Vol. 34, No. 3INFECTION AND IMMUNITY, Dec. 1981, p. 1044-10550019-9567/81/121044-12$02.00/0

Isolation and Characterization of Streptococcus mutansMutants Defective in Adherence and Aggregation

HETTIE MURCHISON, SYLVIA LARRIMORE, AND ROY CURTISS III*Department ofMicrobiology and Institute ofDental Research, University ofAlabama in Birmingham,

Birmingham, Alabama 35294

Received 30 April 1981/Accepted 18 August 1981

A method was developed which enriched for mutants of Streptococcus mutansthat exhibit defects in adherence to glass, aggregation, or both. Mutants wereisolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) aftermutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survivalafter mutagenesis was kept above 1 to 2% to enhance the probability that mutantsresulted from single mutational events. A total of 117 mutants were isolated; theyalso displayed non-wild-type colony morphology on mitis salivarius agar. Thesemutants were examined for (i) adherence and aggregation after overnight growthin sucrose-containing medium, (ii) aggregation of nongrowing cells in the presenceof 200 jig of sucrose per ml or 20 yg of dextran per ml, and (iii) dextranaseproduction on blue dextran agar plates. Although we isolated mutants whichexhibited a variation from the parent strain in only one of the traits tested, themajority of mutants exhibited defects in two or more characteristics. Thirty-eightstable mutants of independent origin were categorized into 13 separate phenotypicgroups.

Streptococcus mutans is a bacterium indige-nous to the oral cavity and is a major factor inthe etiology of dental caries (19). The ability ofS. mutans to adhere to and form aggregations ofbacteria as plaque on the tooth surface is closelyrelated to its ability to cause caries (14). Themechanism by which the colonization of S. mu-tans occurs is incompletely understood. Recentevidence indicates that initial attachment ofthese organisms occurs by means of a tightlybound cell surface protein which interacts withthe glycoprotein pellicle present on the smoothenamel surface (27). Other data suggest that theproduction of water-insoluble glucan from su-crose by extracellular glucosyltransferases(GTF) also facilitates the adherence of thesebacteria (21, 22) and plays an important role inthe formation of the bacterial aggregates whichmake up plaque (8).

Cariogenic wild-type S. mutans strains adheretightly to glass when grown in medium contain-ing sucrose (8), aggregate in the presence ofsucrose or high-molecular-weight dextran (7),and produce colonies which exhibit a distinctcolonial morphology on mitis salivarius (MS)agar (15). These colonies are typically rough,irregular, and hard. However, several mutantsisolated as smooth, soft, colony morphology var-iants on MS agar or medium containing highlevels of sucrose lack the ability to adhere toglass and to form plaque (2, 4, 6, 10, 16, 19). One

such nitrosoguanidine-induced nonadhering mu-tant (C4) produces significantly lower levels ofGTF, water-insoluble glucans, and caries thanare produced by wild-type strains (19).

Closer examination of a greater diversity ofmutants defective in cell surface traits could giveinsight into the number of genes involved in thecomplex mechanisms of colonization ofthe toothsurface by S. mutans. For example, complemen-tation for cariogenicity has been observed be-tween a nonaggregating mutant (UAB165) anda nonadhering mutant (C4) in gnotobiotic rats,but only when the nonaggregating mutant isallowed to colonize the rats for several daysbefore infection with the nonadhering mutant(11). Furthermore, neither of these strains aloneproduces significant levels of caries. These dataalone suggest that at least two independent geneproducts are necessary for wild-type levels ofcaries to occur.To isolate additional mutants, we developed

a method which specifically enriched for mu-tants with defects in adherence, aggregation, orboth. Those strains isolated were examined fordefects in several characteristics which may berelated to the formation of plaque by wild-typestrains. A total of 117 mutants were isolated; themutants varied from the parent strain in colonialmorphology on MS agar (all but one) and in oneor more of the following traits: (i) adherence toglass, (ii) aggregation in the presence of either

1044

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ADHERENCE-DEFECTIVE S. MUTANS MUTANTS

sucrose or dextran, and (iii) dextranase produc-tion. We report here the properties of 38 of thesemutants which we believe were all of independ-ent origin and which could be grouped into atleast 13 phenotypic classes. Ultimately, we hopeto examine genotypic complementation withinand among these phenotypic classes.

MATERIALS AND METHODSBacterial strains and culture media. Two strep-

tomycin-resistant (Strr) parental strains of S. mutanswere obtained from S. Michalek: PS14 Strr (UAB51)and 6715 Strr (UAB50) (5, 12). UAB numbers are ourown laboratory strain designations. UAB62 (Strr Rif')and UAB66 (StrT Spcr) were isolated from UAB51 andUAB50, respectively, as spontaneous antibiotic-resist-ant isolates on brain heart infusion (BHI) agar (DifcoLaboratories, Detroit, Mich.) containing 100lOg ofrifampin per ml or 1 mg of spectinomycin per ml. Allstrains were maintained at -70°C in 1% peptone(Difco) and 5% glycerol and at 4°C on BHI agar slants.Slants were transferred monthly.

Strains were cultured at 37°C in BHI broth (Difco)or partially defined medium (13) supplemented witheither 1% sucrose or 2% glucose. Colony morphologycharacteristics were examined after 48 h of anaerobicincubation (GasPak anaerobe system with an H2 +CO2 atmosphere; BBL Microbiology Systems, Cock-eysville, Md.) at 37°C on MS agar (Difco) and BHIagar.

