Substitutions in the Presumed Sensing Domain of the Bacillus subtilis Stressosome Affect Its Basal Output but Not Response to Environmental Signals

Abstract

The stressosome is a multiprotein, 1.8-MDa icosahedral complex that transmits diverse environmental signals to activate the general stress response of Bacillus subtilis. The way in which it senses these cues and the pathway of signal propagation within the stressosome itself are poorly understood. The stressosome core consists of four members of the RsbR coantagonist family together with the RsbS antagonist; its cryo-electron microscopy (cryoEM) image suggests that the N-terminal domains of the RsbR proteins form homodimers positioned to act as sensors on the stressosome surface. Here we probe the role of the N-terminal domain of the prototype coantagonist RsbRA by making structure-based amino acid substitutions in potential interaction surfaces. To unmask the phenotypes caused by single-copy rsbRA mutations, we constructed strains lacking the other three members of the RsbR coantagonist family and assayed system output using a reporter fusion. Effects of five individual alanine substitutions in the prominent dimer groove did not match predictions from an earlier in vitro assay, indicating that the in vivo assay was necessary to assess their influence on signaling. Additional substitutions expected to negatively affect domain dimerization had substantial impact, whereas those that sampled other prominent surface features had no consequence. Notably, even mutations resulting in significantly altered phenotypes raised the basal level of system output only in unstressed cells and had little effect on the magnitude of subsequent stress signaling. Our results provide evidence that the N-terminal domain of the RsbRA coantagonist affects stressosome function but offer no direct support for the hypothesis that it is a signal sensor.

Two Surfaces of a Conserved Interdomain Linker Differentially Affect Output from the RST Sensing Module of the Bacillus subtilis Stressosome

“…Although single substitutions within the N-terminal, nonheme globin domain were found to increase system output in unstressed cells, they had no effect on the magnitude of subsequent stress signaling (16). These results indicate that the N-terminal domain influences stressosome function but offer no direct support for the hypothesis that it is a stress sensor.…”

“…A QuikChange Lightning kit (Stratagene, La Jolla, CA) was used to introduce missense substitutions into the linker region of the rsbRA gene encoded on integrative plasmid pTG5923 (16); substitution was confirmed by sequencing the entire coding region of each construction. These missense alleles were exchanged for wild-type rsbRA on the chromosome using the I-SceI-mediated method of Janes and Stibitz (20) as described previously (16). The constructed strains also carried a single-copy transcriptional fusion between the ctc promoter and a lacZ reporter to provide an indirect measure of B activity (8).…”

“…Unstressed samples were taken during early exponential growth up to a cell density of 20 absorbance units (Klett-Summerson colorimeter equipped with a number 66 transmission filter), at which point ethanol or NaCl stress was imposed at a final concentration of 4% (vol/vol) or 0.3 M, respectively. Samples were treated essentially according to the method of Miller (30); ␤-galactosidase activity was defined as ⌬A 420 ϫ 1,000 min Ϫ1 mg Ϫ1 of protein (16). Basal activity was the value measured in unstressed cells at 20 absorbance units; stress activation was the difference between this basal value and the maximum activity realized after stressor addition.…”

“…Assays were conducted as described previously (16). Briefly, shake cultures were grown at 37°C in buffered Luria broth medium lacking salt (7), with moderate white-light illumination (3 to 4 mol m Ϫ2 s Ϫ1 ).…”

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

“…2D), the difference in activation ("nhaA2p-lacZ + activity with stress" − "nhaA2p-lacZ + activity without stress") relative to the WT ["relative stress activation" (RSA)] rather than the absolute nhaA2p-lacZ + activity was compared. The same approach (measurement of RSA) has been used before in the Bacillus subtilis GSR to discriminate between the contribution of sensory proteins to the basal activity level and their roles in stress activation (31,32). As shown in Fig.…”