Roles of genes to biofilm formation in Streptococcus mutans

Objective: Streptococcus mutans is considered to be the primary etiological agent of human dental caries, and possess various abilities to colonize and form biofilm on the tooth surface. Many genes associate with the attachment, colonization, aggregation and biofilm formation of S. mutans. We were previously found several genes using DNA microarray, RT-PCR and Com deficient mutants, which associated with com-dependent quorum sensing and biofilm formation, in clinical isolates of S. mutans from 3 years old children and mothers. Further, SMU832, 833 and 1912 were identified among clinical isolates from children but not mothers. In this study, to clear roles of these genes to biofilm formation, the deficient mutants were constructed and compared with the wild type (S. mutans UA159) in the biofilm formation.

Methods: The mutants were constructed by double-crossover homologous recombination via insertion of an erythromycin resistance determinant into each gene. The SMU832, 833 and 1912-deficient mutants of S. mutans UA159 were incubated at 37°C for 6 and 18 hours under 5% CO₂ aerobic conditions in Tryptic Soy Broth without Dextrose (TSB) and TSB with 0.25% sucrose. After incubation, planktonic cells were removed, the well's biofilm were rinsed with sterilized DW, then air dried and stained with 0.25% safranin. Quantification of the stained biofilm was performed by measuring A₄₉₂ absorbance.

Results: The biofilm formation levels were higher significantly in SMU832 and 833-deficient mutants than wild type at 6 hours incubation. However, the biofilm formation levels in these mutants were similar to that in wild type at 18 hours incubation.

Conclusion: Gene expressions of SMU832 (Glycosyltransferase involved in cell wall biogenesis) and SMU833 (hypothetical protein) in the com-dependent quorum sensing system might associate with regulation of biofilm development in early (log) phase but not late (stationary) phase.