Vol. 32, No. 3INFECTION AND IMMUNITy, June 1981, p. 1105-11120019-9567/81/061105-08$02.00/0

Regulation of Extracellular Slime Production by Actinomycesviscosus

TAKASHI OOSHIMA AND HOWARD K. KURAMITSU*

Department ofMicrobiology-Immunology, Northwestern University Medical and Dental Schools, Chicago,Illinois 60611

Extracellular slime polysaccharides produced by two Actinomyces viscosusstrains, T14V and T14Av, were compared. In various media containing glucose,T14Av produced abundant extracellular viscous slime polysaccharide, whereasT14V produced lower levels. Furthermore, fractionation of these polysaccharidesshowed that the two extracellular polysaccharides differed in molecular size andnet charge. Since there was a significant difference in the relative abilities ofchemically defined medium and chemically defined tissue culture medium tosupport slime production by T14Av, the nutritional factors influencing the pro-duction of extracellular slime were examined. Sodium bicarbonate was demon-strated to stimulate both cellular growth and the production of extracellularslime. In chemically defined medium with and without sodium bicarbonate, strainT14Av produced large quantities of viscous slime in glucose and sucrose media.In contrast, relatively low levels of slime were produced in fructose, lactose,raffinose, and inositol media, even though sodium bicarbonate stimulated thegrowth of T14Av in these latter media.

Actinomyces viscosus has been shown to pro-duce extensive plaque deposition, root surfacecaries, and massive bone loss characteristic ofperiodontal disease when inoculated into exper-imental animals (6, 8). Some strains of A. vis-cosus produce extracellular viscous slime poly-saccharide, and a role for the extracellular pol-ymer in plaque formation has been proposed (7,12, 14-16). In a recent investigation using bothvirulent strain T14V ofA. viscosus and avirulentstrain T14Av, it was demonstrated that T14Av(which produces abundant extracellular slime)colonized rat teeth very poorly, whereas T14V(a poor producer of slime) could readily colonizetooth surfaces of the animals (1). Furthernore,this difference in the ability of the two strains tocolonize tooth surfaces may result from the pres-ence of a thick slime layer covering the surfaceof strain T14Av which is absent in strain T14V(1, 2). The cell-associated slime layer may inter-fere with the recently proposed fibril-mediatedattachment ofA. viscosus to saliva-coated toothsurfaces (18). Previous investigations with A.viscosus strains isolated from rodents have dem-onstrated that two of these strains, T6 (14) andNyl (15), produce abundant extracellular slime.Chemical analysis of partially purified slimepreparations revealed that these high-molecu-lar-weight polymers contain large amounts ofglucosamine with lower levels of other sugars(14,15). However, a comparable analysis of slimepolysaccharides produced by human oral iso-lates of A. viscosus has yet to be reported. The

present investigation was initiated to compareextracellular polysaccharide production by theA. viscosus strains T14V and T14Av, which areof human origin. In addition, the regulation ofextracellular hexosamine-rich polysaccharideformation by these two organisms was examinedto identify the environmental factors affectingslime production.

MATERIALS AND METHODSOrganisms and media. A. viscosus T14V and

T14Av were kindly supplied by F. C. McIntire (Uni-versity of Colorado, Denver) and S. Brecher (ForsythDental Center, Boston), respectively. Each organismwas maintained in Trypticase soy broth (TS) (BBLMicrobiology Systems, Cockeysville, Md.) at 4°C androutinely transferred biweekly in TS broth.

Batch cultures of the organisms were grown in TSbroth, Todd-Hewitt broth (Difco Laboratories, De-troit, Mich.), brain heart infusion broth (Difco), chem-ically defined medium (5) supplemented with 0.01%inositol (CD medium), and Dulbecco tissue culture(TC) medium (GIBCO Laboratories, Grand Island,N.Y.). The organisms from stock cultures were inoc-ulated into 10 ml of TS broth and grown for 18 h at37°C. These cultures were harvested, washed withsaline, and used to inoculate the indicated media witha 2% inoculum.Growth conditions. The cultures were normally

incubated at 37°C aerobically as static cultures for theindicated time intervals. Anaerobic growth conditionswere obtained by placing the cultures in the GasPakanaerobic system (BBL).

