INFECTION AND IMMUNITY, Sept. 1986, p. 587-594 Vol. 53, No. 30019-9567/86/090587-08$02.00/0Copyright C) 1986, American Society for Microbiology

Cloning of a Streptococcus mutans Glucosyltransferase Gene Codingfor Insoluble Glucan Synthesis

H. AOKI,t T. SHIROZA, M. HAYAKAWA,t S. SATO, AND H. K. KURAMITSU*

Department of Microbiology-Immunology, Northwestern University Medical-Dental Schools, Chicago, Illinois 60611

Received 24 February 1986/Accepted 3 June 1986

The gtJB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated ona 15.4-kilobase DNA fragment by using a XL47.1 gene library. The activity was catalyzed by gene products of150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble andinsoluble glucans. The enzyme was partially purified from lysates of the XDS-76 clone and synthesized bothtypes of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI ofapproximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguousnucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct genesharing partial homology with gfl was also obtained. The gtfB gene was subcloned into plasmid pACYC184into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximatelocation of the GTF promoter and the direction of gene transcription were also determined. The cloned enzymewas not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in thecytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtJB gene was insertionallyinactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region.After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesiswere identified. These results indicate that the gtJB gene codes for a GTF involved in insoluble glucan synthesisin strain GS-5.

The conversion of dietary sucrose to glucan polymers is animportant cariogenic property of Streptococcus mutans (5).In particular, insoluble glucan production has been shown togreatly increase the ability of these organisms to colonizetooth surfaces. The results from several laboratories havesuggested that multiple glucosyltransferases (GTFs) are in-volved in the synthesis of these polymers (3, 4, 14). Twoseparate GTFs have been identified in S. mutans serotype d(4, 10) and c strains (9, 14, 20), and three enzymes have beencharacterized from a serotype g strain (30). In addition,recent results have suggested that a lower-molecular-weightGTF, gtfA (26), exists in many strains of these organismsand may also play a role in glucan synthesis (25). Takentogether, the results from these investigations suggested thatone of the GTFs, termed GTF-S or dextransucrase, may beresponsible for synthesizing the primer required for anotherenzyme, GTF-I or mutansynthetase, to produce an insolubleglucan polymer.

Serotype c strains, which are the predominant S. mutansserotype isolated from the human oral cavity of manypopulations (1), may synthesize insoluble glucan by tworelated mechanisms. Like other serotypes, these organismscan produce insoluble glucan by the combined action of thepreviously characterized GTF-S and GTF-I (9, 14, 20). Inaddition, under conditions of aggregation (14, 21), the GTF-Sof these organisms can also catalyze insoluble glucan syn-thesis. However, the relative contributions of these twomechanisms to insoluble glucan synthesis under conditionsexisting in the human oral cavity is still unclear. Oneapproach toward resolving this question would be to pro-duce specific mutations in the GTF-S and GTF-I genes of

* Corresponding author.t Present address: Nihon University Dental School, Matsudo,

Japan.

these organisms. Although mutants defective in the latteractivity were previously isolated (13, 24), specific GTF-Smutants were not. In addition, the former mutants have notbeen extensively characterized to determine whether or notsingle mutations are responsible for the observed phenotypictraits. One strategy to produce such mutants involved ini-tially isolating the genes for the respective enzymes byrecombinant DNA techniques for subsequent in vitro muta-genesis, followed by reintroduction of the altered genes backinto serotype c strains by transformation (22). Recently,several laboratories have reported the isolation of S. mutansgenes which express GTF activities (6; Y. Abiko, and H.Takiguchi, J. Dent. Res. 64:737, 1985; H. Aoki, and H. K.Kuramitsu, J. Dent. Res. 64[special issue]:258, 1985). Thisreport describes the isolation and characterization of a GTFgene (designated gtfB) isolated from a serotype c strainGS-5.

MATERIALS AND METHODSMicroorganisms. S. mutans GS-5 was maintained and

grown routinely as previously described (13). Escherichiacoli C600 and C600(P2) were kindly provided by R. Russell,Royal College of Surgeons, Downe, England, and strainWL66 was obtained from Amersham Corp., Arlington Hts.,Ill. XL47.1 DNA was also obtained from Amersham Corp. E.coli strains were grown in L broth (10 g of tryptone [DifcoLaboratories, Detroit, Mich.], 5 g of yeast extract (Difco), 5g of NaCl per liter) with the appropriate antibiotics. Phagestocks were prepared on LB agar plates in soft agar overlays(16).DNA manipulations. Chromosomal DNA from S. mutans

GS-5 was purified essentially as previously described (8;Barletta and Curtiss, personal communication), extractedtwice with phenol, precipitated with ethanol (2 x), anddialyzed against TE buffer (10 mM Tris hydrochloride, 1.0

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FIG. 1. Isolation of the gtfB gene inactivated with an Emr gene.- , Plasmid vectors; *, GS-5 chromosomal DNA; =, Emr genefragment from plasmid pVA891. The relevant restriction sites are B(BamHI), E (EcoRI), H (HindIll), and S (Sall).

mM EDTA [pH 8.0]). Phage DNA was purified from indi-vidual clones as previously described (16). Plasmid DNAwas isolated by the alkaline-sodium dodecyl sulfate proce-dure (16). Restriction digestion by endonucleases and liga-tions was done by following the directions of the suppliers(Bethesda Research Laboratories, Inc., Gaithersburg, Md.and Amersham Corp.).

