Mutational analysis of the adcCBA genes in Streptococcus gordonii biofilm formation.

Streptococcus gordonii, a primary colonizer, is part of the pioneer biofilm consortium that initiates dental plaque development on tooth surfaces. An insertion of Tn917-lac transposon into the adcR gene produced a biofilm-defective phenotype. S. gordonii adcR is a regulatory gene and is part of an operon (adc) that includes three other genes, adcCBA. AdcC contains a putative consensus-binding site for adenosine triphosphate, AdcB is a putative hydrophobic membrane protein, and AdcA is a putative lipoprotein permease. Mutants were constructed by insertional inactivation in each of the three adcCBA genes and their effects on biofilm formation examined. The adcC::spec(R) and adcB::spec(R) mutations displayed a biofilm-defective phenotype, whereas the adcA::spec(R) mutant was biofilm-positive in a static polystyrene microtiter plate biofilm assay. All three mutants formed poor biofilms in a flow-cell system and were competence-defective, suggesting the adc operon plays an important role in S. gordonii biofilm formation and competence.

Role of a nosX homolog in Streptococcus gordonii in aerobic growth and biofilm formation.

Oral streptococci such as Streptococcus gordonii are facultative anaerobes that initiate biofilm formation on tooth surfaces. An isolated S. gordonii::Tn917-lac biofilm-defective mutant contained a transposon insertion in an open reading frame (ORF) encoding a homolog of NosX of Ralstonia eutropha, a putative maturation factor of nitrous oxide reductase. Located downstream are two genes, qor1 and qor2, predicted to encode two putative NADPH quinone oxidoreductases. These three genes are cotranscribed, forming a putative oxidative stress response (osr) operon in S. gordonii. Inactivation of nosX, qor1, or qor2 resulted in biofilm-defective phenotypes. Expression of nosX, measured by the beta-galactosidase activity of the nosX::Tn917-lac mutant, was growth-phase dependent and enhanced when grown under aerobic conditions or in the presence of paraquat. Real-time reverse transcription-PCR revealed that nosX-specific mRNA levels were increased approximately 8.4 and 3.5 fold in biofilm-derived cells grown on plastic and glass, respectively, when compared to planktonic cells. Expression of nosX increased 19.9 fold in cells grown under aerated aerobic conditions and 4.7 fold in cells grown under static aerobic conditions. Two ORFs immediately adjacent to the osr operon encode a putative NADH oxidase (Nox) and a putative thiol-specific antioxidant enzyme (AhpC, for alkyl hydroperoxide peroxidase C). Expression of nox and ahpC was also significantly increased in cells grown under aerated and static aerobic conditions when compared to anaerobic conditions. In addition, nox expression was increased in biofilm cells compared to planktonic cells. These genes may be part of an island that deals with oxidoreductive response, some of which may be important in S. gordonii biofilm formation.

Role of a Streptococcus gordonii copper-transport operon, copYAZ, in biofilm detachment.

Streptococcus gordonii is a pioneer oral bacterium that is associated with the initiation of dental plaque development. Located downstream of the S. gordonii adc operon, which is involved in competence and biofilm formation, were three open reading frames, designated copY, copA and copZ. These open reading frames were homologous to the copYAZ genes in Streptococcus mutans that are involved in copper homeostasis and biofilm detachment. This study examined whether copYAZ genes play any role in the biofilm formation and detachment of S. gordonii. The copY gene encodes a 143-amino acid protein homologous to the negative transcriptional regulator of a copper-transport operon, copA encodes a 748-amino acid copper-transporting P-type ATPase, and copZ encodes a 69-amino acid putative metallochaperone protein in S. mutans. Each open reading frame in the copYAZ operon in S. gordonii was inactivated by insertional mutation and the growth, biofilm formation and detachment of each mutant were examined. S. gordonii copY::specR, copA::specR, and copZ::specR mutants were able to form biofilms on both polystyrene and glass surfaces. However, inactivation of copZ and to a lesser extent copY resulted in phenotypes that were defective in biofilm detachment, which is consistent with previous observations in S. mutans and suggests that the trace element copper might influence biofilm detachment of bacterial biofilms.

Involvement of an inducible fructose phosphotransferase operon in Streptococcus gordonii biofilm formation.

Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3' end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation. Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm. Expression of fruK, as measured by the beta-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in beta-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms.

