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1 UCLA Department of Chemistry and Biochemistry, Los Angeles, CA 90095, USA
2 UCLA California NanoSystems Institute, Los Angeles, CA 90025, USA
3 UCLA Institute for Cell Mimetic Space Exploration, Los Angeles, CA 90095, USA
4 UCLA School of Dentistry, Los Angeles, CA 90095, USA
5 UCLA Molecular Biology Institute, Los Angeles, CA 90095, USA
6 Colgate-Palmolive, Piscataway, NJ 08855, USA
Correspondence
James K. Gimzewski
gim{at}chem.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; O'Toole et al., 2000). These bacterial communities are of particular medical importance due to their increased tolerance to antibiotics (Costerton et al., 1999). With the formation of biofilms such as those produced by Streptococcus mutans (commonly referred to as dental plaque), cell–surface interactions play an enormous role in initial cell colonization, and cell–cell protein interactions largely mediate the growth and continued existence of biofilms (O'Toole et al., 2000). The surface features of micro-organisms, such as those associated with S. mutans, are involved in most of the inter- and intra-species interactions. They play an important role during contact with the environment, organic and inorganic surfaces, as well as host organisms, and thus are pivotal for biofilm formation, virulence and metabolic regulation (Ghuysen & Hackenbeck, 1994).
The function, structure and properties of bacterial cell surfaces are determined by the presence of species-specific proteins, lipids and polysaccharides. Lactic-acid-producing bacteria like S. mutans, Streptococcus sobrinus and Leuconostoc mesenteroides produce specific exopolysaccharides called glucans, which constitute a major feature of the surface of these micro-organisms (Cerning, 1990). Glucans are chemically and physically complex high-molecular-mass homo-polysaccharides composed of a main linear chain of 
-D-glucopyranose subunits. Several different kinds of glucans are known; they are categorized by the sizes and structures of the molecules: e.g. dextrans are principally linked through (1-6) glucosidic bonds, and mutans through (1-3) glucosidic bonds. Glucans are also distinguished by their degree of branching, the types of branch linkage [(1-2), (1-3), (1-4) and (1-6)], the extent of branch chains and the spatial arrangement (Cerning, 1990; Monchois et al., 1999).
S. mutans is known as the principal dental pathogen associated with caries (Loesche, 1986). One of the most important virulence factors of S. mutans is the synthesis of glucan from sucrose (Hamada & Slade, 1980). Glucan synthesis allows the bacteria to firmly attach to the tooth surface and form a biofilm, while the gelatinous nature of glucan retards diffusion of acid produced by the bacteria from fermentable sugars in the dental plaque (Kuramitsu, 1993). This eventually leads to dissolution of the hard enamel surface of the tooth and cavity formation. Research on dental plaque development and the aetiology of dental caries has established the central role of glucans in sucrose-dependent adhesion, and the correlation between sucrose consumption and increased caries rates (Yamashita et al., 1993; Loesche, 1986). In S. mutans these glucans are synthesized from sucrose by the action of three types of glucosyltransferases (Gtfs): GtfB and GtfC synthesize mainly water-insoluble glucans (>85 %) with (1-3) glucosidic bonds (mutan); GtfD forms water-soluble glucans (>70 %) with (1-6) glucosidic bonds (dextran) (Monchois et al., 1999). The contribution of the individual glucans to the cariogenicity has been the subject of several studies, including the identification and characterization of the enzymes and genes encoding the respective Gtfs and showing that the action of all Gtfs is essential for the maximal cellular adherence (Tsumori & Kuramitsu, 1997). Ooshima et al. (2001) demonstrated that the maximal sucrose-dependent adherence can be achieved by adding recombinant Gtfs to S. mutans in a ratio of 5 rGtfB : 0.25 rGtfC : 1 rGtfD. However, most of these studies were conducted under non-physiological conditions or with isolated recombinant proteins.