Cellular concentrations were estimated by measur-ing cell turbidities with a Klett-Summerson colorime-

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1106 OOSHIMA AND KURAMITSU

ter (no. 54 filter). Viscous polysaccharide did not in-terfere with the turbidimetric estimation of bacterialgrowth, since the turbidities of culture samples were

almost the same as those of the same cell suspensionswashed with 1 M NaCl (three times) and suspendedin the original volume of medium. pH adjustments ofthe media were carried out by adding 1 N NaOH or 1N HCl.

Isolation of extracellular slime. Extracellularslime was isolated according to the procedures ofRosan and Hammond (14) with slight modifications.The cells were grown in complex medium (TS, brainheart infusion, or Todd-Hewitt broth) at 37°C for 24h, and the culture supernatant fluids were collectedafter centrifugation at 10,000 x g for 10 min. Threevolumes of acetone was added to the culture super-

natant fluids, and the precipitates formed were allowedto settle at 40C for 18 h. The precipitates were thencollected by centrifugation and suspended with 10%trichloroacetic acid for 4 h at room temperature. Thesuspensions were next centrifuged at 10,000 x g for 10min, and an equal volume of acetone was added to thesupernatant fluids. The precipitates that formed were

again allowed to settle at 4°C overnight and collectedby centrifugation. The precipitates were dissolved indistilled water and dialyzed against distilled water at4°C for 18 h. The slime solutions were finally lyophi-lized, and the dry weights were determined. In chem-ically defined (CD and TC) media, slime was isolatedby modifying the standard procedure; i.e., 3 volumesof acetone was added to the supernatant fluids of the10% trichloroacetic acid suspension, since little precip-itate was obtained when an equal volume of acetonewas added.

Procedures for viscosity measurement. Theviscosities of culture supernatant fluids were measuredby using a Cannon-Fenske viscometer with a centi-stoke range of 0.8 to 4 in a water bath at 18°C. Effluxtimes were measured three times for each sample witha stopwatch. There was little variation in flux rates fora given sample. The increase in viscosity was calcu-lated by subtracting the viscosity value (centistokes)of noninoculated medium from that of the culturesupematant fluid.

Fractionation of extraceflular slime. Samples(15 mg of dry weight) of the lyophilized slime were

fractionated initially by gel filtration chromatographyon a Sepharose 4B column. The column (1.5 by 90 cm)was developed at 4°C with 0.01 M phosphate buffer(pH 7.0), and 4.5-ml fractions were collected. Thehexosamine-rich fractions (T14Av, fractions 6 through13; T14V, fractions 14 through 20) were then pooled,dialyzed against distilled water, and lyophilized. Tofurther purify and compare the polysaccharide frac-tions, samples (10 mg of dry weight) of the partiallypurified slime were subjected to ion-exchange chro-matography on diethylaminoethyl-Bio-Gel A (1.5 by30 cm). Samples were eluted with a gradient from 0 to0.5 M NaCl in 0.05 M tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 7.5). All fractionswere assayed for hexosamine, hexose, and proteincontent.Chemical analyses. The total hexoses were deter-

mined by the anthrone procedure (19); protein was

measured according to Lowry et al. (11). Hexosamine

content was assayed according to Levy and McAllan(10). The optimal conditions for release of hexosaminefrom the slime samples were determined to be incu-bation with 2 N HCl at 100°C for 3 h.

RESULTSMeasurement of slime production. To de-

velop a convenient assay for A. viscosus slimeproduction, we examined initially the relation-ship between the increase in viscosity of theculture fluids and the levels of hexosamine inacetone-precipitated slime samples (Table 1).This relationship was previously suggested byusing A. viscosus Nyl (15). The amounts ofhexosamine in the precipitated slime samplesfrom strains T14Av and T14V were measuredand correlated with viscosity changes of thesupernatant fluids. In strain T14Av, the increasein hexosamine in the slime correlated well withthe increase of viscosity of the correspondingculture fluids. However, although strain T14Vproduced hexosamine-containing precipitablematerial, there was essentially no increase in theviscosity of the culture fluids. Thus, the acetone-precipitable polysaccharide made by strainT14V differed markedly from that produced bythe avirulent strain T14Av. In addition, theseresults indicated that measurements of culturefluid viscosity could be related to extracellularslime production by strain T14Av but not bystrain T14V.

