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 DOI: 10.1177/08959374970110010101

1997 11: 100ADRRobert A. Burne, Yi-Ywan M. Chen and Jana E.C. Penders

Analysis of Gene Expression in Streptococcus Mutans in Biofilms in Vitro  

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ANALYSIS OF GENE EXPRESSIONIN STREPTOCOCCUS MUTANS IN BIOFILMS IN VITRO

ROBERT A. BURNE12*

YI-YWAN M. CHEN1

JANA E.C. PENDERS1

Departments of Cental Researchand 2Microbiology and Immunology

University of RochesterSchool of Medicine and Dentistry601 Elmwood AvenueRochester, New York 14642*to whom correspondence should be addressed

Adv Dent Res U(l):100-109, April, 1997

Abstract—The purpose of this study was to developmethods for the consistent production of biofilms of S.mutans containing reporter gene fusions, and to examine theexpression of genes involved in sucrose metabolism inadherent populations of this organism. Three strains of S.mutans harboring reporter gene fusions to the gene promoterregions of the gtfBC genes, ftf, and scrA were grown in aRototorque biofilm fermenter in a tryptone-yeast extract-sucrose medium. Qwasz-steady-state levels of reporter geneactivity were measured after the biofilms were grown foreither 48 hrs or 7 days. Also, induction of gene expression bythe addition of sucrose to biofilm cells was monitored.Reporter gene activity was measurable from all gene fusionstrains. This study (i) establishes the feasibility of doingdetailed molecular and physiologic studies on immobilizedpopulations of S. mutans, (ii) demonstrates that thepolysaccharide synthesis machinery of S. mutans isdifferentially expressed in biofilms, and (iii) opens the wayfor a more detailed analysis of the environmental signals andsignal transduction pathways governing the regulation ofgene expression by S. mutans cells that are immobilized on asolid surface.

Key words: Glucan, fructan, dental caries, gene fusion, generegulation.

Presented at the 14th International Conference on OralBiology, "Biofilms on Oral Surfaces: Implications for Healthand Disease", held March 18-20, 1996, in Monterey,California, organized by the International Association forDental Research and supported by Unilever Dental Research

The formation of dental plaques, biofilms whichdevelop on the hard surfaces of the oral cavity, is anintegral component of oral health and diseases. Inmost cases, the complex community of micro-

organisms on the teeth is relatively benign, and the normalprotective processes of the host are sufficient to preserve theintegrity of the tissues (Bowden et al, 1979; Theilade, 1990).However, when the diet of the host is especially rich incarbohydrates, dental plaque can become dominated byhighly acidogenic and aciduric bacteria, such as Strepto-coccus mutans and Lactobacillus casei (Bowden et al, 1979;Bender et al., 1986; Bender and Marquis, 1987; Bradshaw etal, 1989; Theilade, 1990). In these circumstances, the phasesof plaque acidification, those in which significantdemineralization of the tooth enamel can occur, outweigh thephases of remineralization, and caries lesions can be initiatedor worsened.

S. mutans is generally recognized as the primary etiologicagent of human dental caries (Hamada and Slade, 1980). Amajor contributor to the pathogenic potential of S. mutans isits ability to produce extracellular polysaccharides fromsucrose. S. mutans uses sucrose to synthesize al ,3- and al,6-linked glucan polymers through the actions of three secretedglucosyltransferases (Gtfs), encoded by the gtfB, gtfC, andgtfD genes (Kuramitsu, 1993). The gtfB and gtfC genes are inan operon-like arrangement and encode enzymes whichproduce water-insoluble glucans, consisting primarily ofa l , 3 linkages (Aoki et al, 1986; Shiroza et al, 1987;Yamashita et al, 1992). It is the water-insoluble products ofthe GtfB and GtfC enzymes which appear to be majorcontributors to adhesion to teeth, and which are essential forthe efficient initiation of dental caries on the smooth surfacesof the teeth (Munro et al, 1991; Yamashita et al, 1993). ThegtfD gene, which is not linked to the gtfBC locus (Perry andKuramitsu, 1989), encodes a gene product that catalyzes theformation of a glucan composed almost exclusively of a 1,6-linked glucose units (Hananda and Kuramitsu, 1989). TheGtfD reaction end-product has a much greater solubility inwater than those of the GtfB- and GtfC-catalyzed reactions.Interestingly, although S. mutans and other oral bacteriaproduce an endo-dextranase, which will attack al,6-linkedglucans (Barrett et al, 1987; Lawman and Bleiweis, 1991),the glucan end-products of cell-free enzyme preparations ofS. mutans are almost completely refractile to digestion byenzymes produced by any known oral bacteria, including S.mutans (Hamada and Slade, 1980). Thus, the principal rolesof the glucans produced by S. mutans are believed to be tofacilitate adhesion and accumulation of the organisms, and toestablish an extracellular polysaccharide matrix which maygive the organisms resistance to the normal mechanicalforces of clearance by the host and which may afford someprotection from host immune and non-immune defenses.