Construction of the S. mutans clone bank. Strain GS-5DNA was partially digested with Sau3AI and mixed withXL47.1 DNA which was completely digested with BamHI ina 1:1 ratio (total DNA concentration was 300 ,ug/ml). TheDNA mixtures were phenol extracted, washed with ether,and ethanol precipitated. The precipitates were dissolvedwith TE buffer and ligated at 12°C for 16 h in the presence ofT4 DNA ligase. After ligation, the mixtures were extractedwith five cycles of phenol extraction, washed with ether, andprecipitated with ethanol.

In vitro packaging of the ligation mixtures was done witha packaging (Promega, Madison, Wis.) system by the direc-tions of the supplier. Samples of the packaging mixture werethen incubated with E. coli C600(P2) cells for 20 min at 37°Cin 10 mM MgSO4. The samples were mixed with 3.0 ml ofLBtop agar and plated on LB agar plates at 37°C for 8 to 16 h.The resultant plaques were harvested by scraping each platewith 3.0 ml of A dilution buffer (10 mM Tris, 10 mM MgSO4[pH 7.5]). The clone bank was stored at 4°C and was used toinfect C600 cells for subsequent screening.

Screening of the GS-5 clone bank for GTF clones. The clonebank was initially screened for GTF clones by examiningplaques for sucrose-hydrolyzing (sucrase) activity essen-tially as previously described (6). Samples of the clone bank(10 ,ul) were mixed with 0.10 ml of mid-log-phase C600 cellsand incubated at 37°C for 15 min in the presence of 10 mMMgSO4. After the addition of 3.0 ml of LB top agar contain-ing 0.1% triphenyl tetrazolium chloride, the infected cellswere overlayed onto LB-1% sucrose agar plates. Afterincubation for 16 h, sucrase-positive clones could be readilydetected by the appearance of a red zone surrounding thepositive plaques.The clone bank was also screened for plaques that ex-

pressed antigens which react with antibody directed againsta partially purified GS-5 GTF fraction, GTF-A (11). Plaquesdeveloped on LB agar plates were blotted to nitrocellulosedisks, incubated with anti-GTF-A, washed, and developedwith the peroxidase-chloronaphthol system (28).

Transformation of E. coli cells. Plasmids and XL47.1 DNAcontaining the GTF inserts were cleaved with the appropri-ate restriction enzyme, ligated, and transformed into C600cells as previously described (16). Transformants were se-lected on LB agar plates containing the appropriate antibi-otic: chloramphenicol (30 ,ug/ml), tetracycline, (12.5 ,ug/ml),or erythromycin (Em) (200 jig/ml).

Transformation of S. mutans. Transformation of strainGS-5 was carried out as previously described (24) by usingthe appropriate plasmid constructs (2.5 ,ug/ml). The trans-formants were isolated on mitis salivarius-em (10 ,ug/ml) agarplates.Enzyme assays. E. coli extracts were prepared and frac-

tionated for isolation of the cytoplasmic, cytoplasmic mem-brane, and periplasmic fractions as previously described (7).

Sucrase activity was determined as previously described(17) in a standard incubation mixture containing 100 mMpotassium phosphate buffer (PB) (pH 6.0), 2% sucrose, 0.2%saline-NaN3, enzyme, and water in a total volume of 0.1 ml.The reaction mixtures were incubated for 30 min at 37°C andanalyzed for reducing sugar formation.GTF activity was determined as previously described (12)

by using sucrose [U-14C]glucose, except that the reactionmixtures were incubated for 1 to 16 h at 37°C. Longerincubation periods (16 h) were necessary for some fractionsto maximize activity (14). Insoluble, soluble, and total glu-can formation were also determined as previously reported(14).

Gel electrophoresis. DNA was analyzed after electropho-resis on horizontal 0.7% agarose gels with TBE buffer (89mM Tris hydrochloride, 89 mM boric acid, 2.5 mM EDTA[pH 8.3]). The gels were stained in ethidium bromide,washed, and photographed as recently described (29).