Cloning and expression of alpha-D-glucosidase and N-acetyl-beta-glucosaminidase from the periodontal pathogen, Tannerella forsythensis (Bacteroides forsythus).

Screening a genomic library of Tannerella forsythensis (Bacteroides forsythus), using synthetic substrates conjugated to a fluorogen, 4-methylumbelliferone identified two glycosidase genes, which encode alpha-D-glucosidase and N-acetyl-beta-D-glucosaminidase, respectively. The alpha-D-glucosidase has a Mr of 81,141 and is homologous to an alpha-D-glucosidase from Bacteroides thetaiotaomicron. The N-acetyl-beta-D-glucosaminidase has a Mr of 87,787 and is homologous to an N-acetyl-beta-D-glucosaminidase in Porphyromonas gingivalis W83.

Involvement of the adc operon and manganese homeostasis in Streptococcus gordonii biofilm formation.

Pioneer oral bacteria, including Streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues. An S. gordonii::Tn917-lac biofilm-defective mutant was isolated by using an in vitro biofilm formation assay. Subsequent inverse PCR and sequence analyses identified the transposon insertion to be near the 3' end of an open reading frame (ORF) encoding a protein homologous to a Streptococcus pneumoniae repressor, AdcR. The S. gordonii adc operon, consisting of the four ORFs adcR, adcC, adcB, and adcA, is homologous to the adc operon of S. pneumoniae, which plays a role in zinc and/or manganese transport and genetic competence in S. pneumoniae. AdcR is a metal-dependent repressor protein containing a putative metal-binding site, AdcC contains a consensus-binding site for ATP, AdcB is a hydrophobic protein with seven hydrophobic membrane-spanning regions, and AdcA is a lipoprotein permease with a putative metal-binding site. The three proteins (AdcC through -A) are similar to those of the binding-lipoprotein-dependent transport system of gram-positive bacteria. Reverse transcriptase PCR confirmed that adcRCBA are cotranscribed as an operon in S. gordonii and that the transposon insertion in S. gordonii adcR::Tn917-lac had resulted in a polar mutation. Expression of adcR, measured by the beta-galactosidase activity of the adcR::Tn917-lac mutant, was growth phase dependent and increased when the mutant was grown in media with high levels of manganese (>1 mM) and to a lesser extent in media with zinc, indicating that AdcR may be a regulator at high levels of extracellular manganese. A nonpolar inactivation of adcR generated by allelic replacement resulted in a biofilm- and competence-defective phenotype. The biofilm-defective phenotype observed suggests that AdcR is an active repressor when synthesized and acts at a distant site(s) on the chromosome. Thus, the adc operon is involved in manganese acquisition in S. gordonii and manganese homeostasis and appears to modulate sessile growth in this bacterium.

Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein.

Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000-300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-beta 1-4-N-acetylglucosamine, galactose and galactose-beta 1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African-American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface.

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes.

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.

Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin.

It has been shown that Fusobacterium nucleatum PK1594 coaggregates with Prophyromonas gingivalis PK1924 through a galactose-binding adhesin. In the present study, attachment of F. nucleatum PK1594 to a variety of mammalian cells was characterized. F. nucleatum PK1594 attached to all eukaryotic cells tested, including human buccal epithelial cells, gingival and periodontal ligament fibroblasts, HeLa cells and murine lymphocytes, macrophages, and polymorphonuclear leukocytes. These attachments were (i) inhibited by galactose, lactose and N-acetylgalactosamine and (ii) inhibited by monoclonal antibody specific for the galactose-binding adhesin of F. nucleatum PK1594. In addition, a coaggregation-defective mutant of F. nucleatum PK1594 (PK2172), which does not exhibit galactose binding activity, did not attach to the mammalian cells. Coaggregation of F. nucleatum PK1594 with P. gingivalis PK 1924 and Actinobacillus actinomycetemcomitans JP2, but not with other bacteria, showed a similar pattern with sugars, monoclonal antibody, and the adhesin-deficient mutant. The results suggest that the attachment of F. nucleatum PK1594 to mammalian cells and its coaggregation with periodontal pathogens are mediated by the same galactose-binding adhesin.

Characterization of a novel N-acetylneuraminic acid-specific Fusobacterium nucleatum PK1594 adhesin.

Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.

Helicobacter pylori adheres selectively to Fusobacterium spp.

