Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S mutans type e antiserum.

S mutans strain MT703 from an active carious lesion in the tooth of a child had type e specificity and showed a cross-reaction with the Lancefield group E cell wall streptococcal polysaccharide antigen. Heat-killed cells MT703 adhered to a glass surface in the presence of CGT MT703 and sucrose. Pretreatment of the cells with anti-MT703 whole cell serums inhibited adherecne. The removal of glycerol teichoic acid antibody and group E antibody from the MT703 serum did not result in a loss of inhibitory activity. Antiserum with or without adsorption significantly inhibited glucan synthesis by CGT from sucrose. Antibodies specific for the polyglycerol phosphate of teichoic acid did not inhibit adherence. Anti-group E serum and serums specific for other types of S mutans, did not show adherence inhibitory activity except for an occasional type c specific antiserum. Antibody specific for the type e antigen produced significant inhibition of the binding of CGT to the MT703 cell wall, and adherence of these cells did not occur. Antibody to CGT inhibited glucan synthesis. Treatment of the cells with dextranase, dextran antibody, or trypsin caused a significant reduction in adherence. The results suggest that the type antigen and dextran on the surface of the S mutans type e cell are functional in adherence, and that these polymers are associated with cell wall protein.

Chemical composition of Streptococcus mutans type c antigen: comparison to type a, b, and d antigens.

Studies of the serotype a, b, and d antigens of S mutans were extended to include the serotype c antigen. Type c antigen was extracted from cells of strain Ingbritt and purified by repeated column chromatography. Two type c antigens fractions were separated by chromatography on DEAE-Sephadex and were designated I and II. On agar gel diffusion, the major components of antigens I and II were serologically identical with the major component of the crude Ingbritt extract. Rhamnose and glucose were present in a 2.4:1 ratio and comprised 98 and 93% of the purified antigens, respectively. The compositions of the c antigens were directly related to the carbohydrate content of the serotype c cell wall. In contrast, purified a, b-II, and d antigens, which are also cell wall polysaccharides contain only trace amounts of rhamnose, a major wall carbohydrate. Studies which used comparative immunoelectrophoresis demonstrated that the purified a, b-I, and d antigens were serologically identical to the antigen preparations that have been used for the immunological classification of these serotypes.

Dextran/glucan binding by Streptococcus mutans: the role of molecular size and binding site in agglutination.

1) S. mutans strains of serotypes a, d and g were strongly agglutinated with soluble glucans and dextran T2000. Homologous glucan did not in all cases produce agglutination. 2) The quantity of low molecular weight dextrans bound (T20 and T70) does not correspond to the agglutination induced by glucan or T2000. 3) The agglutination and binding of high molecular weight glucan by B13 cells was sensitive to heat, trypsin, dextranase, EDTA, SDS and urea, whereas no inhibition of binding of T20 and T70 was seen. 4) Pretreatment of B13 cells with anti-d, or anti-glucan sera, or Con A, RCA I, or RCA II completely inhibited agglutination by T2000 and caused a significant reduction of the binding of glucan. No reduction in the binding of T20 and T70 occurred. 5) An agglutination-negative mutant was agglutinated by sucrose but not by T2000 or high molecular weight glucan. It bound normal levels of T20 and T70. 6) The results indicate that B13 cells possess multiple glucan binding sites and that the site responsible for agglutination consists of both polysaccharide and protein. 7) Inhibition studies on agglutination and adherence using B13 cells indicate that the two processes involve different mechanisms.

Detection of polysaccharide, teichoic acid, and protein antigens in bacterial colonies on an agar surface.

An improved method of using fluorescein-labeled antibody for the detection of polysaccharide, protein, and teichoic acid antigens synthesized by streptococcal colonies on an agar surface is described. The bacteria were grown on the surface of an agar medium contained in the shallow well of an immunodiffusion slide. An agar overlay containing the fluorescein antiserum was dispensed over the colonies, excess antiserum was washed out of the overlay agar, and the fluorescent colonies were observed under an ultraviolet microscope. The shallow well in the immunodiffusion slide prevented the agar from floating loose during washing, and the agar overlay prevented the fragmentation and loss of colonies. The thin layer of agar facilitated microscopic examination and the counting of fluorescent and nonfluorescent colonies. Colonies producing an antigen against which the antiserum was directed could readily be distinguished from colonies not producing the antigen. The specificity of the method was shown by using mixtures of streptococci representing six serological groups and five types. Those not known to possess cross-reacting antigens were specific in their reaction to the fluorescein antibody. Cross-reactions between the group antigens of A, C, and G, as reported previously by fluorescent staining of streptococcal suspensions, were also seen. Group A colonies reacted weakly with fluorescent E antibody and vice versa. The extraction of this antigen with cold trichloroacetic acid indicates it was related to the teichoic acids. Colonies possessing polysaccharide, protein, and teichoic acid antigens gave equally strong fluorescent reactions. This procedure permits detection of the synthesis of antigen which could not be observed by the use of a selective medium; it also eliminates the necessity for subculture of each colony and testing by appropriate serological means. Such a technique has value for studies in classification and biochemical genetics, and should be applicable to other genera of bacteria.

