Vol. 35, No. I, pp. 55-62, 1990 Printed in Great Britain. A.11 rights reserved

0003-9969/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc

Archs oral Bid.

IMMUNOCHEMICAL SEVERAL STRAINS

STUDIES ON LEVANS FROM OF ACTINOMYCES VISCOSUS

P. Z. ALLENand W. H. BOWEN Department of Microbiology and Immunology and Department of Dental Research, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, U.S.A. (Accepted

6 July 1989)

Summary-High molecular-weight levans elaborated by 8 separate strains of Actinumycesviscosus were purified; the interactions of these levans with concanavalin A and anti-fructan myeloma immunoglobulins UPC-10 and J606 were examined by the quantitative precipitin method. Oligosaccharides released from the levans by partial acid hydrolysis were separated by partition chromatography on paper and characterized m situ by selective spray reagents. The liberated oligomers were compared with oligomers of known structure released from levans of Aerobucfer fmunicum and Leuconosroc mesenteriodes 8512 as well as with plant inulin. The fragmentation analysis indicated a structure for Actinomyces levans comprising chains of b(2 -+ 6)-linked fructofuranosyl units joined through multiple (1,2,6)-linked fructosyl branch units. Key words: plaque bacteria, polysaccharides, Actinomyces.

were also compared with oligomers of known structure liberated from Aerobacter levanicum and Leuconostoc mesenteriodes levans as well as with plant inulin.

1NTRODUCTlON AciiPromyces viscosus colonizes the mouth, constitutes a major bacterial component of dental plaque (Ellen, 1976) and is associated with root-surface caries in humans (Sumney and Jordan, 1974). When grown on media containing suc:rose, A. viscosus produces levan, a polymer of D-fructose (Howell and Jordan, 1967; Krichevsky, Howell and Lim, 1969; Miller et al., 1975; Pabst, 1977). Molecular structures for Actinomyces levans have never been determined, but serological studies with anti-fructan myeloma proteins suggest that /I(2 -+ I)- and fl(2 +6)-linked fructofuranosyl units are structural features (Lundblad et al., 1972; Cisar el’ al., 1974; Pabst, 1977; Warner and Miller, 1978; Pa.bst, Cisar and Trummel, 1979). Differences in type of fructofuranosyl linkage, either predominately /?(2 +r 6) or /3(2 --) l), have been found among levans elaborated by different strains of Streptococcus mutans (Corrigan and Robyt, 1979; Ebisu et al., 1975; Rose11 and Birkhed, 1974). Whether similar differences in molecular structure occur among the l‘ructans produced by different strains of A. uiscosus is not known. We have now examined 18 strains of Actinomyces for their ability to synthesize fructan when grown in synthetic medium containing sucrose. High molecular-weight levans produced by 8 strains of A. uiscosus were isolated in purified form and their interactions with the lectin conca.navalin A and with anti-fructan myeloma proteins IJPC-10 and 5606 were studied by the quantitative precipitin method. We have also explored the molecular structure of Actinomyces levans by fragmentation analysis: oligosaccharide fragments released by partial acid hydrolysis were separated by partition chromatography on paper and then characterized in situ by their reaction with selective spray reagents. Two series of oligosaccharide homologues liberated from A. viscosus levans

MATERIALSAND METHODS Actinomyces strains A. viscosus strains ATTC 15987 (T6), Ny-1, GN 431, AN6, Be 64, HlG3, WVU 627 and A. naeslundii 12104 were provided by George H. Bowden, Department of Oral Biology, University of Manitoba. A. uiscosus strains ATTC 15988 (HS6) and ATTC 27044 (W1053) were obtained from the American Type Culture Collection (Rockville, Md., U.S.A.); strains T14V and T14Av were provided by Dr Arthur Eisenberg (Eastman Dental Center, Rochester, N.Y., U.S.A.), while strains OMZ-105E, X602m, MlOO, A. odontolyticus W 1514 and A. naeslundii W752 and X569 were provided by Wayne Little (N.I.D.R., Bethesda, Md., U.S.A.). Strain OMZ-105E is a streptomycin-resistant variant of strain Ny-1. Actinomyces were grown in Trypticase soy broth (Baltimore Biologicals, Md., U.S.A.) and RPMI-1640 synthetic medium (Sigma Chemical Co., St Louis, MO., U.S.A.), supplemented with 1% glucose or 5% sucrose. Levan production and isolation