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VOLUU) S. MUTANS GENE EXPRESSION IN BIOFILMS 101

Strains of S. mutans also produce a single fructo-syltransferase, the product of the ftf gene (Shiroza andKuramitsu, 1988), which catalyzes the cleavage of sucroseand the incorporation of the fructose moiety into a fructanpolymer, composed primarily of 62,1-linked fructose units(Birkhed et aL, 1979). Fructans do not facilitate adhesion oraggregation of mutans streptococci and are relatively short-lived in dental plaque, due largely to enzymatic hydrolysis offructans by fructan hydrolase enzymes of a number of oralbacteria (DaCosta and Gibbons, 1968; Manly andRichardson, 1968; van Houte and Jansen, 1968; Higuchi etaL, 1970; Burne et aL , 1987). These and other findings(Burne, 1991; Burne et aL, 1996) have fostered the belief thatthe extracellular fructans produced by S. mutans and otherdental plaque bacteria function primarily as storagecompounds.

In addition to the ability of S. mutans to use sucrose forexo-polysaccharide production, a large proportion of thesucrose presented to this organism is metabolized directly toacids (Chassy, 1983) that can damage the tooth enamel. Theprincipal route for sucrose uptake is via the product of thescrA gene (Sato et al., 1989), a highly active sucrose-specificEII of the bacterial sugar phosphotransferase system (PTS),which concomitantly phosphorylates the sugar at the 6position of the glucose moiety and internalizes the sugar-phosphate. The Km of ScrA for sucrose is about 10 pmol/L.Intracellular sucrose-6-phosphate is hydrolyzed by a sucrose-6-phosphate hydrolase, encoded by the scrB gene (Sato andKuramitsu, 1988; Kuramitsu, 1993), which also can functionat very low sugar concentrations. High-affinity, high-capacitytransport of sucrose by S. mutans is probably a major contri-butor to acidogenesis and caries formation when sucrose is asignificant component of the host's diet (Kuramitsu, 1993).

The only known natural habitat for S. mutans is on thesurfaces of the teeth. The organisms are not found in themouth until the eruption of the teeth, and a single strain of S.mutans can persistently colonize its host for extremely longperiods (Hamada and Slade, 1980). Thus, S. mutans appearsto be entirely dependent on the "biofilm life style" forpersistence, and the persistence of S. mutans in the humanpopulation implies that it is remarkably well-adapted to thisexistence. The ability of S. mutans to survive and persist inthe oral cavity is undoubtedly linked to its tremendousphenotypic plasticity. Numerous studies have demonstratedthat this organism can alter the expression of a variety ofgenes in response to large and sudden variations inenvironmental pH, oxygen tension, carbohydrate availability,and carbohydrate sources (Hardy et aL, 1981; Rosan et aL,1982; Keevil et aL, 1983; Marsh et aL, 1984; Jacques et aL,1985). These studies revealed that the production of adhesins,the glucan synthetic machinery, and sugar transport, amongother things, were highly responsive to growth conditions.More recently, the use of gene fusion technology has beencoupled with continuous chemostat culture to demonstratethat the expression of the gtfBC and ftf genes was influencedby culture pH, growth rate, and sucrose availability (Wexleret aL, 1993). Thus, it is clear that S. mutans has a tremendouscapacity to modulate the expression of known virulence

TABLE 1

BACTERIAL STRAINS

StrainDesignation

RelevantGenotype

Source(Reference)