Binding of deoxyribonucleic acid by cell walls of transformable and nontransformable streptococci.

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.

[Detection by hemagglutination of antibodies to group A and group E streptococci by the use of O-stearoyl derivatives of their cell wall carbohydrate-grouping antigens].

The streptococcal group A and group E cell wall polysaccharide antigens were extracted with trichloroacetic acid from the cell or cell wall and esterified with stearic acid. The stearoyl derivatives contained 5 to 8% (by weight) of the ester. Sheep or human red blood cells were sensitized with the esterified antigens and were shown to agglutinate in the presence of specific rabbit antisera. Sera from (i) children hospitalized with group A streptococcal respiratory disease and (ii) swine possessing group E streptococcal lymphadenitis were shown to possess antibody titers significantly higher than the controls. The use of the two esterified antigens as controls for each other established the specificity of the reaction in each case. The general shape of the antigen-antibody precipitin curves was not changed when the stearoyl antigens were used; however, the quantitative aspects differed markedly. Oligosaccharides which inhibit the normal antigen-antibody precipitin reaction did not inhibit the hemagglutination reaction. The adsorption of antisera with whole streptococcal cells reduced the hemagglutination titer in relation to the quantity of cells employed. Data are given on the (i) optimal concentration of stearoyl antigen for sensitization, (ii) time of adsorption of antigen to red cells, (iii) use of albumin as diluting fluid, and (iv) condition of red cells. Properties of the esterified antigens and the mechanism of the agglutination reaction are discussed. The results indicate that polysaccharide antigens of other bacteria may be esterified and employed in a similar manner.

Composition and properties of a group A streptococcal teichoic acid.

Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.

Immunochemistry of the streptococcal group R cell wall polysaccharide antigen.

The group R streptococcal group antigen has been shown to be a polysaccharide located at the surface of the cell wall of the organism. The antigen was extracted from cell walls in 0.05 n HCl or 5% trichloracetic acid at 100 C, from whole cells at room temperature in 0.85% NaCl or 0.1 m acetate (pH 5.0), and by sonic oscillation. The antigen is largely destroyed when extracted from whole cells in 0.05 n HCl at 100 C. Acetate is recommended for routine extraction. The antigen extracted by sonic treatment was separated into six immunologically active fractions on diethylaminoethyl-Sephadex. The fractions were found to possess a common antigen which exhibited similar properties on immunodiffusion and immunoelectrophoresis. The purified antigen did not react with any other streptococcal group antisera. Adsorption of group R serum with the antigen removed all antibodies against whole cell antigen extracts of R cells. Chemical and enzymatic analysis of three fractions showed that the antigen was composed of d-glucose, d-galactose, rhamnose, and glucosamine. No significant quantities of phosphorus, glycerol, ribitol, or muramic acid were present. Significant inhibition of the quantitative precipitin determination by d-galactose and stachyose indicated that galactose in terminal alpha linkage was the immunodominant hexose in the antigen. d-Glucose and d-glucosamine possessed a partial inhibitory activity. N-acetyl-d-glucosamine and l-rhamnose did not produce significant inhibition. The results indicate that the R antigen is an immunologically specific structure which serves as a reliable means of identification of these streptococci as a serological group.

Mechanism of the Adherence of Streptococcus mutans to Smooth Surfaces III. Purification and Properties of the Enzyme Complex Responsible for Adherence.