Strains of Actinomyces initially grown in Trypticase soy broth with 1% sucrose were pelleted by centrifugation, washed twice with sterile, 50 mM sodium acetate buffer, pH 6.0, and then inoculated into 1 litre volumes of RPMI-1640 medium containing 5% sucrose. Sedimented cells harvested from 1 litre Trypticase soy broth cultures and then washed were also suspended directly in 50 mM acetate buffer (400ml) containing 5% sucrose and 3500 U potassium penicillin G, as described by Miller et al. 55

56

P. Z. ALLENand W. H.

(1975). RPM1 cultures were grown to stationary phase (48 h) at 37°C while acetate buffer-suspended cells were kept at room temperature for 3 days. After centrifugation, levan-containing cell-free media were passed through a 0.3 pm millipore filter, concentrated about lOO-fold by dialysis against Carbowax-PEG 3350 (Union Carbide, Danbury, Conn., U.S.A.), and then deproteinized by shaking with a mixture of N-butanol-chloroform+aprylic alcohol (1: 5: 2 v/v/v). Levan was precipitated from the separated aqueous phase by adding 3 vol 95% ethanol. About 200-250 mg polysaccharide per litre growth medium or buffer were obtained at this stage of isolation. Polysaccharides were air-dried, solubilized in water and finally purified by passage through a column (80 x 2.5 cm) of Sepharose-6B. Purified levan was recovered by lyophilizing the ketosugar peak fraction emerging in the void volume ( VO)of the calibrated column. High molecular-weight levans (molecular weight > 106) isolated in this way were free of protein (O.D.,,,,, < 0.050, at 10mg dry weight/ml) and gave D-fructose as the only sugar detectable by paper chromatography after acid hydrolysis in 2 N sulphuric acid at 100°C for 3 h. Total carbohydrate was determined from the optical density at 490 nm obtained by the phenolsulphuric acid method of Dubois et al. (1956) with D-fructose as a standard. Ketohexose was determined by the method of Dische and Devi (1960). Oligo and po/ysaccharides

D-Fructose, D-glucose, sucrose, raffinose, stachyose, dextran B512 with average molecular weight of 40,000, dahlia tuber inulin and Aer. levanicum levan were purchased from Sigma Chemical Co. L. mesenteroides levan B512 with 10% nonreducing D-fructose end groups, 68% b(2 +6)-linked D-fructose units and 22% /?(1,2,6)-linked branch points (Lindberg, Lonngren and Thompson, 1973) was provided by Allene Jeanes of the Northern Regional Research Laboratories (Peoria, Ill., U.S.A.). The homologous series of fl(l -+2)-linked fructofuranose oligomers, inulobiose (I,), inulotriose (I,), inulotetraose (I,), inulopentaose (I,), inulohexaose (16) and inuloheptaose (I,), was obtained by mild acid hydrolysis of inulin as described below for Actinomyces levans, separated by partition chromatography on large sheets (46 x 57 cm) of Whatman 3MM paper, then recovered from the paper by elution with water.

