Involvement of an Inducible Fructose Phosphotransferase ... · the fruK::Tn917-lac mutant resulted...

JOURNAL OF BACTERIOLOGY, Nov. 2003, p. 6241–6254 Vol. 185, No. 210021-9193/03/$08.00�0 DOI: 10.1128/JB.185.21.6241–6254.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Involvement of an Inducible Fructose Phosphotransferase Operon inStreptococcus gordonii Biofilm Formation

C. Y. Loo, K. Mitrakul, I. B. Voss, C. V. Hughes, and N. Ganeshkumar*Department of Pediatric Dentistry, Goldman School of Dental Medicine, Boston University,

Boston, Massachusetts 02118

Received 20 May 2003/Accepted 7 August 2003

Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilmformation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated byusing an in vitro biofilm formation assay showed that the transposon insertion is near the 3� end of an openreading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK,and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS)in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the induciblefruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphatekinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependentsugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon inS. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation.Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defectivephenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm.Expression of fruK, as measured by the �-galactosidase activity of the fruK::Tn917-lac mutant, was observed tobe growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose,sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible,similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose,indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene inthe fruK::Tn917-lac mutant resulted in a greater increase in �-galactosidase activity when the organism wasgrown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructosePTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii isintricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, whichmodulates carbon flux in response to environmental nutritional levels, appears to be important in theregulation of bacterial biofilms.

The process of bacterial accumulation and proliferation af-ter initial bacterial adhesion leads to the formation of persis-tent, complex, organized sessile communities on oral surfaces.The multistep process of oral biofilm formation is a complexdevelopmental process initiated by attachment to saliva-condi-tioned oral surfaces of primary colonizers, such as viridansstreptococci (including Streptococcus gordonii), which consti-tute a majority of the cultivable bacteria found in dental plaque(21). Subsequent accumulation and growth of attached bacte-ria result in microcolonies that increase in size and eventuallyform biofilms. Fully developed oral biofilms (dental plaque)are similar to other complex sessile communities that havehighly structured, distinct architecture and physiochemicalproperties (4, 37).

In nutritionally limited environments, such as oral surfaces,biofilm formation may represent a survival strategy (17). In theoral cavity, streptococci depend on sugars as an energy source;the main energy supply is carbohydrates. Thus, these bacteriaare transiently exposed to a mixture of various sugars and liveunder feast-or-famine conditions. Oral streptococci constitute

the dominant acidogenic population in supragingival plaqueand are capable of transporting and fermenting a wide varietyof sugars. The phosphotransferase system (PTS) is the majortransport system for carbohydrates, which are phosphorylatedduring translocation through the membrane. In oral strepto-cocci, the high-affinity phosphoenolpyruvate-dependent sugarPTS is the principal route for transport of most sugars and isprimarily responsible for sugar transport at low sugar concen-trations (43).

Comparative proteome and transcriptome analyses of bio-film and planktonic cells have revealed that various anabolicand catabolic operons are differentially expressed. These oper-ons include operons involved in energy metabolism and in thebiosynthesis, transport, and metabolism of carbon compounds,lipids, and amino acids in Pseudomonas aeruginosa (33, 49),Escherichia coli (34, 41), Bacillus subtilis (36), Bacillus cereus(22), Streptococcus mutans (39), and Listeria monocytogenes(42).

The transition from a planktonic existence to growth in abiofilm occurs primarily in response to environmental cues,including the availability of nutrients. Addition of carbohy-drates to growth medium has been shown to affect biofilmformation by oral streptococci on abiotic surfaces (9, 18, 28,51). In Streptococcus parasanguinis, addition of glucose en-hanced biofilm formation in different types of media (9),

* Corresponding author. Mailing address: Department of PediatricDentistry, Goldman School of Dental Medicine, Boston University,801 Albany Street, Room 215, Boston, MA 02118. Phone: (617) 638-4773. Fax: (617) 638-5033. E-mail: [email protected].

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whereas enriched media inhibited biofilm formation by S. gor-donii (18) and S. mutans (51).

In contrast, glucose repressed biofilm formation by severalspecies of the Enterobacteriaceae (13). In E. coli, this glucoseeffect or catabolite repression is partially mediated by cyclicAMP (cAMP) and cAMP receptor protein. Several othergenes involved in global carbon regulation have been shown tobe important in biofilm formation. A cAMP-independent ca-tabolite repression control protein (Crc), which is a globalcarbon regulator, was found to be necessary for biofilm forma-tion in P. aeruginosa (23). Another global regulatory factor thatinfluences biofilm development in E. coli is the carbon storageregulator CsrA, which serves as both a repressor of biofilmformation and an activator of biofilm dispersal under a varietyof culture conditions. The effects of CsrA on biofilm formationwere found to be mediated largely through regulation of in-tracellular glycogen biosynthesis and catabolism (14). In gram-positive bacteria, the global carbon regulator CcpA (catabolitecontrol protein) may regulate genes required for stable biofilmformation in S. mutans (48) and B. subtilis (36), as loss of CcpAresulted in an approximately 60% decrease in biofilm forma-tion on an abiotic surface. In this report we describe isolationand characterization of an S. gordonii Tn917-lac mutant with amutation in the fructose PTS operon and a biofilm-defectivephenotype, and we provide additional evidence that regulationof carbon flux involving a sugar PTS also plays a significant rolein the development of biofilms.

MATERIALS AND METHODS

Bacteria, media, and chemicals. S. gordonii Challis 2, the rifamycin-resistant(500 �g/ml) strain of S. gordonii Challis (18), was used as the parent strain.Unless indicated otherwise, bacteria were subcultured and maintained routinelyon brain heart infusion (BHI) agar (BBL, Becton Dickinson, Cockeysville, Md.)or Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with0.2% yeast extract (THBYE) at 37°C under anaerobic conditions (VWRbrandanaerobic chamber; VWR, Plainfield, N.J.).

All chemicals were purchased from Sigma (St. Louis, Mo.) or Fisher Scientific(Pittsburgh, Pa.). All enzymes used for DNA manipulations were purchasedfrom Promega (Madison, Wis.) or Fisher Scientific unless indicated otherwise.Oligonucleotide primers were obtained from Invitrogen Life Technologies(Rockville, Md.).

Tn917-lac mutagenesis of S. gordonii Challis. An in vitro microtiter platebiofilm formation assay was performed as previously described (18) by using aminimal defined medium as the biofilm medium (BM). This BM contained 58mM K2HPO4, 15 mM KH2PO4, 10 mM (NH4)2SO4, 35 mM NaCl, 0.8% (wt/vol)glucose, 0.2% (wt/vol) Casamino Acids, 0.1 mM MnCl2 � 4H2O (pH 7.4), filter-sterilized vitamins (0.04 mM nicotinic acid, 0.1 mM pyridoxine HCl, 0.01 mMpantothenic acid, 1 �M riboflavin, 0.3 �M thiamine HCl, and 0.05 �M D-biotin),amino acids (4 mM L-glutamic acid, 1 mM L-arginine HCl, 1.3 mML-cysteineHCl, and 0.1 mM L-tryptophan), and 2 mM MgSO2 � 7H2O.

S. gordonii Challis 2 was mutagenized with pTV32OK (6), and a lacZ fusionlibrary was generated as described previously (19). Round-bottom polystyrene(Falcon 3918) microtiter plates (Becton-Dickinson Labware, Lincoln Park, N.J.)containing 100 �l of BM with erythromycin (10 �g/ml) in each well were inoc-ulated with S. gordonii Challis::Tn917-lac mutants. After 24 to 48 h of incubationat 37°C under anaerobic conditions, bacterial growth and biofilm formation werequantified by measuring the absorbance at 575 nm of each bacterial culture andcrystal violet-stained biofilm, respectively (19). Bacteria from a duplicate platewith corresponding wells having equivalent growth but poor crystal violet stain-ing were used as a biofilm-defective mutant.

