Glycine prevents the phenotypic expression of streptococcal glucan-binding lectin

Glycine has been used extensively in bacterial cell surface research. Some researchers employ glycine in growth media so as to increase the transformability of streptococci during electroporation. Others have found that glycine, similar to wall antibiotics, 'weakens' peptidoglycan. It is now shown that when glycine is incorporated into the growth medium, Streptococcus sobrinus exhibits a diminished ability to aggregate with high molecular weight alpha-1,6-glucan. Growth of the bacteria in either a rich or a chemically defined medium results in a cell population with full lectin (glucan-binding) fidelity. Incorporation of glycine, but not serine or other amino acids, at concentrations of 100-200 mM gives rise to bacteria with lowered lectin activities. Bacteriolytic enzymes were able to lyse bacteria from glycine-grown cultures more readily than from cultures without the glycine supplement. The bacteria produce glucan-binding proteins, including glucosyltransferases, but they do not readily aggregate with added dextran. Furthermore, SDS-PAGE gels of supernatants of growth media (+/-glycine) are similar, suggesting the bacteria do not produce a unique set of proteins. Western blotting with a fluorescein isothiocyanate-labeled dextran probe reveals normal amounts of glucan-binding proteins in glycine-grown streptococci. Glycine may be acting as a type of antibiotic, reducing wall integrity upon which glucan promoted cellular aggregation depends.     

Subinhibitory concentrations of antibiotics affect cell surface properties of Streptococcus sobrinus.

Several antibiotics, at subinhibitory concentrations, caused an increase in the ability of Streptococcus sobrinus to bind alpha-1,6-glucans, whereas other antibiotics decreased glucan binding. In every case, glucan binding was inversely proportional to cell surface hydrophobicity. High levels of glucan-binding activity resulted in low levels of hydrophobicity, whereas low levels of glucan binding caused high levels of cellular hydrophobicity. The results show that low concentrations of antibiotics may modulate lectin and hydrophobin adhesins in streptococci.     

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.     

Modulation of glucan-binding protein activity in streptococci by fluoride

Glucan-binding lectin (GBL) activity of Streptococcus sobrinus was significantly reduced by fluoride in the growth medium. Approximately 1.5 mM fluoride was required for a 50% reduction in GBL activity. In addition to the GBL, several other glucan-binding proteins were reduced when the bacteria were grown in subinhibitory fluoride. Fluoride had no effect on glucosyltransferases (GTFs), enzymes capable of converting sucrose into alpha-1,6-glucans. All the proteins were detected by use of enhanced chemiluminescence (ECL of fluorescein-labeled dextran) and Western blotting of renatured SDS-PAGE gels. The effects of fluoride on the bacteria were abrogated when the manganous ion was included in the growth medium. It thus appears that one mechanism of action of fluoridated water is its effects on glucan-binding proteins. The fluoride may be reducing metabolism of the mangano aquo ion, essential for expression of the glucan-binding proteins.     

Glucan-binding proteins of Streptococcus mutans serotype c.

Three glucan-binding proteins have been isolated from the extracellular fluid cultures of Streptococcus mutans serotype c. These proteins were adsorbed to glucans containing 1,3-alpha or 1,6-alpha bonds and linked to various chromatographic supports: they were eluted from columns by a dextran solution. Glucosyltransferase activity was associated with two of the glucan-binding proteins.     

Fluoride inhibits the glucan-binding lectin of Streptococcus sobrinus

Abstract The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.     

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase).

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.     

Characterization of the product of the gtfS gene of Streptococcus downei, a primer-independent enzyme synthesizing oligo-isomaltosaccharides

The gtfS gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype h) into a bacteriophage vector, was subcloned into a plasmid vector. The gtfS gene products expressed in Escherichia coli were compared to the primer-independent, oligo-isomaltosaccharide synthase in Streptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and Km values. The glucans produced from sucrose by the gtfS gene products are alpha-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar gtfS gene was also detected on chromosomal DNA from S. sobrinus strain AHT.     

