Vol. 61, No. 4INFEcrION AND IMMUNrIy, Apr. 1993, p. 1246-12500019-9567/93/041246-05$02.00/0Copyright © 1993, American Society for Microbiology

Expression of gtfS Is Essential for Normal Insoluble GlucanSynthesis by Streptococcus downei

KEETA S. GILMORE,1 ROY R. B. RUSSELL,2 AND JOSEPH J. FERRETTI'*Department ofMicrobiology and Immunology, University of Oklahoma Health Sciences Center,

Oklahoma City, Oklahoma, 73190,1 and Department of Oral Biology, University ofNewcastle upon Tyne, NE2 4BW, United Kingdom

Received 15 June 1992/Accepted 30 December 1992

The gt7 and gtf genes of Streptococcus downei were investigated to determine the contribution of therespective enzymes to glucan production in the presence and absence of other glucosyltransferases. Extracts ofEscherichia coli expressing cloned gtJS produced a short linear dextran from sucrose which could act as aprimer for insoluble glucan synthesis when mixed with extracts of a strain expressing recombinant gtfI. Toelucidate the contribution ofguS to glucan production by S. downei, a mutant was constructed by insertionallyinactivatinggtS. S. downei (go mutant) colonies exhibited a marked phenotypic change on sucrose-containingmedia and a decreased ability to adhere to glass and produced no detectable water-insoluble glucan. Theseexperiments confirm that expression of gutS is essential for normal insoluble glucan synthesis by S. downei.

The adherence of mutans streptococci is due, in part, tothe production of a group of enzymes called glucosyltrans-ferases (GTFs). These extracellular enzymes cleave dietarysucrose to yield fructose and glucose, the latter componentbeing polymerized into water-soluble and/or water-insolubleextracellular polymers, or glucans, which contribute to theadherence and aggregation of mutans streptococci on thetooth surface (6, 19). Several GTFs and their genes havebeen isolated and characterized from various mutans strep-tococci (1, 2, 5, 7, 8, 10-12). These GTFs vary in the type ofglucan produced and in primer requirement but show a highdegree of amino acid sequence homology (3, 7). Streptococ-cus downei produces four GTFs which are separable bychromatofocusing (19a), two of which have been cloned andsequenced (5, 7).The gtfS gene encodes a 147-kDa GTF which is primer

independent and produces a water-soluble glucan. The glu-can was shown by nuclear magnetic resonance (NMR)analysis to consist of over 90% a-1,6-linked glucosyl resi-dues (7) and to have an average chain length of 25 glucoseunits (20). Insoluble glucan is produced primarily by theenzyme encoded by gtf and is believed to contribute tosucrose-dependent attachment of mutans streptococci tosmooth surfaces (9). The contribution of the water-solubleglucan produced by GTF-S to bacterial adhesion and dentalcaries formation is of considerable interest. It has beenhypothesized that the water-soluble glucan produced byGTF-S plays a role in caries formation by providing theprimer necessary for water-insoluble glucan synthesis byprimer-dependent GTF-I (15, 23). Such interdependencebetween different GTFs has been demonstrated with purifiedenzyme preparations (14, 15, 18, 23), but in vitro experi-ments offer only limited insight into the interrelationshipsbetween GTFs in vivo.

In the present study, the relative contribution of GTF-Iand GTF-S to insoluble glucan synthesis was determined byexamining activities expressed by a heterologous host andby insertionally inactivating gtfS in the native host, S.downei, and observing the effects on insoluble glucan for-

* Corresponding author.

mation and its role in sucrose-dependent colonization. Onthe basis of the available nucleotide sequence information(7), a mutant g9tS strain of S. downei was constructed byinsertional inactivation of the cloned gene in vitro, followedby allelic replacement. The resulting mutant was comparedwith the wild-type strain morphologically for its ability toproduce extracellular polymers and for its ability to adhereto smooth surfaces. By observing differences in these pa-rameters, we were able to determine that soluble glucanproduced by primer-independent GTF-S encoded by thegene g#tS provides the primer for insoluble glucan synthesisby GTF-I.