We chose S. mutans, a biologically and medically relevant bacterial species, to investigate the role that glucans play in the virulence of this organism. The detailed knowledge about the genetic organization of the gtf genes enabled us to construct strains with individual mutations of these genes. The biochemical and biophysical properties of microbial surfaces resulting from the enzymic activity of Gtfs have been extensively studied in numerous bacterial species using techniques such as dynamic light scattering and micro-electrophoresis which require intense manipulation of the cell surface as well as lengthy preparation of the cell (Cerning, 1990; Ryan et al., 1980). However, in vivo techniques such as atomic force microscopy (AFM) (Binnig et al., 1986) provide a novel, nondestructive method for providing insight into critical properties associated with bacterial cells and their related surface proteins. AFM has proved to be a powerful tool not only for imaging of the ultrastructure of bacterial surfaces under in situ conditions, but also for determining the associated mechanical properties and intermolecular forces (Pelling et al., 2005; Rief et al., 1997). Schär-Zammaretti & Ubbink (2003a, b) probed the surface properties of different Lactobacillus strains using AFM to quantify tip–cell-surface adhesion forces and related this study to the ability of the lactobacilli to adhere to surfaces, clustering, auto- and co-aggregation. van der Mei et al. (2000) used AFM to determine cellular stiffness of fibrillated and non-fibrillated strains of Streptococcus salivarius.
Cross et al. (2006) demonstrated that the surface roughness of S. mutans strains harbouring genetic mutations of specific surface proteins correlated with their different cariogenic potential. Together with the mechanical analysis of the cell-surface protein interactions, these studies clearly demonstrate the potential use of AFM for characterization of bacterial surface properties under physiological conditions using intact bacterial cells. As these studies were conducted with uncompromised cells under in situ conditions, the results add substantial new information regarding the cell-adhesion and cariogenic properties of S. mutans and the individual role of the Gtfs in its adhesion properties. Here we report direct measurement of the mechanical properties associated with the cell-surface macromolecules native to S. mutans wild-type and mutant strains as probed by AFM.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and its derivatives were grown in Todd–Hewitt (TH, Difco) medium or on brain heart infusion (BHI, Difco) agar plates. For selection of antibiotic-resistant colonies, BHI plates were supplemented with 800 µg kanamycin ml–1, 15 µg erythromycin ml–1 or 5 µg tetracycline ml–1 (Sigma). All S. mutans strains were grown anaerobically (80 % N2, 10 % CO2 and 10 % H2) at 37 °C. For cloning and plasmid amplification, Escherichia coli cells were grown in Luria–Bertani (LB, Fisher) medium with aeration at 37 °C. E. coli strains carrying plasmids were grown in LB medium supplemented with 25 µg kanamycin ml–1.
Construction of various Gtf– mutants.
The GtfBCD–, GtfBC– and GtfD– mutant strains were constructed by transferring the particular mutations of gtfBC (ErmR) and gtfD (TetR) combined or individually into UA140 (kindly provided by H. Kuramitsu, University of Buffalo, NY, USA). Plasmid and chromosomal DNA was transformed into S. mutans after induction of an exponential culture with a synthetic competence-stimulating peptide (CSP, 1 µg ml–1) (Kreth et al., 2005) for 2 h with the subsequent addition of DNA. After further incubation for 2–3 h the culture was plated on selective media. To generate the gtfB deletional mutant, two fragments corresponding to approximately 1 kb of upstream and downstream sequence of the target gene were generated by PCR using Pfu and Taq polymerase mix (2 : 1 ratio) and the primers gtfB upF (5'-GCTAGCGAGAAGATTGCTGAGCGATC-3'), gtfB upR (5'-ATAGTCTGACGCAGCCAATC-3'), gtfB downF (5'-TGGTCTACAGCTCAGAGATG-3') and gtfB downR (5'-ATGAAGCAACAGATACTGTC-3'). These fragments were cloned into pGEM-T Easy (Promega). All plasmids were extracted and purified from E. coli, digested with appropriate restriction enzymes, gel-purified and ligated to compatible restriction sites of the erythromycin-resistance cassette and the cloning plasmid pBluescript (Stratagene). The gtfC deletional mutant was created by using the same protocol, except that the primers for generating up- and downstream fragments were gtfC upF (5'-TGGAGAACGAGTTCGGATTAAC-3'), gtfC upR (5'-TGACTAAGTGATGACGGCTGTT-3'), gtfC downF (5'-TGTTCAGGCTAAGGGAGAGC-3') and gtfC downR (5'-CCCATTTGTCGGCTTTTCTA-3'). The fragments were ligated with a kanamycin-resistance cassette and the cloning plasmid pBluescript through compatible restriction sites. The resulting plasmids were confirmed via restriction digestion as well as PCR and linearized for transformation into S. mutans. Transformants were selected on Erm plates for gtfB deletional mutant and on Kan plates for gtfC deletional mutant. Correct mutants were confirmed by PCR and their altered ability to form biofilms with sucrose compared to the parental strain.