Construction of region-specific partial duplication mutants (merodiploid mutants) to identify the regulatory gene for the glucan-binding protein C gene in vivo in Streptococcus mutans

The gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans has been identified by random mutagenesis. We analyzed ddag(-) mutants containing the intact gbpC gene and found that these mutants possessed a large and characteristic duplication of a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, we developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690-bp gene responsible for the ddag(-) phenotype was identified within a 60-kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants.     

A new mechanism of action of fluoride on streptococci.

Addition of fluoride to the growth medium of Streptococcus sobrinus resulted in a loss of glucan-binding lectin activity. Upon removal of fluoride, the bacteria regained their ability to bind glucan in about one generation. Chloramphenicol prevented recovery of ability to produce the lectin, showing the requirement for protein synthesis. Fluoride also caused a significant reduction in the tendency of the streptococci to form chains of cells, although the spent medium from fluoride-containing growth media did not dechain control cells. The fluoride thus does not activate autolytic enzymes. Importantly, 2-D electrophoresis and SDS-PAGE revealed several proteins were synthesized in the presence of fluoride that were not synthesized in its absence. It seems possible that fluoride places a stress on the bacteria, causing the synthesis of proteins that may play a role in protecting the cells against the stress. Numerous stress proteins are known for bacteria, including those resulting from heat, enzymes and osmotic shocks. The ability of fluoride to cause loss of glucan-binding may be related to its reported beneficial effects on oral health.     

Substrate specificity of hydrolase activity of the primer-dependent glucosyltransferases from Streptococcus sobrinus.

Four kinds of glucosyltransferases, P1, P2, P3 and P4, were separately purified from the culture supernatant of Streptococcus sobrinus. Their dependencies on primer were analysed. There were two primer-dependent glucosyltransferases (P3 and P4). In the absence of primer 1,6-alpha-D-glucan, P3 was not able to produce glucan from sucrose. However, P3 showed sucrose hydrolase activity, whereas P4 was still able to produce glucan without primer 1,6-alpha-D-glucan. Consequently, glucosyltransferase activity of P4 was incompletely primer-dependent. Both P3 and P4 showed high substrate specificity for sucrose, failing to use melezitose, raffinose, or stachyose as the substrates.     

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.     

Optimization of conditions for the efficient production of mutan in streptococcal cultures and post-culture liquids

The strain Streptococcus sobrinus CCUG 21020 was found to produce water-insoluble and adhesive mutan. The factors influencing both stages of the mutan production, i.e. streptococcal cultures and glucan synthesis in post-culture supernatants were standardized. The application of optimized process parameters for mutan production on a larger scale made it possible to obtain approximately 2.2 g of water-insoluble glucan per 11 of culture supernate--this productivity was higher than the best reported in the literature. It was shown that some of the tested beet sugars might be successfully utilized as substitutes for pure sucrose in the process of mutan synthesis. Nuclear magnetic resonance analyses confirmed that the insoluble biopolymer synthesized by a mixture of crude glucosyltransferases was a mixed-linkage (1-->3), (1-->6)-alpha-D-glucan (the so-called mutan) with a greater proportion of 1,3 to 1,6 linkages.     

Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.     

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS-PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.     

Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.     

Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.     

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.     

Fructosyltransferase activity of a glucan-binding protein from Streptococcus mutans

Streptococcus mutans serotype c produces several extracellular proteins which bind to affinity columns of immobilized glucans. The proteins are three distinct glucosyltransferases and another glucan-binding protein (molecular weight 74000) which is now shown to be a fructosyltransferase. This enzyme is antigenically distinct and genetically independent of two other fructosyltransferases produced by the same organism. A mutant is described which lacks the glucan binding fructosyltransferase and has defective ability to form adherent colonies in the presence of sucrose. Although the production of glucans from sucrose results in the glucan binding protein becoming bound to the bacterial surface, and hence perhaps contributing to adherence, the fructans synthesized by the enzyme do not appear to contribute to this phenomenon.     

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.