Dextranase production was determined on blue dex-tran agar (R. G. Holt, personal communication) whichcontained modified (Holt, personal communication)FMC (29). The minor modifications included the omis-sion of sodium citrate and the lowering of mineral saltconcentrations to prevent precipitation during auto-claving. Vitamins were filter sterilized and added sep-arately just before pouring took place. The assay me-dium contained 440 mg ofKH2PO4, 300 mg ofK2HPO4,3.15 g of Na2HPO4, 2.36 g of NaH2PO4. H20, 9.95 g ofsodium acetate-3H20, 600 mg of (NH4)2SO4, 10 mg ofNaCl, 20 mg of MgSO4. 7H20, 750 ,Lg of MnSO4 H20,200 ,ug of FeCl2, 80 ,Ag of pantothenate hemi-calcium,10 Mg ofp-aminobenzoic acid, 40 ,g of thiamine-hydro-chloride, 20 Mig of nicotininc acid, 80 Mg of pyridoxinemonohydrochloride, 1 Mug of D-biotin, 40 Mg of ribo-flavin, 1 ug of folic acid, 10 mg of adenine, 10 mg ofguanine, 10 mg of uracil, 300 mg of L-glutamate, 5 mgof L-glutamine, 100 mg of L-cysteine, 100 mg of L-tyrosine, 100 mg of L-lysine, 200 mg of L-histidine, 100mg of L-leucine, 100 mg of L-vahne, 100 mg of L-aspartate, 100 mg of L-methiorine, 200 mg of glycine,200 mg of L-alanine, 100 mg of L-serine, 100 mg of L-threonine, 200 mg of L-proline, 200 mg of L-hydroxy-proline, 100 mg of L-phenylalanine, 200 mg of L-tryp-tophan, 2.1 g of Na2CO3, 5.0 g of blue dextran (molec-ular weight, 2,000,000; Sigma Chemical Co., St. Louis,Mo.), 15 g of agar, and 10 g of glucose per liter. ThepH of the medium was adjusted to pH 6.6 with HCl.Each plate contained 25 ml of agar.NA mutagenesis (20). Cells from 5 ml of overnight

BHI broth cultures were sedimented by centrifugationand washed in 5 ml of 0.1 M acetate buffer (pH 4.6).The cells were suspended for 10 min in 0.3 ml of 0.05

M nitrous acid (NA). After treatment, 4.7 ml of lx Amedium (10.5 g of K2HPO4, 4.5 g of KH2PO4, 1.0 g of(NH4)2SO4, 0.5 g of sodium citrate * 2H20 per liter) wasadded, the cells were pelleted by centrifugation, andthe supernatant fluid was discarded. Mutagenized cellswere examined by phase-contrast microscopy to de-termine the effect of NA on chain length and thenwere suspended in 100 ml of BHI broth and incubatedfor 18 h at 37°C.EMS mutagenesis. Cells from 1 ml of overnight

BHI broth-grown cultures were diluted 1:20 in BHIbroth and incubated further at 37°C for 2 h. Ethylmethane sulfonate (EMS) was added at 0.03 ml/2 mlof culture. The culture was mixed with a Vortexblender to dissolve EMS and was then incubated for2 h at 37°C. Mutagenized cells were examined byphase-contrast microscopy to determine the effect ofEMS treatment on chain length. These cells were thenpelleted by centrifugation, washed, suspended in 100ml of BHI broth, and incubated overnight at 37°C.Mutant isolation. A 2-liter glass Erlenmeyer flask

containing 200 ml of PD medium + 1% sucrose wasinoculated with 5 ml of a previously mutagenized BHIbroth-grown culture of UAB62 or UAB66 and incu-bated standing for 24 h at 37°C to stationary phase.During this time, fionmutant cells adhered to the flasksurface or settled to the bottom as bacterial aggre-gates. A 10-ml sample of culture fluid from this 24-hculture was carefully removed by pipette and used toinoculate another 2-liter glass flask containing 200 mlof PD medium + 1% sucrose. This serial subculturewas repeated four times. Finally, 100 ml of BHI brothwas inoculated with 5 ml of culture fluid and incubatedstanding for 18 h at 37°C. Cells from this culture weremixed with a Vortex blender, diluted, and plated onMS agar. Plates were incubated for 48 h anaerobically(BBL GasPaks) at 37°C. Under these conditions, col-ony variations occur which are not readily apparentwhen colonies are incubated under previously de-scribed conditions (14). At least three of each distinctcolony type (including wild type) were picked, purifiedon BHI agar, and characterized as described below.This isolation procedure was repeated at least fourtimes for both UAB62 and UAB66. A similar proce-dure was used twice with U50 during development ofthe final enrichment protocol. Mutants were frozensoon after isolation.Adherence and aggregation of growing cefls.

Glass tubes (16 by 75 mm) containing 5 ml of PDmedium + 1% sucrose were inoculated with each mu-tant and incubated standing for 24 h at 37°C. Eachculture was gently mixed with a Vortex blender for 5s to remove those cells which grew in close contactwith the glass surface but did not actually adhere; theculture fluid was poured off (adherent cells of wild-type strains cannot be removed by being mixed witha Vortex blender). The tubes were scored on a scale of0 to 4+ for adherence (Fig. 1). The culture fluid wasscored on a scale of 0 to 4+ for cell aggregation asfollows: 0, no visible aggregation; 1+, slightly visibleminute clumps of cells in turbid fluid; 2+, easily visiblesmall clumps of cells in turbid fluid; 3+, well-definedclumps of cells in clear supernatant fluid; 4+, verylarge flocculent clumps of cells in clear supernatantfluid. Macroscopic scoring for aggregation was con-

1045VOL. 34, 1981

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1046 MURCHISON, LARRIMORE, AND CURTISS

I

,i

isr

/I.S .FIG. 1. Appearance of different levels of adherence to glass tubes. Adherence was scored as follows: 4+,

thick confluent coat of cells on bottom and sides of tube; 3+, thin confluent coat of cells on bottom and sidesof tube; 2+, thin confluent coat of cells on bottom of tube; 1+, few visible cells adhering to bottom of tube; 0,no visible adherence.

firmed by microscopic examination of the culture fluid(Fig. 2).Aggregation of nongrowing cells. Aggregation

in the presence of either 200 jig of sucrose per ml or 20jig of dextran per ml was examined by the methoddescribed by Gibbons and Fitzgerald (7). Cells grownanaerobically at 37°C in PD medium + 2% glucose for18 h were pelleted by centrifugation, washed twice in0.86% NaCl, and concentrated 10 times in 0.1 M glycyl-glycine buffer (pH 8.6). To the concentrated cells, 200jig of sucrose per ml or 20 jg of dextran per ml was

added, and these cell suspensions were incubated for18 h at 37°C. Strains were scored for aggregation on ascale of 0 to 4+ as described above.Dextranase production. Samples (10 ,l) of over-

night BHI broth cultures of mutant strains were spot-ted on blue dextran agar. Plates were incubated an-aerobically at 37°C for 72 h. Dextranase productionwas indicated by a clear halo surrounding the spot ofgrowth (Fig. 3).

