FIG. 4.

Isolation of deletion mutants by counterselection of the rpsL1 allele. (A) Scheme showing the protocol followed to isolate deletion mutants of the narC gene. Insertion of pS18ΔnarC derivative by homologous recombination is selected on plates with Str (+Str), and the plasmid presence is subsequently counterselected on plates without the antibiotic (−Str). Arrows labeled 4 and 5 correspond to primers FPPnarDIR and O27-32 used in panel B. (B) PCR with primers 4 and 5 of panel A on total DNA from colonies (lanes 1 to 5) selected from panel A that did not show nitrate reductase activity. Lanes P and W correspond to the PCR amplification of the pS18ΔnarC plasmid and to total DNA of the wild-type strain, respectively. Lane M corresponds to DNA size markers. Lateral numbers indicate the sizes in base pairs of the PCR products.