INFECrION AND IMMUNITY, JUlY 1994, p. 2679-2686 Vol. 62, No. 70019-9567/94/$04.00+0Copyright C 1994, American Society for Microbiology

Genetic Analysis of Fructan-Hyperproducing Strains ofStreptococcus mutans

DEANNA L. KISKA AND FRANCIS L. MACRINA*Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia 23298-0678

Received 20 December 1993/Returned for modification 3 March 1994/Accepted 7 April 1994

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans,is thought to contribute to the progression of dental caries. It may serve as an extracellular storagepolysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulationof bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoidcolonies on sucrose-containing agar as a result of increased production of fructan have been discovered. Byusing eight independent isolates, we sought to determine if such fructan-hyperproducing strains representeda genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA wereexamined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared tocorrelate with the serotype of the organism and the geographic site of isolation were evident. Southern blotanalysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains,but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan producedby these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutantsdeficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. ThefiruA4 gene wasinactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strainwhich does not hyperproduce fructan. All of thefru,4 mutant strains were devoid of fructan hydrolase activitywhen levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability tohydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.

Streptococcus mutans, the etiologic agent of smooth-surfacedental caries, produces several extracellular enzymes whichcontribute to the virulence of this organism (30, 39). Theseenzymes include three glucosyltransferases (GtfB, -C, and -D)and a fructosyltransferase (Ftf) which split sucrose to formextracellular polymer (1, 17, 18, 20, 35, 38, 42, 48). Fructosyl-transferase utilizes the fructose moiety of sucrose to synthesizea high-molecular-weight fructan polymer of the inulin typeconsisting mostly of beta-(2-1)-linked fructose residues withsome branching in the 6 position (2, 11, 36, 41). Actinomycesviscosus, Streptococcus salivarius, and Bacillus subtilis alsopossess fructosyltransferase activity and produce extracellularlevan-type fructans composed of a backbone of beta-(2-6)linkages with side chains in the 1 position (2, 11, 43). The highmolecular weight (>106) and extensive branching of thesepolysaccharides result in a large, globular polymer with lowintrinsic viscosity (12, 27, 28, 32). These characteristics wouldlikely prevent its diffusion from the plaque matrix, allowing itto serve as an extracellular carbohydrate reserve (14, 29, 32).The polymer accumulates in dental plaque upon exposure tosucrose (14, 16, 19) and then is catabolized to acid whenenvironmental sugar is depleted (10, 26, 49). This would resultin prolonged exposure of the tooth surface to acid, thuspromoting dental caries. The extracellular fructan hydrolase(FruA) of S. mutans, responsible for the degradation offructans, has been biochemically and genetically characterized(4, 5). The fruA gene encodes a protein of 159 kDa with atypical gram-positive amino-terminal signal sequence. Thefructanase enzyme was most active in hydrolyzing levan, re-leasing fructose as the sole end product (5, 6). This enzymeshowed about one-third the hydrolytic activity against inulin

* Corresponding author. Phone: (804) 786-9728. Fax: (804) 786-9946. Electronic mail address: MACRINA@GEMS.VCU.EDU.

that it showed against levan. Sucrose and raffinose could alsoserve as substrates but were hydrolyzed with much less effi-ciency. The preference of this enzyme for beta-(2-6)-linkedfructan polymers is interesting in that the fructan produced byS. mutans is composed of beta-(2-1) linkages. However, thisparticular activity may be advantageous in the plaque environ-ment where large amounts of levan, synthesized by S. salivariusand other oral microbes, may be present (2, 29, 49). Theimportance of fructans to the pathogenicity of S. mutans wasdemonstrated by using mutants deficient in fructosyltrans-ferase activity. These mutants exhibited decreased cariogenic-ity in the gnotobiotic rat model (30, 39). If fructan functions asa storage compound in the oral cavity, then strains whichcannot degrade this polymer should be less virulent. In otherwords, the inability to degrade fructan was anticipated to bethe biochemical equivalent of the inability to synthesize thispolymer. However, it was surprising that aJiuA-deficient strainof S. mutans showed no significant differences in caries viru-lence compared with the wild-type organism (50). This mayhave been due to the feeding schedule used in the rat cariesstudies or to in vivo complementation of the fruA defect byorganisms colonizing the specific-pathogen-free rats.A number of S. mutans strains have been isolated which

display large, mucoid colonies on mitis salivarius agar due tothe increased production of fructan (7, 13, 15, 33). Thisphenotype is a result of increased transcription of theff gene,which has been demonstrated by chloramphenicol acetyltrans-ferase gene fusions with the ftf promoter region (21). Thefructan-hyperproducing phenotype has been useful in examin-ing the role of fructan in the development of dental caries.Initial caries studies, which demonstrated the importance offtfto the pathogenicity of S. mutans, employed a fructan-hyper-producing strain (39). Thus, we sought to characterize genet-ically several of the known fructan-hyperproducing strains and

