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Journal of Bacteriology, June 2006, p. 3813-3825, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.01845-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Glucan Binding Protein B from Streptococcus mutans

Renata O. Mattos-Graner ,1,#,{dagger} Kristen A. Porter,2,# Daniel J. Smith,1 Yumiko Hosogi,2 and Margaret J. Duncan2*

Department of Immunology,1 Department of Molecular Genetics, The Forsyth Institute, Boston, Massachusetts 021152

Received 2 December 2005/ Accepted 16 March 2006


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ABSTRACT
 
Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division.


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INTRODUCTION
 
Mutans streptococci accumulate on tooth surfaces and are the major etiological agents of dental caries due to their production of and tolerance to acid. Secreted products of mutans streptococci that play a role in bacterial accumulation include glucosyltransferases, their glucan products, and glucan binding proteins. Streptococcus mutans possesses at least four glucan binding proteins (GbpA, GbpB, GbpC, and GbpD), each of which appears to be immunologically and biochemically distinct (references 1, 41, 35, and 39, respectively; reviewed in reference 3). Clinical studies showed that GbpB was the antigen most commonly recognized by antibodies in saliva of young children (42), and the natural immunoglobulin A response to GbpB after initial exposure to S. mutans may modulate infection (30). Also, systemic or mucosal immunization of rats with GbpB or GbpB-derived peptides induced protective immunity to dental caries (43, 44, 45), indicating that GbpB may be involved in virulence of S. mutans.

Apart from modest glucan binding properties (41) and a positive relationship with in vitro biofilm formation (21), the biological function of GbpB in S. mutans is unclear. It shares extensive amino acid homology with putative peptidoglycan hydrolase (PcsB) from S. agalactiae and S. pneumoniae (25, 33). In the S. mutans genome, gbpB is flanked by genes encoding proteins involved in cell shape determination (mreC and mreD), and this gene order is conserved in other gram-positive bacteria (21). Further sequence analyses showed that GbpB contained homology to the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) superfamily, whose members hydrolyze gamma-glutamyl-containing substrates (34). Thus, sequence-derived evidence indicates that GbpB may be involved in cell wall synthesis. Also, a sagA-encoded S. mutans protein that is identical to GbpB appeared to be induced when cells were grown under the stress conditions of high salt or low pH (7). Therefore, the biological function of GbpB remains obscure. To define the biological function of GbpB, we took several approaches that included genetic and expression analyses and isolation and identification of proteins that interacted with GbpB. The data presented here indicate that GbpB is an essential protein involved in cell growth and division, possibly complexed with ribosomal protein L7/L12.


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MATERIALS AND METHODS
 
Construction and analysis of mutants obtained by gene disruption with pRMG2. Strains and plasmids used in this study are described in Table 1, and primer sequences are listed in Table 2. Streptococcus mutans was grown as described previously (21). Constructs for the disruption or allelic exchange of the S. mutans gbpB gene were prepared in Escherichia coli strain DH5{alpha} (Invitrogen, Carlsbad, CA) which was grown in Luria-Bertani (LB) broth or on plates with additions of ampicillin (100 µg/ml), carbenicillin (50 µg/ml), or erythromycin (200 µg/ml) as appropriate to select and maintain plasmids. For gene disruption, a 578-bp internal sequence of gbpB was obtained with primer set 1 (Table 2) and cloned into the EcoRI and BamHI sites of suicide vector pVA891.2, which is a PvuII deletion derivative of pVA891.1 (19). Transformation of S. mutans strains UA130 and GS5 was performed using a modification of the protocol of Perry and Kuramitsu (31). Gene-disrupted transformants were selected for resistance to erythromycin on TH agar and also under conditions known to favor growth of mutants with potential defects in cell wall synthesis, e.g., supplementation with 250 mM potassium chloride as an osmotic stabilizer and suspension in 0.8% TH agar (33, 40). As negative controls, cells not exposed to DNA were plated on the same selective medium.


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  		TABLE 1. Strains and plasmids used in this study


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  		TABLE 2. PCR primers and probes used in this study

Table 2 lists the PCR primer pairs (Invitrogen) used to amplify DNA fragments spanning the junction of the integrating vector and gbpB. Reaction volumes were 50 to 100 µl and contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 50 µM dcoxynucleoside triphosphates, approximately 0.5 µM primers, 0.1 to 0.5 µg template, and 2.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Boston, MA). PCRs were carried out in a Peltier model PTC-200 thermal cycler (MJ Research, Waltham, MA), and thermal conditions varied with the primer sets used. Sequencing of PCR products was carried out as described previously (21).

Electron microscopy. Bacteria were fixed in 4% formaldehyde-glutaraldehyde and postfixed in osmium tetroxide. Dehydrated bacteria were air dried on a carbon disk, sputter coated with palladium/gold, and examined in a JEOL 6400 scanning electron microscope (JEOL, Peabody, MA).

Construction and analysis of mutants obtained by allelic exchange. To generate a mutant by allelic exchange, the gbpB gene (1,653 bp) was amplified with primer set 3 and subcloned in pCR2.1TOPO vector (Invitrogen), generating pCRGB. The Ermr gene from pVA891.2 was amplified using primers with EcoRI linkers (set 7) and inserted into the unique MfeI site of gbpB (at positions 542 to 547 of the open reading frame) in pCRGB to yield pRMG3. A 2.5-kb linear EcoRI fragment containing the gbpB::Erm cassette was obtained from pRMG3, gel purified (QIAGEN gel extraction kit; QIAGEN, Valencia, CA), and used to transform S. mutans strains UA130 and GS5.

