Adhesion of Streptococcus sanguis CH3 to polymers with different surface free energies.

The adhesion of the oral bacterium Streptococcus sanguis CH3 to various polymeric surfaces with surface free energies (gamma s) ranging from 22 to 141 erg cm-2 was investigated. Suspensions containing nine different bacterial concentrations (2.5 X 10(7) to 2.5 X 10(9) cells per ml) were used. After adhesion for 1 h at 21 degrees C and a standardized rinsing procedure, the number of attached bacteria per square centimeter (nb) was determined by scanning electron microscopy. The highest number of bacteria was consistently found on polytetrafluorethylene (gamma s = 22 erg cm-2), and the lowest number was found on glass (gamma s = 141 erg cm-2) at all bacterial concentrations tested. The overall negative correlation between nb and gamma s was weak. However, the slope of the line showing this decrease, calculated from an assumed linear relationship between nb and gamma s, appeared to depend strongly on the bacterial concentration and increased with increasing numbers of bacteria in the suspension. Analysis of the data for each separate polymer showed that the numbers of attached cells on polyvinyl chloride and polypropylene were higher but that those on polycarbonate were lower than would be expected on basis of a linear relationship between nb and gamma s. Desorption experiments were performed by first allowing the bacteria to attach to substrata for 1 h, after which the substrata and attached bacteria were removed to bacterial suspensions containing 10-fold lower bacterial concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of adhesion of the oral bacterium Streptococcus sanguis CH3 to polymers with different surface free energies

The kinetics of adhesion of Streptococcus sanguis CH3 from suspension to polymers with different surface free energies were studied by using three bacterial concentrations (2.5 X 10(7), 2.5 X 10(8), and 2.5 X 10(9) cells per ml-1). Substratum surface free energies (gamma s) ranged from 18 to 120 erg cm-2. The kinetics of bacterial adhesion to these surfaces showed a typical two-step adhesion process, indicating an equilibrium in both steps. In the initial adhesion step (step 1), low equilibrium numbers of adhering bacteria were counted on substrata with surface free energies lower than 55 erg cm-2. A maximal number adhered on substrata with higher surface free energies. At the lowest bacterial concentration tested, the highest number of bacteria were found on substrata with a surface free energy around 55 erg cm-2. For each substratum, step 2 started after a characteristic time interval tau, being short (30 min) for gamma s less than 50 and long (120 min) for gamma s greater than 50 erg cm-2. The relationship between the substratum surface free energy and the number of bacteria adhering at equilibrium after step 2 was similar to, although less distinct than, that during step 1 with a slight indication of a bioadhesive minimum around gamma s = 35 erg cm-2. The results are indicative of a two-step adhesion model, in which step 1 is controlled by macroscopic substratum properties.     

Kinetics of adhesion of the oral bacterium Streptococcus sanguis CH3 to polymers with different surface free energies.

The kinetics of adhesion of Streptococcus sanguis CH3 from suspension to polymers with different surface free energies were studied by using three bacterial concentrations (2.5 X 10(7), 2.5 X 10(8), and 2.5 X 10(9) cells per ml-1). Substratum surface free energies (gamma s) ranged from 18 to 120 erg cm-2. The kinetics of bacterial adhesion to these surfaces showed a typical two-step adhesion process, indicating an equilibrium in both steps. In the initial adhesion step (step 1), low equilibrium numbers of adhering bacteria were counted on substrata with surface free energies lower than 55 erg cm-2. A maximal number adhered on substrata with higher surface free energies. At the lowest bacterial concentration tested, the highest number of bacteria were found on substrata with a surface free energy around 55 erg cm-2. For each substratum, step 2 started after a characteristic time interval tau, being short (30 min) for gamma s less than 50 and long (120 min) for gamma s greater than 50 erg cm-2. The relationship between the substratum surface free energy and the number of bacteria adhering at equilibrium after step 2 was similar to, although less distinct than, that during step 1 with a slight indication of a bioadhesive minimum around gamma s = 35 erg cm-2. The results are indicative of a two-step adhesion model, in which step 1 is controlled by macroscopic substratum properties.     

Adhesion and motility of gliding bacteria on substrata with different surface free energies

The adhesion and motility of several aquatic and terrestrial gliding bacteria on slides differing in their critical surface energies have been examined. In general, adhesion was tenacious on low-critical surface energy (hydrophobic) surfaces and tenuous on hydrophilic surfaces. Gliding was inhibited on very hydrophobic substrata and skittish on very hydrophilic surfaces.     