In addition to the microtiter plate assay, biofilm formation on borosilicateglass coverslips was visualized directly by phase-contrast microscopy (23). Cellsfrom overnight colonies grown on BHI agar plates were inoculated into BM in50-ml Falcon tubes to an A600 of �0.04. Borosilicate coverslips were coated withfilter-sterilized, clarified whole saliva for 1 h at room temperature on a shaker.Then uncoated and saliva-coated coverslips were placed in the Falcon tubes

containing BM inoculated with streptococci and incubated at 37°C under anaer-obic conditions. At each time point, a coverslip was removed and rinsed with BMto remove nonadherent cells. Biofilm bacteria present on the coverslip wereexamined by phase-contrast microscopy by using a Micromaster phase-contrastmicroscope (Fisher Scientific), and images were captured with a Nikon Coolpix950 digital camera (19).

Southern hybridization, localization of transposon insertion site, and se-quence analyses. Chromosomal DNA isolated from S. gordonii Challis 2 and themutant strain were digested with HindIII and transferred onto a nitrocellulosemembrane. Southern hybridization with a digoxigenin-labeled pTV32-OK probewas performed as described previously (19).

The location of the transposon insertion was determined by sequence analysisof the region flanking the transposon. Initially, pBluescript vector and chromo-somal DNA from the mutant were digested with HindIII, purified with a nucle-otide removal kit (Qiagen, Valencia, Calif.), ligated to each other, and used asthe PCR template. A PCR with primers IP917B and PBSSK3 (Table 1) was thenperformed under the following conditions: after an initial denaturation for 2 minat 95°C, 36 cycles of amplification (denaturation for 45 s at 94°C, annealing for45 s at 53°C, and extension for 2 min at 72°C), followed by a final extension for10 min at 72°C. The PCR products were analyzed by agarose gel electrophoresis,purified with a PCR purification kit (Qiagen), and sequenced at the GeneticsCore Sequencing Facility of Boston University by using a model 377 automatedsequencer (Applied Biosystems, Foster City, Calif.). The sequence obtained wascompared with sequences in the GenBank database by using the BLASTXprogram (1) to identify homologous bacterial sequences. The putative proteinsequences encoded by the genes identified were then compared to the sequencesof FruR, FruK, and FruI homologs from different streptococci. Amino acidsequence alignment and phylogenetic analysis were performed by using theAlignX program in Vector NTI (Informax Inc., Bethesda, Md.), which utilizesthe neighbor-joining algorithm (32).

RT-PCR of S. gordonii Challis 2 RNA. In order to determine the genes thatconstitute the fructose PTS operon, reverse transcription-PCR (RT-PCR) wasperformed with total RNA extracted from S. gordonii Challis 2 grown to themid-log phase (A600, 0.3 to 0.4) by using an RNeasy mini kit (Qiagen). Theprimer pairs used for RT-PCR analysis are listed in Table 1. Primers FruR1 andFruR2 are specific for an intergenic region extending from the open readingframe (ORF) encoding hypothetical protein 1 (upstream from fruR) to fruR;primers Fru repressor5� and fruPTS3� are specific for an intergenic region ex-tending from fruR to fruI; primers PTS3 and Hypo2rev are specific for anintergenic region extending from fruI to the ORF encoding hypothetical protein2 (downstream from fruI); and primers PTS3 and Hypo3rev are specific for anintergenic region extending from fruI to the ORF encoding hypothetical protein3. The RT-PCR was performed by using the Access RT-PCR system (Promega)under the conditions that were described previously (19).

Expression of fruK in different environmental conditions. The �-galactosidaseactivity of the biofilm-defective S. gordonii fruK::Tn917-lac mutant was deter-mined by a fluorimetric assay (12, 19) by using 4-methylumbelliferyl-�-D-galac-toside (MUG). Cells were grown in THBYE containing erythromycin, and the�-galactosidase activities of cells harvested at different growth phases over 24 hwere determined. In order to determine the effects of various growth conditionson expression, bacteria were grown in 10 ml of THBYE or BM containingerythromycin at 37°C (unless indicated otherwise) in the anaerobic chamber for18 h. The conditions tested were BHI agar, Todd-Hewitt broth, and THBYEwithout any supplement; THBYE without any supplement grown under aerobicconditions or at 30 or 42°C; THBYE without any supplement adjusted to variouspHs (pH 5, 6, 7 8, or 9); and THBYE supplemented with zinc (1 mM), manga-nese (1 mM), 10% clarified whole human saliva, 10% human serum, NaCl (0.1,0.2, 0.3, 0.4, or 0.5 M), a sugar (fructose, galactose, glucose, lactose, maltose,mannose, sucrose, mannitol, sorbitol, or xylitol) at a concentration of 0.8%(wt/vol), or an amino acid (alanine, arginine, aspartate, cysteine, glutamine,glutamate, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline,serine, threonine, tyrosine, or valine) at a concentration of 100 mM. In subse-quent assays, the growth conditions tested were BM containing 0.8% glucosesupplemented with various concentrations of fructose (0.1, 0.5, 0.8, 2, 5, or 10%[wt/vol]), xylitol (0.1, 0.5, 0.8, 2, 5, or 10% [wt/vol]), fructose 1-phosphate (50�M), fructose 6-phosphate (50 �M), fructose 1,6-biphosphate (1 mM), 10%human serum, and 10% whole saliva. Growth in BM without glucose supple-mented with various concentrations of glucose (0.1, 0.5, 0.8, 2, 5, or 10% [wt/vol]), fructose (0.1, 0.5, 0.8, 2, 5, or 10% [wt/vol]), lactose (0.1, 0.5, 0.8, 2, 5, or10% [wt/vol]), or sucrose (0.1, 0.5, 0.8, 2, 5, or 10% [wt/vol]) or with 0.8% (wt/vol)galactose, 0.8% (wt/vol) maltose, or 0.8% (wt/vol) mannose was also tested. Cellswere also grown in an alternative base medium, 3.5% (wt/vol) tryptone with

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vitamins (TV medium) (3) supplemented with 0.5% (wt/vol) glucose, 0.5% (wt/vol) fructose, or 0.5% (wt/vol) sucrose.

Cells were centrifuged, resuspended in 10 ml of fresh, prewarmed liquidmedium, and grown to the log phase (A600, 0.15 to 0.4) (�3 h). The bacterialgrowth (A600) was recorded, and the cells were washed twice with 5 ml of TESC(10 mM Tris, 1 mM EDTA, 150 mM NaCl; pH 8.0), resuspended in 5 ml ofTESC, and sonicated for 2 min. The cells were placed on ice for 5 min andcentrifuged, and each clear sonicate was transferred to a new tube. For eachsample, 100 �l of the sonicate was transferred to an opaque microtiter plate, andthis was followed by addition of 100 �l of a solution containing 0.4 mg of MUGper ml in dimethyl sulfoxide. This preparation was mixed well and incubated for30 min at room temperature. After addition of 200 �l of 4-methyl-umbelliferonestandards (at concentrations ranging from 0.1 to 3.2 �M in dimethyl sulfoxide),the microtiter plate was read with a fluorescent plate reader (Spectrafluor plus;TECAN GmbH, Salzburg, Austria) at an excitation wavelength of 355 nm and anemission wavelength of 460 nm. A Bradford protein assay (Bio-Rad Laborato-ries, Richmond, Calif.) was performed according to the manufacturer’s instruc-tions to determine the protein concentration in each sonicate preparation. Theactivity was expressed in micromoles of 4-methyl-umbelliferone per milligram ofprotein per minute.