Northern analyses. To confirm that gbpB is transcribed monocistronically, Northern analysis was performed with RNA samples isolated from log-phase cultures of S. mutans UA130. RNA was isolated using an RNeasy kit (QIAGEN), resolved in 1.2% formaldehyde-agarose, and transferred to Hybond N+ membranes (Amersham Biosciences, Piscataway, N.J.). The gel-purified gbpB probe was obtained by EcoRI digestion of pCRGB (Table 1). Hybridization conditions and signal development were performed as recommended for the Enhanced Chemiluminescence gene detection system (Amersham Biosciences).

Northern dot blots were used to determine gbpB expression under stress conditions. Strains 20A3, SJ32, UA130, and UA159 were grown to early exponential phase (A600, 0.2 to 0.3) and then grown in 0.5 M NaCl or pH 5.5 media for 30 min, i.e., the exact conditions described in a previous study (7). Cell pellets were resuspended in 0.5 ml acid phenol-chloroform (5:1; pH 4.5) and 0.5 ml NAES buffer (50 mM sodium acetate pH 5.0, 10 mM EDTA, 10% sodium dodecyl sulfate [SDS] in diethylpyrocarbonate [DEPC]-treated water) together with 0.5 ml glass beads (0.1-mm diameter) and broken in a Mini-BeadBeater disrupter (BioSpec Products, Bartlesville, OK). Following removal of proteins by centrifugation, nucleic acids were dissolved in DEPC-treated water and treated with DNase I according to the manufacturer's instructions (Ambion, Austin, TX). RNA was extracted with acid phenol-chloroform and chloroform-isoamyl alcohol, ethanol precipitated, washed, and dissolved in DEPC-treated water. Each RNA sample (1 µg) was tested for DNA by conventional PCR using primers to amplify a segment of gbpB. For all samples, RNA was resuspended in denaturation solution (500 µl formamide, 162 µl formaldehyde, 100 µl 10x MOPS [morpholinepropanesulfonic acid] buffer) and serially diluted twofold in the same solution. Samples were denatured by boiling and then spotted onto Hybond N+ membranes (Amersham Biosciences) in twofold serial dilutions (2.5 to 0.156 µg/2-µl spot), and SJ32 chromosomal control DNA was also spotted to the membranes (15 to 120 ng/2-µl spot). Samples were UV cross-linked to the membrane. Duplicate Northern dot blot analyses were performed for each stress condition and the corresponding control, and at least two independent experiments were carried out for each growth condition. PCR primers used to amplify DNA probes are shown in Table 2. The expression of control genes gbpA, gbpC, and ldh (the lactate dehydrogenase gene) was also measured. All PCR products were gel purified (QIAGEN) according to the manufacturer's instructions. Chemiluminescent signal detection was performed with CDP-Star detection reagent (Amersham Biosciences). The intensity of each dot was measured using an Alpha Innotech Imager 2200 instrument, and relative expression values were calculated from 3 dilutions of target RNA per sample. Student's t test was performed to determine statistical significance (P < 0.01) in gene expression values in stress versus control conditions, and samples that showed a statistically significant increase in gene expression, i.e., a twofold increase, were considered biologically significant.

Quantitative real-time PCR (QRT-PCR). Further analyses of gbpB expression were carried out by real-time PCR using RNA from strains SJ32 and UA159 that were grown as described above, except that incubations in high-salt or low-pH media were for 60 min. Expression of groEL and dnaK was also measured, since expression of these chaperones was previously shown to be stress responsive (14, 18). RNA was extracted from S. mutans cultures using a MasterPure RNA purification kit (Epicenter, Madison, WI). Each RNA sample (400 pg) was tested for DNA contamination by real-time PCR using groEL primers. Transcription of RNA to cDNA was carried out with an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Triplicate PCRs were carried out in a 96-well plate, and the mixes (20-µl final volume) comprised 4 ng to 4 pg cDNA template, 10 µl SYBR green Supermix (Bio-Rad, Hercules, CA), 0.4 µl forward primer (4 µM), 0.4 µl reverse primer (4 µM), and 8.8 µl water. Reaction cycles in an iCycler instrument (Bio-Rad) were initiated with polymerase activation at 95°C for 10 min and continued with 50 cycles at 95°C for 15 s and 54°C (annealing temperature for gbpB) for 30 s. The annealing temperature for ldh was 62°C, and the temperature for both groEL and dnaK primer sets was 59°C. The change in expression of each gene in experimental versus control conditions was calculated according to an equation of Pfaffl (32). Values were normalized to the reference gene, the lactate dehydrogenase gene (ldh), since there were no significant variations in cycle threshold (CT) at each condition tested. The expression ratio is Formula, where E refers to PCR efficiency, which is calculated by the equation 10(–1/slope) (32). At least two independent experiments were performed for each stress condition.