BOWEN

protein nitrogen procedure of Schiffman (1966). Precipitin determinations were done by adding 40 pg concanavalin A or UPC-10 nitrogen or 17pg 5606 nitrogen to increasing amounts of fructan. The total volume of reactions was adjusted to 0.40ml with saline and mixtures kept at 0°C in an ice bath for 3 days before analysis. Immunodiffusion was done as described by Allen and Pazur (1984). Mild acid hydrolysis and partition chromatography

Preliminary studies had shown that mild acid treatment of A. viscosus levans with 0.5 M acetic acid at 80°C for 30 min gave high yields of oligomers in the di- to octasaccharide range, easily resolved by partition chromatography on paper. For fragmentation analysis, bacterial levans and plant inulin were subjected to these same conditions of hydrolysis at a concentration of IOmg fructan per ml acetic acid. After hydrolysis, samples were recovered by freeze drying. Descending chromatography of hydrolysates was carried out on Whatman 3MM paper with N-propanol+thanol-water (7: 1:2 v/v/v) and N-butanolethanol-water (40: 11: 19 v/v/v) as solvents. Reducing sugars were detected by the silver spray of Trevelyan, Procter and Harrison (1950). Ketosugars were detected by the characteristic blue colour developed by the urea-phosphoric acid spray described by Wise et al. (1955). 2,3,5Triphenyltetrazolium chloride (TTC) was used as a selective spray reagent to distinguish (2,6)-linked fructosides, which manifest 1: 2-enediol isomerization, from (1,2)-linked fructosides, whose ability to reduce TTC is markedly diminished by substitution at the primary hydroxyl group at Cl adjacent to the reducing carbonyl function (Feingold, Avigad and Hestrin, 1956; Zelikson and Hestrin, 1961; Avigad, Zelikson and Hestrin, 1961). Mobility in these solvent systems was expressed as RFruc, the distance migrated by a test sugar relative to the distance migrated by D-fructose. French-Wild plots, log[R,,,,/( 1 - RFruc)] versus degree of polymerization (DP; French and Wild 1953), were constructed for the homologous series of polymers obtained by mild acid hydrolysis of (1,2)-linked fructan (inulin), 8(2,6)-linked fructan (levan BS12E) and various A. viscosus levans. Each homologous series of oligosaccharides generates a straight-line plot whose slope is characteristic for the repeating unit chemical structure.

Antibodies and lectin

Gel filtration

Purified mouse myeloma protein 5606, an IgG, immunoglobulin with strong antifi(2 --) l)- and weak anti-p(2 + 6)-fructofuranosyl specificity (Lundblad et al., 1972; Leiberman et al., 1975) and IgG,, myeloma protein UPC-10 with anti-/I(2 + 6)-fructofuranosyl specificity (Cisar et al., 1974, Vrana, Tomasic and Glaudemans, 1976) were purchased from Litton Bionetics Inc. (Charleston, S.C., U.S.A.). Crystalline concanavalin A was obtained from Pharmacia Chem. Co., Piscataway, N.J., U.S.A.

Oligosaccharides obtained from A. viscosus levans by mild acid hydrolysis were fractionated by gel filtration on a column (82 x 1.5 cm) of Biogel P2. Fractions of 1.7 ml were collected and their sugar content estimated by the phenol-sulphuric acid method. The exclusion volume (I’,,) of the column was determined using dextran B512 with a molecular weight of 40,000. Elution volumes (V,) for reference standards-fructose, sucrose, raffinose stachyose and the homologous series of inulin oligomers, inulobiose (Z2) through inuloheptaose (1,) were determined; their retention coefficients ( VO/V,) were then used to estimate the size and degree of polymerization (DP) of separated unknown oligosaccharides.

Immunochemical methods

Quantitative precipitation of lectin and antibodies by fructans was determined by the micro-ninhydrin

Immunochemical studies on levans from several strains of Acfinomyces uiscosus Table 1. Reaction in agar of mouse anti-fructan myeloma proteins

diffused against the growth medium* used to cultivate various strains of Actinomyces Precipitation with mouse myeloma protein UPC-IO