Helicobacter pylori strains ATCC 43504 and ATCC 43629 were tested for their ability to coaggregate with 79 strains of bacteria representing 16 genera. All except two of the strains were of human origin, and most of the strains were isolated from the oral cavity. The helicobacters failed to coaggregate with all strains except the fusobacteria. Several coaggregations were partially or completely inhibited by lactose. Strong coaggregation was seen with each of four subspecies of Fusobacterium nucleatum and with Fusobacterium periodonticum ATCC 33693, all of human dental plaque origin. In contrast, the helicobacters failed to coaggregate with non-plaque isolates, Fusobacterium mortiferum ATCC 25557 and Fusobacterium ulcerans ATCC 49185. Heat treatment of the fusobacteria inactivated their ability to coaggregate, whereas heating of the Helicobacter partners had no effect, suggesting the presence of an adhesin on the fusobacteria and a corresponding receptor on the helicobacters. The potential ability of H. pylori to colonize the oral cavity by adhering selectively to the ubiquitous fusobacteria gives credence to the possibility that dental plaque may serve as a reservoir for this pathogen outside of the stomach.

Identification of a Fusobacterium nucleatum PK1594 galactose-binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination.

Attachment of Fusobacterium nucleatum to various oral surfaces is mediated by several adhesins anchored on its outer surface. Monoclonal antibodies (MAbs) were prepared and used to identify the putative galactose-binding adhesin of F. nucleatum PK1594. Four unique MAbs, 8G7, 26B9, 28G11, and 29D4, were isolated on the basis of their ability to inhibit coaggregation of F. nucleatum PK1594 with Porphyromonas gingivalis PK1924. All four MAbs were also capable of inhibiting galactose-inhibitable interactions of F. nucleatum PK1594 with other oral gram-negative bacteria and with erythrocytes. Preincubation of F. nucleatum PK1594 with MAb 26B9 or its Fab fragments at concentrations lower than 1 microg/ml resulted in complete inhibition of coaggregation with P. gingivalis PK1924 or hemagglutination. F. nucleatum PK1594 surface components prepared by mild sonication or by extracting whole cells with detergents were subjected to Western blot analysis. None of the MAbs were able to recognize any polypeptide in these experiments. Therefore, detergent extracts of F. nucleatum PK1594 surface components were subjected to three experimental procedures: (i) separation by ion-exchange chromatography and testing of fractions for reaction with MAb 26B9 in an enzyme-linked immunosorbent assay (ELISA), (ii) lactose-Sepharose affinity chromatography and testing of the lactose eluate in ELISA with MAb 26B9, and (iii) immunoseparation with either MAb 26B9 or 8G7. Collectively, the results suggest that the putative adhesin is a 30-kDa outer membrane polypeptide which mediates the coaggregation with P. gingivalis PK1924 as well as other galactose-sensitive interactions of F. nucleatum PK1594.

Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette.

Human oral viridans group streptococci that coaggregate with Actinomyces naeslundii PK606 express surface proteins related to ScaA, the coaggregation-mediating adhesin of Streptococcus gordonii PK488 (R. N. Andersen, N. Ganeshkumar, and P. E. Kolenbrander, Infect. Immun. 61:981-987, 1993). The nucleotide sequence of the 6,125-bp EcoRI insert of pRA1, containing scaA, the gene encoding ScaA, was determined. Six open reading frames (ORFs) were identified. The orientation of four ORFs, two upstream (ORF 1 and ORF 2) and one downstream (ORF 4) of scaA (ORF 3), indicated transcription in one direction, whereas ORF 5 and ORF 6 were transcribed divergently. Computer analysis of the deduced amino acid sequences identified a consensus binding site for ATP (GxxGxGKS) in the putative 28,054-Da protein encoded by ORF 1. ORF 2 potentially encoded a hydrophobic protein of 29,705 Da with six potential membrane-spanning regions. ScaA was 310 amino acids, 34,787 Da, and contained the lipoprotein consensus sequence LxxC, also reported for the ScaA-related proteins SsaB, FimA, and PsaA from Streptococcus sanguis 12, Streptococcus parasanguis FW213, and Streptococcus pneumoniae R36A, respectively. ORF 4 potentially encoded a 163-amino-acid protein of 17,912 Da, which was nearly identical to the downstream adjacent gene products of ssaB, fimA, and psaA. No significant homology with other proteins was found with the putative ORF 5 gene product, a 229-amino-acid protein of 25,107 Da. ORF 6 was incomplete and encoded a protein larger than 564 amino acids. This putative protein had a consensus Zn2+ binding motif, HExxH, found among bacterial thermolysins and mammalian neutral endopeptidases and was 40% identical to a homologous 210-amino-acid region of human enkephalinase. The genetic organization of ORFs 1, 2, and 3 was similar to those of the bacterial periplasmic-binding protein-dependent transport systems of gram-negative bacteria and binding-lipoprotein-dependent transport systems of gram-positive bacteria, and these genes appeared to encode ABC (ATP-binding cassette) proteins. This report describes a cell-to-cell adherence function associated with an ATP-binding cassette.