Pull-down assays. Recombinant GbpB was used as bait in pull-down assays, and gbpB was amplified using primers for mature GbpB that excluded the 27-bp signal sequence and contained 5' BamHI and 3' XhoI restriction sites (Table 2). The 1,290-bp product was amplified from SJ32 chromosomal DNA using Platinum Pfx DNA polymerase (Invitrogen) according to the manufacturer's instructions. After sequence verification, the insert in pGEX-6P-3 (Amersham) was transformed into electrocompetent E. coli BL21 (Novagen). Fusion protein expression was induced in cultures by the addition of 0.2 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and incubation for 2.5 h. Escherichia coli cell pellets were resuspended in phosphate-buffered saline (PBS) with Protease Inhibitor Cocktail Set III (Novagen), sonicated on ice, and then incubated on ice with shaking with 1% Triton X-100 to solubilize the protein. The GbpB-glutathione S-transferase (GST) fusion protein was coupled to glutathione 4B beads (Amersham) and purified according to the manufacturer's protocol. The Porphyromonas gingivalis FimR protein, expressed as a GST fusion (29), was used as a negative control in pull-down assays.

S. mutans SJ32 cell wall and cell membrane proteins were prey in pull-down assays and were isolated by using a modification of a published protocol (13). Briefly, S. mutans SJ32 was grown anaerobically overnight in 1 liter of brain heart infusion broth. Cells were collected by centrifugation and washed three times in PBS containing 1 mM phenylmethylsulfonyl fluoride. Cells were broken in a bead beater (as described above) and centrifuged for 15 min at 2,000 x g to remove glass beads and unbroken cells. The supernatant was centrifuged (173,000 x g, 30 min, 4°C), and the pellet containing cell wall proteins was washed three times in PBS-phenylmethylsulfonyl fluoride. Cell membrane proteins were isolated from the supernatant by centrifugation (320,000 x g, 1 h, 4°C). The pellet containing cell membranes was washed three times by centrifugation. Cell wall and cell membrane extracts were resuspended in PBS, and protein was quantified.

Glucan binding protein B-GST or FimR-GST fusion proteins were mixed with 50 µl bed-volume glutathione-Sepharose 4B beads, 2% bovine serum albumin (BSA), and 143.5 µg of SJ32 purified cell walls and mixed by end-over-end rotation at 4°C overnight. Reaction mixes were centrifuged (500 x g, 5 min) and beads with attached proteins were resuspended in VPEX-500 buffer (24) and then passed through a Spin-x 0.22-µm cellulose acetate filter (Corning Inc., Corning, NY). Beads were washed with VPEX-100 buffer on the filter and then resuspended in 100 µl PBS for on-column removal of the GST tag using PreScission protease (Amersham) according to the manufacturer's protocol. Released proteins were centrifuged through the filter and collected. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized with SYPRO Ruby protein gel stain. A second tube that contained only fusion protein, beads, and BSA and that was incubated as described above was also fractionated by SDS-PAGE and stained and thus provided a comparison for common bands. Potential GbpB-interacting bands were excised and sequenced by liquid chromatography-tandem mass spectrometry at Proteomic Research Services, Inc., Ann Arbor, MI.

Coimmunoprecipitation assays. Chicken anti-GbpB immunoglobulin Y (200 µg) was used in these assays. By use of a Profound coimmunoprecipitation kit (Pierce Biotechnology Inc., Rockford, IL), antibody-coupled AminoLink Plus gel and control cross-linked agarose beads without antibody were prepared according to the manufacturer's instructions. Coimmunoprecipitation assays were carried out with 40- and 60-µg cell wall and cell membrane preparations, respectively, from S. mutans SJ32. The cell fractions were incubated with antibody-coupled and control gels together with Protease Inhibitor Cocktail Set III (Novagen/EMDB Biosciences, Madison, WI) in a final volume of 400 µl and incubated overnight at room temperature with rotation. Samples were prepared for SDS-PAGE according to the manufacturer's protocol. Gels were stained using a GelCode E-Zinc reversible stain kit (Pierce), and the image was recorded. After the destaining procedure, proteins were transferred to membranes which were probed with anti-L7/L12 antibody.


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RESULTS
 
The cloning of gbpB from S. mutans SJ32 and structural features of the encoded protein were described previously (21). Although GbpB was first isolated from culture supernatants, further analysis of clinical isolates and laboratory strains showed that various but significant amounts of the protein remained cell associated (21).

Characterization of transformants obtained by gene disruption with pRMG2. A 578-bp internal fragment of gbpB was cloned into pVA891.2 to generate integrating plasmid pRMG2. After transformation of strain UA130 and 3 to 9 days of incubation, morphologically different Ermr colonies that grew slowly as hard aggregates in liquid media (Fig. 1B) compared to the parent strain UA130 (Fig. 1A) were obtained. The growth of these transformants was not enhanced by use of potassium chloride as an osmotic stabilizer or by plating on agar or in soft agar. Cells of the parent strain grew as chains (Fig. 1C), which is typical of S. mutans, while transformant cells were large and atypically shaped (Fig. 1D), contained abnormal cell division septa, and did not form chains (Fig. 1E and F). Figure 2A shows a hypothetical model for pRMG2 integration at the gbpB locus. Although the transformants did not grow enough to isolate genomic DNA for Southern hybridization, PCR analysis using individual colony lysates as templates was possible. Fragments that spanned the junction of the integrating vector and gbpB were PCR amplified and sequenced. These analyses showed that a representative transformant, MB1, contained a wild-type copy of gbpB (primer set 3 generated the 1.6-kb amplicon shown in Fig. 2B). Primer sets 4 and 5 amplified an additional copy of gbpB that was linked to Ermr (Fig. 2C). Sequence analysis of PCR products confirmed integration of the vector and the gbpB fragment, and from the data we constructed a hypothetical map of the integration and subsequent recombination (Fig. 2D).