Proteins were analyzed on 8% polyacrylamide gels con-taining sodium dodecyl sulfate as described earlier (32). Thegels were either stained with Coomassie blue for detecting

l 2 3 1 2 3 1 2 3

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116

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A B CFIG. 2. SDS-PAGE analysis of XDS-76 lysates. (A) Coomassie

blue staining. (B) Western blotting with anti-GTF-S. (C) Activitydisplayed after incubation with sucrose. Lane 1, Concentratedchromatofocused GTF fraction; lane 2, XDS-76 crude lysate; lane 3,crude XL47.1 lysate.

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proteins and immunoblotted to nitrocellulose followed byreaction with antibody and peroxidase-chloronaphthol (28),or analyzed for GTF activity as previously described byRussell (27), with subsequent periodate Schiff base used tostain both soluble and insoluble glucan synthesizing activi-ties (19).

Southern blot analysis. Fragment transfer to nitrocellulosepaper, hybridization with 32P-labeled probes, washing, andfilm development were carried out as previously described(16) except that 50% formamide buffers were utilized and thefilters were washed at 37°C (approximately 15°C below themelting temperature for bacterial DNA [18]). 32P-labeledprobes were constructed after nick translation with theKlenow fragment (Pharmacia, Inc., Piscataway, N.J.) using[ot-32P]dCTP (Amersham Corp.) as recommended by thesupplier. The gtfl fragments were subcloned into vectorpUC9, and the resultant plasmids were used as probes.

In vitro inactivation of the gtfB gene. The gtJB genecontained on a 4.5-kilobase (kb) PstI-SalI fragment in vectorpUC9 (designated pTS20) was digested with BamHI, and theinternal 1.6-kb BamHI fragment was isolated after agarosegel electrophoresis (Fig. 1). The fragment was ligated toBamHI-cleaved pUC9dH (pUC9 with the HindIll site re-moved after HindIII cleavage, blunt ending with the Klenowfragment, then ligation) to produce plasmid pTS60.The 1.8-kb fragment coding for erythromycin resistance

(Em9) was isolated from agarose gels after BamHI cleavageof plasmid pTS19E. This latter plasmid was constructedfrom the transfer of the blunt-ended Emr fragment derivedfrom pVA891 (15) into pUC19. The Emr fragment was filledin with the Klenow fragment and ligated into the compatiblesite produced in plasmid pTS60 after HindIll cleavage andsubsequent treatment with the Klenow fragment. The result-ant Apr Emr plasmid pTS61 was used as the source of the invitro-inactivated gtJB gene. The linear 3.4-kb gtfB genefragment with an Emr insert was isolated from agarose gelsafter complete digestion of pTS61 with BamHI.

Purification of cloned GTF activity. Lysates (1.5 liters) ofC600 cells infected with XDS-76 were prepared as describedrecently (28). After lysis, cellular debris was removed bycentrifugation at 6,000 x g for 10 min at 4°C. The superna-tant fluids were then treated with ammonium sulfate (516g/liter) and allowed to stand for 18 h at 4°C. The resultantprecipitate was collected by centrifugation at 6,000 x g for10 min and dissolved in 20 mM PB buffer. The dissolvedsample was dialyzed against PB buffer and applied to a

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TABLE 1. Properties of cloned GTF activity

GTF activitya (cpm/16 h per ml of enzyme)

Soluble glucan Insoluble glucanEnzyme

Without With Without Withdextran dextran dextran dextranT10 T10 T10 T10

Crude XDS-76 lysate 2,870 4,890 2,370 2,540Crude MH76 extract 3,500 38,000 9,050 9,200Purified enzyme' 2,500 8,900 2,300 1,200

a GTF activity was measured by using sucrose-[U-14C] glucose.I The pooled concentrated chromatofocused enzyme was used as the

purified enzyme.

hydroxylapatite (Bio-Gel HTP [hydroxylapatite; Bio-RadLaboratories, Richmond, Calif.]) column previously equili-brated with PB buffer and eluted with a linear 20 mM-1.0 MPB buffer gradient and assayed for sucrase and GTF activi-ties (12). The fractions containing significant activity werepooled, concentrated through an Amicon UM-10 ultrafilter(Amicon Corp., Lexington, Mass.), and dialyzed against 20mM PB buffer.The concentrated enzyme sample was next resolved on a

DEAE-Bio-Gel A (Bio-Rad) column with a linear gradientconsisting ofPB buffer-1.0 M NaCl-PB buffer. The fractionscontaining GTF activity were pooled, concentrated throughan ultrafilter, dialyzed against 25 mM imidazole-hydrochlo-ride buffer (pH 7.4), and applied to a chromatofocusingcolumn (Pharmacia). The resin, previously equilibrated withimidazole-hydrochloride buffer, was washed with a 180-mlPolybuffer 74 linear gradient as recommended by the manu-facturer. The fractions containing GTF activity were pooled,concentrated, and dialyzed against 20 mM PB buffer.

