FIG 5.
Effects of degS mutations on RseA and AcrAL222Q levels. (A and B) RseA and MBP (control) were detected by Western blot analysis. Bacterial cultures were grown overnight at 37°C in a roller drum. Samples from two independent cultures, per strain, normalized to OD600, were boiled in a SDS buffer and analyzed by SDS-PAGE. Membrane filters were probed with antibodies against RseA (A) and MBP (B). Relevant genotypes of the strains, as well as positions of RseA, MBP and prestained protein markers are shown. (C and D) Effects of degS mutations on AcrAL222Q levels. Western blot analysis was conducted to observe the effects of degS suppressor mutations on a labile AcrA protein carrying an L222Q substitution. Bacterial cultures were grown overnight at 37°C in a roller drum. Samples from two independent cultures, per strain, normalized to OD600, were boiled in an SDS buffer and analyzed by SDS-PAGE. Membrane filters, carrying samples from the degS-D102V (C) or degS-D320A (D) background, were probed with antibodies against AcrA. Relevant genotypes of the strains used are shown, except that all strains had deletions of tolC and expressed the mutant allele of acrA-L222Q from the chromosome.