Enzymes which possess the ability to cause the adherence of Streptococcus mutans cells to a smooth glass surface were purified 1,100 times by chromatography on agarose gel followed by hydroxylapatite gel. During the purification procedures, the enzymes from strain HS6 (group a) were examined for the synthesis of water-soluble and water-insoluble polysaccharide and the ability to produce adherence. The enzyme preparations producing adherence of the S. mutans cells in the presence of sucrose possessed a molecular size of about 400,000 to 2,000,000 and were composed of approximately equivalent amounts of dextran and levan sucrases and 5 to 30% polysaccharide. The most highly purified preparation contained a negligible amount of contaminating protein as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and gel diffusion. In these three tests, the location of the enzyme responsible for the synthesis of insoluble polymer was detected by embedding or covering the enzyme-containing gel with a layer of sucrose-containing agarose gel and observing the formation of insoluble polymer. During purification the ability of all fractions to produce adherence was parallel with the enzyme activity responsible for the synthesis of insoluble polysaccharide from sucrose. About two-thirds of the sucrase enzyme complex in the S. mutans culture fluid synthesized water-soluble polymer. This complex, obtained by filtration through agarose gel, was smaller in molecular size, lower in sugar content, and did not produce adherence, in contrast to the enzyme complex which possessed adherence activity. The inhibition of the enzyme complex synthesizing soluble polymer required more anti-synthetase serum than that required to inhibit the synthesis of water-insoluble polymer. It is not known whether the lack of adherence activity in this enzyme was due to its smaller size and lower sugar content or the absence of unknown factors which are essential for its activity. The carbohydrate in these enzyme preparations, composed of glucose, may represent a primer molecule and/or a remnant of the polymer synthesized by the enzyme. The enzyme activity was not inhibited by anti-dextran globulin.

Mechanism of adherence of Streptococcus mutans to smooth surfaces. I. Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation.

The mechanism of adherence of Streptococcus mutans to smooth glass surfaces has been studied. The results with both viable and heat-killed cells showed that the process required (i) the synthesis of a water-insoluble dextran-levan polymer by cell-bound enzymes and (ii) the participation of a binding site on the surface of the S. mutans cell. Synthesis of the polymer from sucrose in the presence of the cells was required for adherence, and indicates that an "active" form of the polymer was required. Polymer synthesized by cell-free S. mutans enzymes when added to S. mutans cells did not produce adherence. Purified antibody globulin, specific for the a-d site in the polysaccharide S. mutans group a antigen, completely inhibited adherence. Antibody to the second antigen present in the polysaccharide molecule, the a antigen, did not inhibit adherence. The evidence indicates that adherence did not require an antigenic binding site which might be common to all S. mutans strains. The orientation of the synthetase enzyme(s), antigenic binding site, and dextran-levan polymer on the cell surface is under study.

Production and properties of an extracellular bacteriocin from Streptococcus mutans bacteriocidal for group A and other streptococci.

An extracellular bacteriocidal substance is produced by a serotype c strain of Streptococcus mutans in liquid meduim during the stationary phase of growth. The lethal effect of the substance was demonstrated by the decrease in viable counts of a standardized suspension of group A streptococci in broth. No lysis of affected cells was observed and no changes in appearance of these cells was seen in electron micrographs. The material was effective against certain strains of immmunological groups A, C, D, G, H, L, and O streptococci. It was inactive against strains of S. mutans belonging to the a, b, c, and d serotypes, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The factor was purified 273-fold from the culture fluid by column chromatography. It was sensitive to trysin and Pronase and resistant to catalase. It possessed a molecular weight of more than 20,000 and was not dialyzable. The properties of this substance indicate that it is a bacteriocin. Group A streptococci, which had been treated with antiserum specific for the cell wall group and type antigens, were susceptible to the bacteriocin. Streptococcal strains resistant to the lethal action of the bacteriocin adsorbed the bacteriocin from the solutions, as did the sensitive cells. The bacteriocin was not adsorbed at 0 C.

Properties of a Streptococcus sanguis (group H) bacteriocin and its separation from the competence factor of transformation.

STH(1), a streptocin elaborated by group H streptococcus strain Challis, is lethal for group H streptococcus strain Wicky and is produced maximally during the exponential growth phase of liquid medium cultures. Crude streptocin preparations are resistant to oxidation and display a biphasic pH stability (stability being maximal at pH 5.0 and 10.0). Survivor studies indicate that streptocin-mediated killing is a "one-hit" phenomenon and proceeds rapidly. The streptocin has been purified 50-fold with (NH(4))(2)SO(4) fractionation and Sephadex G100 chromatography and appears to exist in equilibrium between two molecular weight forms. Low ionic strength and neutral pH buffers favor the isolation of the 110,000 molecular weight form, whereas high ionic strength and alkaline pH conditions facilitate isolation of the 28,000 to 30,000 molecular weight form. These findings suggest an association-dissociation relationship between macromolecules of 28,000 to 30,000 molecular weight. Purified STH(1) has no "competence factor" (CF) activity. In addition, CF has no STH(1) activity and displays no inhibitory effect on exponential-phase Wicky cultures as determined by absorbancy measurements. It appears, therefore, that initiation of the competent state for transformation in strain Wicky is not necessarily accompanied by gross alterations in cell growth.