S. mutansSMS101

S. mutansSMS 102

S. mutansIS3AZ4

Harbors ftf:: cat genefusion, parent:UA130

Em-resistant

Harbors gtfBC ::catgene fusion, parent:

UA130. Em-resistant

Harbors scrAr.lacZgene fusion, parent:

GS-5. Em-resistant

M. Hudson(Hudson and Curtiss,

1990)

M. Hudson(Hudson and Curtiss,

1990)

Y. Sato(Sato et aL, 1991)

determinants in response to environmental stimuli commonlyencountered in the human oral cavity. The ability to respondrapidly and efficiently to large changes in its environment hasbeen suggested to be an essential element in the dominanceof S. mutans in cariogenic dental plaque (Burne, 1991).

Despite the significant advances that have been made inour understanding of the physiology and molecular geneticsof S. mutans, virtually nothing has been done to evaluate thephenotypic capabilities and the molecular control of geneexpression in this organism when colonizing a solidsubstratum. Yet a variety of studies has demonstrated thatcells growing in biofilms have phenotypic characteristicsradically distinct from those of their planktonic counterparts(Costerton et aL, 1987). Given the apparent obligate biofilmlifestyle of S. mutans, it seems that studying the organismswhile growing in biofilms will be necessary if a trueknowledge of the pathogenic strategies of this organism is tobe gained. The purpose of this study was to establish methodsto study differential gene expression in sessile populations ofS. mutans and to begin to examine the expression of genesencoding sucrose metabolism enzymes in organisms growingin biofilms.

MATERIALS AND METHODS

Bacterial strains and growth mediaThe bacterial strains utilized in this study are described inTable 1. Strains of S. mutans were maintained on Brain HeartInfusion Agar (BHI) supplemented with 10 pg of erythro-mycin (Em) per mL. Overnight cultures used for inoculationof the biofilm reactor vessel were grown at 37°C in a 5%CO2, aerobic atmosphere in BHI broth with 10 pg of Em permL. For cultivation of cells in biofilms, a tryptone-yeastextract medium (TY; Burne et aL, 1987) supplemented with10 mmol/L sucrose and 10 pg of Em per mL was used(TYS). Unless otherwise indicated, media components wereobtained from Difco Laboratories (Detroit, MI), and

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102 BURNE ET AL. ADV DENT RES APRIL 1997

Effluent(Pump)

Shaft(To motor)

I Media Inlet

Position of Slides (12)

21.2 cm

Top View

Fig. 1—A schematic diagram of the modified Rototorque as utilized in this study.Medium was pumped into the vessel by a peristaltic pump at a constant rate of 60 mLper hour. Effluent was pumped from the top of the vessel as shown. The speed of therotating inner drum was kept constant at 75 RPM. The temperature of the vessel wasmaintained by immersion in a 37°C circulating water bath. Some dimensions of thevessel, the inner drum, and the width of the slides are shown for reference.

chemicals, reagents, and buffers were purchased from SigmaChemical Co. (St. Louis, MO).

Biofilm fermentation conditionsBiofilms of pure cultures of S. mutans were cultivated in aRototorque reactor (Characklis, 1990), which was obtainedfrom Montana State University and was slightly modifiedfrom the original design. Specifically, the modifiedRototorque was roughly 5 cm shorter than the standard in-strument, and the port in the bottom of the vessel was sealed,yielding a vessel with a "working volume" of 0.6 L. Theaverage surface area of the exposed face of one of the 12polystyrene slides in the vessel was 31 cm2. The vessel wasconfigured as depicted in Fig. 1, and lacked a reflux/recirculation loop for planktonic cells. The vessel was filledwith TYS, equilibrated at 37°C, and inoculated with 10 mLof an overnight culture of the recombinant S. mutans strain to

be studied. Medium (TYS) wasintroduced at a rate of 60 mL per hr("Dilution rate", D = 0.1 h 1 )immediately after inoculation. Theinstrument was operated with themotor connected directly to theshaft of the rotating inner drum.Rotation of the inner drum wascontrolled at a speed determined bya rheostat setting of "20" on theinstrument (approximately 75RPM). Analyses of biofilm cellswere conducted after 48 hr or for 7days post-inoculation.

