LuxS-Mediated Signaling in Streptococcus mutans Is Involved in Regulation of Acid and Oxidative Stress Tolerance and Biofilm Formation
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FIG. 1.AI-2 bioassay (left panel) and reporter gene fusion (right panel). For AI-2 bioassays, conditioned cell-free supernatants of S. mutans UA159 grown in TV medium with 0.5% glucose were examined for the capacity to induce light production in V. harveyi BB170 (AI-1 sensor− AI-2 sensor+) by using the method of Surette and Bassler (45). Data are presented as the percentages of AI-2 activity at different growth phases, with the highest activity observed assigned a value of 100%. For reporter gene fusion, the promoter region of luxS was fused with a promoterless chloramphenicol acetyltransferase (cat) gene, and the fusion was integrated into the chromosome by double-crossover recombination using the pBGK2 integration vector. Expression of luxS was then analyzed by CAT assays with cells grown in TV medium (5) plus 0.5% (wt/vol) glucose, and CAT activity was expressed as nanomoles per minute per milligram of protein. O/N, overnight.
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FIG. 2.Effect of LuxS on fruA expression. The expression of cat under the direction of the fruA promoter in wild-type UA159 and the luxS-deficient mutant TW26 were analyzed by CAT assay. All strains were grown in TVI medium (5) with or without inclusion of cell-free supernatants prepared from UA159 (W) and TW26 (S) and harvested for CAT assays when the cells reached the mid-exponential phase of growth (OD600 ≅ 0.3). CAT activity is expressed as nanomoles per minute per milligram of protein.
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FIG. 3.Acid tolerance assay. (A) S. mutans strains UA159, TW26, and TW42 were grown in BHI medium adjusted to pH 7.0. Cultures with an OD600 of ∼0.3 were harvested, washed with 0.1 M glycine buffer, pH 7.0, and subjected to acid killing by incubating the cells in 0.1 M glycine buffer, pH 2.8. For preadapted acid killing, cultures with an OD600 of ∼0.2 were harvested and resuspended in fresh BHI medium adjusted to pH 5.0. Following two additional hours of incubation, cells with an OD600 of ∼0.3 were prepared for acid killing as described above. Survival rate was determined by plating in triplicate on BHI agar plates, and results are expressed as percent survival rate versus time at pH 2.8. (B) Cell-free supernatants from UA159 (W) and TW26 (S) were reconstituted with concentrated TV medium to 0.5×, and their impact on acid tolerance was measured by incubating the cells in 0.1 M glycine buffer, pH 2.8, for 45 min as described above.
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FIG. 4.Sensitivity of S. mutans to hydrogen peroxide. S. mutans strains UA159, TW26, and TW42 were grown to an OD600 of ∼0.3 in BHI medium that was adjusted to pH 7.0. Cells were harvested by centrifugation and resuspended in 0.1 M glycine buffer, pH 7, containing 0.2% (vol/vol) (58.8 mM final concentration) hydrogen peroxide. Surviving cells were plated on BHI plates, and the results were expressed as percent survival versus time.
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FIG. 5.SEM of S. mutans biofilms. Twenty-four-hour biofilms of S. mutans strains UA159 (panels 1, 4, and 7), TW26 (panels 2, 5, and 8), and TW42 (panels 3, 6, and 9) were grown on HA disks that were deposited in 24-well cell culture clusters in semidefined BM (27) with 0.8% (wt/vol) glucose (panels 1 to 3) or 0.5% (wt/vol) sucrose (panels 4 to 9).
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FIG. 6.Impact of cell-free supernatants on biofilm formation. All strains were grown in BM with sucrose (27) with or without inclusion of cell-free supernatants prepared from UA159 (W) or TW26 (S). Biofilms were grown in the wells of a 96-well (flat-bottom) cell culture cluster and stained with 0.1% crystal violet (48).
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FIG. 7.2D gel analysis. S. mutans UA159 and TW26 were grown in BHI medium to an OD600 of ∼0.5. Cell extracts of 200-μg total proteins were subjected to 2D electrophoresis using 2% ampholines at pH 4 to 8. Gels were stained with Coomassie blue. The triangles indicate the internal standard, tropomyosin, with pI 5.2 and a molecular weight of 32,700.
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FIG. 8.Northern blot analysis. Total RNAs were extracted from S. mutans strains UA159, TW26, and TW42 grown in BHI medium at early exponential phase (OD600 ≅ 0.3) or late exponential phase (OD600 ≅ 0.5). Twelve micrograms of total RNA was loaded to each lane, separated by gel electrophoresis, blotted to a nylon membrane, and then hybridized with radioactively labeled gene-specific probes as indicated.
- American Society for Microbiology