TABLE 1. Relationship between the increase ofculture fluid viscosity and the amount of

hexosamine in polysaccharide samples produced byA. viscosus T14Av and T14V0

T14Av T14V

Incuba- Avstion AVi' AHex- AViscos- AHex-

time (h) cosity osamine ity (centi- osaminestokes)' (ug/mI)C stokes) (ug/mi)

0 0 0 0 03 0.03 ND -0.01 0.356 0.05 0.48 -0.01 0.439 0.07 0.72 0 0.6312 0.16 1.23 -0.01 0.3615 0.18 1.81 -0.01 0.7618 0.22 2.68 -0.01 1.13

aEach organism was grown at 37°C, and 10-mlportions were removed at the indicated time intervals.Measurements of viscosity and the amount of hexos-amine in supernatant fluids were carried out as de-scribed in the text.

'All values were calculated by subtracting the vis-cosity value (centistokes) of noninoculated mediumfrom those of the culture supernatant fluid.

c Acetone precipitates of culture supernatant fluid(all values corrected for hexosamine present in acetoneprecipitates from sterile medium). ND, not deter-mined.

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A. VISCOSUS SLIME PRODUCTION 1107

Growth of A. viscosus and extracellularslime production in TS broth. To assess therole of cellular growth on slime production, cellconcentrations of strains T14V and T14Av weremeasured, and the production of extracellularviscous slime was followed by measuring theviscosity of culture supernatant fluids (Fig. 1).In TS broth, strain T14Av produced an abun-dance of slime during the logarithmic phase ofgrowth, but T14V produced no detectable vis-cous slime at any time. Furthermore, slime pro-duction by strain T14Av was coincident withgrowth and ceased when the cells entered theearly stationary phase.Slime production in various media. Strain

T14Av produced significant levels of viscousslime in all media used, with the lowest yields inthe CD medium (Table 2). By both the criteria

02 200

z~~~~~~~~~~t 0.1 voo1

8 24

0 6 12 Is 24INCUBATION TIME (h)

FIG. 1. Growth of A. viscosus and extracellularslimeproduction in TS broth. The cells were culturedin TS broth (100 ml), and cell concentration and theviscosity ofculture supernatant fluids were estimatedas described in the text. Symbols: (0) Viscosity ofT14Av culture supernatant fluids; (0) cell growth ofT14Av; (A) viscosity of T14V culture supernatantfluids; (A) cell growth of T14V.

TABLE 2. Slime production by A. viscosus T14Av invarious mediaa

AViscos- AHexosa-Medium ity (centi- mine (ug/

stokes)' mflY

TS broth 0.22 1.89Todd-Hewitt broth 0.42 2.80Brain heart infusion broth 0.13 3.81CD medium 0.03 1.68TC medium 0.50 3.66

aGrowth conditions, measurements of viscosity,and determination of hexosamine were carried out asdescribed in the text.

'Change in culture viscosity relative to sterile un-inoculated media.

c Increase in culture fluid hexosamine in acetoneprecipitates relative to sterile uninoculated media.

of hexosamine-containing polysaccharide pre-cipitation and viscosity changes, strain T14Vproduced little slime in any medium (data notshown). In each medium, cultures of T14V andT14Av reached nearly maximal cell density atthe time of harvest. Changes in medium viscos-ity produced by growth of strain T14Av did notalways correspond directly with hexosamine-containing polysaccharide precipitation.Nutritional factors influencing the pro-