PCR ligation mutagenesis. PCR ligation mutagenesis with vectorless interme-diates was used to construct fruR, fruK, and fruI deletion mutants (15). A spec-tinomycin resistance gene, spec, amplified from plasmid pSF152, was used as theantibiotic marker insert. PCR amplification of the two flanking regions and theantibiotic marker insert was performed with the appropriate primers (Table 1).PCR products of the 5� and 3� flanking region and the spec cassette were ligatedand used for transformation of S. gordonii Challis 2 as described previously (19).

Primers were designed for insertional inactivation so that allelic replacementresulted in a nonpolar mutation. PCR ligation mutagenesis (15) was used toreplace most of the fruK gene (from position 9 to position 290 of the predicted

303-amino-acid sequence) on the chromosome of S. gordonii with spec. Thismethod generated mutants with a fruK::spec allele. PCR ligation mutagenesis wasalso used to replace most of the fruR gene (encoding positions 28 to 214 of thepredicted 247-amino-acid sequence) and the fruI gene (encoding positions 15 to633 of the 653-amino-acid sequence) with spec in order to generate mutants withfruR::spec and fruI::spec alleles, respectively. The fruK::Tn917-lac biofilm-defec-tive mutant was used for transformation instead of the Challis 2 strain, and thisresulted in the double mutants fruK::Tn917-lac fruR::spec (fruK/fruR) andfruK::Tn917-lac fruI::spec (fruK/fruI). The transformants were then plated onBHI agar containing 1 mg of spectinomycin per ml and incubated at 37°Canaerobically for 3 to 5 days. RT-PCR was performed (19) by using RNAisolated from the fruK::Tn917-lac, fruR::spec, fruK::spec, fruI::spec, fruK/fruR, andfruK/fruI mutants to determine whether these strains had a polar or nonpolarmutation. S. gordonii Challis 2 RNA was use as a control. The primers used wereFruK3 and FruK4, which are specific for an intergenic region extending fromfruK to fruI.

Growth rate and xylitol toxicity assay. The growth rates of S. gordonii strainChallis 2 and the fruK::Tn917-lac, fruK::spec, fruR::spec, and fruI::spec mutantswere assessed by inoculating the strains from overnight THBYE cultures into 10ml of fresh THBYE and growing them at 37°C under anaerobic conditions.Growth was quantified by recording the A600 at regular intervals for 24 h. Thegrowth rates of the double mutants, fruK/fruR and fruK/fruI, were also assessedby the same method.

The sensitivity of these strains to xylitol was assessed by using the method ofWen et al. (47). Overnight cultures were grown in TV medium containing 0.5%(wt/vol) glucose and then diluted 1:10 in prewarmed TV medium containing0.2% (wt/vol) glucose and incubated at 37°C anaerobically. TV medium was usedbecause S. gordonii was not able to grow in TV medium without a carbohydratesupplement, so the experiment would not be confounded by the presence ofsignificant quantities of uncharacterized sugars (3). Growth was monitored by

TABLE 1. Oligonucleotide primers used in this study

Primer Nucleotide sequence (5� to 3�)a Location Application Amplicon

SSP-917B AAC TGT ACC ACT AAT AAC TCA CAA T Tn917-lac PCRPBSSK3 GTA AAA CGA CGG CCA GT pBluescript PCRSpec1 CGA CGC GTC GAA ATC TAT AAA TAA ACT A pSF152 Mutagenesis Spectinomycin cassetteSpec2 CGG TCG ACC GAA ATA ATA AAA CAA AAA A pSF152 Mutagenesis Spectinomycin cassetteFru repressor5� CGA CGC GTC GGG TAC TAC AAA TGA GTT G fruR RT-PCR fruR-fruI (3,330 bp)FruPTS3� CGA CGC GTC GTT ATT YTG ACA ATG GTT T fruI RT-PCR fruR-fruI (3,330 bp)FruR1 CGG GTA CCC GTC AAT ATC AAC TAC TGA AT Hypothetical

protein 1Mutagenesis of fruR,

RT-PCRfruR 5� flanking region

(907 bp)FruR2 CGA CGC GTC GTA TGA ACC AAA CTA TCT A fruR Mutagenesis of fruR,

RT-PCRfruR 5� flanking region

(907 bp)FruR3 CGG TCG ACC GTG TTA AAG TAG CTC CTC T fruR Mutagenesis of fruR fruR 3� flanking region

(740 bp)FruR4 CGC TCG AGC GTA AGA TTT GAC GAG CAT A fruK Mutagenesis of fruR fruR 3� flanking region

(740 bp)FruK1 CGG GTA CCC GAC AGG AGT AAA AAC AGA G fruR Mutagenesis of fruK fruK 5� flanking region

(1,022 bp)FruK2 CGA CGC GTC GGG TTT AAC GTT ACA GTA T fruK Mutagenesis of fruK fruK 5� flanking region

(1,022 bp)FruK3 CGG TCG ACC GTA TTA AAG AAA CAT ATG A fruK Mutagenesis of fruK fruK 3� flanking region

(834 bp)FruK4 CGC TCG AGC GTC AGT TCT TCT GTC TTA C fruI Mutagenesis of fruK fruK 3� flanking region

(834 bp)PTS1 CGG GTA CCC GTT GCT AAG GCA AAG ATA A fruK Mutagenesis of fruI fruI 5� flanking region

(1,053 bp)PTS2 CGA CGC GTC GCT AGC AAC ATA ACG TCT T fruI Mutagenesis of fruI fruI 5� flanking region

(1,053 bp)PTS3 CGG TCG ACC GTT TGG TTG GTG CAG TAG T fruI Mutagenesis of fruI fruI 3� flanking region

(613 bp)PTS4 CGC TCG AGC GCA TTT AGA ACT CCA AAT TAA Hypothetical

protein 2Mutagenesis of fruI frul 3� flanking region

(613 bp)Hypo2rev ATT CGA CCT TGA ATA TGG TTA ACT TGA G Hypothetical

protein 2RT-PCR

Hypo3rev CTC CAA GAT GTG TTC CAA TAA CAC TAC C Hypotheticalprotein 3

RT-PCR

a Engineered restriction sites are indicated by boldface type. The restriction endonuclease recognition sequences are as follows: KpnI, GGT/ACC; MluI, ACG/CGT;SalI, GTC/GAC; and XhoI, CTC/GAG.

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determining the A575 of the cultures. At the early exponential phase (A575, 0.2 to0.3), xylitol was added to a final concentration of 1% (wt/vol), and growth wasmonitored for an additional 22 h. Controls received an equivalent amount ofsterile distilled water or 1% fructose instead of xylitol.

RESULTS

A biofilm-defective mutant was identified by screening anin-frame lacZ fusion library generated following Tn917-lac mu-tagenesis of S. gordonii Challis and was characterized further insubsequent experiments. The transposition was confirmed bySouthern hybridization with Tn917-lac, which has two HindIIIsites. Three hybridizing bands predicted to be present in Hin-dIII-digested DNA from the biofilm-defective mutant wereobserved (Fig. 1A). This confirmed that a single transposoninsertion had occurred in the biofilm-defective Tn917-lac mu-tant.

The ability of the biofilm-defective mutant to form biofilmson microtiter plates was measured. The biofilm assay showedthat biofilm formation by this S. gordonii Tn917-lac mutant(A575, 2.19 � 0.25 [mean � standard deviation]) was reducedby 55.77% compared with the biofilm formation by the S.gordonii Challis 2 strain (A575, 4.94 � 0.10).