Immobilization of S. mutans cells for AFM analysis.
Two millilitres of an overnight culture in BHI medium (5x108 bacteria ml–1) with no antibiotic added, supplemented with 1 % sucrose when indicated, were filtered through an isopore polycarbonate membrane (Millipore) with a pore size of 0.6 µm (i.e. slightly smaller than the diameters of streptococcal cells) to immobilize the bacteria through mechanical trapping, a common technique used to gather AFM images and force measurements of molecular interactions at microbial surfaces (Kasas & Ikai, 1995; Pelling et al., 2004). After filtering, the filter was carefully removed from the filter device and fixed with double-sided tape onto a small Petri dish; 5 ml 50 % (v/v) BHI was added to the dish prior to imaging the sample using AFM, submerging the filter in liquid.
AFM methodology.
All imaging in fluid was conducted using a Nanoscope IV Bioscope (Veeco Digital Instruments) (Fig. 1A). AFM images were collected in contact mode using sharpened silicon nitride cantilevers (OTR4 Veeco probes) with experimentally determined spring constants of 0.02 N m–1 and a nominal tip radius of <20 nm as defined by the manufacturer (Veeco). Probes were kept in their sterile original container as prepared using standard manufacturing techniques (Veeco), examined optically for debris before use and used promptly. Fluid imaging and mechanical measurements were obtained at room temperature (
20 °C), with force measurements recorded at a pulling rate of 1 Hz and the tip taken to be a conical indenter. Height and deflection images were simultaneously acquired; these are both important with regard to microbial cell surface characterization as they yield complementary information. Force–distance measurements were collected on single mechanically trapped S. mutans cells by lowering the cantilever tip toward the cell, pressing against the cell surface and retracting the tip from the cell as shown schematically in Fig. 1(D). The resulting curves, generated from the cantilever displacement, were analysed to reveal the force magnitude and relative cell surface adhesion by monitoring the rupture events revealed in the associated tip retraction traces from the cell surface (Fig. 1D) (Smith et al., 1999). The force-spectroscopy analysis of the wild-type and mutant cells consisted of 100 measurements made on three individual cells in each case (n=300).
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RESULTS |
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), thus yielding quantitative information regarding the mechanics of these glucan-polymer macromolecules. Kreth et al. (2004) demonstrated that the concentration of 1 % sucrose during growth as a static culture is in a saturating range for maximal biofilm adhesion strength. We report results for 1 % sucrose-treated cells compared to control, untreated cells in our experiments. To determine the right time point for probing of S. mutans cells by AFM, we compared cells grown without sucrose to those supplemented with 1 % sucrose at the exponential growth phase (6 h) and at the stationary phase (12 h). Fig. 2 shows the results obtained with S. mutans UA140 wild-type cells. These cells were mechanically trapped, and under a liquid environment (see Methods) force–displacement curves were collected on the cells. The applied force of the tip against the cell was 5 nN. With retraction of the tip from the cell wall a sequence of rupture events was observed, forming sawtooth-like patterns (Fig. 3) revealing the nature of the adhesive interactions between the local cell-surface macromolecules and the AFM tip (Fisher et al., 2000; Sen et al., 2005). In addition, force curves taken on the bare substrate, before and after cellular measurements, showed no rupture events, indicating null adhesion and confirming no contamination of the AFM tip, which may have otherwise caused nonspecific tip–cell-surface binding (Fig. 3). As shown in Table 1, the mean rupture force for S. mutans UA140 wild-type control, 6 h and 12 h sucrose-treated cells was 84.1±156.0, 304.3±281.9 and 375.6±563.2, respectively. A two-sample independent t test was conducted on the mean rupture force obtained for the control, 6 h and 12 h sucrose-treated cases. At the 95 % confidence level, it was found that the population mean for S. mutans UA140 wild-type control cells was significantly different from both the population means for the 6 h and 12 h sucrose-treated conditions (P