Strains that were shown to lack the dextranaseenzyme by the plate method were examined by themethod of Somogyi (25, 26) as modified by Nelson(23). This procedure gives a quantitative measure ofreducing sugars generated from dextran by dextranase.Cultures of UAB66 and its dextranase-negative mu-tants were grown for 18 h in 1 liter of modified FMCmedium (29) + 1% glucose at 37°C. These cultures(cooled to 4°C) were centrifuged 30 min at 8,000 rpm

in a GSA rotor in a Sorvall refrigerated centrifuge.The proteins contained in the cell-free supernatantfluids were precipitated with 70% (NH4)2SO4. Theprecipitate was dialyzed against three changes of 10mM sodium acetate (pH 5.6) for 24 h at 4°C. The totalamount of protein contained in each sample was mea-sured by the Lowry method (17), with bovine serumalbumin as the standard. The assay mixture (1.0 mlvolume) contained 0.04 ml of 1.0 M sodium acetate(pH 5.6), 0.4 ml of 10% dextran T2000, and 0.56 ml ofdiluted protein sample; it was incubated for 1 h at37°C. Somogyi reagent A (1 ml) was added, and thesamples were boiled for 20 min. After the samplescooled to room temperature, 1 ml of Nelson arsen-omolybdate color reagent (23) was added. The result-ing mixture was diluted to 10 ml with 0.5 N H2SO4,and the absorbance was read at 520 nm. One unit ofactivity was defined as 1.0 jimol of reducing sugargenerated per h.

Antibiotic resistance. Mutants were tested forantibiotic-resistance markers by streaking overnightcultures on BHI agar containing 50 jig of rifampin perml, 500 jLg of streptomycin per ml, or 500 jLg of specti-nomycin per ml.

Fermentation. Wasserman tubes containing 2.5 mlof Purple Broth Base (Difco) + 2.4% Thioglycollate(without dextrose or indicator; Difco) and either 1%mannitol, 1% sorbitol, 1% melibiose, or 1% raffinosewere inoculated with 0.1 ml of an 18-h culture of each

INFECT. IMMUN.

t it.,'v !0.V ;-

,. .- 'if Nk

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ADHERENCE-DEFECTIVE S. MUTANS MUTANTS'~~~4+'

3+

MO

Jm

IOtm

0

FIG. 2. Microscopic appearance of cell clumps in culture fluids of strains scoring 0 to 4+ in aggregation(see text). Both growing and nongrowing cells gave similar microscopically visible aggregates for the sameaggregation score determined macroscopically.

FIG. 3. Dextranase production by wild-type andmutant strains grown for 72 h anaerobically at 37°Con blue dextran agar (see text).

strain. Tubes were incubated standing at 37°C andchecked every 24 for 72 h for fermentation (colorchange from purple to yellow).Reversion ofUAB96. To test UAB96 for reversion

to Mel-, the ability to ferment melibiose, we concen-

trated 10 ml of an 18-h BHI broth culture after cen-trifugation in 1 ml of buffered saline plus gelatin (3)and plated it on Purple Broth Base agar + 2.4%Thioglycollate + 1% melibiose. (It should be notedthat the parent of UAB96, UAB62, and other serotypec strains do not grow on a minimal medium such asPD agar containing melibiose, even though they fer-ment melibiose in an enriched medium such as PurpleBroth Base). Plates were screened for Mel' coloniesafter 72 h of anaerobic incubation at 37°C. Mel+ re-vertants were picked, purified on BHI agar, and char-acterized as described above.

RESULTSMutagenesis. One potential problem in mu-

tant isolation with S. mutans has been due togrowth of cells in chains rather than as singlecells. Not only must the conditions for mutagen-esis and subsequent growth allow for genotypicand phenotypic expression within an individualcell, but also, these conditions must allow for theformation of chains comprised of cells of thesame genotype. Therefore, we first examined thelength of chains produced in both BHI brothand PD medium + 2% glucose by phase-contrastmicroscopy. The average chain length in over-night BHI broth cultures was 8 to 10 cells perchain, whereas the average chain length in PDmedium + glucose was 20 to 30 cells per chain.

Next, ideal conditions for mutagenesis were

VOL. 34, 1981 1047

.1 t- +

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1048 MURCHISON, LARRIMORE, AND CURTISS

determined by using either NA and EMS, andthe effects of these treatments on chain lengthwere examined. Under standard conditions, a10-min treatment of BHI broth-grown cultureswith NA reduced chains to one or two cells, withapproximately 1 to 2% survival of colony-form-ing units. Two-hour treatment under standardconditions with EMS did not affect chain lengthwhile resulting in a 10% survival of colony-form-ing units. Thus, cell survival in both cases wassimilar. Cultures of BHI broth-grown strainswere used, and appropriate conditions for mu-tagen treatment and growth after mutagenesiswere determined so that, at the time of platingor enrichment, all cells in a chain were of iden-tical genotype and phenotype. The conditionsused minimized the probability that mutant phe-notypes would be due to more than one muta-tion.Mutant isolation. UAB62 and UAB66 were

mutagenized with NA or EMS. After allowingfor segregation and phenotypic expression byovernight growth in BHI broth, we inoculatedthe cells into PD medium + 1% sucrose in 2-literglass Erlenmeyer flasks. During a 24-h standingincubation, cultures reached stationary phasewhile adherence and aggregation of nonmutantcells occurred. After subculturing in PD medium+ 1% sucrose four times as described above, wediluted the BHI broth-grown cells and platedthem on MS agar. Plates incubated for 48 hanaerobically at 370C were examined for colo-nies which varied from the wild type in mor-phology, because many mutants previously iso-lated with defects in adherence or aggregationdisplay colony morphology variations on MSagar (2, 4, 6, 16, 19). During the enrichmentprocedure, the amount of adherence to the glasssurface in 24-h cultures was noticeably dimin-ished with each successive culture, and the per-centage of colonies with non-wild-type morphol-ogies noticeably increased. After the fourth en-richment cycle, about 70% of all colonies exam-ined showed some colony morphology variationin one or more of several colonial characteristics,such as texture, color, adherence to agar surface,size, and shape (1). Up to three colonies of eachmorphology displayed, including the wild type,were picked, purified, and characterized aftereach mutagenesis and mutant enrichment ex-periment. Those strains which ultimately wereshown to have defects in adherence or aggrega-tion were among those with colony variations,whereas all isolates (except one) displaying wild-type morphology maintained parental adher-ence and aggregation characteristics. Althoughup to three mutants from each mutagenetic ex-periment with a given colony morphology were

