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RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

Figure 3

RsfA inhibits translation during both stationary phase and the transition from rich to poor media.

(A) Growth competition experiment: equal numbers of E. coli wild type and ΔrsfA cells derived from an overnight LB-culture were mixed and grown in rich medium (LB, rich→rich), poor medium (M9, rich→poor) and poor medium plus 2% casamino acids as indicated (rich→poor+aa). Growth was maintained in log phase conditions by regular dilutions in the corresponding media. Shown is the fraction of viable ΔrsfA mutant cells in the total cell population. (B) Wild type and mutant strains were grown overnight in rich medium (LB) and then diluted in rich (rich→rich) or poor M9 medium (rich→poor). The generation time was derived from the slopes of the regression lines made of the points indicating the logarithmic phase. The errors of the generation-time determinations are below ±5%, i.e. generation times of 30 and 32 min are not significantly different. (C) Wild type and mutant strains transformed with a plasmid harboring the gene for RsfA fused with a His-tag under control of the native promoter or the corresponding empty plasmid were grown overnight in rich medium (LB) and then diluted in poor M9 medium. At certain times samples were withdrawn (S1–S6) and the relative amount of RsfA was quantified by Western-blot (represented with bars). S1–S3: samples were analyzed from both strains. S4–S6: samples were analyzed only from wild type (blue) or mutant strain (red). (D) Same as (C) but using a plasmid with a His-tagged RsfA gene under a tac promoter. After ∼3 h incubation in M9 medium 0.2 mM IPTG (final concentration) was added to all strains in order to induce expression from the tac promoter. (E) Viability competition similar to the growth competition described under (A) but in a batch culture without dilution. Red, growth of the mixture of ΔrsfA and WT strains; blue, the fraction (in %) of the mutant strain. (F) Expression of β-galactosidase as reporter to test translational activity of logarithmic and stationary phase cells in WT and ΔrsfA cells induced by 2% arabinose. Induction time was 3 h in logarithmic and 2.5 and 6.5 h in stationary phase. The expression level was derived from the band-intensity on a gel (Coomassie-stained SDS-PAGE).

Figure 3