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TABLE 1. Characteristics of S. mutans strains

designation Serotype Fructan Site of isolation Origin; source; reference

GS5 c No Human carious lesion Massachusetts; H. Kuramitsu; 15LM7 e Yes Human carious lesion Massachusetts; D. Clewell; 15V310 c Yes Human carious lesion Illinois; I. ShklairV403 c Yes Human blood Georgia; R. Facklam; 1373-M3 ca Yes Human carious lesion Alabama; P. CaufieldKPKS2 c Yes Human dental plaque Sweden; B. Rosan; 7MT6801 c Yes Human carious lesion Japan; S. Hamada; 33MT6861 c Yes Human carious lesion Japan; S. Hamada; 33MT6879 c Yes Human carious lesion Japan; S. Hamada; 33a Serotype inferred from biotype.

to create fruA-deficient strains which could be used to assessfurther the contribution of fructan to caries pathogenesis.

MATERIALS AND METHODS

All chemicals were from Sigma Chemical Co. (St. Louis,Mo.) unless otherwise specified.

Bacterial strains and media. The S. mutans strains used inthis study are summarized in Table 1. These strains wereroutinely cultured anaerobically at 37°C on brain heart infu-sion agar plates (Difco Laboratories, Detroit, Mich.). Trans-formants of S. mutans which had acquired antibiotic resistancegenes were selected by growth on brain heart infusion agarplates containing tetracycline (5 jig/ml). Mitis salivarius (MS)agar (Difco) was used to examine colonial morphology result-ing from polymer production. S. mutans was grown in Todd-Hewitt broth (Difco) for the preparation of total cellular DNA.Genetically competent cells were prepared in Todd-Hewittbroth containing 10% heat-inactivated horse serum (GIBCO,Gaithersburg, Md.). Brain heart infusion broth supplementedwith 20 mM DL-threonine and 20 mM DL-alanine was used forsmall-scale preparation of chromosomal DNA. Cells grown forpurposes of extracellular protein isolation were cultivated in3% tryptone-0.1% yeast extract-0.5% fructose (TYF) broth.Escherichia coli HB101 (F- hsd-20 recA13 ara-14 proA2 lacYlgalK2 rpsL20xyl-5 mtl-l supE44 X-) was maintained on Lennoxbroth agar plates (GIBCO) aerobically at 37°C. For theselection of antibiotic-resistant transformants, tetracycline (5,ug/ml) or carbenicillin (30 ILg/ml) was added to Lennox brothagar plates. For plasmid DNA isolation and preparation ofcompetent cells, E. coli was grown aerobically at 37°C inLennox broth.DNA isolation and characterization. Large-scale plasmid

DNA isolation from E. coli was performed by using anion-exchange columns as described in the instructions provided bythe manufacturer (Qiagen Inc., Chatsworth, Calif.). Small-scale plasmid preparations were performed by using themethod of Birnboim and Doly (3). S. mutans chromosomalDNA was isolated by the method of Marmur, modified asdescribed by Schroeder et al. (39). Restriction endonucleasedigestion of plasmid and chromosomal DNA was carried out asdescribed in the instructions provided by the manufacturer(Bethesda Research Laboratories [BRL], Gaithersburg, Md.).Restriction endonuclease digests of chromosomal DNA weretransferred to 0.45-,um-pore-size reinforced nitrocellulose fil-ters (Schleicher & Schuell, Inc., Keene, N.H.) by using themethod of Smith and Summers (44) and analyzed by Southernhybridization (37). The DNA was immobilized on the nitrocel-lulose filters with the Stratalinker 2400 UV cross-linker (Strat-agene, La Jolla, Calif.). Probe DNA was labeled with 50 ,uCi of

[32P]dCTP (3,000 Ci/mmol; ICN Pharmaceuticals, Inc., CostaMesa, Calif.) by using a commercially available nick translationsystem (BRL). When needed, DNA size standards were la-beled with 50 ,uCi of [32P]ATP (4,500 Ci/mmol; ICN) by usinga commercially available 5' terminus labeling system (BRL).Unincorporated radioactive nucleotides were removed by us-ing Sephadex G-50 Mini-Spin columns and the protocol sup-plied by the manufacturer (Worthington Biochemical, Free-hold, N.J.). DNA ligation reactions were performed with T4DNA ligase (BRL) and buffer supplied by the manufacturer.