duction of slime. Since there was a significantdifference in the relative ability of CD and TCmedia to support slime production by strainT14Av (Table 2), it was of interest to determinethe molecular basis for this observation. UnlikeTC medium, CD medium contained no CaCl2,KCl, NaHCO3, choline chloride, or sodium py-ruvate and relatively low levels of NaCl, panto-thenate, folic acid, and pyridoxine. Since anyone of these components could be responsiblefor the difference in the relative capacities of thetwo media to support slime production, each wasadded separately to CD medium, and slime pro-duction was measured. Sodium bicarbonate wasdemonstrated to markedly stimulate both thegrowth of T14Av and the production of extra-cellular slime in CD medium (data not shown).None of the other potential nutrients producedsignificant increases in slime production. In ad-dition, we examined cell growth and slime pro-duction in CD medium containing 44 mMNaHCO3 to determine whether slime productionwas influenced by the growth stage in this de-fined medium. As in TS broth, slime productionparalleled growth in CD-NaHCO3 medium (datanot shown). The increase in hexosamine-con-taining polysaccharide was also paralled by anincrease in culture viscosity. Since the presenceof NaHCO3 stimulated both growth and slimeproduction, the effects of other carbonate salts(Na2CO3 and K2CO3) on cell growth and slimeproduction were evaluated (Table 3). At lowlevels of NaHCO3 (<9 mM), both cell growthand slime production were stimulated. However,at higher concentrations of NaHCO3 (>18 mM),slime production appeared to be differentiallystimulated. Addition of Na2CO3 to CD mediumstimulated both cell growth and slime produc-tion at lower concentrations. However, at highconcentrations of either Na2CO3 or K2CO3 (>18mM), strain T14Av did not grow well. Additionof K2CO3 also stimulated cell growth at lowconcentrations, but the stimulatory effects onslime production were less striking than those ofNaHCO3 and Na2CO3. These results suggestedthat pH or the presence of C02, or both, stimu-lated cell growth and slime production. Subse-quently, we examined the effects of pH in CDmedium without the carbonate salts under aero-

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1108 OOSHIMA AND KURAMITSU

bic and anaerobic conditions (Table 4). Underanaerobic conditions, cell growth and slime pro-duction were both stimulated, and pH 6.9 ap-peared to be optimal for cell growth and slimeproduction. When CD media containing equalconcentrations of NaHCO3, Na2CO3, or K2CO3were adjusted to pH 6.9, maximal cell growth

TABLE 3. Effects ofNaHCO3, Na2CO3, and K2CO3on slime production and cell growth ofA. viscosus

T14Ava

Cell SlimeAddition Concn pH of growth produc-Adiin (mM) medium (AKlett tion

unt)stokes)

None 6.60 110 0.03NaHCO3 4 6.65 355 0.12

9 6.80 390 0.1618 6.85 450 0.2626 6.90 500 0.3235 7.00 550 0.3644 7.05 550 0.40

Na2CO3 4 6.90 410 0.199 7.20 465 0.2918 7.80 350 0.04

K2CO3 4 6.90 380 0.069 7.25 350 0.0918 7.80 120 0

a Strain T14Av cells were grown in 10 ml of CDmedium at the indicated initial pH values for 18 h.

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and slime production occurred in the presenceof NaHCO3. Media containing Na2CO3 or K2CO3at this pH displayed similar levels of growth, butmore slime was produced in the presence ofNa2CO3 than K2CO3 (data not shown).Slime production in the presence of var-

ious sugars. To determine the effects ofvariouscarbon sources on slime production, strainT14Av was grown in CD medium with and with-out 44 mM NaHCO3 in the presence of 1%glucose, sucrose, fructose, lactose, galactose, raf-finose, and inositol (Table 5). In glucose- and

TABLE 4. Effects ofpH on slime production andcell growth in CD medium without bicarbonate

under aerobic and anaerobic conditionsaCell growth Slime production

pH of (AKlett units) (Acentistokes)medium Anaero- Anaero-

Aerobic bic Aerobic bic

6.60 110 186 0.03 0.096.75 110 215 0.04 0.106.90 107 240 0 0.107.05 70 160 0 0.047.30 48 125 0 0.047.70 35 80 0 0.038.10 30 65 0 0.02

a Strain T14Av cells were grown in 10 ml of CDmedium for 18 h at the indicated initial pH values.

TABLE 5. Slime production by A. viscosus T14Av grown in CD medium in the presence of various sugarsa

Sugar NaHCO3 Incubation time Cell growth Slime production vis- HexosamineSugar (44 mM) (h) (AKlett units) cosity (Acentistokes) (Ag/ml)

Glucose + 24 430 0.43 6.93- 24 83 0.02 1.05

Sucrose + 24 430 0.38 5.57- 24 72 0.03 1.05

Fructose + 24 55 0.02 0.41- 24 60 0.02 0.51+ 72 242 0.03 2.00- 72 130 0.02 0.68

Lactose + 24 20 -0.01 0.38- 24 21 0.01 0.63+ 72 310 0.01 1.43- 72 135 0 0.82

Galactose + 24 10 0.03 0.01- 24 13 0.01 0.11+ 72 338 0.04 1.24- 72 135 0.01 0.09

Raffinose + 24 44 0.04 0.44- 24 42 0 0.53+ 72 108 0.12 1.83- 72 91 0.09 1.05

Inositol + 24 25 0.01 0.34- 24 25 0.02 0.38+ 72 150 0.09 0.92- 72 37 0.04 0.51

aMeasurements of cell concentration, viscosity, and the amount of hexosamine precipitable with acetonewere carried out as described in the text.