5606

+ + + + + + + + _ + + + + +

+ + + + + + + + + + + + +

_ +

_ _ +

_

-

Actinomyces viscosus

Strain

OMZ-IOSE X602 Wl-053 T14V T14Av Ml00 HI03 Ny- 1 GN43 I WVU627 AN6 Be64 T6 HS69

Actinomyces naeslundii

Strain

W752 12104 X569

Actinomyces odontolyricus s1514

22 pg nitrogen. Aer. levanicum and L. mesenteroides B512 levans each precipitated 22 pg concanavalin A nitrogen, but dahlia inulin failed to precipitate concanavalin A. Similarly, bacterial levans maximally precipitated 9-11 pg UPC-10 or 5-1Opg 5606 antibody nitrogen, while inulin did not precipitate these anti-fructoside antibodies. Partial acid hydrolysis

*Cell-free culture medium from stationary phase cultures in RPMI-1640 with 5% sucrose, diffused against myeloma immunoglobulin at :! mg/ml. RESULTS

Levan production

Table 1 shows that 13out of 14 strains of A. viscosus and 1 out of 3 strains of A. naeslundii produced levan, as demonstrated by immunoprecipitation with antifructoside immunoglobulins UPC-10 and 5606. Four strains of Actinomyces (GN431, W572, 12104 and Sl514) that failed to produce levan in synthetic RPMI-1640 medium ,~Iso failed to synthesize levan in complex Trypticase soy broth with 5% sucrose. Quantitative precipitation with concanavalin A and anti-fructose immuno,plobulins Text Fig. 1 shows the quantitative course of concanavalin A, UPC-10 and 5606 precipitation with isolated, purified, high molecular-weight levans synthesized by 8 different strains of A. viscosus. Maximum precipitation of concanavalin varied from 9.6 to (A)~oncanovo~in-A

57

(B)

UPC-

Plate Fig. 2 shows the chromatographic separation of oligosaccharides liberated by partial acid hydrolysis of purified levans produced by 8 strains of A. viscosus (T14V, Tl4Av, OMZ-IOSE, W627, H103, Be64, Ny-1, T6). Inulin and Aerobacter levan were included as reference oligosaccharides. Inulin yielded a single, homologous series of b(2 + I)-linked fructofuranose oligomers of increasing unit length (Z,, I,, Id, etc. in Plate Fig. 2). In contrast, however, fragmentation of Aerobacter and Actinomyces levans yielded predominately a recognizably different series of oligomers-the /3(2 + 6)-linked fructofuranoside series of oligosaccharides (L,, L,, L,, etc. in Plate Fig. 2). Like the levan of Aerobacter, Actinomyces levans gave rise to doublets or two series (A and B) of oligomers (Plate Fig. 2 and Table 2). For each degree of polymerization, oligomers of the A series migrated more slowly and were produced in higher yields. To demonstrate release of these two separate, distinct series of homologous oligosaccharides, fructans were partially hydrolysed and the fragments obtained then fractionated, according to molecular size, by gel filtration. Text Fig. 3 shows the separation of A. viscosus Tl4V levan oligosaccharides into oligomers of increasing chain length by gel filtration through a calibrated Biogel P2 column. Isolated fractions containing disaccharide (78-81), trisaccharide (72-75), tetrasaccharide (68-71) and pentasaccharide (64-67) were then examined by partiton chromatography on paper. The disaccharide fraction consisted entirely of levanbiose; inulobiose was not detected in disaccharide fractions obtained from any Actinomyces levan. Plate Fig. 4 shows resolution by paper chromatography, of tri, tetra- and pentasaccharide members of the oligomeric series into two discrete isomers (A and B) of the same molecular size. Silver and urea spray reagents applied to companion chromatographs (Plate Fig. 4) identified both series A and B oligomers as reducing ketosugars. Although IO

Fructon added ( pg I Fig. 1, Quankative precipitation of concanavalin A (A), UPC-10 (B) and 5606 (C) by bacterial levans. Fructans: (A) L. mesenteroides levan B512E; (v) Aer. levanicum; ( q) dahlia inulin. A. uiscosuslevans: (0) T14V; (I)) T14Av; (0) T6; (A) H103; (+) Ny-I; (W) WVU 627; (0) Be64; (0) OMZ-105E.

