Figure 3

Knockout of NMB0551 ( priA ) and complementation. (A) Experimental design for priA disruption by single cross over. The genetic and physical map of the priA gene is depicted above the map of the pDEXpriA region involved in single cross-over and the probe (open rectangle) used in the Southern blot experiments shown in panels B and C. ermC', erythromycin-resistance gene; DUS, DNA uptake sequence; B, BamHI restriction sites; Hf, HinfI restriction sites. (B) Southern blot analysis demonstrating the inactivation of priA. Chromosomal DNAs were extracted from the parental strain B1940 (lane 4) and from three recombinant strains (B1940ΩpriA) transformed with pDEXpriA (lanes 1–3). The HinfI-restricted chromosomal DNAs were analyzed by Southern blot using the ³²P labeled priA-specific DNA fragments cloned in pDEXpriA as probes. The arrows on the right indicate priA-specific fragments whose sizes were deduced on the basis of the relative migration of DNA ladders (bars on the left). (C) Southern blot experiment demonstrating complementation of B1940ΩpriA. B1940ΩpriA was transformed with pACpriA harboring a functional copy of priA, and chromosomal DNAs were extracted from two recombinant strains (B1940ΩpriA/priA+) (lanes 3 and 4). The DNAs from these two strains, from B1940 (lane 1) and from B1940ΩpriA (lane 2) were digested with HinfI and analyzed as in panel B.