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A. VISCOSUS SLIME PRODUCTION 1109

sucrose-containing media, large quantities ofvis-cous slime were produced. In addition, the cellsreached maximal growth within a 24-h period.Sodium bicarbonate stimulated the growth ofT14Av in media containing relatively poor car-bon sources (lactose, galactose, raffinose, andinositol), but the cells produced relatively lowlevels of slime. These results suggest that thepresence of glucose (either as free glucose or asglucose derived from sucrose) in the growthmedium is required for maximal production ofextracellular slime. Therefore, it was of interestto determine the optimal concentration of glu-cose required for slime production in CD-NaHCO3 medium. Slime production measuredas a function of glucose concentration revealedthat optimal growth and slime production ofstrain T14Av were observed at approximately0.6% glucose (data not shown). Above this level,no increase in growth was observed, and only asmall increase in slime production was detected.Preliminary fractionation of T14Av and

T14V slime polysaccharides. The previousresults (Table 1) demonstrated a direct correla-tion between the increase in culture supernatantviscosity and the amount of hexosamine meas-ured in T14Av slime samples in TS broth cul-ture. However, no such direct relationship was

100

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0 50

w

Icr-

C,)

0a-

0

established for T14V hexosamine-containingpolysaccharide. Therefore, we examined the po-tential differences between the T14Av and T14Vpolysaccharides by purification ofboth fractions.Gel filtration chromatography of T14Av slimerevealed that hexosamine-containing fractionswere found predominantly in the void volume ofthe column, whereas hexosamine-containingmaterial from the T14V polysaccharide (peak atfraction 16) was retarded by the column (Fig. 2).After ion-exchange chromatography, the hexos-amine-rich polysaccharide from strain T14Avpassed directly through the column with no sig-nificant retention, whereas the correspondingfraction from T14V was retained and could onlybe eluted by increasing concentrations of NaCl(Fig. 3).

DISCUSSIONThe present investigation has demonstrated

that A. viscosus T14Av produces abundant vis-cous extracellular slime polysaccharide contain-ing hexosamine in a variety of growth mediacontaining glucose. Under most conditions (Ta-ble 2), slime production could be correlated withan increase in the viscosity of culture fluids.Slime production also paralleled the growth ofstrain T14Av and ceased in the early stationary

E

co

I-

zwz0a.

00wa

00

J-J

10 20 30FRACTION

FRACTIONFIG. 2. Gel filtration chromatography on Sepharose 4B of acetone-precipitated polysaccharides of A.

viscosus T14Av and T14V. Acetone-precipitated polysaccharides from T14Av (A) and T14V (B) were obtainedfrom culture supernatant fluids of24-h cultures in TC medium. Samples (15 mg) were applied as described inthe text. Symbols: (0) hexosamine; (0) hexose; (A) protein.

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1110 OOSHIMA AND KURAMITSU

EN..C0'

_ 300I-zwz0a.

00

W 100a-

I

0

0.50

2

0.25 ()0z

FRACTION

BI-

WS10 pool B,02Z2O.L- 0.25z0a.

0

W 0.125o50

4~~~~~~~~~~~~~~~~~~~

O ° 10 20 30 40 5000C FRACTION

FIG. 3. Fractionation of hexosamine-rich polysaccharides of T14Av and T14V on diethylaminoethyl-Bio-Gel A resin. Partially purified polysaccharide fractions obtained after Sepharose 4B gel filtration chroma-tography from T14Av (A) and T14V (B) were chromatographed as described in the text. Symnbols: (0)hexosamine; (0) hex.ose; (A) protein.

