Insertional inactivation of ftsB , ftsC , and mtsA. (A) Schematic showing the arrangement of ftsABCD genes and their neighbors in the MGAS5005 genome. The ftsA, ftsB, and ftsCD genes encode ATP-binding protein, lipoprotein, and permease, respectively. The numbers above the other arrows are the spy numbers assigned to the corresponding open reading frames in the M1 genome sequence (15). (B) Schematic for insertional gene inactivation. The paired solid or dotted arrows under the mutant genome indicate the locations of primers used to confirm the disruption of the gene using PCR and DNA sequencing. (C) PCR confirmation of insertional inactivation of ftsB, ftsC, and mtsA. The picture shows agarose gel analysis of PCR reactions using mutant (lanes labeled by 1) or wild type (lanes labeled by 2) genomic DNA as template and primers at the locations indicated by the solid arrows under the mutant genome in panel B. (D) Western blot showing the absence of FtsB in the ftsB mutant strain (lane 2) and the presence of FtsB in the wild-type (lane 1) and ftsB/pCMVftsB (lane 3) strains. Proteins from 5 × 108 wild-type cells, 5 × 108 ftsB mutant cells, and 4 × 106 ftsB/pCMVftsB were probed with FtsB-specific mouse antiserum. The amount of ftsB/pCMVftsB cells used was 1/125 of that of the wild-type cells. (E) Western blot showing the presence of MtsA in the wild-type strain (lane1) and the absence of MtsA in the mtsA mutant strain (lane 2). Proteins from 6 × 106 wild-type cells and 3 × 108 mtsA mutant cells were probed with MtsA-specific mouse antiserum.