Purification and characterization of Streptococcus mutans group d cell wall polysaccharide antigen.

The Streptococcus mutans group d antigen of strain B13 has been purified and characterized with respect to chemical composition and immunochemical properties. The antigen was extracted from lyophilized cells or cell walls by using 5% trichloroacetic acid at 5 C for 16 h. The antigen could also be extracted with water or 0.01 N HCl at 100 C for 20 min. The antigen was purified by ion-exchange and gel chromatography and was found to contain 96% carbohydrate, 1.6% protein, and 0.3% phosphorus. Characterization by gas chromatography indicated that the polysaccharide was composed of galactose and glucose in a 2:1 ratio. The antigen contained two serologically active sites: one site specific for group d and a second site common to both group d and group a strains. Agar diffusion and immunoelectrophoresis indicated that the two sites existed on a single molecule. The immunological specificity of the group d polysaccharide site depended on a terminal d-galactose. The purified B13 antigen did not react with antisera specific for the glycerol teichoic acid from streptococci. Anti-d serum rapidly agglutinated whole cells, indicating that the antibody receptor sites of the polysaccharide antigen were at the surface of the streptococcal cell.

Mechanism of adherence of Streptococcus mutans to smooth surfaces. II. Nature of the binding site and the adsorption of dextran-levan synthetase enzymes on the cell-wall surface of the streptococcus.

The mechanism of adsorption of the Streptococcus mutans enzymes responsible for the synthesis of insoluble dextran-levan to the S. mutans cell-wall binding sites has been studied. Certain characteristics of these binding sites are presented. The adsorption of these enzymes to the cell surface occurred rapidly without the addition of a source of energy and over a pH range of 3 to 11. The adsorption was inhibited by soluble dextran, probably due to the strong affinity of the polymer to the enzyme. All other polymers and sugars studied showed little or no inhibition. The adsorption was also inhibited by antibody globulin to the a-d immunologically specific group antigen surface polysaccharide of S. mutans and by anti-dextran globulin. The inhibition by anti-a-d globulin is considered to be due to a restriction of access of enzyme to the binding site of the enzyme which may be located in close proximity to the group antigen. On the other hand, anti-dextran globulin appeared to directly inhibit the adsorption by covering the binding site. Dextranase destroyed the binding site and released glucose from the S. mutans cells. These data indicate that S. mutans grown in media containing glucose possesses a small amount of dextran on the cell surface, and that this dextran is, or is a part of, the binding site for enzymes which synthesize the insoluble dextran-levan polymer. Trypsin inhibited the synthesis of insoluble polysaccharide and the adherence of cells. It is not clear in this case that destruction of the binding sites occurred. These data present a partial explanation of the processes which may be concerned in the formation of dental plaque on the smooth surfaces of teeth.

Purification and immunochemical characterization of type e polysaccharide antigen of Streptococcus mutans.

The type-specific antigen of Streptococcus mutans strain MT703, serotype e, has been chromatographically purified and characterized. Two chromatographic fractions were obtained from saline extracts which reacted with both anti-MT703 whole-cell serum and Lancefield group E serum. The major fraction (eI) was identified as a polysaccharide composed of 37% glucose, 56% rhamnose, 5% protein, and 0.3% phosphorus, whereas the minor fraction (eII) contained 66% protein in addition to 10% glucose and 17% rhamnose. The immunological specificity of these antigens was found to be the same by immunodiffusion in agar gel. Another fraction with a negative charge (eIII) reacted with polyglycerophosphate antisera from Streptococcus mutans and Streptococcus pyogenes. For comparison, the MT703 antigen in a hot trichloroacetic acid extract (eA) and the group E antigen from a saline extract of cells of strain K129 (EI) were similarly purified by anionic ion-exchange chromatography. Although the ratio of glucose and rhamnose in eA was 1:0.9 and in eI and eII approximately 1:1.5, reactions of identity were obtained in gel diffusion against specific anti-e serum. This difference in ratio is probably a result of the extraction procedures. Both the type e and group E antisera were reactive with both eI and EI antigens. The adsorption of group E antiserum with MT703 cells removed all E antibody, whereas type e-specific antibody remained after adsorption with K129 cells. These results suggest that eI antigen possesses both e and E specificities, whereas EI possesses E only. These findings were supported by the quantitative precipitin test and immunodiffusion and/or immunoelectrophoretic patterns in agar gel. Methyl-beta-D-glucopyranoside markedly inhibited the precipitin reaction in both type e and group E sera. However, a significantly stronger inhibition by cellobiose of type e serum than of group E serum indicates that a beta-linked glucose-glucose dimer is the predominant antigenic determinant of the e specificity. The presence of both e and E specificities on a single polysaccharide molecule was demonstrated by the use of purified e antigen released from a specific e-anti-e complex. This antigen reacted with a group E-specific serum as well as a type e-specific serum. An examination of five S. mutans type e strains showed the presence of group E specificity also, whereas the I, II, and IV serotypes of group E streptococci only possessed the group E specificity.

