Functional analysis of virulence-associated DNA fragment from Streptococcus mutans

Objectives: To identify and functional analysis a virulence-associated DNA fragment from Streptococcus mutans of serotype c. Methods: The clinical isolates were isolated from dental plaque samples from twenty caries-active subjects [DMFS (the number of decayed, missing, and filled surfaces) °Ý10]. The isolates per subject were bio-, sero-, and AP-PCR typed. One strain representative of each genotype identified was examined for its cariogenic ability including WIG synthesis, adherence to SHA, acidogenicity, and acid tolerance. A virulent S. mutans strain and an avirulent strain was screened. Genomic DNA of the putatively virulent strain was used as tester, and that of the less virulent strain as driver. Subtractive hybridization was applied between them. The subtractive libraries representing sequence differences were obtained. The subtractive fragments that were found specifically in the virulent strain but not in the less virulent strain were identified by dot blotting and then sequenced. BLAST sequence homology analyses were performed. The distributions of tester specific sequences were examined in some virulent strains of S. mutans by Southern blots. One fragment was screened and its encoding gene was knocked out of the chromosome of wild strain of S. mutans. The ability in biofilm formation was compared between wild strain and its mutants. Results: Twenty-seven sequences were specific to the virulent strain genome. Among them, five revealed no homology to published nucleotide sequences while the remaining sequences showed different degrees of homology to known genes of S. mutans strain UA159. SFC7 fragment contained part of threonyl-tRNA synthetase, which could catalyze threonine to connect with corresponding tRNA. It distributed in S. mutans virulent strains, but absent from non-cariogenic strains. It was shown that this enzyme may take part in biofilm formation. Conclusions: Identifying and functional analysis S. mutans virulent strain specific DNA sequence is beneficial to comprehending the cariogenesis of S. mutans.