RESULTS

Isolation of a GTF clone. The XL47.1 clone bank wasinitially screened for the presence of sucrase-positive clonesby growing C600 infected cells in soft agar overlays contain-ing sucrose and triphenyl tetrazolium chloride. In addition,the clone bank was propagated in C600 cells grown inminimal medium containing sucrose and low levels of glu-cose to detect sucrase-positive clones (6). By both screeningtests, a large number of sucrase-positive clones were iso-lated. Most of these appeared to express sucrose-6-

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Fraction NumberFIG. 3. Purification of cloned GTF by chromatofocusing. The pooled GTF active fractions from a DEAE-Bio-Gel A column were

fractionated on a chromatofocusing column as described in the text. A, pH; *, sucrase activity (A.56); 0, protein (A280); A, GTF activity.

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FIG. 4. Restriction map of the XDS-76 GTF gene. A restriction map of the XDS-76 DNA and the indicated subclones was generated byusing the following restriction enzymes: B (BamHI), C (ClaI), E (EcoRI), H (HindIII), P (PstI), S (Sall), X (XhoI). , GS-5 insert; O, X47.1DNA; *, pACYC184.

phosphate hydrolase activity, and one has been furthercharacterized (M. Hayakawa, H. Aoki, and H. K.Kuramitsu, submitted for publication). Several also codedfor gtfA activity (26) and were not further investigated. Inaddition, two clones expressed fructosyltransferase activityand have been recently characterized (29). However, noneof the sucrase-positive clones expressed GTF activity whenassayed by the standard radioactive procedure.

Since it was possible that a GTF-positive clone wouldexpress only weak activity which might not be detectable bythe sucrase screening assay, an immunoblot screening assaywas used to screen the clone bank. Therefore, antibodyagainst a partially purified GTF fraction (11) which synthe-sizes both soluble and insoluble glucans was used to screenthe clone bank. Approximately 20,000 plaques werescreened with anti-GTF-A, and 193 positive clones were

identified. Lysates of each positive clone were assayeddirectly for GTF activity. Only one active clone, XDS-76,was identified.

Characterization of XDS-76. When sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels was

used to observe GTF activity, two white insoluble glucanbands were present in XDS-76 lysates and none were ob-served in control XL47.1 lysates (Fig. 2C). Since antibodyagainst purified GTF-S preparations from strain GS-5 wasavailable (11), it seemed possible to conveniently discrimi-nate between GTF-S and GTF-I expression by XDS-76DNA. The antibody reacted with a single major protein bandof 155 kilodaltons (kDa) after Western blot analysis of crudeGS-5 culture fluids (data not shown). After SDS-PAGE andWestern blotting, it was possible to demonstrate that XDS-76protein bands of molecular weights 150 and 145 kDa reactedwith anti-GTF-S sera and that comparable bands were notpresent in crude lysates of X47.1-infected cells (Fig. 2).However, antisera directed against a purified GTF-I fromanother serotype c strain Ingbritt (kindly provided by H.Mukasa, National Defense Medical College, Tokorozawa,Japan) also reacted with the same GTF bands from XDS-76and the GS-5 culture fluids (data not shown).When the XDS-76 lysate was assayed for GTF activity, it

was observed that both soluble and insoluble glucans weresynthesized (Table 1). As previously determined (12), thesoluble glucan was degraded by dextranase, whereas theinsoluble product was quite resistant to the hydrolase (data

not shown). Neither soluble nor insoluble glucan synthesisrequired the addition of exogenous dextran.

Purification and characterization of the GTF activity. Crudelysates of ADS-76 were subjected to protein purification afterammonium sulfate precipitation, hydroxylapatite chroma-tography, and DEAE-Bio-Gel A anion exchange chromatog-raphy followed by chromatofocusing. The resultant GTFactivity was resolved from most of the contaminant proteins,but several lower-molecular-weight proteins were still pres-ent in the final preparation (Fig. 2A). This latter fractionrepresented an approximate 25-fold increase in the specificactivity relative to the crude lysate. It was of interest that theapparent pl of the purified activity was approximately 5.0,whereas a minor fraction eluted through the column with anapparent neutral or alkaline pl (Fig. 3). Like the crude GTFpreparations, the purified GTF fraction synthesized primer-independent soluble and insoluble glucans (Table 1). How-ever, in contrast to the crude enzyme preparations, thepresence of the primer somewhat inhibited insoluble glucansynthesis by the purified enzyme. Finally, SDS-PAGE anal-ysis of the purified GTF revealed the same active species(150 and 145 kDa) as observed in XDS-76 lysates (Fig. 2).However, the 150-kDa active band was very weak in thepurified enzyme preparation.

