Molecular and biological characterization of gtf regulation-associated genes in Streptococcus mutans

Surface protein antigen (PAc) and glucosyltransferases (GTF) are major adhesive molecules of Streptococcus mutans, though the mechanism of their regulation has not been fully elucidated. To investigate the regulation mechanism, we determined a nucleotide sequence in the upstream region of the pac locus in S. mutans and identified two open reading frames (ORF), designated as orf1 and orf2. Each ORF was inactivated and functional analyses were performed. Western blot analyses revealed that the expression level of PAc was unaffected, while that of cell-associated GTF was diminished in both mutant strains. Furthermore, they showed higher hydrophobicity levels and an impaired sucrose-dependent adherence to smooth surfaces. RNA dot blot analysis demonstrated that transcriptions of the gtfB and the gtfC genes, which encode GTF-I and GTF-SI, respectively, were downregulated, while that of pac was comparable to the wild-type strain. In addition, the GTF activities of the mutant strains were significantly lower than those of the wild-type, though a greater amount of total glucan produced by the mutants was noted in culture supernatants. These findings suggest that orf1 and orf2 are associated with positive regulation of the gtfB and gtfC genes.