MATERIALS AND METHODS

Bacteria and media. The gtJS gene was previously clonedfrom S. downei MFe28, a serotype h strain (7, 8). The clonederythromycin resistance determinant (ermAM) (17) was usedfor in vitro insertional inactivation of gtfS. S. downei strainswere grown in Todd-Hewitt broth or Todd-Hewitt brothsupplemented with 5% sucrose. M17 broth (13) supple-mented with 0.5% (wt/vol) glucose was used to produceelectrocompetent S. downei cells. M17 agar with 0.5%glucose, 0.5 M sucrose, and 50 ,ug of erythromycin per mlwas used to select electrotransformants. Changes in colonymorphology and glucan production were observed on mitissalivarius agar containing 5% sucrose.

Insertional inactivation of gfiS in Escherichia coli. A 6-kbEcoRI restriction fragment containing gtfS was subclonedfrom pMLG60 (7) into pUC8. A 3-kb SphI-SstI restrictionfragment encoding a portion of the catalytic domain and theentire glucan-binding domain was removed (see Fig. 1). Theremaining 5.7-kb restriction fragment contained the pUC8vector and the N-terminal 1-kb portion of gtfS. This 5.7-kbDNA band was separated through low-melting-point agar-ose, excised, and purified by adsorption to glass milk (Gene-Clean, Bio 101, Inc.). The ends of the restriction fragmentand a similarly purified 1.2-kb restriction fragment contain-ing the erythromycin resistance gene, ermAfM, were treatedwith S1 nuclease to obtain blunt ends and ligated overnightat 4°C. The ligation mixture was used to transform E. coli

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XL1-Blue (4), and transformants were selected by growth on

media containing 100 p,g of erythromycin per il.Electrotransformation of insertionally inactivated g8S into

S. downei. A 4.2-kb restriction fragment containing the 1-kb

encoding part of the N-terminal portion of gtfS, the 1.2-kb

ennAM gene, and 2 kb of sequences which flank gtJ on the

S. downei genome was isolated from the above construction

after EcoRI digestion and separation through low-melting-

point agarose. The purified DNA fragment (1 p,g) was

introduced into wild-type S. downei MFe28 by electrotrans-

formation (22). Transformants were selected on M17 agar

supplemented with 0.5% glucose, 0.5 M sucrose, and 50 ,ul of

erythromycin per ml.Characterization of S. downeigjS mutants. To determine if

recombinational displacement of the native S. downei gtfShad occurred, transformatits for which the MIC of erythro-

mycin was 512 ,ug/ml were selected for Southern blot anal-

ysis. Chromosomal DNA (5 p,g) from the mutant and wild-

type strains of S. downei and 1 ng of pUC8 vector DNA

(control) were digested with EcoRI and HindIII, and the

resulting fragments were separated by agarose gel electro-

phoresis. DNA was transferred to nitrocellulose as de-

scribed previously (16). DNA probes, which included

pUC8ennAM, pUC8 harboring the 6-kb EcoRI restriction

fragment that includes gtfS, pUC8 harborinigtfI, and pUC8

as a control, were radiolabeled with [a-3 P]dCTP as de-

scribed in supplier specifications (U.S. Biochemical random

primed DNA labeling kit). Hybridization patterns were

detected by using Kodak SB film.Colony morphologies for wild-type S. downei and gtfS

insertion mutants were compared on mitis salivarius agar

containing 5% sucrose. The ability of these strains to adhere

to glass was determined by culturing the wild-type and

mutant S. downei strains in Todd-Hewitt broth supple-

mented with 5% sucrose, with a sterile glass microscope

slide inserted into the flask. After 48 h of incubation at 37°C,

the microscope slide was removed, rinsed with distilled

water, and examined under low power in a stereomicro-

scope.

Analysis of enzyme interaction. To evaluate the in vitro

production of insoluble glucan, lysates of E. coli JM109(pMLG60) (expressing GTF-S), JM109(pMLG5) (5) (ex-

pressing GTF-I), and JM109 (as a control) were prepared.

Overnight cultures were harvested by centrifugation at

12,000 x g for 10 min at 4°C. The cells were lysed by

extrusion through a French pressure cell, and the lysate was

diluted 1:100 in 0.05 M NaH2PO4 (pH 6.5), 5% sucrose, and

0.01% ethylmercurithiosalicylate in the following combina-

tions: (i) 0.5 ml of JM109(pMLG60) lysate plus 0.5 ml of

JM109 lysate; (ii) 0.5 ml of JM109(pMLG5) lysate plus 0.5 ml

of JM109 lysate; (iii) 0.5 ml of JM109(pMLG60) lysate plus

0.5 ml of JM109(pMLG5) lysate. The mixtures were incu-

bated for 48 h at 37°C and examined for insoluble glucan

production visually and by turbidimetry at 660 nm.

