Fig. 2: Impact of Ala-substituted versions of RsbRA encoded at the native locus on the σB response to 3% ethanol.
From: Stressosome-independent but RsbT-dependent environmental stress sensing in Bacillus subtilis
A Schematic of the allelic replacement strategy, wherein Ala-substituted RsbR was encoded at the native rsbR locus in cells deleted for all other RsbR paralogs and carrying a σB-responsive PrsbV-lacZ reporter. B–D Graphs showing β-galactosidase activity from PrsbV as a proxy for σB activity (measured at 15-min intervals for 3 h) upon stimulation of the general stress response with 3% ethanol. “True WT” (CSS1113) cells carry all four RsbR paralogs, whereas “WT” denotes a strain (CSS1480) with wild-type RsbRA as the only RsbR paralog in the cell. Substitutions (e.g., “E17”) indicate allelic replacement of the native rsbRA gene with Ala-substituted rsbRA as indicated as the only source of RsbR in the cell. All traces are means of biological triplicate experiments, with the shading indicating standard deviation. B Substitutions resulting in increased (>10% increased from wild-type) peak σB activity. C Substitutions resulting in decreased (>10% decreased from wild-type) peak σB activity. D Substitutions resulting in unchanged (within 10% of wild-type) peak σB activity. E Color-coded sequence logo showing the impact of Ala substitutions. Red, decreased; blue, unchanged; green and bold, increased; black, untested; gray, unsubstituted (already Ala). Asterisks appear below a position when the impact of that substitution was different in the complementation than in the allelic replacement, with the color of the asterisk denoting the result from the complementation strategy. F Crystal structure of the dimeric N-terminal region of RsbRA (PDB 2BNL)49 with positions impacting the σB response highlighted using the same color scheme as in panel E. The left panel shows a ribbon model, and the right panels show space-filling models. For simplicity, we highlighted only those positions whose results agreed in the complementation and allelic replacement experiments. Source data are provided as a Source Data file.
