FIGURE 5.

Polyamine addition to polyamine-deficient cells induced a large and rapid increase in RpoS level. rpoS⁺/gadE⁺ cells were grown without any amines to an A₆₀₀ of 1.0; in part of the culture, polyamines (100 μm each of putrescine and spermidine) were added, and the cultures were harvested after 10-, 20-, and 60-min intervals. RNA was isolated as above, ribosomal RNAs were removed, and cDNAs were synthesized using Superscript Vilo. 1.0 ng of cDNA was used for each quantitative real time PCR analyses with rpoS gene-specific primers; gapA gene expression was used as a control to calculate relative transcript level. A and B, the relative rpoS induction after 10 and 20 min (A) and 60 min (B) is shown. C, rpoS⁺/gadE⁺ polyamine mutant cells were grown without any amines, and aliquots of the cultures at different growth phases were treated with 100 μm of putrescine plus 100 μm of spermidine, and cultures were harvested after 20 min incubation at 37 °C. Protein extracts (5 μg/lane) were resolved by SDS-polyacrylamide gel electrophoresis; blotted into PVDF membranes; probed with anti-RpoS antibody, anti-RpoD antibody, or anti-RpoB antibody; and subjected to immunological detections as described in the text. The autoradiograms containing the RpoS and RpoD protein intensities were scanned in Bio-Rad GS-800 densitometer, and the scanned values are presented below each band of RpoS and RpoD. D, rpoS⁺/gadE⁺ cells were grown without any amines to an A₆₀₀ of 1.0, and part of the cultures were divided into 14 parts to treat with different amines or left untreated as indicated for 20 min. Western blot and immune detections were performed as described. The relative ratio of RpoS:RpoD is shown below each band after scanning the Western blot data. E, rpoS⁺/gadE⁺ cells were grown without any amines to an A₆₀₀ of 1.0, and part of the cultures were divided into nine parts to treat with or without amines and different inhibitors as indicated. Rifampicin (RF, 10 μg/ml) and chloramphenicol (CM, 200 μg/ml) were added 10 min before the addition of polyamine to the indicated cultures, and then amines were added as indicated, and samples were harvested after 10- and 20-min intervals. For minus amine cultures, the same amount of inhibitors were used, and samples were harvested after a total time of 30 min. Protein samples were extracted and assayed by Western blot as above, and the relative ratio of RpoS:RpoD is shown below each band after scanning the Western blot data.