Sample preparation and assaysof reporter gene product activityThe levels of activity expressedfrom the gene fusions in biofilmcells at "quasi-steady state" andafter the addition of sucrose to theliquid phase were assessed asfollows. After either 48 hr or 7days, three slides were removedfrom the vessel, and the cells weremechanically dissociated from theslides into a solution of ice-cold 10mmol/L Tris-HCl (pH 7.0)containing 10 ug of rifampicin andtetracycline per mL, so thattranscription and translation,respectively, would be arrested. 'The cells were immediatelycentrifuged at 8000 g for 10 min at4°C. Cells were washed in ice-cold10 mmol/L Tris-HCl (pH 7.8) andthe cell pellets kept on ice until allsamples were ready for processing.Immediately after removal of thefirst three slides, a solution ofsucrose (1.0 mol/L) was added to

the vessel to achieve a final concentration of 0.025 mol/L. At10, 20, and 40 min after the addition of sucrose, threeadditional slides were removed, and the adherent cells werecollected and washed as above.

Cell-free lysates were prepared for the analysis ofchloramphenicol acetyltransferase (CAT) activity asdescribed previously (Wexler et ai, 1993). Briefly, cells weresuspended in 1 mL of 10 mmol/L Tris-HCl, pH 7.8, 10mmol/L hexanoic acid. A one-third volume of glass beads(0.2 mm avg. diam.) was added to the samples, and the cellswere lysed by sonication at 350 W for 8 min, at 30-secondintervals, with intermittent CO2(s) cooling, or by homo-genization in a Bead-Beater (Biospec Products, Bartlesville,OK) at top speed in three one-minute intervals, with coolingbetween intervals. The lysates were centrifuged at 12,000 g at4°C for 15 min and the supernatant fluid collected. CATactivity was measured by the spectrophotometric method of

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VOLll(l)

A

£» •'•

S. MUTANS GENE EXPRESSION IN BIOFILMS

B

103

5

Fig. 2—Microscopic appearance ofS. mutans biofilms. (A-F) Typical appearance of various areas of biofilms formed by S.mutans SMS102 after growth for 7 days. Slides were removed from the vessel, cut to a length appropriate for microscopy, andmounted on a glass slide, with a #1 cover slip placed gently on top of each biofilm. Images were obtained by phase contrastmicroscopy (1000X). As noted in the text, the bio-films were heterogeneous, and one could observe individual streptococci andchains ofS. mutans, microcolonies, larger mats, and relatively dense areas of colonization of the polystyrene slides.

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104 BURNE ET AL. ADV DENT RES APRIL 1997

TABLE 2

CHLORAMPHENICALACETYLTRANSFERASESPECIFIC ACTIVITY MEASURED FROM S. mutans

SMS102 (gtfBCrxat )-48-HOUR BIOFILM GROWTH*

Time (min) afterAddition of Sucrose

(0.025 mol/L)

CATSpecific ActivityUnits/mg Protein

0102040

0.020.150.030.04

* Chloramphenicol acetyltransferase was measured inbiofilm cells which had been grown for 48 hrs in theRoto torque. After 3 of the 12 slides in the vessel wereremoved, sucrose was added to a final concentration of0.025 mol/L. Cells were harvested from 3 additionalslides at each of the indicated time points and enzymeassays conducted as described in the text. Values areaverages of a minimum of triplicate samplings.

Shaw (Shaw, 1979) with use of the colorimetric substrate,5,5'-dithio-bis-nitrobenzoic acid (DTNB, Boehringer-Mannheim, Indianapolis, IN). All assays were performed intriplicate or more, with internal standards containing allreagents except chloramphenicol to account for CAT-independent reduction of DTNB. One unit of CAT activitywas defined as the amount of enzyme needed to catalyze theacetylation of one pmol of chloramphenicol per min. Proteinconcentrations of samples were determined by the method ofBradford (Bradford, 1976) with use of a commerciallyavailable reagent (BioRad Protein Assay, Richmond, CA).

