Bacillus subtilis Stressosome Sensor Protein Sequences Govern the Ability To Distinguish among Environmental Stressors and Elicit Different σB Response Profiles

Abstract

Bacteria use a variety of systems to sense stress and mount an appropriate response to ensure fitness and survival. Bacillus subtilis uses stressosomes-cytoplasmic multiprotein complexes-to sense environmental stressors and enact the general stress response by activating the alternative sigma factor σ^B. Each stressosome includes 40 RsbR proteins, representing four paralogous (RsbRA, RsbRB, RsbRC, and RsbRD) putative stress sensors. Population-level analyses suggested that the RsbR paralogs are largely redundant, while our prior work using microfluidics-coupled fluorescence microscopy uncovered differences among the RsbR paralogs' σ^B response profiles with respect to timing and intensity when facing an identical stressor. Here, we use a similar approach to address the question of whether the σ^B responses mediated by each paralog differ in the presence of different environmental stressors: can they distinguish among stressors? Wild-type cells (with all four paralogs) and RsbRA-only cells activate σ^B with characteristic transient response timing irrespective of stressor but show various response magnitudes. However, cells with other individual RsbR paralogs show distinct timing and magnitude in their responses to ethanol, salt, oxidative, and acid stress, implying that RsbR proteins can distinguish among stressors. Experiments with hybrid fusion proteins comprising the N-terminal half of one paralog and the C-terminal half of another argue that the N-terminal identity influences response magnitude and that determinants in both halves of RsbRA are important for its stereotypical transient σ^B response timing. IMPORTANCE Bacterial survival depends on appropriate responses to diverse stressors. The general stress-response system in the environmental model bacterium Bacillus subtilis is constantly poised for an immediate response and uses unusual stress-sensing protein complexes called stressosomes. Stressosomes typically contain four different types of putative sensing protein. We asked whether each type of sensor has a distinct role in mediating response dynamics to different environmental stressors. We find that one sensor type always mediates a transient response, while the others show distinct response magnitude and timing to different stressors. We also find that a transient response is exceptional, as several engineered hybrid proteins did not show strong transient responses. Our work reveals functional distinctions among subunits of the stressosome complex and represents a step toward understanding how the general stress response of B. subtilis ensures its survival in natural environmental settings.

Keywords: microfluidics; sigma factors; signal transduction; stress proteins; stress response.

FIG 1

(A) Schematic of Bacillus subtilis cell containing stressosomes within the cytoplasm sensing stress and triggering the activation of σ^B. Once activated, σ^B turns on gene expression of the general stress response, including the P_rsbV-mNeonGreen reporter that we used to detect when σ^B is active. (B) Schematics of the stressosome complex in the wild type and stressosomes formed by single RsbR paralogs. Stressosomes are comprised of 20 RsbS proteins (gray) and 20 RsbR dimers (RsbRA in red, RsbRB in green, RsbRC in purple, and RsbRD in orange). All strains in this study contained the ΔytvA deletion of the blue light sensor to prevent activation by fluorescence imaging. (C) Diagram of RsbR paralogs compared to one another, as well as RsbS, with their phosphorylation sites highlighted. RsbR paralogs and RsbS share a conserved C-terminal sulfate transporter and anti-sigma factor antagonist (STAS) domain, which makes up the core of the stressosome. (D) Image of a single cell lineage in a channel in the microfluidic device. The red fluorescent protein (RFP) image shows the cell boundaries, whereas the green fluorescent protein (GFP) image reports on σ^B activity. The yellow box is a 5 × 50-pixel region of interest (ROI) drawn in ImageJ for analysis, from which we obtained a kymograph depicting that ROI over time. The ROI was also used to obtain the mean GFP fluorescence value to plot over time for graph generation. Data curation and analysis were manually performed for each individual cell lineage.

FIG 2

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in a wild-type (WT) strain (MTC1801) and strains containing only RsbRA (MTC1761), RsbRB (MTC1763), RsbRC (MTC1765), and RsbRD (MTC1767) before and after the addition (indicated by vertical dotted line) of 2% ethanol. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Asterisks in the EtOH trace denote peaks that were present in only some lineages corresponding to the same experimental replicate (see also Fig. S1) and hence are considered artifactual.

FIG 3

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in a wild-type strain (MTC1801) before and after the addition (indicated by vertical dotted line) of the indicated stressors. The stressors were 2% ethanol, 0.005% H₂O₂, 500 mM NaCl, and pH shift from 6.5 to 6.25. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Asterisks in the EtOH trace denote peaks that were present in only some lineages corresponding to the same experimental replicate (see also Fig. S1) and hence are considered artifactual.

FIG 4

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in an RsbRA-only strain (MTC1761) before and after the addition (indicated by vertical dotted line) of the indicated stressors. The stressors were 2% ethanol, 0.005% H₂O₂, 500 mM NaCl, and pH shift from 6.5 to 6.25. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Asterisks in the NaCl trace denote peaks that were present in only some lineages corresponding to the same experimental replicate (see also Fig. S2) and hence are considered artifactual.

FIG 5

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in an RsbRB-only strain (MTC1763) before and after the addition (indicated by vertical dotted line) of the indicated stressors. The stressors were 2% ethanol, 0.005% H₂O₂, 500 mM NaCl, and pH shift from 6.5 to 6.25. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Representative individual traces are shown in Fig. S3.

FIG 6

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in a an RsbRC-only strain (MTC1765) before and after the addition (indicated by vertical dotted line) of the indicated stressors. The stressors were 2% ethanol, 0.005% H₂O₂, 500 mM NaCl, and pH shift from 6.5 to 6.25. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Representative individual traces are shown in Fig. S4.

FIG 7

(A) Individual-lineage intensity traces of a stress-responsive P_rsbV-mNeonGreen reporter in an RsbRD-only strain (MTC1767) before and after the addition (indicated by vertical dotted line) of the indicated stressors. The stressors were 2% ethanol, 0.005% H₂O₂, 500 mM NaCl, and pH shift from 6.5 to 6.25. (B) Average intensity of the P_rsbV-mNeonGreen reporter corresponding to the single-cell traces from panel A. Representative individual traces are shown in Fig. S5.

FIG 8

(A) Schematic illustrating the domain architecture of two different RsbR paralogs (RsbRA and RsbRC are shown as examples) and a hybrid between them, comprising the N-terminal variable domain or RsbRC and the C-terminal conserved domain of RsbRA. The schematic also illustrates the nomenclature of the example RsbRC/A hybrid. (B) Average traces of P_rsbV-mNeonGreen reporter in hybrid strains (see strain table for complete genotypes and accession numbers) before and after addition (indicated by the vertical dotted line) of 2% ethanol. High average responses (gray lines) and low average responses (black) were distinguished, with the horizontal dotted line at 135 AU as a visual guide for “strong” and “weak” responses. (C) Individual average traces of P_rsbV-mNeonGreen reporter in the indicated hybrid strains with a strong response (A or C in the N terminus) before and after addition of 2% ethanol. (D) Individual average traces of P_rsbV-mNeonGreen reporter in the indicated hybrid strains with a weak response (B or D in the N terminus) before and after addition of 2% ethanol. (E) Traces of strong-response strains (as in panel B) displayed individually. (F) Traces of weak-response strains (as in panel C) displayed individually.