FIG 3.

Expression of mutant phosphodiesterases in E. coli. Immunoblot analysis was performed on the wild type and on strains with suppressor variants for detection of PdeB (a), PdeC (b), PdeN (c), PdeA (d), PdeI (e), and PdeL (f) carrying 3×Flag tags at their C termini. Proteins were analyzed in the following strains grown to exponential phase: wild type, ΔpdeH mutant (AB607), and csrA mutant (AB958). Suppressor variants of PdeB (a) and PdeC (b) showed strongly increased protein levels indicating derepression of their expression. In contrast, suppressor variants of PdeN, PdeA, and PdeI showed unaltered protein levels in all strain backgrounds tested. Of the 10 PdeL suppressor variants isolated, three were analyzed (G299S, F206S, and F249L). All variants showed increased protein levels in all genetic backgrounds tested. Note that protein levels of PdeC and PdeL differed in different genetic backgrounds.