Bacterial transformation. E. coli HB101 was transformed bythe CaCl2 method (37) using mid-log-phase cells harvested atan optical density of 0.2 to 0.3 at 660 nm. Genetic transforma-tion of S. mutans was performed as described by Lindler andMacrina (22). Antibiotic-resistant transformants were selectedby growth on solid medium containing the appropriate antibi-otic(s).

Preparation of S. mutans extracellular protein. Ten-millili-ter overnight cultures of S. mutans grown in TYF broth wereinoculated to an optical density at 660 nm of 0.05 in 250 ml offresh TYF broth. The cultures were incubated anaerobically at37°C until an optical density at 660 nm of 0.8 was reached.Phenylmethylsulfonyl fluoride and EDTA were added to thecultures to a final concentration of 1 mM, and the supernatantswere harvested by centrifugation at 7,500 x g for 15 min.Acetone precipitation of extracellular protein was performedat 4°C by the method of Scopes (40) with the addition ofacetone to 50% of the culture volume. Precipitated protein wasrecovered by centrifugation at 7,000 x g for 15 min at 4°C. Thepellet was washed with cold 50% acetone and centrifuged at12,000 x g for 10 min at 4°C. The protein pellet was suspendedin 10 mM imidazole-HCl (pH 6.5) and dialyzed overnight at4°C in the same buffer supplemented with 1 mM phenylmeth-ylsulfonyl fluoride and 1 mM EDTA. Samples were stored inaliquots at -20°C for no longer than 2 weeks. Protein concen-trations were determined by the method of Lowry et al. (23)with bovine serum albumin as a standard.

Reducing sugar assay. The fructanase activity of extracellu-lar protein extracts was determined by measurement of freereducing sugar by the photometric assay of Nelson (31).Protein extract (50 ,ug) was incubated with 2 mg of inulin orlevan substrate per ml in 50 mM potassium citrate buffer (pH5.5) for 18 h at 37°C. Copper and arsenomolybdate reagentswere prepared as described by Nelson (31), and 1 ml of coppersolution was added to 1 ml of each sample. The mixture wasboiled for 20 min and cooled to room temperature. Onemilliliter of arsenomolybdate color reagent was added to thereaction mixture, and the optical density of each sample wasdetermined at 520 nm. The amount of reducing sugar liberatedby hydrolysis of fructan was determined by comparison with a

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FRUCTAN-HYPERPRODUCING STRAINS OF S. MUTANS 2681

fructose standard curve. Units of fructanase activity wereexpressed as micromoles of fructose released per milligram ofprotein extract.