Restriction mapping of the XDS-76 insert. The DNA fromXDS-76 was purified from C600 lysates and subjected torestriction mapping (Fig. 4). After the construction of a

restriction map, it could be determined that XDS-76 con-tained a 15.4-kb insert from strain GS-5. Since the GTF mustbe coded by a gene of at least 4.1 kb (corresponding to a150-kDa protein), it was of interest to subclone the gene tofurther characterize the coding region.

Subcloning the Gtf gene. Based on the restriction map ofthe XDS-76 insert, several strategies for subcloning the GTFgene were evaluated. It was observed that an activesubclone carrying plasmid pMH76 could be isolated bycleaving pACYC184 and XDS-76 DNAs with ClaI followedby ligation and transformation into C600. The resulting CmrTcs tranformants then were directly assayed for sucraseactivity, and five active subclones were isolated. One ofthese, MH76, was chosen for further analysis; restrictionmapping of its chimeric plasmid revealed that pMH76 car-ried a 7.7-kb insert from XDS-76 (Fig. 4). Furthermore,subsequent analysis revealed that the entire insert originated

LutXDS-76

pMH76

pTS 5

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from the strain GS-5 fragment. This subclonesized large amounts of visibly detectable inso]when crude extracts were incubated with sucialso of interest that MH76 grew very slowlcontaining sucrose, suggesting that the synthesby the subclone is deleterious to the cells.

In order to isolate a GTF active subclone 4smallest GS-5 insert, XDS-76 DNA was subjectSau3AI digestion and ligated to BamHI-cleavedThe resultant Cmr Tcs transformants were screeability to grow on LB-sucrose agar, and 12 colgrew poorly in this medium were identified. Ea(GTF activity when assayed enzymatically and dsame GTF activity bands after SDS-PAGE (dataRestriction mapping revealed that each carBamHI fragment in common (Fig. 4). The insertpositive subclones ranged in size from 4.5 tcaddition, the GS-5 insert was carried in bothrelative to the vector (exemplified by pTS5 and4), suggesting that the GTF promoter was preinserts.

Subsequent deletion analysis suggested th.promoter was located near the common PstIinsets, since relatively small deletions in this arecomplete loss of enzymatic activity and the disaj

A

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1 2 3 4 1 2 3 4

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FIG. 5. Southern blot analysis of GS-5 chromosorRestriction map of XDS-76 and location of probe fragnTtion enzymes are as described in the legend toApproximate location of the gtfB gene. Probe a, enc(entire gtfB gene, was constructed after Bal 31 dPstI-Sall fragment containing the gtfB gene incorporalpUC9; probe b was obtained by cloning the PstI-Ecinto pUC9; probe c was isolated after ligation of thefragment into pUC9. (B) Hybridization with probe aization with probe b. (D) Hybridization with probe c.DNA was cleaved with HindIII (lane 1), BamHI (lI(lane 3), and PstI (lane 4). Arrows, Fragments genergene partially homologous to gtJB (for the PstI fragmepossible to determine which of the two fragments oithe homologous gene). Molecular size markers (Iindicated at the left.

also synthe-luble glucanrose. It wasly in media;is of glucan

carrying the;ed to partialpACYC184.ned for theirlonies whichch displayed

TABLE 2. Localization of GTF activity in E. coli'

Relative distribution (%)Cell compartment P-Galactosidase Alkaline GTF

phosphatase

Periplasm 1.0 43 2Cytoplasm 91 57 60Cytoplasmic membrane 8 0 38

aCell extracts of MH76 were prepared, fractionated, and assayed asdescribed in Materials and Methods.

lisplayed the GTF polypeptide bands (assayed after Western blotting ofLnot shown). SDS-PAGE gels with anti-GTF-S). In contrast, Bal 31 nu-ried a PstI- clease digestion at the opposite end of the insert led tos in the GTF truncated GTF polypeptides with varying degrees of GTF9.2 kb. In activity (T. Shiroza and H. K. Kuramitsu, manuscript in

orientations preparation). These results strongly suggested that thepTS18; Fig. cloned gene was transcribed from the PstI region toward the,sent on the internal EcoRI site of the common inserts (Fig. 4).

Southern blot analysis. In order to demonstrate that theat the GTF cloned gtjB gene represented a contiguous fragment ofDNAsite of the from the GS-5 chromosome as well as the number of copies

a resulted in of the gene in strain GS-5, Southern blot analysis was carriedppearance of out. Chromosomal DNA was digested to completion with

several restriction endonucleases, and the fragments weretransferred to nitrocellulose paper. Complete digestion witheach enzyme was confirmed by the demonstration that aprobe made from the gtfA plasmid pYA601 (26) hybridizedto only one fragment of chromosomal DNA cleaved with

_ C BamHII, EcoRI, PstI, ClaI, and to two fragments aftercleavage with HindIlI (data not shown). In addition, nohybridization between gtfA and fragments corresponding toportions of the gtJB gene could be detected.When a 32P-labeled probe made from the 4.5-kb fragment

containing the entire gtfB gene, (Fig. 5A, probe a) washybridized to fragments produced from cleavage with a