stocked, in this report we eliminated most ofthese from description, because most but not allmutants with similar colony morphologies werenearly always identical in regard to other phe-notypic traits. Tables 1 and 2 describe the colonymorphologies of the mutants isolated and indi-cate those mutants with identical colony mor-phologies which were found to differ with regardto other phenotypes.Mutant characterization. After isolation,

each mutant was picked, purified, and tested for(i) adherence to glass and aggregation after a 24-h standing incubation at 370C in PD medium+ 1% sucrose, (ii) aggregation in the presence of200 ,ug of sucrose per ml or 20 ,ug of dextran perml, and (iii) production of dextranase on bluedextran agar (Tables 1 and 2). Each test wasperformed at least twice on each mutant toensure that the values were reproducible. Fromthe results of testing mutants for these proper-ties, we determined that those mutants whichhad similar colony morphologies generally dis-played similar traits in all properties tested. Ex-ceptions to this general rule were UAB218 andUAB220 (Table 1) and UAB116 and UAB118(Table 2). All mutants isolated from the sameexperiment that had similar or identical pheno-types for all tested traits were excluded fromTables 1 and 2, as were mutants which ulti-mately proved to be genotypically unstable.Thus, we believe that the 38 mutants describedin Tables 1 and 2 and obtained from 10 differentenrichment experiments were of independentorigin.By the procedures outlined, 67 mutants of

UAB62, 44 mutants of AUB66, and 6 mutants ofUAB50 were isolated and tested for adherenceand aggregation as well as dextranase produc-tion. The results summarized for the 38 pre-sumed independent mutants isolated (Tables 1and 2) indicate that mutants defective in onlyone trait can be isolated, but many of the mu-tants isolated showed defects in two or morecharacteristics.Dextranase activity was measured quantita-

tively by the Somogyi method (23, 25, 26) fordextranase-negative mutants of UAB66. All mu-tants which appeared to be dextranase negativeon blue dextran agar plates (UAB107, UAB113,UAB122, UAB158, and UAB245; Table 2)lacked measurable dextranase activity, as op-posed to UAB66, which produced 13.8 units ofdextranase activity per mg of protein. In addi-tion, UAB119, which showed significantly lowerlevels of activity by the plate method, producedonly 2.6 units of dextranase activity per mg ofprotein, a value significantly lower than thatobtained for UAB66.

INFECT. IMMUN.

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ADHERENCE-DEFECTIVE S. MUTANS MUTANTS 1049

TABLE 1. Colony morphology, adherence, aggregation, and dextranase production of UAB62 mutants

Growth in PD + Aggregation of1% sucroseC nongrowing cells1%sucrose withd: Dextran-

Strain' Morphology on MS agar' ase pro-Sucrose Dextran duction'Adher- Aggrega 20g (20 ~t/

ence tia- (200tgl(0 g

UAB62 ... Rough, irregular, dark blue, slightly adherent, 4+ 3+ 3+ 2+ +hard, convex, shiny, may have liquid exudate.

UAB91 ... Smooth, round or slightly irregular, dark gray,nonadherent, soft, creamy consistency,convex, dull.

UAB95 ... Rough, irregular, dark gray, slightly adherent,hard, convex, with edges of some coloniesslightly crinkled off agar surface

UAB96 . Rough, irregular, blue-gray, nonadherent, soft,convex, may be slightly umbonate.

UAB100 Rough, round, dark gray, very dull,nonadherent, soft, creamy consistency,convex.

UAB102 Rough, round or slightly irregular, light-bluerim with dark center, nonadherent, soft,slightly umbonate.

UAB104 Smooth, slightly irregular, dark gray,nonadherent, somewhat soft, convex, centersunken in rim.

UAB105 Smooth with rough center, round, dark blue-gray, nonadherent, soft, convex and slightlycone shaped.

UAB128 Smooth, round, light-blue rim with slightlydarker center, nonadherent, shiny, grainy,convex with tiny raised center.

UAB134 Rough, round or slightly irregular, light blue,nonadherent, dull, creamy consistency, flat.

UAB140 Smooth, round, blue, nonadherent, shiny, soft,droplike.

UAB153 Smooth, round, dark center with light-blue rim,nonadherent, shiny, creamy, umbonate.

UAB178 Smooth, round, dark-blue center with light-bluerim, nonadherent, shiny, soft, convex

UAB183 Smooth, round, dark gray, nonadherent, soft,raised.

UAB188 Smooth, round, blue-gray, nonadherent, soft,raised with small, slightly raised center.

UAB190 Smooth, round, light blue, nonadherent, shiny,soft, droplike, small.

UAB208 Rough, round, dark gray, nonadherent, shiny,soft, convex.

0 0 0 2+ +

1+ 2+ 2+ 2+ +

1+ 1+ 0 0 +

1+ 1+ 1+ 2+ +

0 0 0 0 +

0 0 0 0 ++

2+ 1+ 0 0 +

3+ 1+ 1+ 2+ +

0 0 0 0 +

0 1+

1+

1+

0 1+ +

2+ 3+ 2+ +

1+ 0 2+ +

0 1+ 0 2+ +

0 1+ 0 2+ +

1+ 1+ 0 0 +

0 3+ 0 1+ +

VOL. 34, 1981

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1050 MURCHISON, LARRIMORE, AND CURTISS

TABLE 1.-Continued

Growth in PD + Aggregation of1%scrs nongrowing cells1% sucroseC withd: Dextran-

Straina Morphology on MS agarb ase pro-Sucrose Dextran ductione

ence tion (2MI)g/ (2I)g/UAB216 Smooth, round, blue, nonadherent, soft, 0 3+ 1+ 1+ +

droplike, minute.