Pulsed-field gel electrophoresis. Agarose blocks of S. mutanschromosome were prepared essentially as described by Oka-hashi et al. (34). S. mutans strains were grown anaerobically at37°C for 18 h in 10 ml of Todd-Hewitt broth. The cultures werediluted 1:50 in 10 ml of Todd-Hewitt broth with 20 mMDL-threonine and incubated anaerobically at 37°C to an opticaldensity at 660 nm of 0.600 (-109 cells per ml). The cells wereharvested by centrifugation at 4,000 x g for 15 min at 4°C andwashed with 10 ml of 10 mM Tris-HCl (pH 8.0)-i M NaCl.The pellets were suspended in 2 ml of the same buffer, and thecells were warmed to 37°C. Two milliliters of 1.2% InCertagarose (FMC Bioproducts, Rockland, Maine) cooled to 37°Cwas mixed with the cells and held at 37°C. The mixture (100 ,ul)was pipetted into each gel mold, and the molds were cooled at4°C for 10 to 15 min. The agarose blocks were placed in sterileEppendorf tubes containing 2 volumes of lysis buffer 1 (10 mMTris-HCl [pH 8.0], 1 M NaCl, 100 mM EDTA [pH 8.0], 0.5%Brij 58, 0.2% deoxycholate, 0.5% N-lauroylsarcosine, 1 mg oflysozyme per ml, 20,g of RNase per ml, 100 U of mutanolysinper ml) and incubated with gentle agitation at 37°C for 18 to 24h. The blocks were transferred to an equal volume of lysisbuffer 2 (0.25 M EDTA [pH 8.0], 1% N-lauroylsarcosine, 1 mgof proteinase K per ml) and incubated at 50°C for 18 to 24 h.If the cell lysis was not apparent (seen as decreased opticaldensity), the blocks were incubated for an additional 24 h infresh lysis buffer 2. The blocks were prepared for restrictiondigestion by incubation in 5 volumes of 10 mM Tris-HCl (pH8.0)-i mM EDTA-1 mM phenylmethylsulfonyl fluoride for 2 hat 37°C with gentle agitation. The buffer was changed, and theblocks were incubated for 18 h at 37°C with gentle agitation.The blocks were washed twice for 2 h with 10 volumes of 10mM Tris-HCl (pH 7.5)-i mM EDTA at 37°C with gentleagitation. Each block was incubated for 18 h with 30 U of NotIin a total volume of 400 pl as described in the recommenda-tions of the manufacturer (BRL). The Pulsaphor Plus (Phar-macia LKB Biotechnology, Piscataway, N.J.) contour-clampedhomogeneous electric field electrophoresis system was used toseparate NotI-digested DNA. One-third of each block wasplaced into the wells of a 0.9% SeaKem LE (FMC Bioprod-ucts) agarose gel. The gels were run in 0.8x TBE buffer (0.08M Tris base, 0.08 M boric acid, 0.16 mM EDTA) at 180 V for24 h with switch times of 40 s. Lambda DNA concatamers(New England Biolabs, Beverly, Mass.) were used as molecularsize standards.

RESULTS

Restriction fragment pattern analysis of fructan-hyperpro-ducing strains of S. mutans. Previously, we had established thatcertain strains of S. mutans produced about 10 times therelative specific activity of fructosyltransferase produced bymany clinical isolates and well-characterized laboratory strains(e.g., S. mutans GS5). This phenotype, which can be demon-strated easily by large, gumdrop-like colonies on mitis saliva-rius agar, has been shown to be the consequence of increasedftf transcription as measured by using chloramphenicol acetyl-transferase gene fusion constructs (21). The large amounts offructosyltransferase enzyme produced by strains of this typewere demonstrated easily by analysis of extracellular proteinpreparations on sodium dodecyl sulfate-polyacrylamide gels(data not shown).Our collection of eight fructan-hyperproducing strains of S.

mutans (Table 1) was examined to determine whether individ-

A B C D E F G H I J

kb

23.0 No

9.4 - p

6.6 - t

4.4 _

Li LW.... J Li L W I1 2 3 4

FIG. 1. Restriction fragment pattern analysis of S. mutans DNA.Total cellular DNA of S. mutans was digested with HaeIII andsubjected to 0.55% agarose gel electrophoresis for 16 h at 40 V. Thegel was stained with ethidium bromide. Lanes: A, lambda HindIllmolecular size standard; B, GS5; C, LM7; D, V310; E, V403; F, 73-M3;G, KPKS2; H, MT6801; I, MT6861; J, MT6879. The numbers belowthe gel indicate the various genetic types of fructan-hyperproducingstrains of S. mutans.

ual isolates could be distinguished by restriction fragmentpatterns. Total cellular DNA from each strain was digestedwith HaeIII and subjected to agarose gel electrophoresis (Fig.1). Lane B contains DNA from S. mutans GS5, and theremaining lanes represent DNA from the fructan-hyperpro-ducing isolates. The restriction endonuclease pattern of GS5was clearly distinct from the patterns of the fructan-hyperpro-ducing strains, exhibiting a larger number of fragments of >7kb in size. Although some similarities were evident in thegenomic fingerprints of the fructan-hyperproducing strains,there was sufficient diversity to allow the strains to be dividedinto four types, designated 1 to 4 (Fig. 1, indicated at thebottom of the photograph). Strain LM7, the single serotype estrain, did not possess the -15-kb fragment which was evidentin all of the serotype c strains. This fragment was first noted byCaufield and Walker (8) in a study of 30 randomly isolatedserotype c/f strains and may provide a convenient geneticmarker for distinguishing these serotypes. The serotype cstrains comprised the remaining three genetic types whichappeared to correlate with the geographic site of isolation.Type 2 strains originated from several regions of the UnitedStates. Strain 73-M3 displayed a slight pattern variation com-pared with the other type 2 strains but was considered to be thesame type because it had additional genetic features in com-mon with these isolates as discussed below. KPKS2 wasisolated in Sweden and designated a type 3 strain. Type 4strains were from Japan and were isolated from a mother (Fig.1, lane H) and her two daughters (Fig. 1, lane I and J). Theidentical restriction fragment patterns of the type 4 strainsindicated a maternal transmission of S. mutans.The strains were also analyzed by contour-clamped homo-