0.00001). However, the population means for S. mutans UA140 wild-type cells after 6 h and 12 h of sucrose treatment were not significantly different. Interestingly, the number of observed events (noe) for the wild-type control cells (noe=6705) was significantly higher than the number observed for the sucrose-treated samples at both time points (noe=88 for 6 h sucrose-treated cells; noe=261 for 12 h sucrose-treated cells) (Fig. 2). The large number of unbinding events observed in the case of the wild-type control cells is most likely due to the presence of several surface proteins, such as Pac and Wap (Bowen et al., 1991; Russell et al., 1995), which under non-sucrose conditions may cause increased tip–cell-surface interactions, whereas after sucrose is introduced into the system glucan chain growth induces tip–cell-surface protein interactions at a tip–cell distance significantly larger than that required for tip interactions with shorter surface proteins, thus yielding fewer overall adhesion events yet much stronger binding. Moreover, observed adhesion events for the sucrose-treated wild-type cells generally showed significantly increased tip-retraction lengths compared to those exhibited by the control cells (Fig. 3). Although variation in tip–cell contact regions play a part in resulting retraction lengths, the increase in retraction length observed for the sucrose-treated cells indicates that the cell-surface macromolecules on the sucrose-treated wild-type cells are generally longer than those of the non-sucrose-treated cells. Together these results indicate that the optimum adhesion for these cells is in the stationary phase, as the sucrose is most likely used up by the cells for the synthesis of the glucan polymers and metabolic purposes.
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. Force–displacement curves were collected on S. mutans UA140 gtfB, gtfC, gtfD, gtfBC and gtfBCD mutant strains under non-sucrose and sucrose conditions. As shown in Table 1, the mean rupture force observed for UA140 gtfB control cells was 46.1±80.6 pN. After treatment with sucrose for 12 h the mean rupture force increased to 53.0±148.3 pN for this mutant strain. The average unbinding force obtained for UA140 gtfC cells before and after treatment with sucrose for 12 h was 42.0±28.5 and 43.9±68.8 pN, respectively (Table 1). Similarly the control mean unbinding force for UA140 gtfD mutant cells was 42.7±22.4 pN; however, the mean unbinding force after treatment with sucrose for 12 h increased significantly to 111.3±128.2 pN (Table 1). Such an increase in the observed mean adhesion force, compared to the other mutant strains, is expected since the GtfD– strain is still able to produce water-insoluble glucans. Control and sucrose-treated mean unbinding forces for S. mutans UA140 gtfBC cells were 35.1±22.4 pN and 53.2±46.0 pN, respectively. With mutation of three specific genes, gtfBCD, the measured average adhesion remained consistently low in both the control and sucrose-treated cases, yielding values of 36.3±17.3 and 37.4±14.2 pN, respectively (Table 1). These observations were anticipated, as mutation of three specific gtf genes is known to abolish sucrose-dependent colonization (Tsumori & Kuramitsu, 1997). Data collected on all cell types were obtained using different tip–cell combinations and provided consistent results in all cases.