phase of growth (Fig. 1). In contrast, virulentstrain T14V produced lower levels of acetone-precipitable polysaccharide containing hexosa-mine which did not affect the viscosity of theculture fluids (Table 1). Comparison of the par-tially purified polysaccharide fractions producedby the two human oral strains revealed that theT14Av slime possessed a higher molecularweight than the comparable T14V polysaccha-ride. In addition, the T14V product behaved likea strongly anionic component, whereas that of

strain T14Av appears to be a neutral or basicpolysaccharide. Based on preliminary chemicalcharacterization of the two polysaccharides, it isnot yet possible to account for the differences innet charge detected after ion-exchange chroma-tography (Fig. 3). In addition, it is not clearwhether the purified fractions are each homo-geneous or consist of mixtures of polysaccharidemolecules. Further chemical characterization ofthe products will be required to explain thechromatographic differences observed.

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A. VISCOSUS SLIME PRODUCTION 1111

Previous results (14) indicated that the extra-cellular slime polysaccharide produced by A.viscosus T6, a rodent strain, consisted primarilyof N-acetylglucosamine with lesser proportionsof other sugars. In addition, another rodentstrain of A. viscosus, Nyl, was demonstratedalso to produce a comparable polysaccharide inthe presence of glucose (15). These rodent slimepolysaccharides are apparently similar in com-position and properties to that of the humanoral isolate strain T14Av. However, since noneof the polysaccharides have been extensivelycharacterized, differences in structure may oc-cur.

Recently, it was reported that strain T14Avproduced little slime polysaccharide when theorganism was cultured in Socransky chemicallydefined medium (13). However, the present in-vestigation revealed that slime polysaccharidewas produced in comparable amounts when theorganism was cultured in CD-NaHCO3 or TCmedium (Tables 2 and 3). The demonstrationthat the addition of NaHCO3 to CD mediumstimulated both growth and slime production bystrain T14Av suggests that a component presentin CD medium may be limiting in Socranskydefined medium for slime synthesis. Similar butweaker effects on slime synthesis were also ob-tained after addition ofNa2CO3 or K2CO3 (Table3). Strain T14Av did not grow in CD mediumcontaining these carbonates at higher concentra-tions (>26 mM), since these concentrations pro-duced relatively high pH levels which retardedthe growth of the organism. That the bufferingaction of NaHCO3 plays an important role instimulating cellular growth and slime productionwas further suggested by the observation thatthe growth of strain T14Av was markedly im-proved between pH 6.6 and 8.1 when the cellswere incubated anaerobically in the GasPak sys-tem (Table 4). Since this anaerobic system con-tinually generates C02, it is suggested that theconversion ofCO2 to HCO3- may aid in bufferingthe growth media. However, these results do notrule out a direct stimulatory effect of CO2 on thegrowth of strain T14Av. These results furtherindicate that maximum growth and slime pro-duction are observed near pH 7.0 (Tables 3 and4). Therefore, the major effect of NaHCO3 ad-dition to CD medium appears to be the stimu-lation of cellular growth, probably as a result ofthe buffering capacity of this compound. Thus,increased production of slime polysaccharideafter NaHCO3 addition is primarily a secondaryeffect of the increase in the growth rate.Howell and Jordan (4) earlier reported that

the addition of NaHCO3 to the growth mediumstimulated both growth and levan synthesis by

rodent strains of A. viscosus. An earlier investi-gation (15) suggested that the production ofslime polysaccharide by A. viscosus rodentstrains was increased in the presence of glucose.However, since this suggestion was based onexperiments involving complex growth medium,the effects of glucose on slime polysaccharidewere not clearly delineated. The present resultswith CD medium have demonstrated the re-quirement for glucose (either as the free sugaror derived from sucrose) in the production ofslime polysaccharide by strain T14Av (Table 5).Glucose can be produced from sucrose in A.viscosus as a result of the action of fructosyl-transferase (EC 2.4.1.10) (12, 17) or invertase(EC 3.2.1.26) (9). However, it is not clear whygrowth of T14Av in the presence of lactoseyielded little extracellular slime, since A. visco-sus strains (9) have f8-galactosidase activitywhich should cleave lactose to glucose and ga-lactose. One possible explanation for this obser-vation is that extracellular free glucose (derivedfrom added glucose or produced from sucrose bythe action of extracellular fructosyltransferase)is a direct precursor of slime polysaccharide andnot intracellular glucose (produced from lactoseby intracellular ,8-galactosidase). In this regard,we are currently investigating the synthesis ofslime polysaccharide in cell-free extracts ofstrain T14Av to determine the nature of theprecursors involved in slime synthesis.

