FIG 6.

Effect of translation inhibitors on in vivo degradation. A single wild-type Synechocystis culture was grown at 30°C to mid-log phase. The induced culture was transferred to 20°C for 2 h to induce CrhR accumulation. The induced culture was divided into four equal aliquots, and either chloramphenicol (+Cm, 250 μg/ml), kanamycin (+Km, 200 μg/ml), or nothing (−Cm and −Km, the two no-antibiotic controls) was added to the individual cultures. Incubation was continued for a further 1 h at 20°C to allow the antibiotic inhibition of translation to take effect. Aliquots were harvested for protein extraction after 0 h (no low temperature induction), 1 and 2 h in the absence of inhibitors, and after 3 h, i.e., after 1 h in the presence of the inhibitors, of cold induction at 20°C. All four cultures were subsequently transferred to 30°C, and aliquots were harvested at 0.5, 1, 2, 3, and 4 h for protein extraction. Proteins were extracted from the aliquots taken at the indicated times, and CrhR (55 kDa) was identified by Western analysis.