geneous electric field electrophoresis by using the restrictionenzyme NotI, which cleaves the streptococcal genome infre-

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2682 KISKA AND MACRINA

A B C D E F G H I J K

kb

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FIG. 2. Contour-clamped homogeneous electric field electrophore-sis analysis of S. mutans chromosomal DNA. Agarose blocks of S.mutans chromosomal DNA were digested with NotI and subjected topulsed-field gel electrophoresis in 0.9% SeaKem LE agarose for 24 hat 180 V. The gel was stained with ethidium bromide. Lanes: A,lambda DNA concatamer molecular size standards; B, GS5; C, LM7;D, V310; E, V403; F, 73-M3; G, KPKS2; H, MT6801; I, MT6861; J,MT6879; K, lambda DNA concatamer molecular size standards. Thenumbers below the gel indicate the various genetic types of fructan-hyperproducing strains of S. mutans.

quently (Fig. 2). Four types of fructan-hyperproducing strainswere again evident and were consistent with the types obtainedwith HaeIII digestion.

Analysis of genomic loci of fructan-hyperproducing strainsof S. mutans. Since the fructan-hyperproducing phenotype hasbeen shown to be due to increased Ftf activity (33), it was ofinterest to examine the genomic organization of the ftf regionin all of the strains. EcoRI-digested total cellular DNA waselectrophoresed, blotted to nitrocellulose, and hybridized with32P-labeled pResAmpHind. This plasmid contained a 4.3-kbinsert of GS5 chromosomal DNA containing the ftf gene andflanking open reading frames (42). An autoradiogram of theSouthern blot is shown in Fig. 3A. The hybridization patternswere identical for all of the strains except MT6801, whichexhibited an additional component that hybridized with theprobe. These hybridization signals may represent a duplicationof the ftf gene or its flanking DNA.Two additional loci, gfB/C and fruA, were examined for

obvious changes in genomic organization. Genetic polymor-phisms in the gtfB and gtfC genes have been shown to occur inclinical isolates of S. mutans as a result of recombinationbetween these highly conserved genes (9, 51). To evaluate theoccurrence of such events, total cellular DNA was digestedwith PstI and subjected to agarose gel electrophoresis. TheDNA was transferred to nitrocellulose and hybridized with32P-labeled pVA1436, which contained an internal HindIIIfragment of the gtfC gene which was also homologous to gtfB(35). The hybridization patterns were identical for all of thestrains, indicating the conserved tandem arrangement of thesegenes in the chromosome (data not shown).

A B C D E F G H I J

EcoRi EcoHI EcoRi HindlilI. I

ORF I FTF-_' ORF 3 ORF0-ORF Z_d -

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PstI EcoRIEcoRi I EcoRl

Fru A - o

I I I ISizes of -- 4.0 kbPst EcoRI (a)fragments

2.36 kb(c)

2.6 kb(bi

FIG. 3. Southern blot analysis of S. mutans genetic loci. Total cellular DNA of S. mutans was digested with appropriate restriction enzymes andelectrophoresed through 0.7% agarose, transferred to nitrocellulose, and hybridized with various nick-translated 32P-labeled plasmids. Photographsof the autoradiograms are shown. Restriction fragments which hybridized with each probe are denoted by lowercase letters, and their sizes aregiven at the bottom of each panel. Arrows indicate the direction of transcription of the relevant genes. (A) S. mutans DNA digested with EcoRIand probed with pResAmpHind. This plasmid carried a 4.3-kb HindIlI fragment of S. mutans GS5 chromosomal DNA which contained theftfgeneand flanking open reading frames (ORFs) (42). (B) S. mutans DNA was digested with PstI-EcoRI and probed with pFRU1, which contained themajority of thefruA gene (4). Lanes: A, 32P-labeled 1-kb molecular size standard ladder; B, GS5; C, LM7; D, V310; E, V403; F, 73-M3; G, KPKS2;H, MT6801; I, MT6861; J, MT6879.