In addition, biofilm formation on borosilicate glass cover-slips was examined 24 h after inoculation. The abilities of thestrains to form biofilms were confirmed by direct visualizationof the biofilms formed on the glass surfaces by phase-contrastmicroscopy (Fig. 1B). After incubation for 1 h, small numbersof cells of both strains were observed, which probably repre-sented reversible attachment of cells to the surfaces, and therewas no obvious difference between the Challis 2 and mutantstrains. After incubation for 24 h, the Challis 2 strain hadformed large clusters of cells that were interspersed withsparsely covered areas. A number of dense microcolonies werealso observed. In contrast, significantly fewer cells of the mu-tant strain had attached, and they formed a scattered patternthat was markedly different from the Challis 2 biofilm; large

areas were devoid of cells. Biofilm formation on coverslipsprecoated with whole saliva was also visualized in order toexamine the role of saliva in biofilm formation in this in vitrobiofilm assay to mimic in vivo conditions. With both the Challis2 strain and the Tn917-lac mutant, no difference was observedbetween biofilm formation on saliva-coated glass surfaces andbiofilm formation on uncoated surfaces.

PCR performed with genomic DNA that was digested withHindIII and ligated with pBluescript resulted in a PCR productwhich was 3 kb long and contained the region 5� to the trans-poson insertion. Sequence analysis of this PCR product re-vealed that transposition had occurred within an ORF thatencoded a protein with a predicted amino acid sequence ho-mologous to the sequence of FruK (Fig. 1B), a fructose-1-phosphate kinase encoded by the fruRKI operon of S. mutans(2, 47). This gene was not identified when Tn916 mutagenesiswas used in a previous screening of biofilm-defective mutantsof S. gordonii Challis 2 (18).

Genetic organization of the S. gordonii fructose PTS operon.The nucleotide sequence of the region 5� of the transposoninsertion was used in sequence similarity BLASTN searches ofthe S. gordonii unfinished genome database (http://www.ti-gr.org). The S. gordonii nucleotide sequence obtained revealedthat the closest homologs of the deduced amino acid sequenceswere proteins encoded by genes of the S. mutans fructoseoperon, which consists of fruR, fruK, and fruI (Fig. 1B). Thisoperon is involved in fructose transport in S. mutans (2, 47).

The three ORFs identified in S. gordonii were designatedfruR, fruK, and fruI. The 741-bp fruR ORF encodes a 247-amino-acid sequence with a predicted molecular mass of 27.5kDa. The deduced amino acid sequence showed a high degreeof homology to the sequences of members of the deoxyribo-nucleoside repressor (DeoR) family of bacterial regulatoryproteins (35).

Analysis of the region 3� of fruR revealed the presence oftwo other ORFs, which were highly homologous to fruK and

FIG. 1. (A) Southern hybridization of HindIII-digested DNA from biofilm-defective S. gordonii fruK::Tn917-lac mutant. Digoxigenin-labeledpTV32-OK containing Tn917-lac (which has two HindIII sites) was used as the probe. Three hybridizing bands were produced with HindIII-digested DNA, demonstrating the presence of a single transposon insertion. (B) Gene organization of the fructose PTS operon. Genes of thefructose PTS operon are shown in grey. fruR encodes a putative transcriptional repressor, fruK encodes a fructose-1-phosphate kinase, and fruIencodes a fructose-specific enzyme II (EIIABC components). Maps of S. gordonii Challis 2 and fruK::Tn917-lac mutant are shown along with thecorresponding biofilm phenotypes on polystyrene, uncoated glass, and saliva-coated glass surfaces as determined directly by phase-contrastmicroscopy. Cells were grown in BM for 24 h at 37°C under anaerobic conditions.


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fruI of S. mutans. The fruK ORF was 909 bp long and started1 bp upstream of the fruR stop codon. The fruK ORF waspredicted to encode a 303-amino-acid protein with a predictedmolecular mass of 32.8 kDa. The Tn917 insertion in S. gordoniifruK::Tn917-lac was at amino acid 210 of FruK.

The fruI ORF was 1,959 bp long and started 1 bp upstreamof the fruK stop codon. This ORF encodes the fructose per-mease FruI, the EII enzyme of this PTS which allows uptake ofextracellular fructose and concomitant phosphorylation of thisfructose into fructose 1-phosphate (2, 47) and has a deduced653-amino-acid sequence with a predicted molecular mass of67.5 kDa. FruI consists of EII A, B, and C domains of phos-phoenolpyruvate-dependent sugar PTSs. The deduced aminoacid sequences of FruR, FruK, and FruI exhibited high levelsof homology with the sequences of streptococcal and lactococ-cal homologs (Table 2).

Located upstream of fruR is an ORF with a stop codon thatis 139 bp upstream of the start codon of fruR, which encodes aputative 751-amino-acid protein designated hypothetical pro-tein 1. The last 133 amino acids in the C-terminal region ofhypothetical protein 1 exhibit homology to a chromosome seg-regation protein of Xanthomonas campestris pv. campestris,which is involved in cell division and chromosome partitioning.An ORF identified downstream of fruI and starting 48 bp fromthe stop codon of fruI encodes a 143-amino-acid protein des-ignated hypothetical protein 2, which is homologous to a con-served hypothetical protein of unknown function in S. mutans.A second fructose PTS operon has been described in S. mutans(47). Likewise, a TBLASTN search of the S. gordonii unfin-ished genome database revealed a second homolog of FruK(data not shown).

Sequence and phylogenetic analyses of the fructose PTSoperon. The genetic organization, the phylogenetic relation-ship, and an amino acid alignment of the putative FruR, FruK,and FruI proteins were examined. Multiple alignments of allthree proteins from S. gordonii, S. pneumoniae, S. mitis, S.mutans, S. equi, S. agalactaciae, and S. pyogenes suggested thatthey have extensive homologies.

Catabolite repression occurs when enzymes involved in themetabolism of complex carbon and energy sources are re-pressed by glucose, as their functions are unnecessary whenthere are abundant, readily metabolized alternatives, such asglucose. Analysis of the nucleotide sequence 5� of the fruRORF resulted in identification of a catabolite responsive ele-ment (CRE) 7 bp downstream from the putative �10 pro-

moter sequence (Fig. 2A). The CRE has a 14-bp palindromicsequence in concordance with the consensus sequence TGWAANCGNTNWCA (46), with one nucleotide mismatch. A10-bp direct repeat with one mismatch was present 4 bp up-stream from the putative �35 box of the promoter (Fig. 2A),and this repeat is probably also involved in regulation of theoperon. A homologous direct repeat in the promoter region ofthe fruR homolog in S. mutans, fxpA, which is part of thefxpABC fructose and xylitol transport operon in S. mutans, hasbeen proposed to be involved in binding of a trans-acting factor(2).

The first gene in the S. gordonii fructose PTS operon, fruR,encodes FruR, the putative regulator protein of the operon.FruR has significant similarity (Fig. 2B) with transcriptionalregulator proteins of bacterial carbohydrate catabolic operonsbelonging to the DeoR family (45) and the same structuralcharacteristics as other DeoR proteins, with a highly conservedN-terminal region that contains a helix-turn-helix motif atamino acid residues 38 to 56 which is probably involved inDNA binding.