Glucan isolation. To isolate total glucan, soluble and

insoluble, produced in supernatants of late-log-phase S.

downei and S. downei (gtfS mutant) cultures, the cells were

removed by centrifugation at 16,000 x g for15 min at 4°C.

The culture fluid was passed through a 0.45-,m-pore-size

filter unit (Corning). To 1 ml of culture filtrate, 99 ml of

sucrose-phosphate buffer was added and glucan was formed

during 24 h of incubation at 37°C.Insoluble glucan was collected by centrifugation for 30

min at 16,000 x g at4°C. The pellet was dissolved in 10 ml

of 1 M NaOH, and the insoluble glucan was precipitated by

the addition of 10 ml of 1 M sodium acetate. The insoluble

EcoRI Svp HkII HkdIlls V Id EcoRIEcoIIO 1 2 3 4 5 6 Ib

EIRI

IA

so vsd ECORI

FIG. 1. Recombinational displacement of the gtJS structuralgene by the ermnAM determinant. (A) An internal 3-kb SphI-SstIrestriction fragment was excised from gtJS. (B) After removal of thecohesive termini, the 1.2-kb ernnAM determinant was ligated to theremaining gtS flanking sequences. The in vitro insertionally inacti-vated gtfS determinant was reintroduced into S. downei, whererecombination resulted in the displacement of the native determi-nant in the mutant S. downei (gS mutant).

glucan was collected by centrifugation at 48,000 x g at 4°Cfor 15 min. The pellet was washed three times in 10 ml ofdistilled water and collected by centrifugation. The pelletswere washed further with 70% and 95% ethanol to facilitatedrying.

Soluble glucan was precipitated from the supernatant afterremoval of insoluble glucan. Three volumes of 95% ethanolwas added, and the glucan was allowed to precipitate at-20°C overnight. Precipitated glucan was collected by cen-trifugation at 16,000 x g for 15 min at 4°C. The glucan wassuspended in 100 ml of water and reprecipitated twice asdescribed above. After the third precipitation, the glucanpellet was air dried. Soluble and insoluble glucan synthe-sized in vitro by the mixture of E. coli lysates expressingGTF activities were purified similarly. The isolated glucanswere then analyzed by13C-NMR as described previously (7).

RESULTS

Inactivation of gtjS in S. downei. A pUC8 clone containingthe 6-kb EcoRI fragment encoding gtfS was used to con-struct a substitution mutation in g#tfS (Fig. 1). Cleavage ofthis plasmid with SphI and SstI produced a 3-kb restrictionfragment containing the catalytic and glucan-binding activi-ties of gtJs and a 5.7-kb restriction fragment containing thepUC8 vector, 1 kb of the N-terminal portion ofgtfS, and 2kbof sequences which flank g#tS on the genome (Fig. 1). The5.7-kb restriction fragment lacking most of the g#fS gene wasisolated and ligated to a 2-kb restriction fragment containingthe ernAM gene and used to transform E. coli JM109. Afterverification of the construction by restriction analysis, the4.2-kb EcoRI fragment harboring the erythromycin resis-tance determinant flanked by residual gtfS and genomicsequences was introduced into S. downei MFe28 by electro-transformation. The electrotransformed cells were selectedon media containing 50,ug of erythromycin per ml. Trans-

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HindlII

6.0-kb _

EcoRI

_VI __r _.

0.75-kb

FIG. 2. Southern hybridization of S. downei genomic DNA.Genomic DNA from S. downei and S. downei (gS mutant) washybridized with various probes to confirm the predicted genomicdisplacement of gtJS. Lanes: A, F, I, and L, Bacillus subtilisbacteriophage SPP1 digested with EcoRI (molecular size standard),containing an additional 100 pg of EcoRI-digested pUC8 DNA as a

positive hybridization control (2.7 kb); B, S. downei MFe28 genomicDNA digested with HindIII; C, S. downei (gtfS mutant) genomicDNA digested with HindIII; D, G, J, and M, S. downei MFe28genomic DNA digested with EcoRI; E, H, K, and N, S. downei (gtSmutant) genomic DNA digested with EcoRI. Lanes A, B, C, D, andE were hybridized with radiolabeled DNA from a pUC8 clone of the6-kb S. downei genome containing the gfS gene and flankingsequences. Lanes F, G, and H were hybridized with radiolabeledDNA derived from a pUC8 clone of gtf. Lanes I, J, and K werehybridized with radiolabeled pUC8 DNA as a control. Lanes L, M,and N were hybridized with radiolabeled DNA derived from a pUC8clone harboring the macrolide-lincosamide-streptogramin B resis-tance ennAM gene.

