Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.     

Cell-surface proteins of Streptococcus sanguis associated with cell hydrophobicity and coaggregation properties

Incubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: being degraded when cells were incubated with protease; being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.     

The hydrophobicity of Streptococcus salivarius strain HB and mutants deficient in adhesion to saliva-coated hydroxyapatite

The hydrophobicity and adhesion to saliva-coated hydroxyapatite of Streptococcus salivarius HB and the mutants HB7, HBV5 and HBV51 were measured. The mutants HB7 and HBV51 showed a significant reduction in adhesion to salivacoated hydroxyapatite and hydrophobicity compared with the mutant HBV5 and the parent strain. This supports the view that hydrophobic interactions are important for bacterial attachment in the oral cavity and is in contrast to previous studies on the hydrophobicity of these strains.     

Expression of the surface properties of the fibrillar Streptococcus salivarius HB and its adhesion deficient mutants grown in continuous culture under glucose limitation

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0.1 to 1.1 h-1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19.5 +/- 3.8 nm at high growth rate to a minimum of 12.3 +/- 4.8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15.6 kDa. Protein bands at 70, 56, 40.5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.     

Cell-surface charge and cell-surface hydrophobicity of collagen-bindingAeromonas andVibrio strains

Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A. caviae, A. sobria, Vibrio cholerae, and V. anguillarum strains. These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the α1+α2 chains) and gelatin immobilized on latex beads. Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties. There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen. The expression of surface properties was dependent on the culture media. There was no relationship between binding to immobilized collagen and binding to soluble ¹²⁵I-labeled collagen. Particle-agglutination reactivity differed when using various collagen-coated microbead preparations. There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures. The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran. Under these conditions, the α1+α2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads. For latex beads passively coated with collagen preparations, the α1+α2 collagen-chain mixture was more hydrophobic than gelatin and native collagen. We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads. In this regard, carboxy-modified latex beads coated with an α1+α2 collagen-chain mixture gave the best results. Received: 9 January 1995 / Accepted: 30 May 1995     

A comparison of the adhesive properties and surface ultrastructure of the fibrillar Streptococcus sanguis 12 and an adhesion deficient non-fibrillar mutant 12 na

Streptococcus sanguis 12 and a naturally occurring mutant, 12 na, were screened for their ultrastructure and adhesive properties in vitro. Negative staining showed that Strep. sanguis 12 carried three types of surface structure. The majority of cells carried long fibrils that in different batches ranged in length from 80 to 207 nm, and shorter fibrils which were 51mD0 PT 15mD7 nm long. Both types of fibrils were primarily located at the poles of the cells. Occasionally cells were seen that carried fimbriae, which are structurally distinct from fibrils, and were 3mD4 nm wide and <1mD μm long. Strain 12 na carried no detectable surface structures. Ruthenium red staining revealed that both strains carried a loose, amorphous, extracellular polysaccharide layer attached to the cell wall. Streptococcus sanguis 12 na was 83% less adhesive than strain 12 in a saliva-coated hydroxyapatite assay, and 50% less adhesive in a buccal epithelial cell adhesion assay. In contrast, strain 12 na was more sensitive to aggregation by parotid saliva than strain 12, and both strains were equally aggregated by whole saliva. The cell surface hydrophobicity of the two strains was similar. Extraction of surface proteins by sodium lauroyl-sarcosinate followed by sodium dodecylsulphate polyacrylamide gel electrophoresis demonstrated that Strep. sanguis 12 expressed more high mol.wt proteins on its surface than strain 12 na. Using immunogold labelling, the fibrils of strain 12 labelled well with antiserum directed against the long fibrils, but so did the cell surfaces of both Strep. sanguis 12 and 12 na. High molecular weight proteins and cell surface fibrils may be associated with adhesion in this strain.     

A comparison of the adhesive properties and surface ultrastructure of the fibrillar Streptococcus sanguis 12 and an adhesion deficient non-fibrillar mutant 12 na

Streptococcus sanguis 12 and a naturally occurring mutant, 12 na, were screened for their ultrastructure and adhesive properties in vitro. Negative staining showed that Strep. sanguis 12 carried three types of surface structure. The majority of cells carried long fibrils that in different batches ranged in length from 80 to 207 nm, and shorter fibrils which were 51.0 +/- 15.7 nm long. Both types of fibrils were primarily located at the poles of the cells. Occasionally cells were seen that carried fimbriae, which are structurally distinct from fibrils, and were 3-4 nm wide and less than 1.0 micron long. Strain 12 na carried no detectable surface structures. Ruthenium red staining revealed that both strains carried a loose, amorphous, extracellular polysaccharide layer attached to the cell wall. Streptococcus sanguis 12 na was 83% less adhesive than strain 12 in a saliva-coated hydroxyapatite assay, and 50% less adhesive in a buccal epithelial cell adhesion assay. In contrast, strain 12 na was more sensitive to aggregation by parotid saliva than strain 12, and both strains were equally aggregated by whole saliva. The cell surface hydrophobicity of the two strains was similar. Extraction of surface proteins by sodium lauroyl-sarcosinate followed by sodium dodecylsulphate polyacrylamide gel electrophoresis demonstrated that Strep. sanguis 12 expressed more high mol.wt proteins on its surface than strain 12 na. Using immunogold labelling, the fibrils of strain 12 labelled well with antiserum directed against the long fibrils, but so did the cell surfaces of both Strep. sanguis 12 and 12 na. High molecular weight proteins and cell surface fibrils may be associated with adhesion in this strain.     