UAB218 Smooth, round, gray, nonadherent, soft, convex, 0 1+ 0 2+ +small.

UAB220 Same as UAB218 1+ 3+ 0 2+ +

UAB225 Smooth, round or slightly irregular, blue-gray, 4+ 3+ 0 2+ +nonadherent, shiny, soft, umbonate.

UAB229 Smooth, slightly irregular, dark gray, 0 2+ 1+ 3+ +nonadherent, soft and liquid, umbonate.

UAB230 Smooth, round, gray, non-adherent, shiny, soft, 0 2+ 0 2+ +raised.

aUAB91 through UAB105 were isolated from experiment 1 and were EMS induced. UAB128 throughUAB153 were isolated from experiment 2 and were EMS induced. UAB178 through UAB190 were isolated fromexperiment 3 and were NA induced. UAB208 through UAB230 were isolated from experiment 4 and EMSinduced.

b Morphology on MS agar after 48 h of anaerobic incubation at 37°C as described by Benson (1).c Glass tubes of PD + 1% sucrose were inoculated and were incubated standing at 37°C for 24 h; each was

scored for adherence to the tubes and aggregation present in the supernatant fluid.d Aggregation of washed cells after addition of sucrose or dextran (7).e Overnight cultures (10 ul) were spotted on blue dextran agar and incubated anaerobically at 37°C for 72 h.

Values for the production of dextranase by mutant strains are described as follows: +, dextranase equal toparent; ++, more dextranase than parent; ±, less dextranase than parent; -, no dextranase.raised.

On blue dextran agar plates, UAB104 seemedto produce a higher level of dextranase activitythan that produced by its parent, UAB62 (Fig.3, Table 1), and these strains were also analyzedby the Somogyi method for dextranase activity.However, less than one unit of dextranase activ-ity per mg of protein was detected in the culturesupernatant fluids of each strain with this assay,and these levels were not sufficient for makingaccurate comparisons.Reversion. Aside from the tests performed

to determine adherence or aggregation, all mu-tants were routinely screened for retention ofantibiotic resistance markers and fermentationcharacteristics. All mutants isolated retained re-sistance markers, and all except UAB96 con-formed to the fermentation characteristics of theparent strains (Table 1). UAB96, a mutant ofUAB62, did not ferment melibiose even after 72h and fermented raffinose slowly by 72 h. SinceUAB96 also displayed reduced adherence and aunique colony morphology on MS agar, it wasnecessary to determine whether these character-istics were the result of one or several mutations.Mel' revertants of UAB96, which occurred at a

frequency of 2.0 x 10-9 per colony-forming unit,were picked, purified, and checked for fermen-tation characteristics. All revertants had re-gained the fermentation characteristics ofUAB62 with respect to melibiose and raffinose,whereas each maintained the morphological, ad-herence, and aggregation properties of UAB96.Thus, the mutant phenotype of UAB96 was theresult of at least two separate mutations.

DISCUSSIONThe ability of S. mutans to colonize teeth is

dependent on its ability to adhere and aggregatein the form of plaque. Although these two char-acteristics have been shown to be independentof each other (5, 21, 22, 27), it is evident thatthere may be aspects common to both. It washoped that examination of mutants defective inthese characteristics might given some insightinto the relatedness of these traits.Many of the mutants previously isolated with

defects in adherence or aggregation have beenisolated as colonial variants on MS agar (2, 4, 6,16, 19). It has been suggested that the rough,hard, adherent morphology of wild-type strains

INFECT. IMMUN.

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

TABLE 2. Colony morphology, adherence, aggregation, and dextranase production of UAB66 mutants

Growth in PD + Aggregation of1%surose' nongrowing cells1% sucrose' withd: Dextran-

Straina Morphology on MS agarb ase pro-Sucrose Dextran duction'Adher- Aggrega- (200 yg/ (20 Jg/

ence tion (20u/(0/g

UAB66 ... Rough, irregular, dark blue, adherent (colony 4+ 3+ 3+ 4+ +sunk into agar), very hard, umbonate.

UAB56 Smooth, round, blue-gray, center slightly darkerthan rim, nonadherent, slightly convex withraised center.

UAB107 Smooth, irregular, dark gray, nonadherent, softsoupy consistency, entire colony submergedin an surrounded by clear sticky fluid, raised.

UAB113 Rough, round or slightly irregular, blue-gray,nonadherent, soft, creamy, clear fluidsurrounding colony, raised.

UAB116 Rough, round, blue-gray, nonadherent, soft,grainy, convex.

UAB118 Same as UAB116.

UAB119 Rough, round, dark blue, adherent, hard, partof colony submerged, droplike with depressedcenter.

UAB122 Rough, round, blue-gray, nonadherent, dulledge with smooth shiny center, convex.

UAB158 Rough, irregular, round, adherent, slightly softrim, slightly umbonate.

UAB165f Same as UAB66.

UAB201 Rough, round, dark gray, nonadherent, soft,droplike.

UAB232 Rough, round, blue-gray, nonadherent, soft,raised with slight cone shape.

UAB234 Rough, round, dark blue, nonadherent, soft,droplike with slightly flattened top.

UAB237 Rough, round, dull blue rim with shiny darkblue center, nonadherent, soft, convex.

UAB240 Smooth, round with an erose margin, brown,nonadherent, soft, flat with slightly raisedcenter.

UAB241 Very rough, round with an erose margin, blue,nonadherent, dull, soft, convex.

UAB245 Smooth, round, gray, nonadherent, soft andclear liquid completely surrounding colony,convex.

