FIG. 6.

Western blot analysis of CcdA41 and CcdA. (A) Analysis of CcdA41 and CcdB produced by the wild-type and by the tld deletion mutants. SG22622 and its derivatives containing pULB2208 (ptac-ccdA41-ccdB) were grown in LB medium containing the appropriate antibiotic and 0.5 mM IPTG. At an OD₆₀₀ of 0.2 to 0.3, 1-ml samples were collected and analyzed by Western blotting with anti-CcdA (upper panel) or anti-CcdB (lower panel) antibodies. Strain genotypes are as follows: 1, wild type; 2, ΔtldD; 3, ΔtldE; 4, ΔtldD ΔtldE; 5, Δlon; and 6, Δlon ΔtldD ΔtldE. (B) Analysis of CcdA stability in a tld deletion mutant. SG22622 and its derivatives (Δlon and ΔtldD ΔtldE) containing pULB3565 (ptac-ccdA) were grown in LB containing the appropriate antibiotic to an OD₆₀₀ of 0.2 to 0.3. IPTG (0.5 mM) was added to induce CcdA expression for an hour. Spectinomycin was then added (200 μg per ml of culture) to block protein synthesis, and 1-ml samples were collected at the times indicated. Samples were treated and analyzed by Western blotting with anti-CcdA antibodies.