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Production of extracellular and cell-associated glucosyltransferase activity by Streptococcus mutans during growth on various carbon sources.

The production of extracellular and cell-associated glucosyltransferase activity by Streptococcus mutans strain GS-5 was examined during growth on various carbon sources in a chemically defined medium. S. mutans cells produced glucosyltransferase activity only during logarithmic growth when glucose, fructose, mannitol, or sorbitol was the sole carbon source. Cells growing on mannitol or sorbitol produced approximately half as much extracellular glucosyltransferase activity as cells growing on glucose, although the proportions of the glucosyltransferase activity capable of synthesizing insoluble glucans were similar. Cells growing on fructose produced slightly more extracellular glucosyltransferase activity than cells grown on glucose, yet the proportion of the glucosyltransferase activity capable of synthesizing insoluble glucans was again similar to glucose cultures. S. mutans cells growing in the presence of both glucose and mannitol displayed diauxic growth and initial preferential utilization of glucose. Glucosyltransferase enzyme production occurred only during the phases of cell growth in the presence of the two carbon sources. The cell-associated glucosyltransferase activities of glucose-, fructose-, mannitol-, and sorbitol-grown cells were relatively low, yet all the cells were capable of adherence to glass in the presence of sucrose. When glucose-containing cultures of S. mutans were supplemented with sucrose, extracellular glucosyltransferase activity first became cell associated and then appeared to become inactivated, presumably due to the accumulation of insoluble glucans.

Carbohydrate Metabolism

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

Acid Anhydride Hydrolases

Cellular adherence, glucosyltransferase adsorption, and glucan synthesis of Streptococcus mutans AHT mutants.

Streptococcus mutans AHT mutants M1, M2, and M13 failed to adhere to a glass surface, whereas mutants M9 and M35 exhibited decreased and increased adherence, respectively, as compared with the parent strain, when grown in sucrose broth. Extracellular glucosyltransferase prepared from glucose-grown cultures of the adherent strains (wild type, M9, and M35) induced adherence of heat-killed cells of the homologous and heterologous streptococcal strains as well as of Escherichia coli K-12 and uncoated resin particles. The glucosyltransferase was adsorbed on all the streptococcal cells and glucan-coated resins, but not on E. coli cells and the uncoated resins. Glucosyltransferase from the nonadhering mutants (M1, M2, M13) neither was significantly adsorbed on nor induced adherence of any of the cells and resins. Cell-free enzymes from the glucose-grown adherent strains produced water-soluble and water-insoluble glucans, whereas those from the nonadhering mutants produced only water-soluble glucans. Small amounts of alkali-soluble, cell-associated glucan were recovered from the sucrose-grown nonadhering mutants. Thus, the relative proportions of glucosyltransferase isozymes elaborated by the S. mutans mutants, insofar as they affect the physico-chemical properties of the glucans produced, seem to determine the adherence abilities of the cells. The adsorption of glucosyltransferase on glucan molecules on the cell surface is not required for the adherence of S. mutans, but de novo glucan synthesis is important in the adherence process.

Adhesiveness

Preparation of glucosyltransferase from Streptococcus mutans by elution from water-insoluble polysaccharide with a dissociating solvent.

Glucosyltransferase (EC 2.4.1.5) was obtained by dissociation from water-insoluble polysaccharide in the presence of 6 M guanidine-hydrochloride. Water-insoluble polysaccharide was synthesized by cell-free culture supernatants from Streptococcus mutans strain 6715. Gel filtration of the glucosyltransferase on a column of 8% agarose in phosphate buffer, followed by filtration on a column of 4% cross-linked agarose in 6 M guanidine-hydrochloride, gave a 23-fold enrichment of the enzyme. The enriched glucosyltransferase preparation contained 22% carbohydrate and eluted at a position corresponding to a molecular weight of 422,000. Polyacrylamide gel (5%) electrophoresis of this preparation revealed two regions which stained for protein, formed water-insoluble polysaccharide in the presence of sucrose, and precipitated with antisera directed to crude glucosyltransferase preparations. The guanidine-eluted enzyme could be primed by 5 X 10(-5) M dextran T10 (molecular weight, 10,000). High-molecular-weight glucan and a possible glucan-binding protein were also obtained after the final gel filtration step (4% cross-linked agarose) in addition to glucosyltransferase.