Since strains T14V and T14Av are similar inmany biochemical properties (2, 12), it is ofinterest to examine the relationship betweenthese two human oral isolates. Since T14Avproduces abundant slime polysaccharide andT14V does not, it is difficult to envision how aspontaneous mutation in strain T14V results inmutants (T14Av) which synthesize abundantpolysaccharide (3). Moreover, recent results sug-gest that strain T14Av possesses at least onemembrane-associated enzyme activity involvedin slime polysaccharide synthesis which is ab-sent in strain T14V extracts (T. Ooshima and H.K. Kuramitsu, unpublished data). Thus, T14Avand T14V may represent two different strains ofA. viscosus containing multiple genetic and bio-chemical differences, or T14V may be a sponta-neous mutant of T14Av selected for its ability tocolonize tooth surfaces more effectively (1). Fur-ther investigation will be required to distinguishbetween these possibilities.

ACKNOWLEDGMENTThis work was supported by Public Health Service grani

DE-05141 from the National Institute of Dental Research.

LITERATURE CITED1. Brecher, S. M., J. van Houte, and B. F. Hammond.

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1978. Role of colonization in the virulence of Actino-myces viscosus strains T14-Vi and T14-Av. Infect. Im-mun. 22:603-614.

2. Cisar, J. O., A. E. Vatter, and F. C. McIntire. 1978.Identification of the virulence-associated antigen on thesurface fibrils of Actinomyces viscosus T14V. Infect.Immun. 19:312-319.

3. Hammond, B. F., C. F. Steel, and K. S. Peindl. 1976.Antigens and surface components associated with vir-ulence of Actinomyces viscosus. J. Dent. Res. 55A:19-25.

4. Howell, A., Jr., and H. V. Jordan. 1967. Production ofan extracellular levan by Odontomyces viscosus. Arch.Oral Biol. 12:571-573.

5. Janda, W. M., and H. K. Kuramitsu. 1976. Regulationof extracellular glucosyltransferase production and therelationship between extracellular and cell-associatedactivities in Streptococcus mutans. Infect. Immun. 14:191-202.

6. Jordan, H. V., and B. F. Hammond. 1972. Filamentousbacteria isolated from human root surface caries. Arch.Oral Biol. 17:1333-1342.

7. Jordan, H. V., and P. H. Keyes. 1964- Aerobic gram-positive, filamentous bacteria as etiologic agents of ex-perimental periodontal disease in hamsters. Arch. OralBiol. 9:401414.

8. Jordan, H. V., P. H. Keyes, and S. Bellack. 1972.Periodontal lesions in hamsters and gnotobiotic ratsinfected with actinomyces of human origin. J. Perio-dont. Res. 7:21-28.

9. Kiel, R. A., J. M. Tanzer, and F. N. Woodiel. 1977.Identification, separation and preliminary characteri-zation of invertase and ,B-galactosidase in Actinomycesviscosus. Infect. Immun. 16:81-87.

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10. Levy, G. A., and A. McAllan. 1959. The N-acetylationand estimation of hexosamine. Biochem. J. 73:127-132.

11. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

12. Pabst, M. J. 1977. Levan and levansucrase ofActinomycesviscosus. Infect. Immun. 15:518-526.

13. Powell, J. T., W. Fischlschweiger, and D. C. Birdsell.1978. Modification of surface composition of Actino-myces viscosus T14Av and T14V. Infect. Immun. 22:934-944.

14. Rosan, B., and B. F. Hammond. 1974. Extracellularpolysaccharides of Actinomyces viscosus. Infect. Im-mun. 10:304-308.

15. Van der Hoeven, J. S. 1974. A slime-producing micro-organism in dental plaque of rats, selected by glucosefeeding: chemical composition of extracellular slimeelaborated by Actinomyces viscosus strain Nyl. CariesRes. 8:193-210.

16. Van der Hoeven, J. S., F. H. M. Mikx, K. G. Konig,and A. J. M. Plasschaert. 1974. Plaque formation anddental caries in gnotobiotic SPF Osborne-Mendel ratsassociated with Actinomyces viscosus. Caries Res. 8:211-223.

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