0 4+

0

4+ 4+ +

2+ 3+ 3+ -

0 2+ 4+ 4+ -

0 0 0 0 +

0 4+ 4+ 4+ +

4+ 2+ 1+ 0

4+

4+

3+ 4+ 4+

2+ 4+ 2+

4+ 3+ 0 0 +

4+ 3+ 0 0 +

0 3+ 2+ 2+ +

4+ 3+ 1+ 0 +

0 2+ 1+ 1+ +

0 2+ 1+ 2+ +

0 2+ 0 0 +

0 3+ 4+ 4+ -

a UAB56 was isolated in experiment 1 from UAB50 (before selection of Spc' to yield UAB66) and was NAinduced. UAB107 through UAB122 were isolated from experiment 2 and were EMS induced. UAB157 throughUAB158 were isolated from experiment 3 and were EMS induced. UAB201 was isolated from experiment 5 andwas NA induced. UAB232 through UAB245 were isolated from experiment 6 and were EMS induced.

b Same as footnote b of Table 1.'Same as footnote c of Table 1.d Same as footnote d of Table 1.'Same as footnote e of Table 1.f UAB165 was isolated by R. Hull (13) and was NA induced.

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1052 MURCHISON, LARRIMORE, AND CURTISS

on MS agar (14) is the result of the productionof high levels of extracellular water-insolubleglucans and that those mutants defective in glu-can synthesis will have an altered colony mor-phology that is generally smooth, soft, and non-adherent (19). In partial support of these beliefs,we noted after each successive enrichment cyclefor nonadhering, nonaggregating mutants thatthe numbers of colony morphology variants in-creased. Furthermore, by this procedure, all col-ony morphology variants tested were subse-quently shown to have defects in adherence,aggregation, or both, whereas all colonies withwild-type morphology (ca. 40) that were testedretained parental adherence and aggregationcharacteristics. If, however, a reduction in glucansynthesis predisposes a smooth, soft colonymorphology, then mutants such as UAB95 (Ta-ble 1), which produced slightly variant, rough,hard colonies but were also defective in adher-ence, must adhere less well for some reasonother than an inability to produce high levels ofinsoluble glucans. Such mutants might have hada defective attachment protein but producednormal levels of glucan and thus maintainedmost wild-type colonial characteristics. Like-wise, another mutant isolated in this laboratoryby other means, UAB165, produced rough colo-nies identical to wild type but was defective inits ability to aggregate in the presence of sucroseor dextran (Table 2). This mutant has beenshown to produce levels of glucan comparable tothat produced by the wild type (11). These char-acteristics of UAB165 demonstrate that somedefects affecting aggregation may be independ-ent of glucan production and may not result inan altered colony morphology.Other mutants that we have isolated also dem-

onstrated that attempts to correlate colony mor-phology with specific defects may be simplistic.For instance, two smooth-colony variants,UAB128 and UAB225 (Table 1), adhered atnormal levels and only exhibited a defect insucrose-induced aggregation and thus must haveproduced glucan at levels sufficient for bacterialadherence and accumulation to occur. Further-more, UAB218 and UAB220 (Table 1) producedsmooth colonies identical to each other on MSagar, but these strains exhibited different adher-ence and aggregation properties. Therefore, ourresults are compatible with past observationsthat most S. mutans mutants with altered abil-ities to adhere, aggregate, or both will oftenexhibit non-wild-type colony morphologies onMS agar but did not demonstrate any correla-tion between a given phenotypic defect and aspecific colony morphology.

Unlike other workers (4, 6, 16, 19), we avoidedusing the mutagen nitrosoguanidine, because it

is known to produce multiple, clustered muta-tions (20). Our mutant enrichment protocol per-mitted the recovery of many mutants that dis-played a variety of defects in adherence, aggre-gation, and dextranase activity. A total of 38mutants that were stable in character are de-scribed in this report. These mutants came fromeither different mutant enrichment experimentsor from the same experiment but displayed dif-ferences in colony morphology, adherence, ag-gregation, dextranase activity, or any combina-tion thereof. Thus, we believe each mutant to beof independent origin. These 38 mutants (Tables1 and 2) can be categorized into at least thirteenphenotypic groups (Table 3). We assumed thatmost phenotypes were due to single mutationsin view of the survival after mutagenesis, al-though one mutant, UAB96, was shown to haveits Mel- phenotype caused by a mutation thatwas not also responsible for its adherence andaggregation defects. We also believe, based ontarget size, that most mutations probably oc-curred in structural genes affecting the synthesisof a single enzyme or a structural protein ratherthan in regulatory genes that would influencethe synthesis of two or more structural geneproducts. Of course, mutations in structuralgenes could have a polar effect on expression ofother structural genes in the same transcrip-tional unit, if such types of gene arrangementand control are common in S. mutans. Thecurrent inability to genetically manipulate S.mutans precludes definitive resolution of thesepossibilities. Nevertheless, it seems most prob-able that each contribution of phenotypic prop-erties (Table 3) is due to a defect in a singleprotein.

In terms of adherence, the mutants obtainedindicated indirectly that some gene productswere uniquely necessary (Table 3, group 1),whereas other gene products were needed forboth adherence and aggregation (groups 2 and3). Group 2 mutants appeared to be defective insynthesizing glucans from sucrose, because ad-dition of dextran to nongrowing cells permittedaggregation. Group 3 mutants, on the otherhand, were totally defective in adherence andaggregation, and one could speculate that theylack some glucan binding proteins. However,because GTF can bind glucans, it is simplest topostulate that group 2 mutants were due tomutations that abolished the synthetic abilitybut not the glucan-binding ability of GTF,whereas group 3 mutants had mutations thatabolished both functions of GTF.Much debate has arisen concerning the role of

dextranase in S. mutans. One hypothesis is thatthis enzyme is necessary for the formation ofwater-insoluble glucans from water-soluble glu-

INFECT. IMMUN.