2 3 4 variety of enzymes, positive bands compatible with therestriction map of gtfB were detected (i.e., 1.6- and 1.9-kbHindlll fragments hybridized with the probe; Fig. 5B). The6.6-kb HindIII fragment represents the carboxyl terminalportion of the gtJB gene and downstream sequences on theGS-5 chromosome, since probe b but not the amino terminalprobe c also reacted with the same size fragment. In addi-tion, in each case an additional positive fragment wasdetected (4.5-kb HindIII, 3.2-kb BamHI, 4.6-kb EcoI, and7.0-kb PstI fragments). This indicated that either a portion ofthe gtJB gene contained a "scrambled" fragment (anoncontiguous piece of chromosomal DNA inserted duringligation of the GS-5 fragments into the XL47.1 vector) or that

D another homologous gene existed on the GS-5 chromosome.nat DNA. (A That the latter was responsible for the additional hybridiz-

lents. Rest(ic) able fragment was indicated by the utilization of additionalFig. 3. - probes from different portions of the gtj$ gene. When probesompassing the b and c (Fig. SA), composed of the 1.6-kb BamHI-BamHIeletion of the and the 2.0-kb PstI-EcoRI fragments, respectively, wereted into vector hybridized to the restriction fragments, one additional bandooRI fragment in addition to those compatible with the restriction maps was1.6-kb BamHI again detected (Fig. 5C and D). Therefore, a scrambledi-(C) Hybrid- fragment did not exist within the 3.8-kb cloned regionaCnhr2)moEomRal encompassing the b plus c regions. If such a fragmentmaed2)EroRIth existed, probe a and either probe b or c, but not both probesrated from thents, it wasnot b and c, would detect this extra band. A noncontiguousriginated from fragment of chromosomal DNA was also not present in thekilobases) are remaining portion of the gtJB gene (not encompassed by

probes b and c), since all three probes hybridized with only

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FIG. 6. Activity staining of GTF subclones. Concentrated sam-ples (10 x by ammonium sulfate precipitation) were resolved on

SDS-PAGE, followed by activity staining with PAS as described inthe text. Samples: 2 ,ul (lane 1) or 5 ,ul (lane 2) of GS-5 culture fluids;2 ,ul (lane 3) or 5 RI1 (lane 4) of XDS-76 lysate; 2 ,u1 (lane 5) or 5 ,u1(lane 6) of MH76 extracts. The lower-molecular-weight positiveband (84 kDa) in the GS-5 culture fluids was caused byfructosyltransferase. Numbers on right and left are molecular sizemarkers, in kilodaltons.

two PstI fragments. If such scrambling had occurred at theCOOH-terminal region of the gtfB gene (deduced from thelocalization of the promoter as described above), probe awould have hybridized with three fragments: the gtJB frag-ment, the homologous gene fragment, and the noncontigu-ous fragment from the GS-5 chromosome.

Location of GTF expressed in E. coli. Since the GTFs are

synthesized as extracellular enzymes in GS-5, it was ofinterest to determine the cellular location of the enzyme insubclone MH76. Fractionation of the cells revealed thatessentially none of the GTF activity traversed the E. colimernbrane and localized in the periplasmic space (Table 2).Most of the activity was associated with the cytoplasmiccompartment (60%), whereas a large amount was also foundassociated with the cell membrane (38%). The synthesis ofinsoluble glucan by the membrane-associated GTF mightexplain why all of the subclones containing intact gtJB genesgrew poorly on sucrose-containing media.

Activity staining of subclone extracts. When extracts ofMH76 were subjected to SDS-PAGE followed by activitystaining, it was observed that two bands of activity corre-

sponding to the same molecular weights (150 and 145 kDa)observed in XDS-76 lysates could be detected (Fig. 6). Inaddition, a third band of activity corresponding to a molec-ular size of approximately 135 kDa was observed in theMJ176 extracts and was barely detectable in the ADS-76lysates. However, this latter lower-molecular-weight en-zyme could be readily detected after periodate Schiff basestaining. Likewise, all of the twelve GTF active subclones(pTS5, pTS18, etc.) displayed the same activity bands as didMH76. No difference in the number and intensity of the GTFbands was noted in the presence or absence of dextran T10in the reaction mixtures. Concentrated culture fluids of

strain GS-5 displayed a single major band of GTF activity ofapproximately 155 kDa and a barely detectable band at 135kDa (Fig. 6). Inclusion of the protease inhibitorphenylmethylsulfonyl fluoride did not affect this pattern.These results suggested that the product of the gtfB genemay have undergone different degrees of proteolysis in E.coli extracts (MH76 and XDS-76), compared with the GS-5extracellular compartment.