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FRUCTAN-HYPERPRODUCING STRAINS OF S. MUTANS 2683

A B C D E F G H I J

FIG.4~~~~~~~.Hybidzaio of S mua DN witl 9 S mutan

32P-labeled..pVA1..7d, wic cntie an interna.....lHldIIEcR

LM7; soD, V30 ,V4.3;,7-3 , KPS2 H, MT8 I,MT8L

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pFRU1(5),which contained the majorityof.. te fA..e..

(Fig.....3B).StrainsMT6 , MT661, and M oT

(FIG.3B,4lanesHytobJ)rexhibation inS.mtsD wthe hybridiztatons

pattero whichappeaedtoriroeslultsefnrome wthe ninrdcktionsofatePslabeorEcoRI9sit wintche conserved 2.-kfntrnagmeint.IEonRth

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seAqsuEc Rldigest (it 9hchRIBasmH electropholromtherouca-trasei,diplyered tonitrocefaulose, and t hybridized withPnltaled ,which containedth III-EcoRVfaeof the ine

tionseqencefS19elmetwhtgashothybiiedatoradiogrami-BamhI-ndigeste totac2-aelluardN (Fig.4).claFive genomicdfadra,Gments,

(Fig.DV3B 0;StrinV403; 1,73MT6861, and2H MT6801; ofMType1;

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(Fragment) pateransceal distinct from61 tanMT67ofth type2 4slaepernstrain S.mutans.pSine fruct is thougt ntroduction as an

dsturin srainsmaye iave aoseleved23fragment.tintheyoralcitydeis o theiincrseasedn yntlhsis of thie polmer. it wasIeSta-lished pretvioul tha iatvatinc ofthis fimgene in thefructan-hyperproducingstra(insV, dr-amaial reduced thcviualince ofthoanisinathEgeneotobtic ofratent insersmodelu3).Telementh defiasetencizoednrol of fructanmi-

hyesedprtiular strainS, frutan-dhV4cnt mtarnctseraed. costruce d.splayed fere pate was that with win

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Isrinliatvtoofruinfructan-hyperproducingstan,3PlbedpA90whcstrinsoo.mtnssqunc Sinent frcanisbridized to fuctonI-asHI-diextedtoalcellularstrgDcmoNd(1i.4).29 32ve genomctfangmyerprohyrduizedstrinsmyhaISI9 in thelerctivehyperproducingthetralinsvittype to athoughinrasedifernthssopthiternerIwasevdnwihstrab-lished peIoulthaddicivtion,hsstrin posesed ft genek plasmidfructnhyperasproductingL7strainV43daaichall redurcdtionviragmencofthioatrglanlismtincth gnoobotithatdftentype carisoaes

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FIG. 5. FruA insertional inactivation strategy. A 2.36-kb PstI-EcoRI fragment was removed from pFRU1 and cloned into thePstI-EcoRI sites of pUC19 to create pVA1819. pVA1819 was digestedwith HincII to allow insertion of a HincII fragment from pVA981,which contained a streptococcal tetracycline resistance marker. Theresultant plasmid, pVA1972, was linearized with AvaII and trans-formed into S. mutans. Allelic exchange recombination was selected byresistance to tetracycline.

2.36-kb PstI-EcoRI fragment, internal to the fruA gene, wasremoved from pFRU1 and cloned into the PstI-EcoRI restric-tion sites of pUC19 to create pVA1819. A 3.5-kb HincIIfragment, containing a streptococcal tetracycline resistancedeterminant, was removed from pVA981 (47) and cloned intothe HinclI restriction sites of pVA1819. The resultant plasmid,pVA1972, was linearized with Avall and transformed into twofructan-hyperproducing strains, V403 and KPKS2, as well asGS5, which served as a control strain. The allelic exchangeevent, resulting in the insertion of the tet gene within thefuAcoding sequence, was selected by tetracycline resistance andconfirmed by Southern blot analysis with pFRU1 (Fig. 6).Digestion of the parental genomes with PstI-EcoRI resulted inthree fragments which hybridized with the probe. The 2.36-kbPstI-EcoRI fragment (Fig. 6, arrow) represents the clonedfragment on pVA1819 into which the tetracycline resistance