formants were characterized with respect to the MIC oferythromycin for each strain. Two transformants for whichthe MIC was 512 ,ug/ml, suggesting the stable presence of theerythromycin resistance determinant in the genome, wereselected for further study.Chromosomal DNA isolated from the high-level erythro-

mycin-resistant transformants and wild-type S. downei weredigested separately with EcoRI and HindIII. Comparison ofthe DNA banding patterns, or fingerprints, showed thetransformants to be derived from S. downei (data notshown). Southern blot analysis confirmed the recombina-tional displacement of 3 kb of the gtfS structural gene inthe erythromycin-resistant mutants (Fig. 2). As shown byHindIII digestion, a 750-bp restriction fragment containedwithin the recombinationally displaced region was absent inthe genome of S. downei (gtJS). Moreover, the 6-kb EcoRIrestriction fragment which containedgtfS was reduced to 4.2kb as a result of the loss of the displaced gtS structural genesequences and the acquisition of the ermAM determinant inthe genome of this mutant, as was predicted. Becausehomology exists between gtfS and gtf, gt7 was used as aprobe in control experiments to ensure that gtfl was unaf-fected by recombination and that the observed phenotypewas attributable only to the loss ofg#fS. The results showedthat gtfl was unaffected in the mutants. Finally, hybridiza-tion to the ernAM probe demonstrated the presence of theerythromycin resistance determinant within a genomicEcoRI fragment identical in size to that resulting from invitro substitution of ernmAM for gtfS sequences.

Phenotypic changes in S. downei (gJS mutant). S. downeiWS mutant) displayed a considerably different colonialmorphology from that of the wild type when cultivated onmitis salivarius agar containing sucrose (Fig. 3). Wild-type S.

downei colonies were observed to be small and hard, withcrusty mounds of insoluble glucan. In contrast, S. downei(gtfS mutant) colonies were large and smooth, with nodetectable mounds of glucan.

Plaque formation on glass slides was also markedly alteredfor S. downei (gtfS mutant) (Fig. 3). In contrast to thesmooth film of glucan and cells formed on the surface of theslide by the wild-type strain, S. downei (gtJS mutant) ad-hered sparingly but in clumps. Moreover, S. downei (gSmutant) adhered less tenaciously than the wild type and waseasily dislodged from the slide by curettage.

Interaction of GTF-S and GTF-I in glucan production by S.downei. To test the hypothesis that GTF-S plays a centralrole in glucan production by providing the primer for syn-thesis of insoluble glucan by GTF-I, the effect of mixing E.coli lysates containing GTF-S or GTF-I activity on insolubleglucan synthesis was determined. After 48 h of incubation,insoluble glucan produced by a mixture of lysates wasreadily observable, whereas little insoluble glucan was pro-duced by a mixture of the GTF-I-containing lysate and acontrol E. coli JM109 lysate (Fig. 4). As expected, noinsoluble glucan could be detected in a reaction containing amixture of the GTF-S lysate with the JM109 control lysate.Turbidimetric measurements revealed that the addition ofthe GTF-S lysate resulted in an approximate 10-fold increasein insoluble glucan synthesis by GTF-I. (The turbidimetricvalues [in optical density at 660 nm] for the different lysateswere as follows: GTF-S plus control, 0.002; GTF-I pluscontrol, 0.016; GTF-S plus GTF-I, 0.155.)The structures of glucans produced in culture supema-

tants of S. downei wild type and S. downei (gtfS mutant) andby lysates of E. coli JM109 harboring gtfl and gtfS determi-nants were compared by 13C-NMR spectral analysis. Thecomposition of glucan synthesized by E. coli(pMLG60),expressing GTF-S, was determined to consist of more than90% a-1,6-D-glycopyranosyl residues (7). Analysis revealedthat the glucan produced by the mixture of E. coli JM109lysates harboring gtfl and gtfS was similar to that synthe-sized by wild-type S. downei supernatants and that theinsoluble glucan fraction contained a mixture of ao-1,6-linkedresidues and a-1,3-linked residues. Of the glucan producedby the mixture of recombinant enzymes, 25% consisted ofa-1,6-linked residues, in close agreement with 32% observedfor glucan synthesized by S. downei. From supematants ofS. downei (gS mutant) and from the recombinant enzyme,no glucan, either soluble or insoluble, was detected by themethod used.