P. 2. ALLENand W. H. BOWEN

58

Table 2. Characterization by paper chromatography of oligosaccharides released from fructans by partial acid hydrolysis

Fraction

Inulin

TTC’

AU. levanicum

A. viscosust

Levan# B512

Reaction with spray Ag

Urea

TTC

Relative mobility (RFruc)§ N-propanoi-ethyl

o-Fructose Disaccharide Trisaccharide Trisaccharide Tetrasaccharide Tetrasaccharide Pentasaccharide Pentasaccharide Hexasaccharide

A B A B A B

acetate-H,0

+

1.oo

1.oo

1.00

0.62 0.39

-

0.26

-

0.15

-

0.71 0.37 0.43 0.19 0.23 0.08

0.70 0.36 0.45 0.17 0.23 0.09

0.12

+ + + + + + + +

+ + + + + + + +

+ + + + + -

-

0.11 0.03

0.71 0.38 0.45 0.19 0.24 0.09 0.12

1.00 0.75 0.49 0.57 0.31 0.39 0.20 0.24 0.14

+ + + + + + + + +

+ + + + + + + + +

+ + + + + +

N-butanol-ethanol-H,O

D-Fructose Disaccharide Trisaccharide Trisaccharide Tetrasaccharide Tetrasaccharide Pentasaccharide Pentasaccharide Hexasaccharide Heptasaccharide Octasaccharide

B A B A B

(7:1:2)

1.oo

(4O:ll:lO)

1.00

+

1.00

1.00

0.74 0.52

_

0.39

-

0.29

-

0.21 0.15

_

0.73 0.47 0.54 0.30 0.37 0.19 0.22 0.12

0.73 0.47 0.53 0.31 0.34 0.19 0.23 0.13

0.11

*TTC = 2:3:5_triphenyltetrazolium chloride reagent for selective detection of enediols. iIdentical results obtained with levans from A. viscosus strains: T14V, T14Av, OMZ-IOSE, Ny-I, H 103 and WVU627. $Levan B512 from L. mesenteroides. §&ruc = ratio of distance migrated by test sugar relative to that of D-fructose.

Text levan from of A.

Fig. 3 and Plate Fig. 4 present only data from T14V, similar findings were obtained for levans the OMZ-IOSE, X602, Tl4Av and Ny-I strains

inulin series (Table 2) gave a straight line with a significantly less steep slope than that given by other fructoside series examined.

viscosus.

Characterization by mild acid

of oligosaccharide

fragments

released

Ohgosaccharides released by mild acid hydrolysis were separated and characterized by partition chromatography on paper. As shown in Table 2, characterization included RFruc values determined for two different solvent systems, and reactivity with selective spray reagents. As indicated in Table 2, whereas series A oligomers were TTC-reactive, series B oligomers did not manifest enediol isomerization and showed no reactivity with TTC to yield triphenylformazans. Plots of log[R,,,/(l - RFruc)] versus oligosaccharide unit length (DP) produce separate straight lines for A. viscosus oligosaccharides A and B. Plots for Aer. levanicum oligosaccharides A and B were identical to those of the corresponding Actinomyces oligomers and their lines could be superimposed. Plots of data obtained for oligomers of the

DISCUSSION

Of our 14 strains of A. viscosus, only a single strain (GN431) failed to produce levan, as detected by immunoprecipitation with anti-fructan antibodies (Table 1). Similarly, two strains of A. naeslundii and one of A. odontolyticus failed to produce levan (Table 1). Whether this failure can be attributed to a loss or inactivation of levan sucrase structural genes is not known. Each of our 8 Actinomyces levans, obtained in purified form, precipitated with the lectin concanavalin A and with anti-fructan myeloma proteins UPC-10 and 5606. However, the levans showed marked, individual quantitative differences in their extent of precipitation (Text Fig. 1). Quantitative differences in the ability of polysaccharides to precipitate concanavalin A or myeloma proteins have been attributed to differences in the specific solubility of