Coaggregation: specific adherence among human oral plaque bacteria.

Nearly all human oral bacteria exhibit coaggregation, cell-to-cell recognition of genetically distinct cell types. Clumps or coaggregates composed of the two kinds of cells are formed immediately upon mixing two partner cell types. Members of all 18 genera tested exhibit lactose-reversible coaggregation. Many of these interactions appear to be mediated by a lectin on one cell type that interacts with a complementary carbohydrate receptor on the other cell type. A lactose-sensitive adhesin has been isolated from Prevotella loescheii PK1295, and it exhibits the adherence properties observed with whole cells. Other adhesins have been identified and the genes for some of them have been cloned and sequenced. One Streptococcus sanguis adhesin is a lipoprotein that appears to have a dual function of recognizing both a bacterial carbohydrate receptor and a receptor in human saliva. Carbohydrate receptors for some adhesins have been purified from five oral streptococci, and they specifically block the coaggregations with the streptococcal partners that express the complementary adhesins. Coaggregation offers an explanation for the temporally related accretion of dental plaque and bacterial recognition of mucosal surfaces. Early colonizers of the tooth surface coaggregate with each other and late colonizers of the tooth surface coaggregate with each other, but with few exceptions, early colonizers do not recognize late colonizers. Furthermore, bacteria that colonize mucosal surfaces coaggregate with each other, indicating the high degree of specificity of coaggregation in the oral bacterial population.

Cloning of the Streptococcus gordonii PK488 gene, encoding an adhesin which mediates coaggregation with Actinomyces naeslundii PK606.

Coaggregation between Streptococcus gordonii PK488 and Actinomyces naeslundii PK606 is mediated by a 38-kDa streptococcal protein, designated ScaA. The gene, scaA, which encodes this protein has been cloned into Escherichia coli. A genomic S. gordonii PK488 library (in Lambda ZAP II) was screened with anti-S. gordonii immunoglobulin G absorbed with S. gordonii PK1804, an isogenic coaggregation-defective mutant of strain PK488. A positive recombinant phage was isolated, and a phagemid designated pRA1 was obtained which contained a 6.6-kb insert. Expression of scaA from pRA1 and from a subcloned internal 2.1-kb fragment was observed. The absorbed antiserum cross-reacted with a 34.7-kDa protein, SsaB, from S. sanguis 12, also a coaggregation partner of A. naeslundii PK606. Absorbed antiserum to S. gordonii PK488 and antiserum to SsaB both reacted with 38-kDa proteins in supernatants from mildly sonicated preparations from 11 other coaggregation partners of A. naeslundii PK606. Putative adhesin genes were identified in each of these coaggregation partners by Southern analysis of their genomic DNA with the cloned 2.1-kb fragment as a probe. A 30-base oligonucleotide probe based on the sequence of ssaB of S. sanguis 12 hybridized in an identical manner. These data extend the notion that most of the viridans streptococci that coaggregate with actinomyces are capable of expressing ScaA-related proteins.

Saliva-binding protein (SsaB) from Streptococcus sanguis 12 is a lipoprotein.

Two lipoprotein consensus sequences (Leu-X-X-Cys) are found in the presumptive signal peptide region (positions 12 to 15 and 17 to 20) of saliva-binding protein (SsaB) from Streptococcus sanguis 12. Three analogs of SsaB containing Cys-->Gly mutations were constructed by site-directed mutagenesis of pSA2, the recombinant plasmid expressing SsaB. [3H]palmitate was incorporated into SsaB only when the native Cys-20 residue was present. These data show that SsaB is a lipoprotein and that Cys-20 is the critical site for acylation.