The second gene, fruK, codes for a fructose-1-phosphatekinase (FruK), which belongs to the phosphofructokinase PfkBfamily of carbohydrate kinases (50). FruK uses ATP to phos-phorylate fructose 1-phosphate in the cytoplasm to fructose1,6-biphosphate. The third gene, fruI, encodes the fructosepermease FruI, which allows uptake of extracellular fructoseand concomitant phosphorylation of this fructose into fructose1-phosphate. A palindromic region was observed upstream offruK (TGATTATTCTTGG GCAGGTAAAACA; 34 basesupstream of the start codon) and also upstream of fruI (GGGAGTAGCTTGCG GAACAGCAACTACC; 68 bases up-stream of the start codon). These sequences may be fru oper-ator sequences recognized by the helix-turn-helix motif ofFruR.

The third gene, fruI, codes for the fructose permease of thePTS. PTS sugar-specific permeases consist of three domains;two hydrophilic domains, IIA (formerly enzyme III) and IIB,possess the first and second phosphorylation sites, respectively,and a hydrophobic IIC domain is not phosphorylated but con-tains several membrane-spanning �-helices, forms the translo-cating transmembrane channel, and provides the sugar-bindingsite. IIB is phosphorylated by phospho-IIA before the phos-phoryl group is transferred to the sugar substrate. The IICdomain catalyzes the transfer of a phosphoryl group from IIBto the sugar substrate (2, 16, 44).

The sequence upstream of fruR was identified as ORF1,which codes for a hypothetical protein. The sequence down-stream of fruI was identified as ORF2, which codes for anotherhypothetical protein. RT-PCR performed with RNA from theChallis 2 strain and primers Fru repressor5� and FruPTS3�(Table 1), which are specific for a 3.33-kb region that spans thefruR and fruI intergenic region, produced a product that wasapproximately 3,330 bp long (Fig. 3). This product spans thefruR and fruI ORFs, and its size coincides with the predictedamplicon size. RT-PCR performed with primers FruR1 andFruR2 (which are specific for a region extending from theORF encoding hypothetical protein 1 to fruR), primers PTS3and Hypo2rev (which are specific for a region extending fromfruI to the ORF encoding hypothetical protein 2), and primersPTS3 and Hypo3rev (which are specific for a region extending

TABLE 2. Homology of the proteins encoded by the S. gordoniifructose PTS operon (FruR, FruK, and FruI), the proteins of otheroral streptococci, and LacR, LacC, and FruA of Lactococcus lactis

subsp. lactis

Organism

% Identity (% similarity) with thefollowing S. gordonii proteins:

FruR FruK FruI

Streptococcus pneumoniae 69.6 (81.4) 86.1 (90.4) 81.6 (90.2)Streptococcus mitis 69.0 (81.0) 87.2 (91.1) 81.6 (90.2)Streptococcus agalactiae 53.2 (66.9) 74.6 (82.5) 77.7 (85.7)Streptococcus mutans 58.4 (72.4) 74.3 (82.2) 75.8 (83.7)Streptococcus pyogenes 54.3 (70.4) 72.3 (82.8) 77.2 (83.9)Lactococcus lactis subsp. lactis 42.0 (57.2) 53.7 (66.8) 67.0 (79.8)


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from fruI to the ORF encoding hypothetical protein 3) pro-duced no PCR products (Fig. 3). All the primer pairs (Table 1)used in the RT-PCR were also used in standard PCR ampli-fications with the S. gordonii Challis 2 DNA, which producedPCR products of the predicted sizes (data not shown). Theseresults demonstrate that fruR, fruK, and fruI are cotranscribedas a single operon, while the genes encoding hypothetical pro-teins 1, 2, and 3 are probably not cotranscribed. Therefore, thefructose PTS operon in S. gordonii consists of only three ORFs,fruR, fruK, and fruI.

Regulation of fruK expression in response to changes inenvironmental conditions. The effects of growth phase andvarious environmental conditions on the expression of fruKwere examined by measuring the �-galactosidase activity of S.gordonii fruK::Tn917-lac grown in medium supplemented witha variety of nutrients and under various conditions. The initial�-galactosidase assays performed with cells grown in THBYEwith various supplements and under various conditions did notreveal any significant differences in expression except for the

differences observed after addition of 10% human serum or10% whole saliva, which resulted in upregulation of fruK ex-pression (data not shown). As THBYE contains various sugars,in all subsequent assays we used a base medium without anysugar so that the effects of various sugars on fruK expressioncould be evaluated. Two different base media, BM and TVmedium, were used for comparison. The fruK::Tn917-lac mu-tant was not able to grow in BM without glucose supplementedwith 0.1% sucrose, 0.1% glucose, or 0.1% lactose. Growth ofthis mutant was inhibited in BM containing 0.8% glucose sup-plemented with 5 or 10% xylitol.

Assays in which cells grown in BM were used indicated thatthe expression of fruK was significantly increased in the pres-ence of fructose, sucrose, and xylitol (Fig. 4). Expression offruK was also upregulated in the presence of 10% humanserum and 10% whole saliva, while none of the other condi-tions tested had a significant effect on expression (data notshown). The level of fruK induction by fructose increased withan increase in the concentration of fructose in the medium,

FIG. 2. (A) Alignment of the promoter region of S. gordonii fruR with the fruR homologs in S. pneumoniae (accession no. NP345362), S. mitis,S. mutans (accession no. NC004350), S. pyogenes (accession no. 002737), and S. agalactiae (accession no. NC004116) determined by using theAlignX program in the Vector NTI software (Informax). Streptococcal sequences without accession numbers were obtained from the unfinishedmicrobial genome database (www.ncbi.nlm.nih.gov). Nucleotides that are identical and conserved are indicated by dark grey and light greybackgrounds, respectively, and start codons are enclosed in boxes. In S. gordonii, direct repeat regions (DR) are indicated by horizontal arrows,while the CRE, putative ribosome binding site (rbs), putative �35, and putative �10 regions are underlined. (B) Phylogenetic tree of S. gordoniiFruR and other bacterial sugar PTS regulatory proteins. The phylogenetic relatedness dendrogram was constructed on the basis of amino acidsequence similarities by using the AlignX program in the Vector NTI software (Informax Inc.), which utilizes the neighbor-joining algorithm (32).The reliability of the topology was estimated by performing 100 bootstrap trials, and the bootstrap values are expressed in percentages at branchpoints (www.genebee.msu.su). Accession numbers follow the species names, and sequences without accession numbers were obtained from theunfinished microbial genome sequence database (www.ncbi.nlm.nih.gov).


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which suggests that there is a fructose-titratable promoter sys-tem that results in different levels of gene expression. None ofthe other sugars tested (galactose, glucose, lactose, maltose,mannose) induced fruK activity, suggesting that fructose, su-crose, and xylitol are the only sugars capable of inducing fruKexpression in S. gordonii. To investigate whether fruK is regu-lated by other mechanisms, such as catabolite repression byanother sugar, the bacterium was grown in BM containingfructose supplemented with glucose at a final concentration of0.8%. The resulting activities of the cells were lower the activ-ities after induction with fructose in the absence of glucose,indicating that glucose is able to catabolite repress fruK expres-sion (Fig. 4).

DNA binding of Cra, the fructose repressor in E. coli, hasbeen shown to be disrupted by a low concentration (50 �M) offructose 1-phosphate and a high concentration (5 mM) offructose 1,6-biphosphate (25). As growth was inhibited in thepresence of 5 mM fructose 1,6-biphosphate, cells were grownin fructose 1,6-biphosphate (1 mM), fructose 1-phosphate (50�M), or fructose 6-phosphate (50 �M). Expression assays in-dicated that only fructose 1-phosphate upregulated fruK ex-pression (Fig. 4), suggesting that fructose 1-phosphate is alsoan inducer of the fructose operon in S. gordonii.

