Identification of Streptococcus mutans genes involved in fluoride resistance by screening of a transposon mutant...

Fluoride has been used as an effective anticaries agent for more than 70 years, which might result in the emergence of fluoride-resistant strains. However, the fluoride resistance mechanism and the cariogenic properties of fluoride-resistant mutant for cariogenic bacterial species Streptococcus mutans remain largely unknown. We describe here the construction and characterization of a mariner-based transposon system designed to be used in S. mutans, which is also potentially applicable to other streptococci. To identify genetic determinants of fluoride resistance in S. mutans, we constructed a library of S. mutans transposon insertion mutants and screened this library to identify mutants exhibiting fluoride resistance phenotype. Two mutants were found to carry transposon insertion in two different genetic loci (smu.396 and smu.1291c), respectively. Our subsequent genetic study indicates the fluoride-resistant phenotype for the mutant with the insertion in smu.1291c is resulting from the constitutive overexpression of downstream operon smu.1290c-89c, which is consistent with the previous reports. We also demonstrate for the first time that the deletion of smu.396 is responsible for the fluoride-resistant phenotype and that the combining of smu1290c-89c overexpression and smu.396 deletion in one strain could attribute an additive effect on the fluoride resistance. In addition, our results suggest that the biological fitness of those fluoride-resistant mutants is reduced compared to that of wild-type strain. Overall, our identification and characterization of genetic determinants responsible for fluoride resistance in S. mutans expand our understanding of the fluoride resistance mechanism and the biological consequence of the fluoride resistance strains.