Measurements of 6-galactosidase activity in permeabilizedcells of S. mutans were made by means of a modification ofthe method of Miller (1972). Briefly, cells were re-suspendedin Z buffer (Miller, 1972), on ice, sonicated for 15 s todisperse the cells, and 1/10 vol of toluene was added. Thesamples were vortexed immediately for 15 s, and the cellswere collected by centrifugation at 13,000 g for 10 min at4°C. Cells were re-suspended in Z buffer, and B-galactosidaseactivity was measured with o-nitrophenyl B-D-galactopyranoside (ONPG) as substrate. Unit definitions wereas described by Miller (1972), and B-galactosidase activitywas normalized to the optical density at 600 nm (O.D.600) ofthe suspension of permeabilized cells.

Preparation of cells for microscopyFor the imaging of biofilms, cells were cultured as above.Slides were removed from the vessel and briefly dipped inroom temperature dH2O for removal of adventitiously boundcells. The polystyrene slides with the biofilms wereimmediately cut into sections of about 5 cm and mounted ona glass microscope slide. A #1 glass slide was gently placed

over the biofilm populations, and the adherent populationswere examined by phase-contrast microscopy in an ACAS570 image analysis system equipped with an Olympusinverted microscope.

RESULTS

Growth of recombinant Streptococcus mutans strainsin the RototorqueThe growth conditions described in "Materials and Methods"were utilized for the experiments described below. In thiscase, no control of pH was maintained, and the liquid phaseof the vessel routinely attained pH values of 5.0-5.5. Thebiofilms formed had features in common with some otherbiofilm-forming bacteria grown in in vitro model systems(Characklis et al., 1990), i.e., films consisted ofmicrocolonies, large mats, and aggregates, and there wasconsiderable heterogeneity in biofilm depth and spatialarrangement of micro-organisms (Fig. 2). The time todevelopment and appearance of the S. mutans biofilms wasconsistent among experiments. Within 24-48 hours, thebiofilms had achieved wet biofilm thicknesses ranging from10 to 50 um, as ascertained by microscopic techniques(Characklis, 1990). After 7 days, areas of the biofilm attainedwet thicknesses in excess of 100 um, but smallmicrocolonies, monolayers, regions of intermediatethickness, and uncolonized areas were still present on theslides.

Assay of gene fusion activity of recombinant5. mutans strains growing in in vitro biofilmsThe gene fusion strains utilized in these studies have beendescribed previously (Hudson and Curtiss, 1990; Sato et al.,1991; Wexler et al, 1993). Basically, all of the gene fusionsand strains were similarly constructed, as detailed in Fig. 3.For strains SMS 101 and SMS 102, fragments of cloned DNAharboring the ftf and gtfBC promoter regions, respectively,were cloned 5' to a promoterless cat gene from aStaphylococcus sp. The gene fusions were constructed invitro and introduced into E. coli on a plasmid, which couldnot replicate in streptococci. Plasmid DNA with theappropriate configuration was recovered from E. coli andused to transform S. mutans UA130 (Hudson and Curtiss,1990). In the case of the scrA gene fusion, the promoterregion of scrA was cloned 5' to a promoterless lacZ gene,which was derived from E. coli. The fusion was thentransferred into S. mutans strain GS-5 to create strain IS3AZ4(Sato etaL, 1991).

Once it was established that the recombinant strains wouldconsistently form biofilms with similar microscopicappearances, studies were undertaken to examine theexpression of the gtfBC, ftf, and scrA genes in biofilm cells,and to assess the inducibility of expression of these genesfollowing addition of sucrose. Biofilms that had been grownfor either 48 hr or 7 days were examined.

S. mutans SMS102 (gtfBC::cat)Biofilms of S. mutans SMS 102 were grown in the modified

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VOLll(l) S. MUTANS GENE EXPRESSION IN BlOFILMS 105

Rototorque. After 48 hr,samples were taken, andCAT activity wasmeasured (Table 2). Theamount of CAT expressedfrom the gtfBC promoterwas readily measurableand was of a magnitude inthe general rangepreviously reported instrain S. mutans SMS 102grown in batch culture(Hudson and Curtiss,1990), or in cells grownin glucose-limited,continuous culture at pHvalues of 7.0 or 6.0, D =0.1 h"1 or 0.3 h"1 (Wexleret al, 1993). The ability toinduce further expressionof the gtfBC operon withsucrose was observed, asassessed by CAT activitymeasured after theaddition of sucrose to afinal concentration of 25mmol/L to the liquidphase. When CATactivity was measured instrain SMS 102 aftergrowth in the Rototorquefor 7 days (Table 3),specific activity wassignificantly higher thanat the 48-hour time point.Moreover, inducibility bysucrose was observedafter the addition ofsucrose to a finalconcentration of 25 mM.

