Genetic Engineering of S. mutans UA159

S. mutans UA159 strain (ATCC 700610) was used in this work. Todd–Hewitt broth (Thermo Fisher Scientific Inc., Mississauga, ON, Canada) supplemented with 0.3% (w/v) yeast extract (THYE) was used for bacterial cultivation. S. mutans cells were cultivated at 37 °C with 5% CO₂. A nonpolar allelic replacement technique was used to generate the deletion mutants in the UA159 wild-type (WT) strain [20 (link)]. For the construction of the vector for toxin overexpression, the full-length coding region of the relE40 gene was PCR-amplified using UA159 genomic DNA and a PCR product cloned under the control of the constitutive promoter P₂₃ into the E. coli Streptococcus shuttle plasmid pIB166 [21 (link)] harboring a chloramphenicol resistance cassette. The construct was then transferred into the UA159 WT strain via natural transformation [22 (link)].