Chromatography, Gel

Effect of oral administration of glucosyltransferase antigens on experimental dental caries.

The effect of oral administration of soluble antigen preparations containing glucosyltransferase on dental caries in hamsters was studied. Immunization was accomplished by feeding glucosyltransferase for 21 to 27 consecutive days. This immunization regimen resulted in the formation of salivary antibody, which was detected by functional inhibition of enzymatic activity and by a modified enzyme-linked immunosorbent assay. A serum response also occurred in two of the three experiments performed. After infection with cariogenic Streptococcus mutans strain 6715, glucosyltransferase-fed hamsters had significantly fewer S. mutans cells recoverable from molar surfaces on six of nine occasions, compared with buffer-fed control groups. Hamsters orally immunized with glucosyltransferase also always had lower mean caries scores and mean numbers of lesions than comparably infected sham-immunized groups. The results of this study suggest that significant protection from experimental dental caries can be accomplished by oral administration of soluble antigen preparations containing glucosyltransferase.

Animals

Studies on the collagen glucosyltransferase activity present in platelets and plasma.

1. Collagen glucosyltransferase was demonstrated to be associated with pig platelets by using a specific assay for the synthesis of [(14)C]glucosylgalactosylhydroxylysine. 2. This enzyme from pig platelets required denatured collagen as substrate and the reaction was not inhibited by the presence of triple-helical collagen. These observations indicate that the platelet enzyme cannot form either an enzyme-substrate complex or an enzyme-inhibitor complex with triple-helical collagen. 3. Platelets were fractionated by sucrose-density-gradient centrifugation after either lysis by a glycerol-loading technique or homogenization. Assays of subcellular fractions for collagen glucosyltransferase activity indicated that the enzyme was localized predominantly in the cytosolic fraction and less than 5% of the activity was associated with the membrane fractions. 4. Enzyme assays were carried out on platelet-rich plasma and platelet-poor plasma prepared from pig and human blood. These analyses indicated that most of the collagen glucosyltransferase activity of platelet-rich plasma was in a soluble form and only about 10% was associated with platelets. 5. Comparative studies on the enzyme activity in plasma and platelets of various animal species revealed marked variation, with the guinea pig exhibiting the highest activity. In most cases there was a correlation between the activity found in platelets and plasma, but little species variation was noted in enzyme amounts detected in bone-marrow preparations. 6. The results described here are discussed in the context of the proposal that collagen glucosyltransferase might play a role in mediating collagen-platelet adhesion.

Animals

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.

Glucan 1,4-alpha-Glucosidase

Immunogenic properties of the glucosyltransferase from Streptococcus sanguis OMZ 9: kinetic study of inhibition by antibodies.

An anti-glucosyltransferase serum was prepared against a pure enzyme preparation from Streptococcus sanguis OMZ 9, which synthesized both soluble and insoluble dextran. Sera, crude gamma globulins, and antibody fractions obtained after gel filtration on a Bio-Gel P200 column were used to study enzyme-antibody interactions. A strong inhibition of glucosyltransferase activity was obtained only with the purified antibody fraction. Kinetics studies showed that the anti-glucosyltransferase antibodies acted as noncompetitive inhibitors with respect to the substrate (sucrose). The addition of primer dextran in the reaction mixture during preincubation produced a diminution of the inhibition, and the antibodies acted as mixed type inhibitors with respect to dextran. The simultaneous addition of dextran and antibodies can protect the enxyme against antibody inhibition.

Antibodies, Bacterial

Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases.

