synthesized the ca 92,000 Da ~ly~ptide both in vitro and in minicells, which is probably derived from the spa=41 protein, in spite of the different orientation. This suggested that the expression of spaA 1 was independent of the orientation of the HincII fragment in the vector. Given the orientation of the HincII fragment in pYA746, there is no known promoter in pACYCl84 in close proximity to it from which a fusion polypeptide could be synthesized (Sttiber and Bujard, 1981). Therefore, the truncated 92,000 Da polyppetide could be a protein synthesized solely from the 2.82 kb H&c11 fragment. On the other hand, pYA745, which included the HincII fragment in the same orientation as that in pYA726, gave only a 3000 Da larger polypeptide than that synthesized by pYA746 (Fig. 2, lane 2). We therefore inferred that the promoter for spaA 1 is close to the Hi&I,, the gene should be read from left to right on the map, and the 3000 Da difference in size comes from the 0.19 kb distance between HincII, and Eco R&s To localize the spaA 1 promoter more precisely, pYA746 DNA was digested with AuaI and mixed with E. coli RNA polymerase. The 1.25 kb AvaI fragment of pYA746 was found to bind to E. coli RNA polymerase (Fig. 4, lane 4). Another approach to define the location of the gene for the spaA 1 dete~inant of the cloned fragment was done in an immunoprecipitation of the gene products. The 48,000 Da polypeptide synthesized in the minicells harboring pMD759 containing the 1.25 kb Auai insert was immunoprecipitated by anti-.spaA antiserum (Fig. 3B, lane 2). On the other hand, the

4.27 kb-long BamHI,-HincII, fragment harbored by pYA741 did not synthesize any spaA l-related polypeptides either in vitro or in the minicell system (Fig. 2, lane 8; and data not shown in the minicell system) nor did the 0.44 kb HincII,-AuaI, fragment of pMD749 (Fig. 2, lane 6; and Fig. 3B, lane 3) or the 1.13 kb AuuII,-HincII, fragment of pMD750 (Fig. 2, lane 7; and Fig. 3B, lane 5). The 1.19 kb HincII fragment of pYA743 and the 1.18 kb BarnHI,EcoRI, of pYA740 also did not synthesize any spuA l-related polypeptides (data not shown). These results confirm the direction of the gene for

of the spaA 1 gene

1253

the spaA determinant located on the 3.75 kb AvuI,-BumHI, fragment and that an internal spuA 1 promoter recognized by E. coli RNA polymerase seemed to be located close to the AvuI, site within the 1.25 kb AuuI fragment of the cloned DNA. The results indicated above also demonstrate that transcription initiation for the insert DNA of S. sobrinus was autonomous, since expression of the l~,~ Da ~ly~ptide was inde~ndent of o~entation with respect to any possible plasmid promoter. Although it is tempting to suggest that the identified - 10 and -35 sequences observed directly upstream of the open reading frame represent a true S. sobrinus promoter, formal proof awaits mapping of the 5’ end of the transcript from S. sobrinus to determine the precise starting point of transcription. These studies are presently under way in our laboratory. Subclone pMD759, which contained the 1.25 kb AvaI fragment, was strongly immunoreactive with anti-spaA antiserum in the immunoprecipitation assay (Fig. 3B, lane 2). This is a direct demonstration that an im~rtant component of the antigenic determinants recognized by the anti-spaA antiserum is situated within the amino-terminus of the spuA 1 protein. We have not determined whether one or more antigenic sites may exist in the carboxyt-terminal fragment of the spuA 1 gene. Because subclones with DNA specifying the carboxyl-terminus of the protein would not express any polypeptides, we cannot exclude the possibility of some antigenic determinants also situated in the carboxyl-terminal fragment of the spaA 1 gene. In future studies it will be of interest to determine precisely which portions of the spaA 1 peptide are directly involved in the antigen-antibody reaction. Further experiments with highly specific labeled antiserum may be needed to conclusively show the major antigenic determinant of spuA 1 protein. Curtiss et al. (1983) showed that the spuA protein

was necessary for sucrose-induced aggregation of this microorganism. Moreover, Douglas and Russell (1985) investigated that antigen I/II is involved in the sucrose-independent adherence to saliva-coated hydroxyapatite. However the region of polypeptide on spuA molecule expressed these biological functions are still remain to be elucidated so far. Thus recombinant polypeptides expressed by several subclones obtained in this report will be useful material for further studies of biological properties of spaA molecule. Furthermore, since pYA759 coded a 48,O~ Da antigenic determinant of spaA I, the small molecular weight-product of this clone might also permit production of substantial quantities of effective immunogen in inducing protection against dental caries. project was initiated by S. Saito in the laboratory of Professor Roy Curtiss III at the University Acknowledgements-This

of Alabama in Birmingham (U.S.A.) and the pYA plasmid series were constructed there. We thank Professor Curtiss and our colleagues in his laboratory for helpful scientific discussion and technical advice. This work was supported in parts by grants from the Ministry of Education, Science, and CuIture of Japan to S. Saito. This work was also supported by grants from Japan Foundation for Health Sciences, and from Authorized and Commissioned by the President of Nihon University.

SHIGENOSAITO et al.

1254 REFERENCES

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