Figure 1
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  		FIG. 1. Phenotypes of transformants obtained by integration of pRMG2. (A) Parent strain UA130 growing in liquid medium. (B) A representative transformant growing under the same condition. (C) Scanning electron micrographs showing chains of cells of parent strain UA130. (D and E) Abnormally shaped and larger transformant cells. (F) Transformant cells with altered septa.


Figure 2
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  		FIG. 2. Analysis of representative transformants obtained by integration of pRMG2 into the S. mutans genome. (A) Predicted integration of pRMG2 into the genome by Campbell-like homologous recombination within gbpB. PCR primer sites are depicted as arrows. Primers sets are described in Table 2. Figure 2, S. mutans genome; shaded boxes, gbpB and Ermr genes; straight lines, vector sequences. (B) PCR amplicons (1.6 kb) generated with primer set 3 in UA130 (wild type [WT]) and representative transformant MB1. (C) Amplification with primer sets 4 and 5 to confirm linkage of gbpB with Ermr in transconjugants; the wild-type DNA template was used as a negative control. (D) Map of pRMG2 integration deduced from sequencing of PCR amplicons. (E) Western blot of cell extracts from transformants (1 to 3) probed with anti-GbpB antibody. Control fractions from the parent were cell extract (WTa) and culture supernatant (WTb).

Although GbpB is a secreted protein found in culture supernatants, it is also cell associated, and the amounts of secreted protein versus cell-associated protein vary from strain to strain (21). Western blotting of cell extracts from transformants (MB1 to -3) showed that they produced normal GbpB and also coexpressed larger, immunologically cross-reacting proteins of 100 to 200 kDa that were not produced by the parent (Fig. 2E). We speculate that these aberrantly sized proteins may compete with the activity of the native protein and compromise its function, limiting growth of the transformants. The severe growth defects of the transformants precluded isolation of DNA or RNA for further analyses. Interestingly, when S. mutans strain GS-5 was used as the recipient for pRMG2 integration, the transformants obtained grew as well as the parent, contained a wild-type copy of gbpB as well as a disrupted copy, showed normal expression of the protein by Western blotting, and did not produce the larger cross-reactive proteins (data not shown).

Characterization of transformants obtained by allelic exchange. Figure 3A shows the predicted recombination that incorporates the gbpB::Ermr cassette into the genome of S. mutans UA130. Erythromycin-resistant transformants grew normally on agar and in liquid medium and were indistinguishable from the parental strain. Analysis by PCR with primer sets 2 and 6 showed that all transformants contained the gbpB::Ermr cassette, which was not detected in the parent strain (amplicons from representative transformants are shown in Fig. 3B). However, PCR with primer set 3 revealed that a wild-type gbpB copy (1.6 kb) was retained in the transformants (Fig. 3C). From sequence data, we deduced the genomic arrangement within the transformants shown in Fig. 3D; this genomic arrangement was confirmed by Southern blot analysis of genomic DNA after digestion with HindIII, which cuts within the GbpB open reading frame (ORF) (Fig. 3E).


Figure 3
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  		FIG. 3. Analysis of S. mutans UA130 transformants obtained by allelic exchange. (A) Predicted replacement of the wild-type gene by the disrupted gbpB by double crossover recombination. (B) PCR amplicons generated with primer sets 2 and 6 demonstrated linkage of gbpB and Ermr in transconjugants (MC). Amplicons were also detected in pRMG3, from which the transforming fragment was isolated, but not in the parent strain. WT, wild type. (C) A second wild-type copy of gbpB was obtained using primer set 3. (D) Map of gbpB region after recombination and regeneration of wild-type gbpB, deduced from PCR and sequence data. (E) Southern blot analysis of HindIII-digested transformant genomic DNA probed with gbpB confirmed the presence of two copies of the gene; the larger fragment also hybridized with the Ermr probe, proving it to be the mutated version. (F) Western blots of culture supernatants of transformant and parent strains indicated expression of GbpB.

By Western blotting, the transformants showed normal production of GbpB protein in culture supernatants (Fig. 3F) and cell extracts (data not shown), and the same result was obtained when strain GS-5 was used as the transformation recipient. One explanation for these data is that the purified linear gbpB::Ermr-transforming fragment was contaminated with undigested pRMG3 that integrated via Campbell-type recombination and generated duplications. To test for this possibility, PCR was carried out on the transforming DNA with pRMG3-specific primers (M13 forward and reverse paired with Ermr primers); no contamination of the linear gbpB::Ermr fragment was detected (data not shown).

