Fig. 1: Impact of Ala substitutions in the putative sensing region of RsbRA on the σB response using a complementation strategy.
From: Stressosome-independent but RsbT-dependent environmental stress sensing in Bacillus subtilis
A Schematic of the B. subtilis general stress response. Environmental stress is thought to be sensed by stressosome complexes composed of a mixed population of 40 RsbR paralogs (different shades of red) and 20 RsbS proteins (gray) that sequester RsbT proteins (blue). Stress sensation leads to release of RsbT, which then activates downstream steps leading to release of σB. Energy stress is sensed separately, by the proteins RsbQ and RsbP, but the final steps leading to σB release are the same. B Schematic of the RsbRA protein showing the region of residues from 11 to 58 (red) that was subjected to individual Ala substitutions. Mutant rsbRA genes were added back at amyE together with rsbU to a strain background lacking all rsbR genes, ytvA, rsbP (to deactivate the energy stress pathway), and rsbU. The strain carried a σB-responsive PrsbV-lacZ reporter to assess the general stress response. C Cartoon of predicted results, with a nonfunctional or unstable RsbRA predicted to yield a constitutive σB response even without stress (green line), and a sensing-dead version of RsbRA unable to activate in the presence of stress (red line). The black line is the same wild-type data shown in other figure panels, showing the mean of biological triplicate experiments, with the shading indicating standard deviation. D–F 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. WT indicates complementation of the reporter strain with rsbU and wild-type rsbRA at amyE as the only source of RsbR in the cell (CSS744; black trace). No RsbRA or U indicates the uncomplemented recipient strain (CSS716; gray trace). Substitutions (e.g., “T38A”) indicate complementation of the reporter strain with rsbU and Ala-substituted rsbRA as indicated at amyE as the only source of RsbR in the cell. All traces are means of biological triplicate experiments, with the shading indicating standard deviation. D Substitutions resulting in increased (>10% increased from wild-type) peak σB activity. E Substitutions resulting in decreased (>10% decreased from wild-type) peak σB activity. F Substitutions resulting in unchanged (within 10% of wild-type) peak σB activity. G Color-coded sequence logo showing the impact of Ala substitutions. Red, decreased; blue, unchanged; green and bold, increased; black, untested; gray, unsubstituted (already Ala). Source data are provided as a Source Data file.