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ADHERENCE-DEFECTIVE S. MUTANS MUTANTS 1053

TABLE 3. Phenotypic grouping ofmutants

Expression of phenotypic traitsa RepresentativeGroup muReresntstative'Adh AggG AggS AggD Dex mutant strainsb

1 - + + + + 1182 - - - + + 913 - - - - + 102,1164 - + + + - 2455 + (+) + + - 1226 + (+) (-) _ (-) 1197 + + - - + 201, 1658 + + - + + 2259 + + (-) - + 23410 (-) (-) - - + 105, 9611 - (+) - - + 24112 (-) + - + + 22013 - + - (+) + 208a Adh, Adherence; AggG, aggregation during

growth; AggS, aggregation with sucrose under non-growth conditions; AggD, aggregation with dextranunder nongrowth conditions; Dex, dextranase activity;+, wild-type phenotype; (+), intermediate or nearlywild-type phenotype; (-), nearly mutant phenotype;-, mutant phenotype.

bMutants derived from the PS14 strain UAB62(Tables 1 and 3) are identified by boldfacing, whereasmutants derived from the strains 6715 Strr and UAB66are not foldfaced. Only single representatives of eachsubline best reflecting each phenotype are listed.

cans by GTF (9, 28). Another theory is that theratio ofGTF to dextranase regulates the activityof GTF during the formation of plaque (9). Ineither case, the presence of the enzyme wouldbe vital for wild-type adherence and accumula-tion. Of the five dextranase-negative mutantsdiscussed here, three strains were unable to ad-here (UAB107, UAB113, and UAB245; Table 2)and two maintained wild-type adherence(UAB122 and UAB158; Table 2). UAB119 pro-duced reduced levels of dextranase activity butmaintained the ability to adhere. If the firsthypothesis were correct and dextranase is nec-essary for the formation of water-insoluble glu-cans, it seems unlikely that any dextranase-neg-ative mutants displaying wild-type adherencecould have been isolated. If, however, it is thelevel of the enzyme which is necessary to regu-late the activity of GTF, then those dextranase-negative mutants that still maintained the abil-ity to adhere may produce an enzyme that isaltered in such a way that it is unable to effi-ciently degrade dextrans but has sufficient activ-ity to permit GTF to initiate water-insolubleglucan synthesis. Thus, mutants in group 4 (Ta-ble 3), which were unable to adhere, could lackdextranase or produce a totally defective dex-tranase and be unable to produce the insolubleglucans necessary for adherence, but could stillbe able to aggregate because the dextran bindingsites (7, 18) would be unaffected. On the other

hand, mutants in group 5 (Table 3) could pro-duce a defective dextranase which lacks theability to degrade dextrans extracellularly butwhose presence still affects the GTF activity.Such a mutation may be in a gene that codes fora protein necessary for the processing and mod-ification of dextranase (and possibly other cellsurface proteins) during its transit from cyto-plasm to cell surface. Mutants such as those ingroup 6 (Table 3), which exhibited reduced dex-tranase activity and defective aggregation, maybe the result of a defect in the cell surface whichnot only affects the effective transport of dex-tranase but also affects sucrose and dextranbinding as well. Another possibility is that thereduced dextranase activity is due to a mutationin a gene for a protein that normally associateswith and is necessary for dextranase activity.This protein could be associated with the dex-tran binding site and could have an additionalfunction necessary for aggregation.

Obviously, the formation of plaque involvestwo separate phenomena: the adherence of bac-teria to the tooth surface and the aggregation ofcells to form a bacterial matrix over the adheringcells (21, 22, 27). From the aggregation datacompiled on the mutants isolated, it is evidentthat the ability of some strains to aggregatewhile growing in the presence of sucrose maynot be related to the ability of that strain toundergo aggregation under nongrowth condi-tions in the presence of exogenous sucrose ordextran (groups 7, 8, 9, and 10; Table 3). Undergrowing conditions, proteins which are involvedin the aggregation process are present; but inthe case of washed nongrowing cells of mutantstrains that have had sucrose or dextran added,most extracellular proteins that were not tightlybound to the cell are gone (7). Such weak asso-ciations of cell surface proteins might be due toa variety of genetic defects, including those thatwould alter nonprotein cell wall componentssuch as lipoteichoic acid or carbohydrate anti-gens. Weak associations of cell surface enzymesor of sucrose or glucan-binding proteins (8, 18,21, 22) may explain the existence of mutantswhose cells adhered well and aggregated in grow-ing cultures (AggG+) but were unable to aggre-gate with sucrose or dextran (Table 3; groups 7,8, and 9). The isolation of these mutants, as wellas those in group 1, also emphasizes that'adher-ence and aggregation can be separately affectedby mutation. These hypotheses are not sufficientto explain the existence of mutants in groups 10,11, 12, and 13. Again, the fact that these mutants,like those in groups 2 and 3, possessed mutationsaffecting adherence as well as aggregation withsucrose or dextran indicates that these traitsprobably have factors in common. However, mu-

VOL. 34, 1981

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

1054 MURCHISON, LARRIMORE, AND CURTISS

tants in groups 10, 11, 12, and 13 were still ableto aggregate in growing cultures, and AggG+, inthis case, may be independent of sucrose avail-ability or the ability of the cells to synthesizeglucans.One additional point should be made based on

the summary information in Table 3: namely,with the exception of phenotypic groups 3 and10, all other groups contained mutants only fromeither the PS14 strain UAB62 or from the 6715-derived strains UAB50 and UAB66. This factsuggests that the phenotypic types and distri-bution are nonrandom. Insufficient numbers ofmutants of both strains were isolated for theelaboration of the genetic relatedness or differ-ences between PS14 and 6715.

In conclusion, the wide variety of mutantsencountered during this study may in the futureallow us to clarify the role (if any) of factorssuch as dextranase, aggregation, and glucan pro-duction in the virulence of S. mutans. Certainly,further study of these and other mutants isnecessary to clarify many ofthe questions raised.Since there is no current method for geneticanalysis of S. mutans, it is impossible to deter-mine the actual number of genes involved inspecifying the various attributes necessary forplaque formation. But by examining a broadspectrum of mutants as opposed to single iso-lates, we have been able to show that traits of S.mutans such as adherence and aggregation,which have been shown to have factors in com-mon, are probably independently controlled. Weare now beginning to examine in vitro comple-mentation of these mutants for both adherenceand aggregation as a method for determininghow many genes may be involved in the ex-

pressed phenotypes, and several strains are

being monitored for their virulence in gnoto-biotic rats. We are also examining selected mu-tants more closely for levels of soluble and in-soluble glucans, intracellular as well as extracel-lular dextranase, and other proteins nornallyassociated with the S. mutans cell surface.

ACKNOWLEDGMENTSWe express our gratitude to Richard Hull, Jeffrey Hansen,

and Robert Holt for their advice and help. We also thank PatPierce, Anne Galloway, and Brenda Gosnell for assistance inthe typing of this manuscript.