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). Interestingly, the data indicate a significant shift in the percentage of tip–cell-surface rupture events occurring at lower-range forces under both sucrose and non-sucrose conditions for the wild-type as compared with the mutant strains. For example, at a rupture force of 20±5 pN the percentage of observed events for the wild-type cells was 28 % when untreated and 9 % when treated with sucrose; however, the percentage of observed events for both UA140 gtfB and gtfC was significantly greater, with observed values of 68 % for gtfB and 38 % for gtfC under non-sucrose conditions and 52 % for both mutants under sucrose conditions (Table 2). Moreover, at higher-range forces the percentage of observed tip–cell-surface rupture events increased for the wild-type in the presence of glucose. The direct comparison between the overall increase in rupture events between GtfB– (possessing GtfC and GtfD activity) and GtfC– (possessing GtfB and GtfD activity) reveals that the GtfB– mutant is still able to increase sucrose- vs non-sucrose-mediated events, although the increase is not as strong as seen in the wild-type (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(1-3) and (1-6) glucosidic polymers and form a biofilm on surfaces such as tooth enamel, plastic and glass (Hamada & Slade, 1980; Kreth et al., 2004). The purpose of this study was to determine the contribution of the individual glucan-polymers to molecular-level interfacial properties of this micro-organism, such as cell adhesion. We have used AFM (Rief et al., 1997) to probe the mechanics of these macromolecules. AFM has become a pivotal imaging technique for the ultrastructure of bacterial surfaces under in vivo and in situ conditions and has been used to determine the mechanical properties and molecular forces of bacterial cells (Cross et al., 2006; Schär-Zammaretti & Ubbink, 2003a, b; van der Mei et al., 2000; Zhu et al., 2006). The force-measuring aspect of AFM unveils the inter- and intra-molecular forces (van der Aa et al., 2001) through recording the force–displacement curves as illustrated by the schematic in Fig. 1(D). An AFM probe is brought close to the cell surface, indented into the cell surface, and then retracted from the surface. Macromolecules, such as glucan-polymers, attach to the tip during extension of the probe towards the cell surface. The piezoelectric transducer retracts the probe from the cell surface, causing an extension of the attached macromolecule(s) as the tip–cell distance is increased. The force required to separate the probe from the cell-surface molecules is determined by the bending of the AFM cantilever (Morris et al., 2004). Upon separation of the tip and cell-surface macromolecules, rupture events occur, often forming sawtooth-like patterns representative of the sequential stretching, unfolding and breakage of the cell-surface macromolecules, thus revealing the adhesive nature of the biomolecules native to the cell surface (Fig. 1D). Mechanical properties of macromolecules at the microbial surface are commonly probed by studying the tip–cell interactions observed in force spectroscopy experiments (van der Aa et al., 2001). Tip–cell-surface transient and local interactions serve as a reasonable model for probing adhesive properties at the surface of microbial cells (Pelling et al., 2005; van der Aa et al., 2001; Zhu et al., 2006). This technique has commonly been applied to measure forces associated with DNA, polysaccharides and proteins (Pelling et al., 2005; Rief et al., 1997; van der Aa et al., 2001). Although AFM has garnered much interest in the last decade due to its ability to successfully probe the morphological and mechanical properties of living cells, there still remain some limitations with the technique. For example, sample preparation, cantilever flexibility and speed are significant aspects of AFM that need to be addressed to aid in further understanding of cellular structure–function. The soft and elastic propeties of live cell surfaces still remain problematic for obtaining atomic- or nanometre-resolution images; moreover, immobilization of the live cell is critical for examination by AFM. The current state of AFM allows measurement of biological processes in situ; however, an increase of scanning speed with less applied force is desirable for reliably examining biological phenomena in real time. Introduction of increasingly fine, less damaging and less sticky probes remains to be addressed by manufacturers. Despite these limitations, AFM remains one of the best techniques for high-resloution, low-force, quantitative analysis of live cells. Thus, we have used AFM to probe one of the most important virulence factors of S. mutans, its ability to synthesize glucans from sucrose via three Gtfs. Using the force spectroscopy component of AFM we were able to quantify the mechanics associated with particular cell-surface macromolecules, glucans, for S. mutans UA140 wild-type and isogenic mutant strains under physiological conditions.