The data above suggested that gbpB mutants were unstable, with the implication that the gene product has an essential function. These results also could be explained if gbpB itself is not essential but is part of an operon that contains other vital downstream genes. In this case, mutations within gbpB could have polar effects on their expression and lead to lethality. Previously, we used computer analyses to determine that the DNA sequence 5' to the GbpB open reading frame contained promoter motifs and that the 3' sequence contained a hairpin terminator structure (21). By Northern analysis, we show here that gbpB mRNA is monocistronic (Fig. 4), confirming previous data (7, 21). While we conclude that the faint band observed in the Northern analysis (approximately 2.3 kb) is a background signal due to the high sensitivity of the detection system, we recognize the possibility of a hypothetical transcript comprising gbpB and prsA, the next downstream gene that encodes a phosphoribosyl pyrophosphate synthase that would also be 2.3 kb. An in silico analysis of the intergenic sequence between gbpB and prsA indicates a putative transcription start at –23 from the ATG start of the PrsA-encoding sequence, suggesting that they are not transcribed in a unit. A more likely operon is gbpB together with upstream genes mreC and mreD, because of their potential roles in cell shape determination and cell wall biosynthesis (6, 15, 48) and the conservation of the mreC-mreD-gbpB gene order in S. pneumoniae and other gram-positive species (21); however, this transcript would be 2.63 kb and larger than the faint band.


Figure 4
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  		FIG. 4. Northern blot analysis of gbpB transcripts from S. mutans UA130. A schematic map of the gbpB region is depicted above the blot. Genes are represented by arrows that indicate the direction of transcription. Northern blot lanes: M, RNA size markers; 1, RNA purified from a log-phase culture of UA130; 2, an RNA transcript that hybridized with gbpB was approximately 1.3 kb, consistent with monocistronic transcription. nt, nucleotide.

Expression of gbpB under stress conditions. Previously, we showed that among 44 distinct genotypes of S. mutans, there were up to 10-fold differences in GbpB production (21), and in this study we show that attempts to mutate gbpB had different results in different strains. To investigate whether gbpB expression in response to environmental stress was also strain dependent, we measured gene expression in several strains under conditions that were shown to induce transcription of gbpB in strain GS5 (7). Stress conditions were 30- or 60-min incubations of log-phase cultures in high salt (0.5 M NaCl) or low pH (5.5) at 37°C (7), and gbpB expression was measured by Northern dot blot assays and QRT-PCR. In Northern dot blot assays, expressions of gbpC and gbpA were used as positive and negative controls, respectively, since gbpC was reported to be induced under stress conditions (35) and gbpA was reported as constitutive (2). In QRT-PCR experiments, we also measured the expression of groEL and dnaK to test whether the growth conditions induced known stress responses (14, 18).

Dot blot assays showed that in all strains tested there was a trend toward increased expression of gbpB during incubation in high salt (Fig. 5A), although the values were statistically significant only in strains SJ32 (P = 0.002) and UA159 (P = 0.001), which showed approximately twofold increases in expression of gbpB. On the other hand, low pH conditions did not significantly affect gbpB expression levels (Fig. 5B). With regard to the expression of control genes, the amounts of gbpA and gbpC mRNA produced also appeared to be strain dependent; however, there were no indications that these genes were responsive to stress conditions (data not shown).


Figure 5
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  		FIG. 5. Relative expression of gbpB under stress and control conditions. Values were calculated from Northern dot blot intensities of triplicate spots. (A) Expression of gbpB in response to incubation with 0.5 M NaCl. Strains in which gbpB induction was statistically significant are shown with calculated P values. (B) Expression of gbpB in response to incubation at pH 5.5.

Expression of gbpB in response to stress conditions in three independent experiments was further quantified by QRT-PCR; the results of one representative assay are shown in Table 3. Two S. mutans strains were selected for these experiments, and the increased sensitivity of the method revealed differences in gene expression, and hence response, between the strains. In UA159 there was a decrease in gbpB expression when cells were grown at pH 5.5 and in high salt. By comparison, in SJ32, gbpB expression increased twofold in high salt but was no different at low pH. Further, groEL and dnaK both had threefold increases in SJ32 when the strain was grown at 0.5 M NaCl and twofold increases when grown at pH 5.5; in UA159, however, there was no change in either groEL or dnaK expression when grown at 0.5 M NaCl, but there were decreases in both genes when cells were grown at pH 5.5. The increase of dnaK expression in SJ32 when cells are grown at a lower pH is similar to results obtained by Jayaraman et al. (14), who found that dnaK expression increased 1.8-fold both in mRNA slot blot assays and in Western blot assays when S. mutans strain UA159 was acid shocked from pH 7.0 to pH 5.0 (14). The decreases in target genes gbpB, groEL, and dnaK in UA159 when incubated at pH 5.5 shows not only that gene expression is strain dependent but also that this condition was not optimal for the induction of stress-responsive genes.