Expression of fruK was observed only between 4 and 8.5 h ofgrowth in the assay, which corresponded to the early to mid-exponential growth phase of S. gordonii (Fig. 5A). The cellsreached maximum growth by this time. When cells were grown

in an alternative base medium (TV medium) supplementedwith glucose, fructose, or sucrose, the expression of fruK wasalso observed to be higher in the presence of fructose andsucrose (Fig. 5B). These results were similar to those obtainedwhen BM was used.

Construction and characterization of fruR, fruK, fruI, fruK/fruR, and fruK/fruI mutants. In order to confirm the biofilm-defective phenotype identified in S. gordonii fruK::Tn917-lac,an insertionally inactivated fruK gene was constructed by allelicexchange. The fruR gene was inactivated in the S. gordoniiChallis 2 and fruK::Tn917-lac strains to confirm that fruR en-codes a negative regulatory protein. The fruI gene was alsoinactivated in both the Challis 2 and fruK::Tn917-lac strains toinvestigate the role of FruI in biofilm formation and fructosetransport. Mutants with either a fruR::spec or fruI::spec alleleand two double mutants, fruK/fruR and fruK/fruI, were alsogenerated by the same method. PCR confirmed that integra-tion of the spec gene in fruR, fruK, or fruI in the chromosomehad occurred as predicted. PCR was also used to confirm theintegration of spec in fruR or fruI in the fruK::Tn917-lac mutant(data not shown).

RT-PCR with primers spanning the fruK and fruI intergenicregion resulted in a PCR product when RNA isolated fromeither S. gordonii Challis 2 or fruK::Tn917-lac was used as thetemplate (Fig. 3), indicating that the transposition resulted ina nonpolar fruK::Tn917-lac mutation. RT-PCR was also usedto confirm that the fruK/fruR mutation constructed was non-

FIG. 3. RT-PCR analysis of RNA extracted from the S. gordonii Challis 2, fruK::Tn917-lac, and fruK/fruR strains. (A) Organization of thefructose PTS operon and adjacent genes, locations of primers, and predicted size of the successfully amplified RT-PCR product. See Table 1 forthe primers used. The vertical arrow indicates the position of the Tn917-lac insertion in the biofilm-defective mutant. hypot. prot., hypotheticalprotein. (B) RT-PCR products obtained by using total RNA extracted from the Challis 2 strain, the fruK::Tn917-lac mutant, or the fruK/fruR doublemutant as the template. Lane 1, 1-kb DNA marker; lane 2, Challis 2 RNA with primers FruR1 and FruR2; lane 3, Challis 2 RNA with primersPTS3 and Hypo2rev; lane 4, Challis 2 RNA with primers Fru repressor5� and FruPTS3�; lane 5, Challis 2 RNA with primers PTS3 and Hypo3rev;lane 6, Challis 2 RNA with primers FruK3 and FruK4; lane 7, fruK::Tn917-lac RNA with primers FruK3 and FruK4; lane 8, fruK/fruR RNA withprimers FruK3 and FruK4.


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polar (Fig. 3). Similarly, RT-PCR was used to confirm that thefruR::spec and fruK::spec mutants were nonpolar (data notshown).

Expression of fruK in the two double deletion mutant strains,the fruK/fruR and fruK/fruI strains, was assessed by using cellsgrown in TV medium or BM. With the fruK/fruR mutant,expression of fruK was enhanced to a much larger degree whenthis organism was grown in TV medium in the presence offructose (Fig. 5B) than when the fruK::Tn917-lac mutant wasused. Similarly, growth of the fruK/fruR mutant in BM supple-mented with fructose in the absence of glucose (Fig. 6) resultedin a greater response to fructose induction, probably due to therelief of repression by fruR. On the other hand, the fruK/fruImutant grown in TV medium (Fig. 5B) or BM (Fig. 6) did notexhibit elevated fruK expression in the presence of fructose.This is probably because inactivation of fruI affected the trans-port of fructose into the cytoplasm, resulting in no induction offruK.

To investigate whether glucose is a catabolite repressor offruK in the fruK/fruR mutant, this strain was grown in BMcontaining fructose supplemented with glucose. The resultingactivities of the cells were lower than the activities when thecells were grown in fructose in the absence of glucose (Fig. 6),indicating that fruK expression was affected by catabolite re-pression of glucose in the fruK/fruR mutant. None of the bio-film-defective mutant phenotypes could be rescued by additionof fructose 1-phosphate, fructose 6-phosphate, or fructose 1,6-biphosphate at various concentrations, indicating that alterna-tive pathways are involved in biofilm formation.

The effects of insertional inactivation on biofilm formationwere assessed. Biofilm formation by the fruK::spec mutant(A575, 0.847 � 0.211[mean � standard deviation]) and biofilmformation by the fruI::spec mutant (A575, 1.196 � 0.083) weresimilar to biofilm formation by the S. gordonii fruK::Tn917-lacmutant (A575, 2.185 � 0.250), indicating that both fruK and fruIare involved in biofilm formation. The fruI mutant and bothfruK mutants were defective in biofilm formation compared tothe Challis 2 strain (A575, 4.94 � 0.10) (Fig. 7A). On the otherhand, the fruR::spec mutant was still able to form a biofilm(A575, 3.16 � 0.36). The second biofilm formation assay per-formed on glass coverslips by using phase-contrast microscopyproduced similar results (Fig. 8), with the Challis 2 and fruR::spec mutant strains able to form biofilms. The two doubledeletion mutant strains, the fruK/fruR and fruK/fruI strains,were also defective in biofilm formation on both polystyrenesurfaces (Fig. 7A and 8) and uncoated and saliva-coated glasssurfaces (Fig. 8). The biofilm phenotypes of the fruK and fruImutants suggest that these two genes are important for biofilmformation.

Growth rate and sensitivity to xylitol. The growth rates ofthe S. gordonii Challis 2 strain and the fruK::Tn917-lac,fruR::spec, and fruK::spec mutants in THBYE were found to besimilar, indicating that the growth rate was not affected by themutations. On the other hand, S. gordonii fruI::spec and thedouble mutants fruK/fruR and fruK/fruI all grew at lower ratesthan the Challis 2 strain and the three other nonpolar mutants(Fig. 7B), indicating that fruI probably plays a role in thegrowth of S. gordonii, and the double mutations affected bac-

FIG. 4. Effects of different concentrations of various sugars, human serum, and saliva on fruK expression. The biofilm-defective S. gordoniifruK::Tn917-lac mutant was grown in BM containing different concentrations of various sugars, human serum, and saliva at 37°C under anaerobicconditions as planktonic cells. The �-galactosidase activities of the fruK-lacZ transcriptional fusion were quantified by using MUG (12). All assayswere performed in triplicate, and means and standard deviations are shown. MU, 4-methyl-umbelliferone.


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terial growth. However, the final growth yields of the fruI::spec,fruK/fruR, and fruK/fruI mutants after 24 h of growth appearedto be the same as those of Challis 2 and the fruK::Tn917-lac,fruK::spec, and fruI::spec mutants.