Gene fusion constructs

Parent Fusion Strain

gtfB gtfc

ftf

'scrA scrA

FgtfBC

PftfIK

*scrAKH

cat

cat

lacZmmmr

i

>

i

>:|:|:|:::::::::::::J

Strain construction

gene gene

Reporter Intact Gene

Fig. 3—Schematic diagram of the construction of the gene fusion strains ofS. mutans. Thedetails of the gene fusion construction and integration into the S. mutans chromosome can befound in the text. The key point is that all of the fusions were integrated by single-cross-overrecombination, resulting in an insertion-duplication event of the region ofhomology. Inestablishing the gene fusions in this manner, the reporter gene was integrated in near-unit copy,yet near-normal transcription and translation of the target structural gene would likely occur.

S. mutans SMS 101(ftfi:cat)CAT activity in S. mutans strain SMS 101 was examined asfor strain SMS 102. After growth for 48 hr, levels of CATactivity measured in biofilm-growing cells from the ftf::catpro-moter fusion were on the order of 0.1-0.3 Units/ mgprotein (Table 4). Again, this value was comparable withthose that have been measured in continuous chemostatculture (Wexler et al., 1993) and in batch culture (Hudson andCurtiss, 1990). Conversely, when cells carrying the ftfr.catgene fusion were cultured for 7 days, these organismsproduced very low levels of CAT activity (Table 5). How-ever, ftf gene expression in these circumstances could behighly induced by sucrose, some 650-fold.

S. mutans IS3AZ4 {scrA::lacZ)S. mutans strain IS3AZ4 was also examined after growth for

48 hr. LacZ activity (Table 6) in biofilm-grown cells wascomparable with that reported for batch-cultured micro-organisms when sucrose was present (Sato et al., 1989).However, unlike the gtf and ftf genes, further induction of thescrA::lacZ gene fusion was not observed after the addition ofsucrose to a final concentration 0.025 M. A seven-day timepoint was not taken for the IS3AZ4 strain.

DISCUSSION

Research that has focused on the phenotypic plasticity andcapacities of S. mutans and the effects of environmentalinfluences, such as carbohydrate source and growth pH, ongene expression in this organism has been conducted almostexclusively in planktonic populations of the bacteria. In thisstudy, we have demonstrated the feasibility of studying gene

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106 BURNE ET AL. ADV DENT RES APRIL 1997

TABLE 3

CHLORAMPHENICALACETYLTRANSFERASESPECIFIC ACTIVITY MEASURED FROM S. mutans

SMS1Q2 (gtfBC::cai)—l DAYS7 BIOFILM GROWTH*

Time (min) afterAddition of Sucrose

(0.025 mol/L)

CATSpecific ActivityUnits/mg Protein

0102040

0.390.721.141.35

Chloramphenicol acetyltransferase was measured inbiofilm cells which had been grown for 7 days in theRototorque. Otherwise, experiments were performedas described in the text and in the legend to Table 2.

expression in adherent populations of S. mutans. Using amodified Rototorque for cultivation of pure biofilm culturesof recombinant strains of S. mutans harboring gene fusions togenes involved in polysaccharide synthesis and sucrosemetabolism, we have shown that, in the presence of sucrose,biofilms of these bacterial strains form consistently. After thebiofilms are allowed to form, gene fusion activity is readilyquantifiable. Sufficient cells were obtained for highlyrepeatable measurements after 48 hr, and actually, visiblefilms form readily within 12-24 hrs under the cultureconditions utilized, so that a more detailed kinetic analysis ofgene expression as a function of biofilm age and thicknesscould be undertaken. Although not ideal for manyapplications, the Rototorque has a number of advantages forthe study of environmental influences on gene expression inbiofilms, including the ability to collect large quantities ofcells, to modulate a variety of chemical and physicalparameters, and amenability to growth of mixed populationsof organisms (Characklis, 1990). For microscopic imagingpurposes, the slides provided a fairly large sample size, sothat many fields could be viewed quantitatively from a singlesample. Along these lines, we have been able to imagebiofilms of S. mutans not only by phase contrast, but also byusing vital dyes and fluorogenic substrates for 6-galactosidase, and by scanning confocal laser microscopy,collecting fine structure and quantitative information.Analysis of the gene fusion strains of S. mutans not only byenzyme assay, but also by these more sophisticated imagingtechniques, will be useful in providing detailed informationabout gene expression in biofilm populations as a function ofbiofilm age and spatial and temporal heterogeneity.

