Fig 4.

Protein stability test of CodY and RecA in Newman and Newman ΔclpC strains by Western blotting. A portion of the gel stained with Coomassie blue was used as a loading control below each blot. Western blots (n = 3) were quantified by densitometry, normalized to one of the stained protein bands, and analyzed by a one-way analysis of variance. (A) CodY stability was tested after inducing the stringent response by adding mupirocin to mid-log-phase cultures at time zero (T = 0*). Chloramphenicol was added 30 min after mupirocin addition. Cells were harvested immediately after chloramphenicol addition (labeled as 0) and 0.5, 1, 2, and 3 h thereafter. A codY-deleted strain and a codY-complemented strain, denoted − and +, respectively, were also included as controls. There was no significant difference between results at the different time points. (B) RecA stability was tested after SOS induction by adding mitomycin C at T = 0*. Chloramphenicol was added 15 min after mitomycin C addition. Cells were harvested as described for panel A. The RecA level was significantly lower (*, P < 0.05) in preinduced samples (T = 0*) than at other time points for both strains.