Chemical composition and immunological specificity of the streptococcal group O cell wall polysaccharide antigen.

The group O streptococcal group antigen was shown to be a polysaccharide located in the cell wall of the organism. The antigen could be extracted by one of several methods: (i) 0.5 n NaOH at 37 C, (ii) phenol-water (50:50) at 68 C, (iii) 0.2 n HCl at 100 C, or (iv) 10% trichloroacetic acid at 4 C. The last method yielded more polysaccharide with less protein contamination. The polysaccharide was purified on diethylaminoethyl-Sephadex A-25 and Sephadex G-200. It was composed of two-thirds glucosamine and galactosamine, and the remainder glucose plus galactose. Rhamnose, glycerol, ribitol, and muramic acid were absent. Total phosphorus and amino acids were each less than 0.1%. N-Acetyl-beta-d-glucosamine exerted a strong inhibition of the precipitin reaction and is considered the immunodominant sugar. Glucosamine and glucose possessed a partial inhibitory activity. Galactose and galactosamine were essentially negative. No evidence of cross-reactivity was found between the O polysaccharide and group A and L polysaccharides, and group A and Staphylococcus aureus teichoic acids, which posesss N-acetylglucosamine specificity. The release of limited quantities of N-acetyl-glucosamine from its terminal location by enzyme, and glucose by acid hydrolysis, indicates a limited number of side chains in the O antigen. The glucosamine is in acid-stable linkage in the polysaccharide. Glucose was not released by beta-glucosidase and probably does not occupy a terminal position. The O antigen is the only known streptococcal polysaccharide antigen which does not contain rhamnose. The effect of these factors on the immunological specificity is discussed. O serum, after adsorption with the purified polysaccharide, was used to demonstrate the presence of protein antigens in acid extracts of cells from each of the nine strains examined. These antigens may represent type antigens. Two of these strains, originally described as group O, did not contain the O polysaccharide.

Peptidoglycan composition and taxonomy of group D, E, and H streptococci, and Streptococcus mutans.

The qualitative and quantitative composition of the peptidoglycan from the cell wall of groups D, E, and H streptococci, and Streptococcus mutans, was determined. In group D, S. faecalis and the closely related species S. liquefaciens and S. zymogenes were separated from S. faecium and the closely related species S. durans on the basis of their peptidoglycan composition. A relationship among S. bovis, S. equinus, and some strains of S. mutans was indicated by the presence in each of a similar peptidoglycan containing threonine. Threonine was released from the S. mutans polymer as a threonine-lysine dipeptide. Hydrolysis of the dipeptide at 100 C for 24 hr in 6 n HC1 was required to break the peptide bond. Motile group D streptococci possessed a peptidoglycan of the same composition as S. faecium. Group E and H strains were also similar in the composition of their peptidoglycan. The results demonstrate that peptidoglycan composition can be used to (i) aid in the division of members of an immunological group into subgroups, and (ii) indicate a relationship between members of the same genus which are not related on an immunological basis.

Immunological and chemical studies of the streptococcal group O protein type antigens.

Two antigens present in group O streptococci have been described and are designated as type antigens. No other antigens are present in extracts of group O cells in significant quantity. Antigen I is a cell wall protein, and II is a nucleoprotein of undetermined cell location. The immunological specificity of the latter resides only in the protein. Both antigens have been extracted from the cell with water or 0.2 n HCl (pH 2) at 100 C, and antigen I can be removed by trypsin. After purification, pH 2 was found to destroy the serological activity of each antigen. Antgens I and II and also the O polysaccharide antigen can be distinguished on a single agar-gel diffusion plate with group O whole-cell antiserum. N- and C-terminal amino acid analyses and total amino acid composition prove that I and II are separate molecular species. Each possesses properties which separate them from the streptococcal M, R, and T proteins. Antigen I separated into three fractions, each possessing serological specificity, on a diethylaminoethyl-Sephadex column at pH 8.6 during an increase in buffer molarity. The protein is considered to possess a multiple unit structure. The results indicate that the O streptococci are limited to two serological types. Group O whole-cell antisera, after adsorption with a strain not containing the O polysaccharide, can now be prepared for the positive identification of the types of group O streptococci.