Funds for this work were provided by Public Health Servicegrants DE-62491 and DE-02670 from the National Institute ofDental Research.

LITERATURE CITED

1. Benson, H. J. 1973. Microbiological applications, 2nd ed.,p. 145. W. C. Brown Co. Publishers, Dubuque, Iowa.

2. Bulkacz, J., and J. H. Hill. 1977. Glucosyltransferasefrom Streptococcus mutans and a non-plaque formingmutant. Arch. Oral Biol. 22:119-123.

3. Curtiss, R., HI. 1965. Chromosomal aberrations associ-

ated with mutations to bacteriophage resistance inEscherichia coli. J. Bacteriol. 89:28-40.

4. de Stoppelaar, J. D., K. G. Konig, A. J. M. Plas-schaert, and J. S. van der Hoeven. 1971. Decreasedcariogenicity of a mutant of Streptococcus mutans.Arch. Oral Biol. 16:971-975.

5. Fitzgerald, R. J., P. H. Keyes, T. H. Stoudt, and D. M.Spinell. 1968. The effect of dextranase preparation onplaque and caries in hamsters, a preliminary report. J.Am. Dent. Assoc. 76:301-304.

6. Freedman, M. L., and J. M. Tanzer. 1974. Dissociationof plaque formation from glucan-induced agglutinationin mutants of Streptococcus mutans. Infect. Immun.10:189-196.

7. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran-induced agglutination of Streptococcus mutans and itspotential role in the formation of dental plaques. J.Bacteriol. 98:341-346.

8. Gibbons, R. J., and M. Nygaard. 1968. Synthesis ofinsoluble dextran and its significance in the formationof gelatinous deposits by plaque-forming streptococci.Arch. Oral Biol. 13:1249-1262.

9. Guggenheim, B., and J. J. Burckhardt. 1974. Isolationand properties of a dextranase from Streptococcus mu-tans OMZ 176. Helv. Odontol Acta. 18:101-103.

10. Hamada, S., S. Tai, and H. D. Slade. 1978. Binding ofglucosyltransferase and glucan synthesis by Streptococ-cus mutans and other bacteria. Infect. Immun. 21:213-220.

11. Hirasawa, M., H. Kiyono, T. Shiota, R. A. Hull, R.Curtiss, HI, S. M. Michalek, and J. R. McGhee.1980. Virulence of Streptococcus mutans: restoration ofpathogenesis of glucosyltransferase-defective mutant(C4). Infect. Immun. 27:915-921.

12. Ikeda, T., and H. J. Sandham. 1971. Prevalence ofStreptococcus mutans on various tooth surfaces in Ne-gro children. Arch. Oral Biol. 16:1237-1240.

13. Ikeda, T., T. Shiota, J. R. McGhee, S. Otake, S. M.Michalek, K. Ochiai, M. Hirasawa, and K. Sugi-moto. 1978. Virulence of Streptococcus mutans: com-parison of the effects of a coupling sugar and sucrose oncertain metabolic activities and cariogenicity. Infect.Immun. 19:477480.

14. Jordon, H. V., B. Krasse, and A. Moller. 1968. Amethod of sampling human dental plaque for certain"caries-inducing" streptococci. Arch. Oral Biol. 13:919-927.

15. Krasse, B., H. V. Jordan, S. Edwardsson, I. Svens-son, and L. Trell. 1968. The occurrence of certain"caries-inducing" streptococci in human dental plaquematerial. Arch. Oral Biol. 13:911-918.

16. Kuramitsu, H. K. 1976. Properties of a mutant of Strep-tococcus mutants altered in glucosyltransferase activity.Infect. Immun. 13:345-353.

17. 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.

18. McCabe, M. M., R. M. Hamelik, and E. E. Smith. 1977.Purification of dextran-binding protein from cariogenicStreptococcus mutans. Biochem. Biophys. Res. Com-mun. 78:273-278.

19. Michalek, S. M., T. Shiota, T. Ikeda, J. M. Navia, andJ. R. McGhee. 1975. Virulence of Streptococcus mu-tans: biochemical and pathogenic characteristics of mu-tant isolates. Proc. Soc. Exp. Biol. Med. 150:498-502.

20. Miller, J. H. 1972. Experiments in molecular genetics, p.125-138. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

21. Mukasa, H., and H. D. Slade. 1973. Mechanisms ofadherence of Streptococcus mutans to smooth surfaces.I. Roles of insoluble dextran-levan synthetase enzymesand cell wall polysaccharide antigen in plaque forma-tion. Infect. Immun. 8:555-562.

INFECT. IMMUN.

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ADHERENCE-DEFECTIVE S. MUTANS MUTANTS 1055

22. Mukasa, H., and H. D. Slade. 1974. The mechanism ofadherence of Streptococcus mutans to smooth surfaces.II. The nature of the binding site and the adsorption ofthe dextran-levan synthetase enzymes on the cell wallsurface of the streptococcus. Infect. Immun. 9:419-429.

23. Nelson, N. 1944. A photometric adaptation of the Somo-gyi method for the determination of glucose. J. Biol.Chem. 153:375-380.

24. Newbrun, E. 1978. Cariology, p. 46-65. The Williams &Wilkins Co., Baltimore.

25. Somogyi, M. 1945. A new reagent for the determinationof sugars. J. Biol. Chem. 160:61-68.

26. Somogyi, M. 1945. Determination of blood sugar. J. Biol.

Chem. 160:69-73.27. Staat, R. H., S. D. Langley, and R. J. Doyle. 1980.

Streptococcus mutans adherence: presumptive evi-dence for protein mediated attachment followed byglucan-dependent cellular accumulation. Infect. Im-mun. 27:675-681.

28. Staat, R. H., and C. F. Schachtele. 1974. Evaluation ofdextranase production by the cariogenic bacteriumStreptococcus mutans. Infect. Immun. 9:467-469.

29. Terleckyi, B., N. P. Willett, and G. D. Shockman.1975. Growth of several cariogenic strains of oral strep-tococci in a chemically defined medium. Infect. Immun.11:649-655.

VOL. 34, 1981

on October 8, 2020 by guest

http://iai.asm.org/

Dow

nloaded from