Plate 1 Fig. 2. Chromatographic separation of oligosaccharides obtained by mild acid hydrolysis of levans from A. viscosus strains Ny-1, Be 64, T6, OMZ-IOSE, T14V, T14Av, H103 and W627. Lanes labelled A. lev. and inulin contain respectively Aer. levanicum levan and dahlia inulin hydrolysates. I,, I,, I, etc. identify p(1 --t 2)-linked series of inulin oligomers of increasing chain length. L,, L,, L, etc. identify B(2 -+ 6)-linked series of oligomers. Pairs of arrowheads point to doublets obtained for each member of the oligosaccharide series liberated from Actinomyces and Aerobacter levans. Solvent: N-propanol-ethyl acetate-H,0 (7: 1: 2, v/v/v); Trevelyan silver spray.

Immunochemical

studies

on levans from several

Plate

I

strains

of Actinomyces

uiscosus

59

60

P. Z. ALLEN and W. H. BOWEN

urea

Plate 2

Immunochemical studies on levans from several strains of Actinomyces uiscosus

20

0

40 Fraction

60

60

100

NO.

Fig. 3. Separation of Actinomyces T14V levan oligosaccharides on Biogel P2 column (1.5 x 82cm). Arrows show exclusion volume (L’,,) and elution positions (V,) of known reference oligomer standards used to estimate molecular size and degree of polymerization. Fractions of 1.7 ml/tube were collected at a flow rate of 5 ml/h. Ordinate shows optical density at 490nm of 20~1 samples of each fraction developed for total sugar by the phenol-sulphuric acid method.

protein-polysaccharide complexes formed by the interaction (So and Goldstein, 1969; Allen and Pazur, 1984). Furanose sugars with the 1,4-anhydro-D-arabinitol moiety provide the appropriate disposition of hydroxyl groups on C-3, C-4 and C-6 or C-l, C-3 and C-4 of the /I-D-fructofuranosyl unit to permit interaction of levan with concanavalin A binding sites (So and Goldstein, 1969). Thus, precipitation of Actinomyces levans with concanavalin A [Text Fig. l(A)] would be expected, mediated either by fi(2 + 6)- or /?(2 -+ I)-linked fructofuranosyl residues, or by both. Similarly, precipitation of fructans with UPC-10 and 5606 antibodies [Text Fig. l(B) and (C)] would be mediated by 11(2-6)and /I(2 + I)-linked fructosyl units respectively. Although interactions with fructan-binding proteins thus provide evidence for the occurrence of /?(2 +6)- and/or fi(2 + l)linked fructofuranosyl units, these interactions provide only limited information about molecular structure. Precipitation (Table 1; Text Fig. 1) could be mediated through (1,2,6)-linked fructofuranosyl units at branch points, as well as by /?(2 -+6)- or b(2 + I)-linked units. Thus, precipitation reactions do not distinguish highly branched from linear molecules. Moreover, such reactions fail to distinguish the (1,2,6)-h nked branch point from a linear series of recurrent or successive adjacent p(2 + 6)- or /?(2 + I)-linked residues. Fragmentation analysis has been used in elucidation of the molecular structure of levans from Strep. mutans (Corrigan and Robyt, 1979), Strep. saliuarius (Hancock, Marshall and Weigel, 1976) and Aer. leuanicum (Zelikson and Hestrin, 1961), so