Isolation and some properties of extracellular glucan-producing strains of human oral Streptococcus salivarius

A total of eighteen strains of Streptococcus salivarius, which formed rough gelatinous, rough mucoid or smooth mucoid colonies on sucrose agar media, were isolated from the saliva and tongue dorsum of adults. All of the isolates produced glucans as well as fructans from sucrose. The bulk of the glucans was synthesized by the extracellular enzyme fraction and was water insoluble, whereas most of the fructans were synthesized by the cell-associated enzyme fraction and were water soluble. All strains formed microbial deposits on wire and glass surfaces when cultured in sucrose broth, but their sucrose-dependent adhesion was apparently looser than that produced by a cariogenic S. sobrinus strain. The rough gelatinous colony forming strains possessed a greater ability to synthesize water-insoluble glucans and produced heavier deposits with higher cohesion. Preliminary studies showed that the S. salivarius of such characteristic forms of colony were detected primarily in the saliva and tongue dorsum: the smooth mucoid colony formers appeared to predominate in the tongue coat and the rough mucoid and rough gelatinous colony formers were prominent in saliva. Isolation of these S. salivarius from dental plaques was low.     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Lack of correlation between fibrils, hydrophobicity and adhesion for strains of Streptococcus sanguis biotypes I and II

Fifteen strains of Streptococcus sanguis biotype I and eight strains of Streptococcus sanguis biotype II with peritrichous fibrils, tufts of fibrils or a mixture of fibrils and fimbriae on the cell surface, were tested for their ability to adhere to saliva coated spheroidal hydroxyapatite (S-SHA) in a radiolabelled assay. S. sanguis I strains adhered better than S. sanguis II strains and peritrichously fibrillar strains generally adhered better than tufted strains. There was no correlation between the density of fibrillation and adhesion. The only highly adherent strain of S. sanguis II carried fimbriae in addition to fibrils. No correlation was observed between cell surface hydrophobicity as measured by phase partitioning with hexadecane and adhesion to S-SHA.     

Physico-chemical surface characteristics and adhesive properties of Streptococcus salivarius strains with defined cell surface structures

Physico-chemical surface characteristics and adhesive properties of a series of mutants of Streptococcus salivarius HB with defined cell surface structures were determined. Zeta potentials showed no relation either with the presence or absence of specific antigens on the bacterial cell surface, or with the adhesive properties of the cells. Hydrophobicity was assessed by surface free energy determination from measured contact angles, by adsorption to hexadecane and by hydrophobic interaction chromatography. Generally, the progressive removal of fibril subclasses from the cell surface resulted in a reduced hydrophobicity. However, specific fibrillar subclasses appeared to contribute to surface hydrophobicity to widely different extents. Bacterial adhesion to polymethylmethacrylate increased with increasing hydrophobicity of the mutants. However, adhesion to a more complex biological substratum, such as saliva-coated hydroxyapatite, correlated only partly with hydrophobicity. The organism, deprived of most of its fibrillar surface structures, clearly showed the least adhesion to hydrophobic ligands, to both polymethylmethacrylate and saliva-coated hydroxyapatite, and had a significantly higher surface free energy than the other mutants and the parent strain.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.     

Purification and properties of glycogen phosphorylase from Streptococcus salivarius

Glucose-grown cells of Streptococcus salivarius have been shown to contain a polyglucose phosphorylase which had maximum activity in the stationary phase of growth. Despite the fact that activity in crude cell-free extracts was two- to threefold greater in the presence of corn dextrin than with oyster glycogen, subsequent purification (200-fold) of the enzyme from the soluble fraction of the organism by protamine sulfate treatment, ammonium sulfate fractionation (30–50%), ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 demonstrated that this dextrin/glycogen activity was associated with a single enzyme. Since glucose-grown cells of S. salivarius are known to synthesize a typical glycogen polymer, the enzyme was named: glycogen phosphorylase. The purified enzyme preparation was devoid of phosphoglucomutase and ADP-glucose pyrophosphorylase, but contained a small amount of ADP-glucose: α-1,4 glucan transferase activity. The enzyme was stable at ?10 °C in the presence of 0.2 m NaF, while the pH optimum for the enzyme was 6.0 both with glycogen and with dextrin. With the purified enzyme, corn dextrin was the best primer, both in the direction of synthesis and in the direction of phosphorolysis, being 1.8–1.9 times more effective than purified S. salivarius glycogen. When the enzyme was assayed in the direction of glycogen synthesis, a K_m value of 3.4 mm was obtained for glucose-1-P, while the values for S. salivarius glycogen, oyster glycogen and corn dextrin were 25, 42, and 40 mg/ml, respectively. In the direction of phosphorolysis, K_m values were 20 mm for P_i obtained with oyster glycogen, 25 mm for P_i with corn dextrin, and 20 mg/ml and 26 mg/ml for oyster glycogen and corn dextrin, respectively. Present data suggests no involvement of -SH groups in enzyme catalysis, while the enzyme was inhibited by divalent ions with the severest inhibition being observed with Ca²⁺, Zn²⁺ and Fe²⁺. The two ion chelators, EDTA and EGTA, had no effect on enzyme activity.     

A comparison of 2 methods for determining the hydrophobicity of Streptococcus groups A and B

A simple method for the evaluation of the hydrophobic properties of streptococci, pathogenic for humans, by their adherence to polystyrene and a modified method for measuring their hydrophobic properties by their sorption on hexadecane have been developed. The results obtained in the evaluation of the hydrophobic properties of streptococci by these two methods have been compared and the complete correlation of these results in classifying the cultures as hydrophobic and hydrophilic has been shown. For the first time the differences in the hydrophobic properties of different strains of group B streptococci have been established.     

Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface

Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology.     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.