Insertional inactivation of S. mutans GS-5 GTF activity.Since the cloned enzyme synthesized both soluble andinsoluble glucans and reacted with both anti-GTF-S andanti-GTF-I, it was not clear which enzyme was coded by thegtJB gene. To unequivocally identify the gene product, thecloned gene was inactivated after the insertion of an Emrgene fragment to specifically inactivate the cloned geneproduct in strain GS-5 after subsequent transformation.The linearized gtfl gene containing the Emr gene substi-

tuting for an internal HindIll fragment derived from pTS61(Fig. 1) was transformed into strain GS-5. Approximately98% of the resultant Emr tranformants displayed the smoothcolonial morphology on mitis salivarius agar plates that ischaracteristic of mutants defective in insoluble glucan syn-thesis (13). When these tranformants were assayed directlyfor GTF activity and compared with the wild-type activities,it was observed that the transformants displayed markedlyreduced insoluble glucan synthesizing activity. One typicaltransformant, LN61, displayed only 28% of the insolubleglucan synthetic activity of strain GS-5 (Table 3). Thisactivity was not increased by the addition of primer dextranT10 to the assay mixtures. In contrast, the smoothtransformant synthesized higher levels of soluble glucan,compared with the parental organism. The smooth trans-formants also displayed reduced sucrose-dependent coloni-zation of glass surfaces when assayed in an in vitro system.Finally, when chromosomal DNA was extracted from LN61and transformed into parental GS-5, more than 99% of theresultant Emr transformants displayed the smooth colonialmorphology on mitis salivarius agar plates. This indicatedthat this latter property was dependent upon the insertion ofthe Emr gene into the chromosomal GTF-I gene.

DISCUSSIONThe present results indicate that a gene coding for a GTF-I

activity synthesizing insoluble glucan in strain GS-5 has beencloned. This conclusion is contrary to that of our previousreport regarding this gene derived from earlier data (H. Aokiand H. K. Kuramitsu, J. Dent. Res. 64:258, 1985), whichsuggested that the gtJB gene coded for GTF-S activity. Thislatter conclusion was based primarily on the observationsthat the cloned gene product reacted with anti-GTF-S seraand that the purified cloned enzyme synthesized water-soluble glucans. However, the results of the present com-

TABLE 3. Insertional inactivation of the gtfB genea

GTF activity (cpm/0.10 ml per A6w)Soluble glucan Insoluble glucan

StrainWithout With Without Withdextran dextran dextran dextranT10 T10 T1o T10

GS-5 370 509 183 236LN61 1,511 1,189 51 22

a Parental GS-5 and Emr smooth transformant LN61 were grown to mid-logphase in 5.0 ml of Todd Hewitt broth. After centrifugation, 0.10-ml sampleswere assayed for GTF activity as described in Materials and Methods.

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CLONING OF AN S. MUTANS GLUCOSYLTRANSFERASE GENE 593

munication demonstrate that the cloned enzyme also reactedwith antisera directed against a purified serotype c GTF-Ienzyme (20). Either the GTF-S and GTF-I enzymes of strainGS-5 share sufficient cross-reactivity to be detected afterWestern blotting but not by immunodiffusion (20), or one ofthe two enzymes used to prepare the antibodies might becontaminated with small amounts of the other enzyme.

Furthermore, the pl of the purified cloned enzyme (pH 5.0)resembles that estimated for the GTF-I activity of strainGS-5 and is quite distinct from the alkaline pl estimated forthe corresponding GTF-S activity (14).The observation that the gtJB gene product synthesized

both water-soluble and insoluble glucans did not allowdifferentiation between a GTF-S and GTF-I gene product.Recent results have indicated that serotype c GTF-S en-

zymes can also synthesize insoluble glucan under conditionsof enzyme aggregation (14, 21). In addition, the purifiedGTF-I from another serotype c strain synthesizes bothsoluble and insoluble glucans (20). Therefore, the nature ofthe glucan product synthesized by the cloned gene enzyme

alone could not be used to unequivocally identify the natureof the enzyme.

In addition, crude extracts of E. coli MH76 containing thesubcloned gene produced large amounts of insoluble glucanrelative to soluble glucan. It is not clear why the purifiedenzyme and crude phage lysates synthesized lower ratios ofinsoluble glucan/soluble glucan relative to the crudesubclone extracts (Table 1). It may be possible that the crudeand purified XDS-76 GTF-I may be altered in a different waythan is the enzyme present in crude MH76 extracts. Cur-rently, further attempts to purify the enzyme and its iso-zymes from subclone MH76 are in progress.