A method of identifying plasmids containing genes responsible for synthesis of nucleotide sugar:lipopolysaccharide glycosyltransferases is described. Hybrid ColE1 plasmids containing random fragments of the chromosome of Escherichia coli K12 were introduced into an indicator strain of Salmonella typhimurium which lacks UDP-glucose:lipopolysaccharide glucosyltransferase I due to an rfaG mutation. Plasmids capable of correcting the transferase defect were identified by their ability to convert the bacteriophage sensitivity pattern of the recipient strain from Ffm-sensitive to Ffm-resistant. Analysis of the lipopolysaccharide of the S. typhimurium/ColE1 hybrid strains and assay of cell extracts defined the new enzyme activities. Two plasmids were identified which carried the rfaG+ gene; one of these plasmids also contained genetic information for a second glucosyltransferase, the E. coli glucosyltransferase II, which normally is not present in S. typhimurium.

DNA, Recombinant

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.

Animals

Glucosyltransferase activity in kidney fractions of normal and streptozotocin-diabetic rats.

Glucosyltransferase (UDPglucose: galactosylhydroxylysine-basement membrane glucosyltransferase), an enzyme specifically involved in collagen synthesis, was measured in various kidney fractions of normal, diabetic and underfed rats, using as basis the incorporation of radioactivity into protein during incubation with UDP[U-14C]glucose and alkali-soluble fetal calf-skin collagen. Three criteria of enzyme activity were compared: A, total radioactivity of the washed protein precipitate; B, this figure minus activity incorporated in the absence of the collagen acceptor; and C, radioactivity incorporated into the mixed amino acid fraction, collected by elution with dilute NH4OH from a Dowex 50 resin column after alkaline hydrolysis of the protein. Method A was found satisfactory using whole medulla or isolated glomeruli, since the average proportions of total protein radioactivity recovered in the NH3 fraction were 0.81 and 0.87, respectively, and the deviations were small. There was a larger and variable proportion of nonspecific incorporation using whole cortex. Incubation of a control set of sample without added collagen was found to be unnecessary (Method B). Per mg protein, medulla and glomeruli had more enzyme than did whole cortex. In diabetes, activity was enhanced in the 10,000 X g supernatant fraction of cortex, as previously reported. However, the increase associated with diabetes was even more consistent in the medulla, averaging 3-fold in the 10,000 X g pellet fraction. No increase was found in isolated glomeruli in diabetes. Also, no increase was seen in the kidneys of non-diabetic rats with body weight similar to that of the diabetics.

Animals

Metabolism of the polysaccharides of human dental plaque. Part II. Purification and properties of Cladosporium resinae (1 leads to 3)-alpha-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci.

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.

Cladosporium

Improved purification procedure for the extracellular D-glucosyltransferase from Streptococcus mutans 6715.

The exocellular D-glucosyltransferase from Streptococcus mutans 6715 has been highly purified with minimal loss of enzymic activity. The organisms were cultured in trypticase soy-broth that had been treated with invertase and filtered through an ultrafilter fitted with a membrane having a cut-off molecular weight at 10,000. To the growth medium was added Tween 80, which prevented the enzyme from aggregating. The final step in the purification employed insoluble, streptococcal dextran as an affinity support. Two D-glucosyltransferase activities were detected, viz., one that did not adsorb to the insoluble dextran and one that did. The enzymic fraction that had adsorbed to the insoluble dextran in the affinity column was strongly inhibited by added insoluble dextran.

Glucosyltransferases

Effect of salts on water-insoluble glucan formation by glucosyltransferase of Streptococcus mutans.

The formation of water-insoluble glucan by extracellular glucosyltransferase from Streptococcus mutans 6715 found to be greatly stimulated by various mono- or divalent cations. An enzyme preparation, obtained by ethanol fractionation, was able to catalyze the formation of water-insoluble glucan from sucrose in the presence of monovalent cations above 100mM or divalent cations above 20 mM at neutral pH. As the concentration of monovalent and divalent cations was reduced to below 10 mM and 1 mM, respectively, the formation of insoluble glucan decreased to a negligible amount. High concentrations of these cations were found to stimulate the formation of insoluble glucan in the following ways: (i) it increased the activity of total glucosyltransferase up to 1.6- and 2.7-fold in the absence and presence of a primer dextran, respectively, and (ii) it changed the formation of soluble glucan to insoluble. It was postulated that one of the essential factors for the formation of insoluble glucan would be to keep more than two water-soluble glucan chains close to enzyme aggregates and that such interaction could be enhanced by the presence of high cation concentrations.

