FIG. 3.
NAD+-dependent ligA of M. smegmatis can be substituted with NAD+-dependent ligA of E. coli or ATP-dependent ligase of enterobacteriophage T4. SCO strains carrying both ligA and ΔligA (Fig. 2) were enriched with ligAEc (A) or ligT4 (B) controlled by a Pami promoter. DCO mutant strains resulting from the processing of these SCO strains were both wt (ligA) and mutant type (ΔligA). The genotype of selected DCO strains was confirmed by PCR (A and B) and Southern hybridization (C) analyses. Numbers above the lanes of each gel represent the following samples: 1, 1-kb DNA ladder; 2, M. smegmatis wt control; 3, p2NILΔligA plasmid; 4, SCO mutant strains; 5, DCO mutant strains carrying wt ligA; 6, DCO mutant strains carrying ΔligA. In the Southern hybridization shown in C, 6A and 6B represent the complementation by ligAEc and ligT4, respectively. Note that the middle PstI recognition site (A) was present within the deleted region of ligA (ΔligA), thus causing the ΔligA band detected by Southern hybridization to be larger than the wt ligA band. The Southern hybridization probe was amplified using the 3′ undeleted end of ligA.