Nucleotide sequence of a gene coding for a saliva-binding protein (SsaB) from Streptococcus sanguis 12 and possible role of the protein in coaggregation with actinomyces.

The nucleotide sequence of a 2.9-kb streptococcal DNA fragment which codes for two proteins with MrS of 36,000 (Streptococcus sanguis adhesin B [SsaB]) and 20,000 has been determined. The ssaB gene is 927 bp and codes for a 34,684-Da protein. The open reading frame coding for the 20-kDa protein is 489 bp and codes for a protein of 17,885 Da. The SsaB protein has a putative hydrophobic 19-amino-acid signal sequence resulting in a 32,620-Mr secreted protein, whereas the 20-kDa protein has no signal sequence. Both proteins are hydrophilic, and neither appears to have a hydrophobic membrane anchor sequence in the carboxy-terminal region. A DNA sequence homology of 73% exists between the cloned fragment containing the ssaB gene from S. sanguis 12 and the cloned fragment containing the type 1 fimbrial gene of S. sanguis FW213 (J.C. Fenno, D.J. LeBlanc, and P. Fives-Taylor, Infect. Immun. 57:3527-3533, 1989). Amino acid comparisons of the SsaB and type 1 fimbrial proteins show 87% homology, indicating a close similarity of the two proteins. Antiserum raised against the cloned SsaB protein cross-reacts with a 38-kDa protein identified from Streptococcus gordonii (S. sanguis) PK488 which was proposed to mediate coaggregation with Actinomyces naeslundii PK606 (P.E. Kolenbrander and R.N. Andersen, Infect. Immun. 58:3064-3072, 1990). The SsaB adhesion may play a role in oral colonization by binding either to a receptor on saliva or to a receptor on actinomyces.

Cloning of a Streptococcus sanguis adhesin which mediates binding to saliva-coated hydroxyapatite.

Chromosomal DNA from a salivary aggregating strain of Streptococcus sanguis 12 was partially digested with PstI and ligated into the plasmid vector pUC18 and transformed into Escherichia coli JM83. A total of 1,700 recombinant clones of E. coli were examined by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or S. sanguis 12 surface fibrils. Five clones which reacted with one or the other antiserum were shown to be unique by Western blotting (immunoblotting) and restriction endonuclease digestion. One recombinant plasmid pSA2 expressed two proteins with Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated SsaB. Both proteins were purified to homogeneity by Sephadex G-75 and ion-exchange chromatography. The proteins were present in mutanolysin digests of whole-cell lysates of S. sanguis 12 and in the non-saliva-aggregating variant 12na and the hydrophilic variant 12L. Polyclonal antiserum raised against the SsaB protein reacted strongly with the cell surfaces of S. sanguis 12 and 12na but not with that of 12L. SsaB inhibited the adhesion of S. sanguis 12na to saliva-coated hydroxyapatite, indicating that the adhesin mediates the binding to the pH-sensitive receptor.

Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein.

Cell surface fibrils could be released from Streptococcus sanguis 12 but not from strains 12na or N by freeze-thawing followed by brief homogenization. Fibrils were isolated from the homogenate by ultracentrifugation or ammonium sulfate precipitation. Electron microscopy demonstrated the presence of dense masses of aggregated fibrils in these preparations. Under nondenaturing conditions, no proteins were seen in polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS)-PAGE analysis revealed a single band stained with Coomassie blue and periodic acid Schiff stain with a molecular weight in excess of 300,000. The protein has been given the name long-fibril protein (LFP). The molecule was susceptible to digestion with subtilisin, pronase, papain, and trypsin, but was unaffected by chymotrypsin or muramidases. Attempts to dissociate the protein into smaller subunits with urea, guanidine, sodium thiocyanate, and HCl were unsuccessful. Gel filtration on a column of Sephacryl S-400 in the presence of 2% SDS resulted in elution of the protein at the void volume. Antibody raised against the LFP excised from an SDS-PAGE gel reacted with long fibrils on the surface of strain 12 and with isolated fibrils by an immunogold labeling technique. Monoclonal antibody reactive with LFP in SDS-PAGE also reacted with fibrils present on the cell. Antisera raised against the fibrils inhibited adherence to saliva-coated hydroxyapatite.

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.