Fig 1.
Stress sensing in B. subtilis.
A. Energy and environmental stress activate distinct branches of the stress-sensing pathway. Energy stress activates RsbP (P) via an unknown process requiring RsbQ (Q), whereas environmental stress activates RsbU (U) via the RsbU activator RsbT (T). RsbP and RsbU are both phosphatases that dephosphorylate the anti-anti-sigma-factor RsbV (V), causing the anti-sigma factor (and RsbV kinase) RsbW (W) to switch partners and thereby free σB to direct the transcription of the general stress response regulon. B. Environmental stress is sensed by cytoplasmic stressosomes, large, well-characterized protein complexes consisting of RsbR (R) and RsbS (S) proteins that capture RsbT (T) proteins to keep stress signaling in an off state. Upon stressor exposure, RsbR activates the kinase activity of RsbT, leading to the phosphorylation of RsbR and RsbS proteins while RsbT is freed from the stressosome to activate RsbU. The phosphatase RsbX (X) dephosphorylates RsbR and RsbS to return the system to its resting state.
Fig 2.
Energy stress elicits an amplitude-modulated response.
A. A kymograph is shown of an MTC1801 cell lineage growing in a microfluidic channel before and after the addition of 40 μM CCCP (dashed line) to induce energy stress via ATP depletion. B. Single-cell intensity traces of a stress-responsive PrsbV-mNeonGreen reporter before and after (dashed line) the addition of CCCP at the indicated concentrations. DMSO was used as a vehicle control for the medium switch, and a ΔrsbP mutant (MTC1906) controlled for non-energy-stress responses. The traces were manually curated to eliminate cell lineages with cell-death events or other anomalies; comparisons of mean traces for curated and uncurated populations are shown in S2 Fig. C. Overlaid average intensity traces of the cell populations in B, showing different CCCP concentrations (top) and the controls (bottom). D. Distributions of signal increases in single cells from before stress exposure to their maximal peak values. NS, not significant; all other pairwise comparisons (except for DMSO vs. ΔrsbP) were significant (p < 4 x 10−6).
Fig 3.
Environmental stress levels elicit different response profiles in wild-type cells.
A. Schematic of the four RsbR paralogs and RsbS, showing variable and conserved domains with RsbT-phosphorylatable S/T residues (orange). Wild-type cells presumably have mixed populations of RsbR proteins (different shades of blue) in their stressosomes. Stress leads to RsbT release and subsequent σB activation (also see Fig 1). B. Kymographs of single representative wild-type (MTC1801) cell lineages growing in microfluidic channels before and after addition of the indicated ethanol concentrations (dashed line) to induce environmental stress. C. Single-cell intensity traces of a stress-responsive PrsbV-mNeonGreen reporter before and after (dashed line) the addition of ethanol at the indicated concentrations. D. Overlaid average intensity traces of the cell populations in C showing different ethanol concentrations, together with the average trace of a control ΔrsbU (environmental stress-negative) strain upon 2% ethanol exposure. The traces were manually curated to eliminate cell lineages with cell-death events or other anomalies; comparisons of mean traces for curated and uncurated populations are shown in S3 Fig. E. Distributions of signal increases in single cells from before stress exposure to their maximal peak values. All unmarked pairwise comparisons were significant (p < 3 x 10−6).
Fig 4.
The four RsbR paralogs exhibit distinct response profiles in response to an identical stress.
A. Kymographs of individual cell lineages of strains containing RsbRA, RsbRB, RsbRC, or RsbRD as the only RsbR in the cell (MTC1761, 1763, 1765, and 1767, respectively). Ethanol was added at t = 0 (dashed lines). B. Single-cell intensity traces of a stress-responsive PrsbV-mNeonGreen reporter the single-RsbR strains responding to the addition of 2% ethanol (dashed line). C. Average intensity traces of the cell populations shown in B, grouped to highlight similarities and differences. The traces were manually curated to eliminate cell lineages with cell-death events or other anomalies; comparisons of mean traces for curated and uncurated populations are shown in S4 Fig. D. Distributions of signal increases in single cells from before stress exposure to their maximal peak values; only strains showing a sharp and transient initial response were evaluated. NS, not significant.
Fig 5.
Responses of RsbRA- and RsbRC-only cells to different ethanol concentrations.
A. Kymographs of individual cell lineages of strains containing RsbRA or RsbRC as the only RsbR in the cell (MTC1761 or 1765, respectively). Ethanol was added at the indicated concentrations at t = 0 (dashed lines). B. Single-cell intensity traces of a stress-responsive PrsbV-mNeonGreen reporter before and after (dashed line) the addition of ethanol at the indicated concentrations with the indicated strains. C. Overlaid average intensity traces of the cell populations in B showing different ethanol concentrations. The traces were manually curated to eliminate cell lineages with cell-death events or other anomalies; comparisons of mean traces for curated and uncurated populations are shown in S5 Fig. D. Coefficient-of-variance plot comparing the RsbRA-only strain at 4% ethanol with the RsbRC-only strain at 2% ethanol. The cell populations in B were used for the computation.
Fig 6.
Individual cell lineages show stereotyped and stochastic response profiles in RsbRA-only and RsbRC-only strains, respectively.
A. Intensity traces of a stress-responsive PrsbV-mNeonGreen reporter in an RsbRA-only strain (MTC1761) before and after (dashed line) the addition of ethanol at the indicated concentrations. Representative example cells are shown. B. Intensity traces as in A but with an RsbRC-only strain (MTC1765). Example cells were chosen to highlight the range of different observed responses. C. Annotated trace of Cell 5 from B with associated kymographs of the cells during response events.
Table 1.
Strains used in this study.