Carbohydrate Metabolism

Properties of Streptococcus mutans grown in a synthetic medium: binding of glucosyltransferase and in vitro adherence, and binding of dextran/glucan and glycoprotein and agglutination.

The influence of culture media on various properties of Streptococcus mutans was investigated. Strains of S. mutans (serotypes c, d, f, and g) were grown in a complex medium (Todd-Hewitt broth [THB]) or a synthetic medium (SYN). The SYN cells, in contrast to THB cells, did not bind extracellular glucosyltransferase and did not produce in vitro adherence. Both types of cells possessed constitutive levels of glucosyltransferase. B13 cells grown in SYN plus invertase-treated glucose possessed the same level of constitutive enzyme as THB cells. In contrast to THB cells, the SYN cells of seven serotype strains did not agglutinate upon the addition of high-molecular-weight dextran/glucan. Significant quantities of lower-molecular-weight (2 x 10(4) or 7 x 10(4)) dextran and B13 glucan were bound by SYN cells. SYN cells agglutinated weakly in anti-glucan serum (titers, 0 to 16), whereas THB cells possessed titers of 32 to 256. Evidence for the existence of a second binding site in agglutination which does not possess a glucan-like polymer has been obtained. B13 cells grown in invertase-treated THB agglutinated to the same degree as normal THB cells. The nature of this site is unknown. SYN cells possess the type-specific polysaccharide antigen. B13 cells did not bind from THB a glycoprotein which reacts with antisera to the A, B, or T blood group antigens or which allows agglutination upon the addition of dextran. The results demonstrate that S. mutans grown in a chemically defined medium possesse markedly different biochemical and biological activities than cells grown in a complex organic medium.

Agglutinins

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Animals

Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

The glucosyltransferase (UDP-glucose galactosylhydroxylsine collagen glucosyltransferase, EC 2.4.1.?.) was purified 50-fold from calf arterial tissue by ammonium sulfate precipitation, gel filtration and electrofocusing. The purified enzyme has a molecular weight of 72 000 and a requirement for Mn2. It resolves into two activity peaks when submitted to electrofocusing (isoelectric point at pH 4.2 and 8.1) or disc electrophoresis and exhibits a double pH optimum (pH 8.3 and 9.9). The enzyme was found to transfer glucose from UDP-glucose to the denatured forms of citrate-soluble calf skin collagen (I), the alphal chain (II) and the beta12 component (III) derived from it, and of an acetic-acid-souble collagen preparation (IV) obtained from alkali-treated calf arterial tissue. The Km values for the substrates were 1.67 X 10(-4) (I), 6.3 X 10(-4) (II), 3.3 X 10(-4) (III) and 2.8 X 10(-4) mol/l (IV), indicating that the enzyme has the greatest affinity for the calf skin collagen. The glucose transferred to hydroxylysine-linked galactose residues may be released subsequently by the action of a specific alpha-glucosidase purified from bovine spleen. The results support the assumtion that the glucosylation step in the course of the (pro-)-collagen biosynthesis depends on special structural features of the substrate and may be controlled by a specific alpha-glucosidase.

Animals

Partial purification and properties of glucosyltransferase from Streptomyces aureofaciens.

Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase from Streptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 microM for 1,2-dihydroxy-9,10-anthraquinone (alizarin) 110 microM; the maximum rate of glucosylation reaction was 5.32 mumol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 degrees C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with alpha-hydroxyl groups was about 1/8 of the values obtained with beta-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.

Anthraquinones