The fructose PTS of oral streptococci is thought to be re-sponsible for the phosphorylation and uptake of xylitol (40).Xylitol is a naturally occurring five-carbon sugar alcohol andenters the bacterial cell as the nonmetabolizable xylitol 5-phos-phate via the same PTS as fructose. As xylitol 5-phosphate isnot catabolized, it accumulates and/or undergoes a futile phos-phorylation-dephosphorylation cycle and becomes toxic to thecell (40, 47). The xylitol sensitivities of the Challis 2 strain and

the fruK::Tn917-lac, fruR::spec, fruK::spec, fruI::spec, fruK/fruR,and fruK/fruI mutant strains were compared. After 4 h, theincreases in the A575of the Challis 2 (0%) and fruR::spec(0.9%) cultures were minimal in the presence of 1% xylitol,whereas the increases in the A575 of the fruK::Tn917-lac(7.5%), fruK::spec (5.9%), fruI::spec (3.4%), fruK/fruR (30.9%),and fruK/fruI (31.5%) cultures were greater. After 24 h, thegrowth differences among the strains were minimal. Growth ofthe S. gordonii Challis 2, fruK::Tn917-lac, and fruR::spec strainswas more sensitive to inhibition by 1% xylitol, whereas growthof the fruK::spec, fruI::spec, fruK/fruR, and fruK/fruI strains wasless sensitive to xylitol inhibition, suggesting that a nonfunc-

FIG. 5. Effects of various growth conditions on fruK expression. Cells were grown at 37°C under anaerobic conditions in various liquid media.The �-galactosidase activities of the fruK-lacZ transcriptional fusion were quantified by using MUG (12). All assays were performed in triplicate,and means and standard deviations are shown. (A) Relationship between growth phase and fruK expression. The biofilm-defective fruK::Tn917-lacmutant was grown in THBYE. Growth and �-galactosidase activity were determined at 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8.5 and 24 h. (B) Effectof growth in TV medium (3) containing various sugars on fruK expression. The fruK::Tn917-lac, fruK/fruR, and fruK/fruI mutant strains were grownat 37°C under anaerobic conditions. MU, 4-methyl-umbelliferone.


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tional fruK or fruI gene increased S. gordonii resistance toxylitol.

DISCUSSION

Bacteria possess complex regulatory networks that are ableto detect the frequent and abrupt changes that occur in theenvironment and to adapt their metabolism rapidly to suchfluctuations; these networks include transport systems thattransmit information about the nature, concentration, and di-versity of the external energy sources (16, 24, 31). Fructose isa major constituent of the human diet and can be liberatedfrom sucrose by glucosyltransferases and from fructans by fruc-tanases of oral streptococci. A biofilm-defective mutant of S.gordonii::Tn917-lac revealed that a gene with a high degree ofhomology to fruK, which is part of the fructose PTS operon ofS. mutans (2, 47), is required for biofilm formation. Althoughthe S. gordonii fruK::Tn917-lac isolate exhibited a biofilm-de-fective phenotype on polystyrene, glass, and saliva-coated glasssurfaces, these in vitro assay environments may not be repre-sentative of the in vivo environment.

The S. gordonii fructose PTS operon, consisting of the fruR,fruK, and fruI genes, was subsequently identified and was foundto be homologous to the S. mutans fructose PTS operon, amember of the carbohydrate PTS family involved in fructoseand xylitol transport in S. mutans (47). RT-PCR confirmed thatfruR, fruK, and fruI are cotranscribed in S. gordonii.

In the biofilm-defective S. gordonii::Tn917-lac mutant, thetransposon interrupted fruK, the second gene in the fructosePTS operon. fruK encodes a fructose-1-phosphate kinase,which phosphorylates fructose 1-phosphate to fructose 1,6-biphosphate. The first gene in the S. gordonii fructose PTS

operon, fruR, encodes FruR, the putative regulator protein ofthe operon, which belongs to the DeoR family of regulators,which include transcriptional regulators of several carbohy-drate catabolic operons. The third gene, fruI, encodes the fruc-tose permease FruI. The EII enzymes encoded by fruI of S.mutans (47) and fruA of E. coli (26) appear to function asfructose and xylitol transporters as inactivation of these genesconferred resistance to growth inhibition by xylitol. Similarly,S. gordonii fruK::spec, fruI::spec, fruK/fruR, and fruK/fruI mu-tants were more resistant to growth in xylitol.

Expression studies showed that fructose, sucrose, and xylitolwere the only sugars tested that were able to induce fruKexpression. Expression of fruK was observed only during theexponential growth phase. The results also indicated that therewas a fructose-titratable promoter system with different levelsof gene expression. This is consistent with previous findingsobtained with S. mutans, which showed that expression of fruIfrom the fructose operon was upregulated in response togrowth on fructose or sucrose (47). In this study, the inductionof fruK expression by fructose was inhibited by the presence ofglucose, indicating that glucose is able to catabolite repressfruK expression in S. gordonii.

RT-PCR results showed that the Tn917-lac insertion intofruK was nonpolar. A previous study showed that Tn917 inser-tion can disrupt the expression of a particular gene and at thesame time allow outwardly directed transcription initiatingfrom within the transposon, resulting in complete expression ofother individual genes of the Staphylococcus epidermidis icaoperon (8). In the nonpolar fruK::Tn917-lac mutant, a secondmutation created in fruR relieved the repression of the fructosePTS operon, as demonstrated by �-galactosidase assays of thefruK/fruR double mutant grown in the presence of fructose,

FIG. 6. fruK expression of the two biofilm-defective double mutants, fruK/fruR and fruK/fruI. Cultures were grown at 37°C under anaerobicconditions in BM containing different levels of fructose, with and without glucose. The �-galactosidase activities of the fruK-lacZ transcriptionalfusion in the two double mutants were quantified by using MUG (12). All assays were performed in triplicate, and means and standard deviationsare shown. MU, 4-methyl-umbelliferone.


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confirming that FruR is indeed a transcriptional repressor ofthe fructose PTS operon in S. gordonii. In contrast, a secondmutation in fruI in the nonpolar fruK::Tn917-lac mutant notonly failed to relieve the repression of the operon but alsoresulted in a reduced response to induction by fructose, asinactivation of FruI probably affected fructose transport intothe cell.

In the oral cavity, oral streptococci can use various carbo-hydrates as single sources of carbon and energy and must selectthe most appropriate energy source(s) from a constantlychanging environment (43). The presence of a CRE in thepromoter region of fruR suggests that catabolite repression isone of the regulatory mechanisms of the fructose PTS operon.The operator consensus sequence of the CREs, which governcarbon catabolite repression, was first identified in the B. sub-tilis �-amylase gene amyE (46). The S. gordonii fructose PTSoperon may have regulatory mechanisms that are similar tothose of the fructose PTS operon in S. mutans (2) and otheroral streptococci that have homologous CRE sequences. ThisCRE sequence is homologous to an amyO palindromic se-

quence, the CcpA binding site of B. subtilis (11). CcpA isinvolved in catabolite repression in gram-positive bacteria (38)and in biofilm formation in S. mutans (48).

In addition to sugar transport and phosphorylation, PTSsare also involved in transcriptional regulation, catabolite re-pression, inducer exclusion, enzyme activity, and chemotaxis(43). Three mechanisms of bacterial catabolite repression havebeen described, the CcpA mechanism in gram-positive bacte-ria, a cAMP-independent mechanism, and a mechanism de-pendent on the cAMP receptor protein in E. coli (29).

Fructose is the only sugar found in nature that feeds directlyinto glycolysis, the central pathway of carbohydrate metabo-lism. Cra, the catabolite repressor activator formerly known asFruR in E. coli, is a member of the LacI-GalR family oftranscriptional regulatory proteins and is the catabolite repres-sor-activator in the cAMP-independent system (30). Cra mod-ulates the direction of carbon flow by transcriptional activationof the genes encoding enzymes concerned with oxidative andglyconeogenic carbon flow and by repression of the genes con-cerned with fermentative carbon flow (25). The cytoplasmicglycolytic intermediates fructose 1-phosphate and, to a lesserextent, fructose 1,6-biphosphate are inducers of the fructoseoperon and counteract both the repressive and the activatingeffects of Cra by displacing Cra from the DNA (25, 30).