Tip–cell-surface transient and local interactions were analysed for information elucidating the adhesive properties associated with S. mutans UA140 wild-type and gtf mutant strains. Our findings indicate, as expected, that the wild-type cells exhibit increased adhesion as compared to the mutant strains. Furthermore, when cells are grown for 12 h in the presence of sucrose the average local adhesion strength increases dramatically. However, the average number of observed rupture events for S. mutans wild-type significantly decreased when cells were exposed to sucrose as compared to the non-sucrose-treated cells. These findings indicate that the glucan macromolecule chains present on the cell surface undergo significant growth when cells are exposed to sucrose for a prolonged period. It appears that the lengthy glucan chains form tip–cell-surface interactions further away from the cell surface; thus the number of unbinding events decreases in the presence of longer glucan chains as interference with the tip occurs at an earlier stage than that caused by cell-surface macromolecules similar to those noted in the control cells. Thus, the attachment of S. mutans to the enamel surface of the tooth in the presence of sucrose may be mediated exclusively by the synthesized glucans. Furthermore, the observed decline in the number of rupture events is accompanied by a dramatic increase in the overall adhesion strength, thus suggesting a more uniform surface structure. Consistent with this observation is the downregulation of expression of the wapA gene, encoding the surface protein WapA, in the presence of sucrose. The surface properties of a WapA– mutant, clearly distinguishable when grown without sucrose, become similiar to the wild-type when cells are grown in the presence of sucrose (Zhu et al., 2006). Once the sucrose-independent attachment process is completed, S. mutans can switch to a stronger adhesion via glucans when sucrose becomes available.
Interestingly, the AFM method used revealed a possible additional feature of the Gtfs. When comparing the mean rupture force for the wild-type to that of the triple mutant UA140 gtfBCD (84.1 pN vs 36.3 pN) it becomes evident that the proteins themselves contribute to the adhesion between the cell surface and the cantilever tip. Although the cantilever material is different from the tooth surface, this suggests that the enzymes could be involved in the initial attachment of the cell to the tooth surface.
Furthermore, previous studies have demonstrated that the three Gtfs are required to promote full adhesion to the tooth surface (Tsumori & Kuramitsu, 1997). However, the extent to which the Gtfs contribute to the adhesion varies. GtfC seems to play a more prominent role in the adhesion event, as demonstrated by the heterologous expression of Gtfs in Streptococcus milleri. Only GtfC was able to promote significant sucrose-dependent attachment to smooth surfaces (Fukushima et al., 1992). This result was further supported in an in vitro biofilm model by Tsumori & Kuramitsu (1997). In our results, the strain expressing GtfC and GtfD (UA140 gtfB) showed a slight increase in the mean rupture forces in the presence of sucrose, while the strain expressing GtfB and GtfD (UA140 gtfC) showed an insignificant increase in the mean rupture forces. The GtfC- and GtfD-expressing strain was still able to adhere to a glass test tube, whereas the GtfB- and GtfD-expressing strain failed to adhere (data not presented). These results suggest that the broader distribution of rupture events in the GtfB– mutant, as shown by the relatively high standard deviation, is important for attachment (53.0±148.3 pN vs 43.9±68.8 pN in the GtfC– mutant). Future work employing functionalized tips may be useful for further analysis of the contribution to adhesion made by each individually expressed Gtf. The coating of a functionalized tip with saliva could change curve characteristics, as shown for S. mutans strain ATCC 25175 (van Hoogmoed et al., 2006).
The method used to probe S. mutans cells under the conditions described may not detecting solely the interactions of the cantilever tip with the glucans, but also with additional proteins associated with the glucans. A well-described group of proteins interacting with glucans are the glucan-binding proteins or Gbps. Four different types of Gbps (GbpA to GbpD) have been identified in S. mutans. Some of them are involved in biofilm morphology and cell aggregation (Banas & Vickerman, 2003). Of particular interest for this study is the binding ability of GbpC to the water-soluble glucan synthesized by GtfD (Matsumoto et al., 2006). The GtfD mutant is still able to adhere to glass and showed a mean rupture force of 111.3 pN. This is about threefold less than the wild-type. However, the GtfBC– mutant only expressing GtfD could not adhere to the glass surface and the mean rupture force did not increase substantially after sucrose addition. This suggests that the water-soluble glucans somehow require the presence of the water-insoluble glucans to provide full adhesion. If this binding is mediated by GbpC then the role of GbpC as well as the role of the other Gbps in sucrose-dependent adhesion needs to be addressed in future research.
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ACKNOWLEDGEMENTS |
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Edited by: P. E. Kolenbrander
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Received 27 February 2007;
revised 3 May 2007;
accepted 9 May 2007.
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