DISCUSSIONThe sticky insoluble glucan produced by the combined

activities of streptococcal GTFs is thought to be of impor-tance in the development of dental plaque. In S. downei,GTE-I-mediated glucan synthesis requires the presence of aprimer (5, 7, 8). GTF-S has been hypothesized to act as asource of intrinsic primer for the synthesis of glucan byGTF-I (15, 21), so it was of interest to determine thestructural and functional relationships between GTF-S andGTF-I. In this study, we compared the glucan produced byeither GTF-S or GTF-I alone with that produced whenGTF-S and GTF-I activities were both present. We alsoinvestigated the effect of blocking gtfS expression in S.downei and observed marked differences in the properties ofthe glucan made, as well as in colony morphology andsucrose-dependent adherence, when compared with wild-type S. downei.

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FIG. 3. Colonial morphology of S. downei MFe28 (A) and S. downei (gtJS mutant) (B) on mitis salivarius agar and plaque formation onglass by S. downei MFe28 (C) and S. downei (gtJS mutant) (D) when grown in sucrose containing Todd-Hewitt broth. S. downei (gt mutant)colonies are larger and smoother than those of S. downei MFe28 because of the absence of insoluble glucan on the colony surface.Magnification, x5.

The observed differences in colony morphology betweenwild-typeS. downei and S. downei (gt mutant) suggest thatthe absence of GTF-S has a major impact on the productionof insoluble glucan. This impact was confirmed to be directlyrelated by comparing the insoluble glucan produced byeither GTF-S or GTF-I with that produced when GTF-S andGTF-I activities exist together (Fig. 4; and see above). Theaddition of GTF-S enhanced the production of insolubleglucan by GTF-I by approximately an order of magnitude.Additionally, no insoluble glucan could be detected from S.downei (gtS mutant) by "3C-NMR spectral analysis. As a

consequence of the reduced insoluble glucan produced in theabsence of GTF-S activity, g#tS-deficient S. downei adheredless frequently and with less tenacity to glass surfaces thandid the wild-type strain (Fig. 3).Although the insoluble glucan produced by GTF-I has long

been thought to be the major contributor to adherence incolonization of the tooth surface by mutans streptococci,this study provides evidence that GTF-S plays a pivotal rolein the ability of mutans streptococci to produce insolubleglucans. The results of this study demonstrate that one rolefor GTF-S in S. downei is, in fact, to provide a primer

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A B C

FIG. 4. Insoluble glucan synthesized from sucrose in vitro byextracts of E. coli containing GTF-S plus GTF-I (A), GTF-I (B), andGTF-S (C) activities.

necessary for the synthesis of insoluble glucan by GTF-I.Although GTF-S is required for the efficient production ofinsoluble glucan in S. downei, such a role does not appear tobe required for strains of Streptococcus mutans (11) andremains to be demonstrated for other mutans streptococcalgroups.

S. downei MFe28 produces two GTFs capable of solubleglucan synthesis in addition to that encoded by gtfS. Thegene for one of them has recently been cloned (12). Thebiological roles of these additional GTF species are presentlyunknown, although collectively they would presumably con-tribute to the deposition of dental plaque on the toothsurface. Nevertheless, the change in colony morphology andadherence properties and the lack of glucan detectable byNMR from S. downei ( mutant) suggests that the activ-

ities of these uncharacterized GTFs may also be influencedby the expression of GTF-S.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grant DE08191 from the National Institutes of Health. K.S.G. was supportedby a fellowship from the Phillips Petroleum Foundation, Bartles-ville, Okla.We thank Glen Dryhurst and Eric Enwall for performing the NMR

analysis and Rodney K. Tweten for assisting in the computer analysis.

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