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A

1 2 3 4 5 6 7

B

1 2 3 4 5 6 7

4 c 1-__*

Psti Hlncil Hlncil EcoRlEcoRI I I I I Ecol

FruA ---4

L- I l-4 kb 2.36 kb -2.6 kb

(a) (c)

GS5 GS5fruA 403 403fruA KPKS2 KPKS2fruARI

Strains

(b)

50 -eIntegration of tetracyclineresistance marker

Psti EcoRIEcc

EcoRII FrnRIOn F.-V

_1s Tc -lo.

I I I I _-4 kb 0.8 kb 4.5 kb -2.6 kb

(a) (c2) (cl) (b)

FIG. 6. Analysis of allelic exchange events involving fruA. Totalcellular DNA of wild-type and tetracycline-resistant strains wascleaved with PstI-EcoRI, electrophoresed through 0.8% agarose, andtransferred to nitrocellulose. (A) Blot hybridized with 32P-labeledpFRUI, which contained the majority of theJ;uA structural gene; (B)blot hybridized with 32P-labeled pVA981, which contained the tetra-cycline resistance determinant. Photographs of the autoradiogram areshown. Lanes: 1, 32P-labeled lambda HindIll; 2, GS5; 3, GS5 fruA; 4,V403; 5, V403 fiuA; 6, KPKS2; 7, KPKS2 fruA. Diagrams of the fruAlocus and the chromosomal integration event are shown below theautoradiograms. The PstI-EcoRI fragments which hybridized with theprobes are indicated by lowercase letters, and their approximate sizesare noted below the linear map.

marker was inserted. This fragment was not present in thetransformants and was replaced by two fragments of -4.5 and0.8 kb, consistent with the insertion of the tetracycline resis-tance determinant within the JfuA gene at the HinclI sites. APstI-EcoRI digest of parental and transformant DNA was alsohybridized with pVA981, which carried the tetracycline resis-tance determinant. Two fragments were detected in the trans-formants which were identical in size to the two uniquefragments evident in the transformant DNA hybridized withpFRU1. This confirmed the integration of the tet gene withinfi-uA.

Biochemical analysis offiuA-deficient strains. Inactivationof the fiuA gene in the transformants was confirmed biochem-ically by measuring the ability of acetone-precipitated extra-cellular enzymes to release reducing sugar from fructan sub-strate. FruA exohydrolytically cleaves both levan and inulin torelease free fructose which can be quantitated by a colorimet-ric assay. Figure 7 shows the fructanase activity of eachparental strain and itsfiuA-deficient counterpart. There was no

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FIG. 7. Analysis of fructanase activity of wild-type and fruA-deficient strains. Acetone-precipitated extracellular protein prepara-tions were incubated with either levan (A) or inulin (B) for a period of18 h. The amount of fructose present at the conclusion of theincubation period was measured by a reducing sugar assay as describedin Materials and Methods. Each solid bar represents the mean of threeindependent experiments. Error bars indicate standard deviations.

detectable fructan hydrolase activity in the fruA-defectivestrains when levan was used as a substrate. However, in thecase of inulin, only in the GS5 fruA strain was activityeliminated. The two fructan-hyperproducing fruA-deficientstrains still retained more than 85% of the fructan hydrolaseactivity for inulin, suggesting the presence of a second fructa-nase enzyme in these strains which possessed activity specificfor inulin.

DISCUSSION

Fructan-hyperproducing strains of S. mutans are unique inthat they synthesize copious amounts of fructan polymer as aresult of increased transcription of the ftf gene (21). AnFtf-deficient fructan-hyperproducing strain has demonstratedreduced smooth-surface and sulcal caries formation in the

