FIG. 4.

luxS knockout in S. mutans. (A) Illustration of the knockout procedure. Plasmids containing cloned fragments of S. mutans DNA as well as the erythromycin cassette were cut using the indicated restriction sites and then ligated into a linearized pUC19 backbone. The resulting construct was linearized with the unique AatII site and transformed into S. mutans for double crossover. (B) Confirmation of crossover event. In lanes 1 to 4, wild-type DNA was amplified with primers: internal to luxS, the erythromycin cassette, upstream external luxS plus erythromycin, or downstream external luxS plus erythromycin. In lanes 5 to 8, luxS mutant DNA was amplified using the same primer combinations. External luxS primers bind to sites that were not subject to crossover. (C) AI-2 production was assayed to confirm that activity was lost in the mutant.