Fig. 6.

Generation of markerless in-frame deletion mutants. The bacteriocin-encoding genes nlmA, nlmD, and nlmC were all targeted for unmarked in-frame deletions by the use of IFDC2. After the second transformation, 10 randomly selected p-Cl-Phe-resistant clones were PCR amplified using primers flanking the targeted gene. (A) For nlmA, the expected wild-type amplicon is approximately 1.6 kb, while an in-frame deletion mutant should be approximately 1.4 kb. (B) For nlmD, the expected wild-type amplicon is approximately 1.3 kb, while an in-frame deletion mutant should be approximately 1 kb. (C) For nlmC, the expected wild-type amplicon is approximately 1.3 kb, while an in-frame deletion mutant should be approximately 1.1 kb. These experiments were performed 3 times with similar results. (D) Three markerless in-frame nlmA, nlmD, and nlmC deletion triple-mutant strains were independently constructed. Each of these strains was tested with PCR to confirm the presence of all 3 mutations. The expected amplicons are identical to the single mutations described above. (E) A total of 5 wild-type S. mutans strains were used for the construction of nlmA in-frame deletions. The identities of the parent strains are listed below the corresponding amplicons.