to obtain further structural information, we fragmented Actinomyces levans by partial acid hydrolysis, characterized the separated oligosaccharide fragments (Plate Figs 2 and 4 and Text Fig. 3) and compared them with one another as well as with fragments of known structure liberated from inulin, Aer. Ieuanicum and L. mesenteroides B 512 levans (Table 2). Inulin consists of a linear chain of fl(2 + l)linked fructosyl units: as shown in Plate Figs 2 and 4 and indicated in Table 2, its partial acid hydrolysis yields a single series of homologous oligomers of increasing /?(2 + I)-linked fructosyl unit chain length. Apart from a small percentage of non-reducing end groups linked only through C-2, Strep. salivarius, Aer. Levanicum and L. mesenteriodes B 512 levans consist entirely of chains of (2 +6)-linked fructosyl units joined through multiple (1,2,6)-linked branch points. (Lindberg et al., 1973; Zelikson and Hestrin, 1961; Hancock et al.. 1976). Partial acid hydrolysis of these levans yields two series (A and B) of oligosaccharides chromatographically distinct from inulin oligomers (Plate Fig. 2 and Table 2). Each series constitutes a distinct family of homologous oligomers for which a French-Wild plot of DP versus log[R,,,/(l - RFruc)] gives a separate straight line with a slope characteristic for the family. Chemical studies on Aerobacter and Streptococcal levans have shown that series A consists of linear chains of fi(2 -+6)-linked fructosyl units, while B series oligosaccharides comprise a sequence of /?(2 -+ 6)linked residues terminating in an intact (I ,2,6)-linked branch unit (Zelikson and Hestrin, 1961; Hancock et al., 1976). The two homologous series of structural isomers can be distinguished from one another by the ability of the A series to undergo 1:2-enediol isomerization and react with triphenyltetrazolium. Both A and B series homologues are readily distinguished from p(2 + I&linked oligomers on the basis of their relative mobilities (RFruc), listed in Table 2, and their French-Wild plots. All our Actinomyces levans yielded, upon partial acid hydrolysis, mixtures of oligosaccharides identical to fragments released from levans of the T14V and OMZ-105E strains shown in Plate Fig. 2 and Table 2. Inulobiose and higher homologues of the b(2 + l)linked fructosyl series of inulin oligomers did not occur among oligosaccharides liberated from any Actinomyces levan. All such levans resembled one another in molecular structure and did not have portions of structure consisting of successive, adjacent /I(2 -+ I)-linked fructosyl units such as are found in Strep. mutans levan (Corrigan and Robyt, 1969). Thus, interaction of Actinomyces levans with 5606 myeloma protein must be mediated through (1,2,6)linked residues rather than by sequences of p(2 + l)linked units. The two homologous series of oligosaccharides (A and B) identified in partial hydrolysates of Actinomyces levans were identical to

Plate 2 Fig. 4. Descending paper chromatographs of Biogel P2 column fractions (78-8 I), (72-75), (6&67) shown in Fig. 3. I*. 1;, I4 etc. mark positions of oligoinulins (inulobiose, inulotriose, inulotetraose etc). Solid arrows identif!/ members A and B and two homologous series of oligolevans obtained from T14V levan. Ag: paper spra.yed with silver to identify reducing sugars; urea: paper sprayed with urea-phosphoric acid to identify ketosugars. OB3511-o

61

P. Z. ALLENand W. H. BOWEN

62

the corresponding oligosaccharides released from the reference Aer. levanicum and L. mesenteroides B512 levans (Table 2). Our chemical and immunochemical data thus indicate a structure for A. viscosus levans made up entirely of chains of /3(2+6)-linked fructosyl residues joined through multiple (1,2,6)-linked branch units. Acknowledgement-This work was supported by National Institute of Dental Research Grant 5 PSO DE07003 to the Rochester Caries Research Center.

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Ebisu S., Kato K., Kotani S. and Misaki A. (1975) Structural differences in fructans elaborated by Streptococcus mutans and Strep. salivarius. J. Biochem. 78, 879-887. Ellen R. P. (1976) Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity. fnfect. Immun. 14, 1119-1124. Feingold D. S., Avigad G. and Hestrin S. (1956) The mechanism of nolvsaccharide nroduction from sucrose. Biochem. J. 64,. 351-361.

*

French D. and Wild G. M. (1953) Correlation of carbohydrate structure with papergram mobility. J. Am. them. Sot. 75, 2612-2616. Hancock R. A., Marshall K. and Weigel H. (1976) Structure of the levan elaborated by Streptococcus salivarius

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