In this study, the gene fusions were integrated into thechromosome by single-cross-over recombination.Establishment of the gene fusions in this manner wasparticularly important for biofilm experiments. In particular,when the reporter genes are integrated as detailed in Fig. 3,transcription and translation of the structural gene by its

TABLE 4

CHLORAMPHENICOL ACETYLTRANSFERASESPECIFIC ACTIVITY MEASURED FROM S. mutansSMS 101 (ftf::cat)^S-HOUR BIOFILM GROWTH

Time (min) afterAddition of Sucrose

(0.025 mol/L)

CATSpecific ActivityUnits/mg Protein

0102040

0.060.140.070.08

Experiments were performed as described in the text andin the legend to Table 2.

cognate regulatory elements remain intact. In this case,normal or near-normal levels of the gtf ftf, and scrA geneproducts should be produced, and normal expression patternsshould be observed. Maintaining the integrity of the sucrosemetabolism and polysaccharide synthesis pathways wasessential in these experiments, since perturbation of thesepathways would have a dramatic impact on growth andbiofilm formation. As in our past work with chemostat-growncells harboring gene fusions (Wexler et ai, 1993), it wasnecessary to include erythromycin to maintain selectivepressure for strains carrying the fusion. In the presence ofantibiotic, the stability of the gene fusions did not seem to beproblematic, because when biofilm cells were removed fromthe vessel, dispersed, and plated on media with and withoutEm, no differences in cell numbers were observed, indicatingthat the gene fusions were not lost at an appreciablefrequency. This conclusion is further supported by theobservations with the gtfBC fusions, which expressed higheractivity after prolonged passage (Table 3).

We had previously demonstrated that expression from theftf and gtfBC promoters was further induced by the additionof 25 mmol/L sucrose to steady-state, glucose-limitedcultures of S. mutans, as measured by CAT activity in strainsSMS 101 and 102, respectively (Wexler et al., 1993). Despitethe fact that the biofilm cells were cultured in the presence of10 mmol/L sucrose, the ability of S. mutans to increaseexpression from the ftf and gtfBC promoters in response toadded sucrose was retained. Unfortunately, the pathways forinduction of gtf or ftf have not yet been completely defined.Without a more detailed understanding of the cis- and trans-acting elements governing expression of these genes, withouta much more comprehensive study of physiologic factors thatinfluence gtf and ftf expression, and in the absence of aknowledge of how environmental signals are transmitted tothe regulatory molecules, the dominant influences whichgovern the polysaccharide synthesis machinery in biofilm-growing S. mutans cells cannot be predicted.

The level of reporter-gene-specific activity measurablefrom the cells growing in biofilms after 48 hrs was generally

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VOLll(l) S. MUTANS GENE EXPRESSION IN BIOFILMS 107

TABLE 5 TABLE 6

CHLORAMPHENICOL ACETYLTRANSFERASESPECIFIC ACTIVITY MEASURED FROM S. mutans

SMS1Q1 (ftf::cai)—l DAYS* BIOFILM GROWTH

6-GALACTOSIDASE SPECIFIC ACTIVITYMEASURED FROM S. mutans IS3AZ4 (scrA: :lacZ)—

48-HOUR BIOFILM GROWTH*

Time (min) afterAddition of Sucrose

(0.025 mol/L)

CATSpecific ActivityUnits/mg Protein

0102040

0.0010.0700.0520.000

Experiments were performed as described in the text andin the legend to Table 3.