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  		TABLE 3. Expression of gbpB and control genes under stress conditions measured by QRT-PCR

Pull-down assays for proteins that interact with GbpB. Further information on the cellular function of GbpB was sought through identification of interacting proteins. For the first of these approaches, pull-down assays, recombinant GbpB was expressed as a GST fusion protein. Figure 6A shows a silver-stained sample of the fusion protein after SDS-PAGE with protein and relevant breakdown products indicated. The GbpB-GST fusion protein was coupled to glutathione-Sepharose beads and incubated with a cell extract from S. mutans SJ32 that also contained BSA to prevent nonspecific interactions with GbpB. Recombinant FimR, a response regulator protein from Porphyromonas gingivalis that was also expressed as a GST fusion protein, was used as a negative control for protein interaction (Fig. 6B). Protein complexes were retained on and eluted from a filter membrane and then fractionated by SDS-PAGE and silver stained (Fig. 6A). A number of proteins that could potentially interact with GbpB were detected; however, we could account for these either by size or after sequencing. The largest was the GbpB-GST fusion protein (86 kDa); this was followed, in order of decreasing size, by the GST dimer (50 kDa) and the GST monomer (26 kDa). Several similarly sized bands were also detected in the negative control pull-down assay with FimR; these were FimR- and GST-related protein products (Fig. 6B). A band of 14.4 kDa was considered to uniquely interact with GbpB and after sequencing was identified as ribosomal protein L7/L12.


Figure 6
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  		FIG. 6. Pull-down assay with GbpB. (A) Silver-stained SDS-PAGE gel showing purified GST-GbpB that was used in the assay and the proteins recovered from the pull-down fraction. The indicated 14.4-kDa protein was excised and sequenced. (B) Control pull-down assay carried out with GST-FimR from P. gingivalis.

Coimmunoprecipitation assays to identify proteins that interact with GbpB. We used coimmunoprecipitation to further substantiate the finding that L7/L12 interacted with GbpB. Cell wall and cell membrane fractions were isolated from S. mutans SJ32 cell extracts by differential centrifugation to conserve GbpB-interacting protein complexes. The presence of GbpB or L7/L12 in these fractions was verified by Western blotting (Fig. 7A and B, respectively). Anti-GbpB antibody was coupled to a gel matrix column, and cell wall or cell membrane fractions were passed through the column to capture GbpB and proteins with which it was complexed. Cell fractions were also passed through a control column that contained uncoupled gel. Protein complexes retained on the gel matrix were dissociated by boiling, separated by SDS-PAGE, and blotted to polyvinylidene difluoride membranes that were probed with anti-L7/L12 antibody. The zinc-stained SDS-PAGE gel of dissociated proteins (Fig. 7C) showed that comparatively small amounts of S. mutans cell wall or cell membrane proteins bound nonspecifically to the uncoupled gel matrix (Fig. 7C, lanes C), while larger amounts bound to the anti-GbpB antibody-coupled matrix (Fig. 7C). Western blot analyses showed that the proteins dissociated from the antibody-coupled columns included anti-GbpB antibody, GbpB, GbpB-interacting proteins, and GbpB degradation products (data not shown). Blots of these proteins were also probed with anti-L7/L12 antibody (Fig. 7D), revealing that GbpB in the cell membrane fraction was complexed with the 12.4-kDa L7/L12 protein, while GbpB in the cell wall was complexed with a larger 43-kDa protein that interacts with the L7/L12 monoclonal antibody. These interacting proteins were not detected in protein fractions from uncoupled columns (Fig, 7D, lanes C). According to a previous report in the literature, this 43-kDa protein is elongation factor Ts (EF-Ts), and this cross-reactivity was observed in several streptococcal strains, including S. mutans (16).


Figure 7
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  		FIG. 7. Coimmunoprecipitation assays. (A) Western blot of S. mutans cell wall (CW) and cell membrane (CM) proteins probed with anti-GbpB antibody. (B) Western blot of the same fractions probed with anti-L7/L12 antibody. (C) Zinc-stained SDS-PAGE of either cell wall or cell membrane proteins dissociated from uncoupled columns (lanes C [control]) and from anti-GbpB antibody-coupled columns. (D) Western blot of dissociated proteins after probing with anti-L7/L12 antibody.


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DISCUSSION
 
Although the immunodominance and immunoprotective capacity of GbpB have been demonstrated (45), the function of the protein in S. mutans remains unknown. Glucan binding protein B was originally identified and isolated through its ability to bind to dextran polymers (41); however, this activity is relatively modest. Two lines of evidence propose different functions for GbpB. Amino acid homology to the CHAP superfamily, whose members hydrolyze gamma-glutamyl-containing substrates, indicates a role in peptidoglycan synthesis and possibly cell division (8, 21, 33, 34). However, gene expression studies suggested function in a general stress response (7). In this study, we pursued several different approaches, including genetics, gene expression, and protein interaction studies, to clarify the cellular function of GbpB.

