Figure 3.

Verification of region of difference 2 (RD2) deletion and complementation in strains M28ΔRD2 and RD2^Comp, respectively. Total DNA from the parental (M28), RD2 deletion mutant (M28ΔRD2), and complemented mutant (RD2^Comp) strains was isolated and used in polymerase chain reaction (PCR) analysis with 15 pairs of primers (RD1 to RD30). A, Distribution of the primers and expected PCR products across RD2. Note that primers RD1 and RD30 are located outside of RD2. B, Generated PCR products for the 3 tested strains. Note that there is a 1-kb size difference between the RD1/2 PCR product of the parental and RD2^Comp strains, and this is due to the presence of a 1-kb insertion sequence that flanks RD2 in MGAS6180 but not in the complemented strain. C, Western blot analysis of cell wall–associated R28 expression in the 4 indicated GAS strains. Note the characteristic ladder pattern of R28 [33]. The membrane was stained prior to Western blot analysis, to serve as a loading control.