FIG. 1.

Transient increases in the level and synthesis rate of RpoH upon heat shock. (A) Cells of wild-type A. tumefaciens (GV3101) were grown in complete medium (●) or synthetic medium (▪) to the logarithmic growth phase at 25°C and shifted to 37°C at time zero. Samples were taken at intervals, mixed with an equal volume of 2× SDS sample buffer, boiled for 2 min, and analyzed by SDS-PAGE (12.5% polyacrylamide) and immunoblotting with anti-RpoH antiserum essentially as described previously (28). The RpoH band was quantified by densitometry and normalized to the value at 25°C. (B) Log-phase cells grown in synthetic medium at 25°C were shifted to 37°C. Samples taken at the times indicated were pulse-labeled with [³⁵S]methionine for 2 min and treated with trichloroacetic acid, and RpoH was immunoprecipitated, resolved by SDS-PAGE (12.5% polyacrylamide), and quantified as described previously (28). The rate of RpoH synthesis thus obtained was normalized to the value at zero time. (C) Synthesis rate of DnaK was determined by growing and pulse-labeling cells with [³⁵S]methionine as described above for panel B. The cells were treated with trichloroacetic acid and then analyzed by SDS-PAGE (7.5% polyacrylamide) . Radioactivity associated with the DnaK band was determined with a phosphorimager and normalized to the value at zero time.