FIG. 2.

Construction of a ΔrelA mutation in Y. pestis KIM6⁺. (A) A DNA fragment encoding a cat-sacB cassette flanked by long regions homologous to genes just upstream and downstream of relA is introduced into Y. pestis KIM6⁺(pKD46). Selection on media containing chloramphenicol yields a strain in which relA is replaced by the cat-sacB cassette. The positions of oligonucleotide primers, such as relA-U1, relA-D1, relA-V1, and Cm-V, are indicated. (B) A DNA fragment containing the relA233 deletion and flanking regions adjacent to relA is introduced into the Y. pestis KIM6⁺(pKD46) cat-sacB strain. After selection on media containing sucrose, the cat-sacB cassette is replaced by the fragment containing the ΔrelA233 deletion.