Previous results from this laboratory have also indicatedthat the partially purified GTF-I activity of strain GS-5 was

dependent on the addition of exogenous dextran (14). How-ever, both the homogeneous GTF-I from serotype c strainIngbritt (20) and the cloned enzyme are apparently primerindependent. In fact, the addition of primer inhibited insol-uble glucan synthesis by the purified enzyme (Table 1). It islikely that the addition of (x-1,3-glucose units to the solubleprimer shifted the product glucans to a more water-solubleform. These differences may also result from different typesof processing which might occur in the culture fluids of S.mutans strains relative to E. coli extracts. The molecularweight of the GS-5 GTF-I was estimated as 155 kDa (14),that of the active GTF-I fragment from strain Ingbritt was

estimated as 99 kDa (20), and the cloned enzyme exhibitedmolecular weights of 135 to 150 kDa.The most convincing evidence to indicate that the gtJB

gene coded for a GTF-I activity came from the in vivoinactivation experiments. Inactivation of the gtfB gene ofstrain GS-5 after transformation with the altered gene clearlyresults in a major decrease in insoluble glucan-synthesizingactivity and a corresponding loss of sucrose-dependentcolonizing activity. Since a low level of insoluble glucan-synthesizing activity still remains in the tranformants, it ispossible that more than one GTF-I gene is produced inserotype c strains or conversely, that this residual activityresults from aggregated GTF-S activity (14, 21). Attempts toisolate other putative GTF genes in strain GS-5 are continu-ing in this laboratory to evaluate these possibilities.The possibility that some of the properties of the cloned

gtfB gene which differed from the GTF-I of GS-5 resultedfrom scrambling of the GTF-I gene during the constructionof the clone bank was eliminated by the results of theSouthern blotting experiments. These results indicated that

the nucleotide sequences encompassing the GTF gene werecontiguous on the GS-5 chromosome. Furthermore, theseresults suggested the presence of another gene sharinghomology with the gtJB gene. This additional gene did notappear to be merely a duplication of the gtJB gene, since itsapparent restriction map differed significantly from that ofgtjB (Fig. 5). It is possible that this latter gene could beanother closely related GTF-I gene and may be responsiblefor the residual insoluble glucan synthesizing activity de-tected after insertional inactivation of the gtJB gene. Alter-natively, this gene might code for GTF-S activity and couldexplain the cross-reactivity of the gtJB gene product withboth anti-GTF-I and anti-GTF-S. Furthermore, the detectionof a few (<1%) Emr rough colonies displaying wild-typeinsoluble glucan synthetic activity after transformation withpTS61 could also be explained by assuming that the Emrgene inserted into the homologous gene and not into gtfB. Itwill be of interest to isolate the homologous gene from theL47.1 clone bank by using probes constructed from theputative fragments of this gene (Fig. 5). Furthermore, arecent report (M. Pucci and F. Macrina, personal communi-cation) indicated that a GTF gene probe from S. mutansLM-7 also detects two distinct genes from this organism.

Localization experiments (Table 2) suggested that thecloned GTF enzyme was not able to traverse the E. colicytoplasmic membrane. However, a significant portion ofthe GTF activity was isolated that was associated with themembrane fraction. In fact, a higher proportion of GTFactivity (>38%; Table 2) may be associated with the mem-brane, since a substantial amount of the periplasmic enzymealkaline phosphatase is found associated with the cytoplas-mic fraction with the cell fractionation protocol utilized inthe present investigation. Since transcription of the GTFgene in MH76 appears to originate from its own promoter,these results suggest that the intact signal sequence (2) ispresent on the chimeric plasmid. It may be possible that theGTF signal sequence may allow association of the clonedenzyme with the E. coli cytoplasmic membrane but does notallow the heterologous protein to traverse this barrier. It is ofinterest that recent results from this laboratory (29) suggestthat the majority of a cloned GS-5 fructosyltransferaseexoenzyme can be secreted into the periplasmic space of E.coli C600.The availability of the cloned gtJB gene from GS-5 now

makes it possible to examine its role in cariogenicity ingreater detail. Currently, the relative position of the gene onthe chromosome is being determined following transfer tothe mapping vector pVA891 (15) followed by transformationas was done recently to map the gtfA gene (23). In addition,the nucleotide sequence of the gene and its regulatoryregions will be determined to explore the fine structure of theenzyme and investigate the regulation of its expression in S.mutans. Finally, the gtJB gene will be compared with theother GTF genes cloned from GS-5 in order to determine therelationship between these enzymes.

ACKNOWLEDGMENTS

We acknowledge the advice and suggestions of R. R. B. Russell,G. Dougan, P. Morrissey, R. Barletta, and R. Curtiss III in variousaspects of this investigation and the technical assistance of L.Nilsen.H.K. acknowledges the support of a Wellcome Travel Grant to

visit the laboratories of R. R. B. Russell and G. Dougan. Thisinvestigation was supported in part by Public Health Service grantsDE-06080 and DE-03258 from the National Institutes of Health.

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594 AOKI ET AL.

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