Results of our expression assays showed that fructose1-phosphate is an inducer of the S. gordonii fructose operon.Another DeoR family regulator, LacR from the lac operon inLactococcus lactis, is induced by tagatose 6-phosphate (44).Fructose 1-phosphate has also been proposed to be a regulatorof the DNA binding of FruR in Spiroplasma citri to a directrepeat in the promoter region of fruR (10).

Why would the fruK mutation affect biofilm formation in S.gordonii? Phosphorylation of the internalized fructose is a pre-requisite for catabolism by glycolysis, and the fructose needs tobe phosphorylated by a cytoplasmic sugar kinase. Since bothfruK and fruI mutations in S. gordonii were biofilm defective,we speculate that the substrates of both fruK and fruI play arole in biofilm formation. The Cra protein in enteric bacteria isa global regulator of carbon and energy metabolism (25). Sim-ilar regulation in S. gordonii would require that a fruK mutationperturbs the cellular balance of fructose 1-phosphate and fruc-tose 1-6 biphosphate so that the signal involved in the biofilmphenotype is altered. Inactivation of fruR, which relieved re-pression of the fructose PTS operon, did not affect the biofilmphenotype, suggesting that the fructose 1-phosphate and fruc-tose1 6-biphosphate balance was not affected and hence thebiofilm phenotype was retained. On the other hand, fructose1-phosphate and fructose 1-6 biphosphate did not have signif-icant effects on biofilm formation by the Challis 2 or mutantstrains. The substrates may have been ineffective because theywere not transported into the cell or because the concentra-tions examined were too low.

On the other hand, the disruption of biofilm formation bythe fruK::Tn917-lac mutant may indicate that FruK has anadditional function which is important in biofilm formation.Therefore, it is more likely that FruK could play a role intwo-component signal transduction by acting as a kinase. Thismay occur via cross-regulation, which involves the phosphory-lation of a response regulator of a two-component regulatorysystem by a different regulatory system (for example, the phos-

FIG. 7. (A) Bacterial growth and biofilm assay of the S. gordoniiwild type and the fruK::Tn917-lac, fruK::spec, fruR::spec, fruI::spec,fruK/fruR, and fruK/fruI mutant strains in BM. Assays were performedby using BM and polystyrene plates under anaerobic conditions. Allassays were performed in triplicate, and means and standard deviationsare shown. (B) Growth curves for the S. gordonii wild type and thefruK::Tn917-lac, fruK::spec, fruR::spec, fruI::spec, fruK/fruR, and fruK/fruI mutant strains in 10 ml of THBYE over 24 h at 37°C underanaerobic conditions. Growth was measured by determining the A575.


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phorylation of a response regulator by a kinase other than ahistidine protein kinase). In E. coli ANCC22, two histidineprotein kinase-encoding genes, phoR and creC, are requiredfor activation of their cognate response regulator, PhoB. In arecent study the workers found that a phosphofructokinasefrom Streptococcus thermophilus suppressed the phoR creC mu-tation in E. coli ANCC22, probably through a mechanism in-volving the production of acetyl phosphate (5), which mayprovide a regulatory link between glycolytic activity and two-component signal transduction regulation.

It has been demonstrated previously that cell-to-cell signalingis involved in biofilm formation by S. gordonii (18). Insertionalinactivation of luxS, which is responsible for AI-2 synthesis,disrupted the ability of S. gordonii to form a mixed-speciesbiofilm with a LuxS-null strain of Porphyromonas gingivalis,suggesting that LuxS-dependent intercellular communication

is essential for biofilm formation by nongrowing cells of P.gingivalis and S. gordonii (20). S. gordonii AI-2 was also foundto regulate aspects of carbohydrate metabolism, as fruA (whichcodes for the fructan �-frutosidase precursor), gtfG (the glu-cosyltransferase gene), and rgg (which positively regulatesgtfG) were downregulated in the S. gordonii luxS mutant (20).The AI-2 signaling molecule of E. coli significantly upregulatedfrwC, which encodes a fructose-like EII component of a PTS inE. coli (7) with a high level of homology to the EIIC domain(32% identity, 50% similarity) of FruI of the fructose PTS in S.gordonii. FrwC is one of the three silent gene clusters thatencode silent backup fructose PTSs with unknown physiolog-ical functions in E. coli (27). Alternatively, a mutation in fruKmay have affected the expression of other surface proteins thatcould influence initial adhesion and biofilm formation.

A common theme in microbial development involves an

FIG. 8. Maps of the S. gordonii fruK::spec, fruR::spec, and fruI::spec mutants and fruK/fruR and fruK/fruI double mutants. The biofilm phenotypesof the strains on polystyrene, uncoated glass, and saliva-coated glass surfaces were observed directly by phase-contrast microscopy and are shownon the right. Cells were grown in BM for 24 h at 37°C under anaerobic conditions.


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input of environmental cues that results in an output of analtered physiological state or behavior (23). The transitionfrom a planktonic existence to a sessile existence occurs inresponse to environmental factors, including the availability ofnutrients. Regulatory proteins and signaling pathways play arole in the transduction of environmental signals and precipi-tation of developmental changes that allow a bacterium toadapt to its environment. In P. aeruginosa, nutritional cues areintegrated by the catabolite repression control (Crc) protein aspart of a signal transduction pathway, which regulates biofilmformation (23). The P. aeruginosa Crc represses the levels ofenzymes involved in mannitol and glucose catabolism in thepresence of succinate and is involved in the expression of pilA,the pilin structural gene. Crc may be part of a signal transduc-tion pathway that can sense and respond to nutritional signals,such as carbon availability, and thereby may play a role in thebacterium’s transition from planktonic growth to biofilmgrowth.

Transcriptome analysis of B. subtilis biofilms suggests thatnonoptimal growth conditions, such as catabolite repression,starvation, and possibly high cell density, stimulate biofilmformation (36). In addition, biofilm formation by B. subtilis(36) and E. coli (13) is inhibited in the presence of a rapidlymetabolized carbon source, such as a higher concentration ofglucose or fructose. The correlation between the induction ofglucose-repressed genes and biofilm formation also suggeststhat biofilm formation is subject to catabolite repression (36).

The fructose PTS of oral streptococci, as well as a number ofother bacteria, is thought to be responsible for the phosphor-ylation and uptake of fructose (47). When S. gordonii encoun-ters high levels of extracellular fructose, the fructose PTSoperon could function as a sensory mechanism to enable theswitch from a sessile phenotype to a planktonic phenotype,thus facilitating the dispersal and spread of the bacterium.Alternatively, FruK may play a role in cross-regulation of atwo-component signal transduction system by phosphorylationof a response regulator.

The ability of the PTS proteins to modulate biofilm forma-tion points to the possibility that specific applications of basicPTS research could be used in combating infections by patho-genic bacteria. Rational drug design in which PTS proteins areused may be possible as PTSs are present only in prokaryotes.Further characterization of the fructose PTS operon shouldprovide insight into the molecular mechanisms of biofilm for-mation by oral streptococci and a possible role in the patho-genicity of these organisms. Interference with the function(s)of the fructose transport system described here is potentially anovel approach to develop methods for the prevention ofstreptococcal biofilm formation.

ACKNOWLEDGMENTS

DNA and protein sequences used in this study were retrieved fromthe National Center for Biotechnology Information genomic BLASTpages that contain sequences of completed and unfinished microbialgenomes (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).

This work was supported by PHS grant RO1-DE13328 from theNational Institute of Dental and Craniofacial Research.

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