Adhesion and motility of gliding bacteria on substrata with different surface free energies.

The adhesion and motility of several aquatic and terrestrial gliding bacteria on slides differing in their critical surface energies have been examined. In general, adhesion was tenacious on low-critical surface energy (hydrophobic) surfaces and tenuous on hydrophilic surfaces. Gliding was inhibited on very hydrophobic substrata and skittish on very hydrophilic surfaces.     

Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Trypsin-susceptible cell surface characteristics of Streptococcus sanguis

The adherence of Streptococcus sanguis to saliva-coated hydroxylapatite was markedly reduced by treatment of the cells with trypsin. In Scatchard plots of adherence data, protease-treated S. sanguis did not exhibit the characteristic positive slopes, suggesting that trypsin prevented cooperative interactions between the cells and artificial pellicle. Trypsin also reduced the tendency of S. sanguis to bind to hexadecane and to octyl-Sepharose. When sodium dodecyl sulfate was used to elute S. sanguis from columns of octyl-Sepharose, it was observed that the elution profiles of trypsin-treated cells were more complex than those of control cells. Water and salts were incapable of removing the cells from octyl-Sepharose. The results suggest that adherence to saliva-coated hydroxylapatite, binding to hexadecane and to octyl-Sepharose depend on trypsin-susceptible cell surface molecules.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Transformation of Streptococcus sanguis to intrinsic penicillin resistance

A series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.     

Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.

Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.     

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.     

Interaction of hydroxyapatite and protein-coated hydroxyapatite with Streptococcus mutans and Streptococcus sanguis

The present study showed that S. mutans and S. sanguis behaved like negatively-charged particles in their interaction with hydroxyapatite in vitro. Phosphate in the system inhibited bacterial uptake by apatite, whereas calcium increased the uptake. A layer of acidic protein inhibited the uptake of bacteria by hydroxyapatite. The opposite was true when a basic protein was first adsorbed to the apatite. A saliva film on the apatite decreased the uptake of bacteria, supporting the view that acidic proteins are selectively adsorbed by hydroxyapatite from saliva. The results indicate clearly that electrostatic forces may be involved in bacterial interaction with tooth surface.     

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.     

Biosynthesis of oligosaccharide-lipid in Streptococcus sanguis

An oligosaccharide-lipid containing N-acetyl d-glucosamine (GlcNAc), l-rhamnose, and d-glucose was synthesized when the particulate enzyme from Streptococcus sanguis was incubated with UDP-GlcNAc, TDP-rhamnose, and UDP-glucose. The incorporation of d-glucose into the lipid was dependent on the preincorporation of l-rhamnose, which in turn was dependent on that of GlcNAc. This indicates that the order of sugar incorporation is GlcNAc, l-rhamnose, and d-glucose. The synthesis of GlcNAc-lipid was stimulated twofold by ATP and was inhibited strongly by UDP and slightly by UMP, CDP, and TDP, but not by all other nucleoside diphosphates and nucleoside monophosphates tested. A [gamma-(32)P]ATP labeling experiment indicated that some acceptor lipid was present in nonphosphorylated form. The acid and alkaline stabilities of the GlcNAc-lipid were similar to those of glycosyl undecaprenylphosphate, and the thin-layer chromatographic mobility of the lipid was slightly faster than that of the mannosylphosphorylundecaprenol. The molar ratio of phosphate to GlcNAc in purified GlcNAc-lipid was found to be 0.96:1. These results suggested that the GlcNAc was attached to the lipid moiety, presumably undecaprenol, by phosphodiester bonds. The incorporation of l-rhamnose into the lipid was inhibited by UDP and UMP, respectively, in a manner similar to the incorporation of GlcNAc. This suggested that the oligosaccharide was also linked to the lipid moiety by phosphodiester bonds.     

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.     

Inhibition of the cooperative adhesion of Streptococcus sanguis to hydroxylapatite

The adhesion of Streptococcus sanguis to hydroxylapatite is a process involving several adhesins and receptors. Binding isotherms and Scatchard plots of the adhesion suggest that cooperative interactions occur at low cell densities. It was found that sulfolane, a hydrophobic-bond diluent, was capable of inhibiting the cooperative adhesion of S. sanguis to saliva-coated hydroxylapatite beads. Sodium thiocyanate, a chaotropic agent, inhibited not only cooperative adhesion, but also the adhesion thought to result from noncooperative interactions. It is suggested that strong chaotropic agents may not only inhibit adhesin-receptor complexes, but also may influence the secondary/tertiary structures of interacting species.