We attempted to mutate the S. mutans gbpB gene either by disruption with Campbell-type integration of a plasmid vector or by allelic exchange with the mutated gene on a linear DNA fragment. Although over 100 viable transformants of normal and abnormal growth phenotypes were analyzed at the DNA and protein levels, we were unable to obtain a bona fide gbpB mutant. All the viable mutants analyzed showed identical amplicon patterns with several primer sets used for screening (Fig. 2 and 3); thus, they were all merodiploid mutants. These results suggest that GbpB function is essential for growth and thus that gbpB is a vital gene. All the transformants we selected contained both mutant and wild-type copies of gbpB and expressed the native protein as recognized by specific antibody. Multiple integration of plasmid vectors is one possible mechanism that would yield transformants of this type. Such recombination patterns have been described previously for S. mutans, but only after transformation with vectors that contained chimeric DNA fragments providing multiple recombination sites (36). We reasoned that this was not the situation with gbpB-directed plasmid integration because the region of homology was relatively short, so the frequency of multiple recombinations would be vanishingly small. Incorporation of the linear gbpB::Ermr fragment into the genome required two recombinations (double crossover) that theoretically should not generate duplications. However, all 55 transformants obtained were gbpB merodiploids; we propose that this could occur if the allelic exchange occurred at a replication fork, resulting in mutant and wild-type genes on separate DNA strands, and was followed by stress-induced illegitimate recombination between the two strands to retain both mutant and wild-type alleles. Unfortunately, we were unable to map the site of pRMG2 integration because the transformants were only transiently viable, and PCR analysis was performed using only cell aggregates as templates (Fig. 1B). Sequencing analysis of the PCR products (Fig. 2B and C) revealed that the mutations were very unstable, with pRMG2 vector sequences that were integrated in the chromosome (Fig. 2D) having undergone several recombinations, as identified by vector deletions (data not shown), although the intact Ermr was always retained. In contrast, deletion of a nonessential gene, e.g., gbpA, was successfully inactivated by allelic replacement in strain UA130 (12), and gbpA and gbpC were inactivated by Campbell-like insertion without duplication in S. mutans strain MT8148 (20). Merodiploid formation is indicative of a cellular response to prevent the inactivation of an essential function and occurred, for example, in Bacillus subtilis after a double crossover recombination that would have deleted the essential ilvA gene (28).

By use of an allelic exchange strategy similar to ours, three gbpB mutants were isolated in the strain GS-5 without osmotic protection (8). In that study, gbpB was named sagA, for surface antigen A. As revealed by the electron microscopy analysis, the sagA mutants had morphologies very similar to those of the transformants we obtained after the integration of pRMG2 in strain UA130 (Fig. 1D, E, and F); however, there were major differences with regard to growth and viability between sagA mutants in the GS-5 background and transformants in UA130. The transformants we isolated formed tight aggregates that were only transiently viable, i.e., could not be subcultured, and analyses were limited to DNA and protein isolated from the aggregates. This phenotype was also similar to that of pscB mutants of S. agalactiae that were obtained under conditions of osmotic stabilization (33). Although we attempted to protect UA130 transformants from osmotic lysis with 250 mM KCl, we still could not recover a bona fide mutant; however, we cannot exclude the possibility that 250 mM KCl either was toxic or provided insufficient protection. Sorbitol (500 mM) previously was used as a stabilizer for pscB mutants but was not used in the present study because S. mutans can utilize sorbitol, and there is evidence that this carbon source inhibits S. mutans metabolism (46). Apart from these differences, group B streptococcus pscB mutants also showed poor viability and a high rate of reversion to wild-type morphology and growth phenotype (D. Rheinscheid, personal communication). Additionally, deletion mutants could be not be obtained for pcsB, the gbpB homologue in S. pneumoniae (25), another species whose genome is closely related to S. mutans (9). In contrast, the GS-5-derived sagA mutants grew well enough for DNA to be extracted for Southern blots and could even be transformed for complementation analysis. Several reports established that GS-5, a long-established laboratory strain, differed genetically from more-recent isolates, most notably in genes for surface proteins, including a nonsense mutation in the gene for glucan binding protein C (36, 37), a frame shift in the gene encoding surface protein Pac (22, 37), and deletions in glucosyltransferase genes (10). To some extent, the results of Chia et al. (8) and those presented here may be reconciled by considering the differences between strains, especially in genes that potentially affect the assembly of the cell surface architecture. That we were unable to isolate gbpB null mutants with our copy of GS-5 may be a further reflection of changes that occur in strains maintained in different laboratory collections. Studies with S. pneumoniae demonstrated that deletion of virulence genes can yield mutants with very distinct phenotypes dependent on the genetic background of the strain (4), and it has become evident that a significant part of the genome within the same Streptococcus species can be highly variable (47).

We used different strains of S. mutans to compare expression levels of gbpB under conditions the same as those under which SagA was identified as a general stress response protein. By Northern dot blot analysis, we showed that for strains UA130 and 20A3 there were no significant increases in gbpB expression in response to high salt or low pH (Fig. 5). On the other hand, strains SJ32 and UA159 showed upregulation of gbpB in response to osmotic stress (Fig. 5), but this was confirmed by QRT-PCR only with SJ32 (Table 3). Variability in gbpB transcription between strains is consistent with our previous observation that protein production is also strain dependent. Both the osmotic and acid stress conditions induced expression of groEL and dnaK in strain SJ32 but not in UA159, suggesting that stress thresholds are also strain dependent under the conditions tested. In fact, the analysis of the control genes (gbpC, groEL, and dnaK) showed that stress conditions were not ideal. Although gbpB was shown to be stress induced in GS-5 (7), we used UA159 as a reference strain because gbpC has a nonsense mutation in GS-5 and thus is not expressed in this strain (37) and because expression of groEL and dnaK was previously described for UA159 (14, 18). Additionally, because of our inability to inactivate gbpB in the GS-5 background, in contrast to the results of a previous study (8), we raised the hypothesis that strains might have undergone genetic modifications during maintenance in the different laboratories.

