FIG. 5.

Effects of substitution of the recB_ET-37 allele for the wild-type recB allele in N. meningitidis strain MC58 on UV₂₅₄ radiation sensitivity, DNA repair of UV-induced damages, and mutability. (A) Southern blot experiment demonstrating allelic replacement. Genomic DNAs were extracted from the UVR strain MC58 (lane 1); from the UVS strains MC58-1, MC58-2, MC58-3, and MC58-4 (transformed with the recB_ET-37 allele) (lanes 2 to 5, respectively); and from BF2 (source of the recB_ET-37 allele) (lane 6) and were treated with Sau3AI. The digested DNAs were analyzed by Southern blotting using the ³²P-labeled recB-specific probe (Fig. 3A). The arrow on the right indicates the diagnostic restriction fragment of the allelic replacement. The relative lengths of migration of DNA molecular markers and their weights are shown on the left. (B) UV₂₅₄ radiation sensitivity of meningococcal strains MC58, MC58-1, and MC58-2. (C) Repair of UV-induced damages assayed in a marker repair experiment. Rifampin-resistant strains MC58^Rif and MC58-1^Rif were grown to late logarithmic phase and irradiated with a UV₂₅₄ germicidal lamp at different fluences (indicated on the abscissa), as detailed in Materials and Methods. Then the DNA was extracted from irradiated bacteria and nonirradiated controls after a postirradiation period of 20 min at 37°C under nongrowth conditions to allow DNA repair to occur. The DNA was used to transform MC58 to rifampin resistance. Relative transforming activities from irradiated and nonirradiated cells are reported on the ordinate. (D) Spontaneous and UV-induced mutability to rifampin resistance in MC58 and MC58-1. Mutation frequencies were determined in nonirradiated or in UV₂₅₄-irradiated bacteria at different fluences as described in Materials and Methods. Values are reported as logs of frequencies of mutation to rifampin resistance. Error bars are indicated.