of a magnitude similar to that of those that we and othershave previously measured from these same gene fusions, ineither batch or continuous chemostat culture. However, whenbiofilms of S. mutans SMS 101 and 102 were grown for 7days, significant differences in expression of CAT activitywere noted when comparisons were made with cells grown insuspension or in the 48-hour biofilms. In the case ofSMS101, extremely low levels of CAT activity weremeasurable prior to the addition of sucrose—some 10-100-fold lower than previously measured in planktonicpopulations. The addition of sucrose to 25 mmol/L resulted inrapid induction of the ftfr.cat fusion, followed by a rapidreturn to very low levels of CAT. The behavior of the gtfBCgene fusion in SMS 102 was markedly different under thesame conditions. Specifically, the baseline levels of CATactivity were higher—by some 10-70-fold—than we havemeasured in continuous culture. Nevertheless, induction bysucrose of the gtfBC promoter, as measured by CAT specificactivity, was about three-fold, comparable with resultsobtained in chemostat culture. Two important points here arethat it seems unlikely that the observations made withSMS 101 were attributable to a high proportion of dead cells,since roughly half the cells stain as live under theseconditions (not shown), and the high level of inducibility tovalues previously observed in planktonic populationsdemonstrates that the cells are responsive and capable ofexpressing high levels of CAT from the ftf promoter.Moreover, the levels of CAT from the gtfBC promoter weremarkedly higher than those at the 48-hour time point,indicating that cell death or CAT instability likely cannotaccount entirely for the low activity seen in seven-daybiofilms of SMS 101. Second, it does not seem that low pHalone can account entirely for the altered levels of CATmeasured in seven-day biofilms. Although the pH of theliquid phase was near 5.0, a value lower than we have utilizedin continuous culture, batch-cultured S. mutans SMS 101 and102 readily attain pH values of 5.0 and below, but do notdisplay similar expression patterns.

The most relevant question regarding the gene expression

Time (min) afterAddition of Sucrose

(0.025 mol/L)

6-GalactosidaseActivity

Miller Units/OD600

01020

17.918.114.5

6-Galactosidase was measured in biofilm cells which hadbeen grown for 48 hrs in the Rototorque. After 3 of the 12slides in the vessel were removed, sucrose was added to afinal concentration of 0.025 mol/L. Cells were harvestedand enzyme assays conducted as described in the text.Values are averages of a minimum of triplicate samplings.

patterns in seven-day biofilms seems to be: "What are theimportant signals sensed by these organisms that influenceexpression of gtf and ftf!". Hudson and Curtiss (1990) hadpreviously shown that there was stimulation of gtfBCpromoter activity, but not ftf expression, following adsorptionof the strain to hydroxylapatite beads, suggesting thatadsorption or contact phenomena might modulate gtf geneexpression. However, it seems unlikely that the geneexpression patterns observed in this study were due solely togrowth on a solid surface, or one would predict that therewould be no differences in 48-hour and seven-day culturesthat were not readily explainable by cell death. Rather,additional specific signaling events or other environmentalfactors probably influence the baseline level of expression ofthe/f /and gtfBC genes, and the ability of S. mutans toregulate these genes differentially in response to a rapidincrease in the concentration of sucrose available in theenvironment. The potential complexity of the influences ongene expression in biofilms is remarkable and may includelocal pH effects, O2 tension, reducing environment, contactphenomenon, osmolality, and other physiochemicalinfluences, as well as the production of molecules by theadherent micro-organisms that function in a manner that isspecific and concentration-dependent. It is also critical tonote that there are tremendous differences between modelsystems which use non-leaching surfaces and pure cultures ofbacteria, and naturally occurring biofilms such as dentalplaque, which is composed of well over 100 speciescolonizing a semi-porous, leaching surface.

Clearly, growing cells on a surface, under fairly crowdedconditions, may more closely mimic conditions in dentalplaque. However, much work must still be done to dissect thephenotypic capacities of planktonically growing S. mutans,and to determine what biofilm phenomena, if any, occur in a"surface-specific" fashion. Likewise, a much more completeunderstanding of the biofilm environment, the stimuli to

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BURNE ET AL. ADV DENT RES APRIL 1997

which organisms are exposed, and the sensors and signaltransduction pathways in S. mutans and other plaque bacteriawill be needed before a full appreciation of the impact ofbiofilm growth on gene expression can be dissected in arational fashion.

ACKNOWLEDGMENT

This study was supported by United States Public HealthService Grant #DE09878.

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