Orthologs of GbpB are found in a wide range of gram-positive bacteria that includes other Streptococcus, Bacillus, Listeria, and Enterococcus species, denoting a fundamental cellular function. The most thorough functional analysis was carried out with PcsB, the GbpB homolog found in S. pneumoniae (25, 26). It was established that expression of PcsB was regulated by the VicRK two-component system, which is essential for viability and also involved in the development of virulence (25, 49). Down-regulation of pcsB expression led to defects in growth and cell size and shape (25). Subsequent work established that the CHAP domain of PcsB was essential for function as a putative murein hydrolase and that neither GbpB from S. mutans nor PcsB from S. agalactiae could substitute for the PcsB function in S. pneumoniae (26). Most recently, it was found that phosphorylated VicR bound to the promoter of pcsB (27), and we identified the consensus binding sequence (TGTNAN-N5-NGTNANA) in the gbpB promoter (not shown), strengthening the relationship between the two genes. This consensus binding site was present in the same position with respect to the GbpB start codon in six S. mutans strains for which sequence data was available, including GS-5, suggesting that gbpB expression could be similarly regulated in these strains by the vicKR system. In S. mutans, defects in vicK (the sensor kinase) lead to down-regulation of gbpB, glucosyltransferase genes (gtfB, gtfC, gtfD), and a fructosyltransferase gene (ftf), possibly contributing to the observed altered biofilm phenotype (38). Because a strain with a mutation in the vicR response regulator could not be isolated, it was concluded that the null mutation was lethal (38), and our data would suggest that this lethality resulted from loss of activation and, hence, expression of gbpB. It was reported that in S. mutans, VicR bound to the promoters of gtfB, gtfC, and ftf, and because of the similarity in the consensus sequence, we anticipate that VicR will also bind to the gbpB.

The results that GbpB interacted with ribosomal proteins L7/L12, and possibly EF-Ts, and that these interactions occurred in cell membrane and cell wall fractions provide a potential new insight into biological function of GbpB and its homologues. It could be reasoned that, in spite of the precautions taken, outer surface fractions might be contaminated with intracellular proteins that may bind nonspecifically to GbpB. However, we recovered consistent amounts of GbpB and L7/L12 in membrane and cell wall fractions (data not shown), which suggests that they were not contaminants. Several nonspecific proteins might be recovered in pull-down assays, but comparisons of GST-GbpB with the negative control GST-FimR protein revealed several noncoincident bands, and GbpB interaction with L7/L12 was further confirmed in coimmunoprecipitation assays. Although GbpB was first purified from culture supernatants of strain SJ32, we later found that several strains contained significant amounts of cell-associated protein that was extractable with 8 M urea (21). Both L7/L12 and EF-Ts were identified in culture supernatants of group A streptococci of the M1 and M3 serotypes (17). Furthermore, the same proteins were found in surface extracts of S. oralis and, by analogy with work with E. coli, it was suggested that elongation factors may also act as chaperones (50). Previous studies with E. coli have suggested that the interaction of ribosomal proteins with cell surface proteins may be necessary to coordinate division of the cell wall with growth rate (5). More recently, it was demonstrated that in Lactobacillus johnsonii La1, EF-Tu functions as an adhesin for intestinal epithelial cells, and it was found on the cell surface of L. johnsonii by immunogold staining and detected by Western analysis in surface extracts of other lactobacilli and bifidobacteria (11).

The data presented are consistent with a model in which GbpB, L7/L12, and EF-Ts are components of a "divisome" protein complex involved in cell wall synthesis and expansion (23). Defined steps in the murein biosynthetic pathway are carried out in different compartments of the cell. UDP precursors are synthesized in the cytoplasm, and preformed disaccharide-pentapeptide is linked to a lipid carrier for translocation across the cytoplasmic membrane. Outside the cytoplasmic membrane, disaccharide pentapeptide is transferred to the growing point of the peptidoglycan by transglycosylation and transpeptidation reactions by the coordinated activities of murein synthases and hydrolases. Other chemical modifications of the cell wall, e.g., addition of teichoic acids, were previously described as essential for the growth of gram-positive bacteria and involved in the activity of murein hydrolases (24). Our working hypothesis is that GbpB functions in an assembly of proteins, possibly including L7/L12 and EF-Ts, that is part of a biosynthetic pathway with cytoplasmic, cell membrane, periplasmic, and cell wall components. Thus, the essential role of GbpB in S. mutans and its homologs in other pathogenic streptococci, together with the immunodominant properties, further increases interest in this protein as a target for an anticaries vaccine.


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ACKNOWLEDGMENTS
 
We thank Tsute Chen for constructive discussions and Ziedonis Skobe (Forsyth Institute) and Maria Erikson (Department of Cell Biology, Harvard Medical School) for assistance with electron microscopy.

This study was supported by Public Health Service grant R37 DE-06153 (D.J.S.) and by FAPESP (grant 99/08278-9 and 03/0136-3) to R.O.M.-G.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Genetics, The Forsyth Institute, 140 Fenway, Boston, MA 02115. Phone: (617) 262-5200, ext. 8344. Fax: (617) 262-4021. E-mail: mduncan{at}forsyth.org. Back

# These authors made equal contributions to this work. Back

{dagger} Present address: Department of Microbiology and Immunology, Piracicaba School of Dentistry, University of Campinas, Sao Paulo, Brazil. Back


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Journal of Bacteriology, June 2006, p. 3813-3825, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.01845-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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