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FRUCTAN-HYPERPRODUCING STRAINS OF S. MUTANS 2685

gnotobiotic rat model (39). Therefore, these strains may beuseful in ascertaining the importance of fructans in the devel-opment and progression of dental caries. It was the goal of thisstudy to genetically characterize several known fructan-hyper-producing isolates of S. mutans and to create fruA-deficientmutants which could be evaluated for virulence in the ratmodel.To determine if the fructan-hyperproducing isolates repre-

sented a genetically homogeneous group of organisms, restric-tion fragment patterns of total cellular DNA were examined byusing pulsed-field and conventional gel electrophoreses. Fourgenetic types which served to distinguish the strains bothserotypically and geographically emerged from this analysis.Restriction fragment pattern analysis was used by Caufield andWalker (8) to examine 30 randomly isolated serotype c/f(biotype I) S. mutans strains. All of the serotype c/f isolatespossessed a 15-kb fragment which was also present in theserotype c fructan-hyperproducing strains of this study. Con-sistent with this observation, the single serotype e isolate, LM7,did not exhibit this fragment. In the Caufield and Walkerstudy, only those strains isolated from mother-infant pairsdisplayed identical restriction fragment patterns (8). This was

also the case with the Japanese isolates of this study, confirm-ing the utility of this procedure in establishing maternaltransmission of S. mutans. The strains of type 2 appeared topossess identical restriction fragment patterns although thesestrains were isolated from three different states in the UnitedStates. These strains also carried multiple copies of an inser-tion sequence element (IS199) in the chromosome. Thiselement was first discovered in strain V403 (24, 25) and doesnot appear to be widely disseminated among clinical isolates ofS. mutans (7a), a characteristic which indicates that thesestrains probably shared an evolutionary origin. The pattern ofIS199 hybridization to strain 73-M3 revealed by Southern blotanalysis indicated a genomic rearrangement of these elementscompared with the pattern of hybridization to strains V403 andV310. This may account for the slight variation seen in therestriction fragment pattern of 73-M3. Transposition of theseelements into a different chromosomal site has been shown to

result from selective pressure on the organism (25).The genomic organization of several extracellular enzyme

loci was conserved in all of the strains. However, the presence

of an additional restriction site was evident within the fiuAgene of the type 4 strains. This alteration occurred within the2.36-kb PstI-EcoRI fragment of theJiuA gene which is knownto contain several regions that are homologous to other,B-fructosidases and may define the active site of the protein(4). The nucleotide change(s) necessary to create a new

restriction site in the fruA gene may be significant in light ofthis fact.To evaluate the contribution of fructan to the dental caries

process, we sought to construct fiuA-deficient fructan-hyper-producing strains which could be tested for virulence in an

animal model system. The insertion of a tetracycline resistancedeterminant within the coding region of the jfuA gene was

confirmed by Southern blot analysis and measurement offructan hydrolase activity. All of thefiuA-deficient strains weredevoid of fructanase activity against levan; however, the two

fructan-hyperproducing strains tested retained the ability to

hydrolyze inulin, suggesting the presence of a second enzyme

with specificity for inulin. The fact that two fructan-hyperpro-ducing strains from different genetic types exhibited this phe-nomenon suggests that this is not an anomalous result. Atruncated FruA protein of approximately 518 amino acidscould result from the insertional inactivation strategy used inthis study. If this form of FruA were still active against inulin,

then extracellular protein preparations from fruA-deficientGS5 should have exhibited detectable activity. However, theGS5 fruA mutant was unable to hydrolyze inulin. In addition,the ratio of levanase to inulinase activity in the wild-typeorganisms was much lower with the fructan-hyperproducingstrains, particularly V403. This is inconsistent with reports of amuch greater activity of the enzyme toward levan substrate (6,50). The existence of a second fructanase enzyme in S. mutanswould not be surprising. S. salivarius, which synthesizes alevan-type fructan polymer, produces two fructan hydrolases,one with specificity for levan and a second nonspecific enzymewhich can hydrolyze both levan and inulin (45, 46). The factthat S. salivarius synthesizes large amounts of levan andpossesses an enzyme specific for its degradation is significantfrom a metabolic standpoint. Fructan-hyperproducing strainsof S. mutans synthesize large quantities of inulin, therebypossibly requiring an inulin-specific enzyme for efficient deg-radation. The isolation and characterization of this putativeenzyme are being pursued by this laboratory and are necessarybefore jfuA-deficient fructan-hyperproducing strains can betested in the rat dental caries model.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grantDE04224 to F.L.M.We thank Robert Burne for providing pFRU1, Howard Kuramitsu

for providing pResAmpHind, and Shigeyuki Hamada for providing S.mutans MT6861, MT6801, and MT6879. Timothy Stedman and Greg-ory Buck are gratefully acknowledged for their instruction and assis-tance with pulsed-field gel electrophoresis. We thank Arunsri Brownfor excellent technical assistance.

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