Genetic evidence for a phosphorylation-independent signal transduction mechanism within the Bacillus subtilis stressosome.

The stressosome is a 1.8 MDa cytoplasmic complex that controls diverse bacterial signaling pathways. Its role is best understood in Bacillus subtilis, where it activates the σB transcription factor in response to a variety of sharp environmental challenges, including acid, ethanol, heat or salt stress. However, details of the signaling mechanism within the stressosome remain uncertain. The core of the complex comprises one or more members of the RsbR co-antagonist family together with the RsbS antagonist protein, which binds the RsbT kinase in the absence of stress. As part of the response, RsbT first phosphorylates the RsbRA co-antagonist on T171 and then RsbS on S59; this latter event correlates with the stress-induced release of RsbT to activate downstream signaling. Here we examine the in vivo consequence of S59 phosphorylation in a model strain whose stressosome core is formed solely with the RsbRA co-antagonist and RsbS. A phosphorylation-deficient S59A substitution in RsbS blocked response to mild stress but had declining impact as stress increased: with strong ethanol challenge response with S59A was 60% as robust as with wild type RsbS. Genetic analysis narrowed this S59-independent activation to the stressosome and established that significant signaling still occurred in a strain bearing both the T171A and S59A substitutions. We infer that S59 phosphorylation increases signaling efficiency but is not essential, and that a second (or underlying) mechanism of signal transduction prevails in its absence. This interpretation nullifies models in which stressosome signaling is solely mediated by control of RsbT kinase activity toward S59

Genetic evidence for a phosphorylation-independent signal transduction mechanism within the Bacillus subtilis stressosome.

The stressosome is a 1.8 MDa cytoplasmic complex that controls diverse bacterial signaling pathways. Its role is best understood in Bacillus subtilis, where it activates the σB transcription factor in response to a variety of sharp environmental challenges, including acid, ethanol, heat or salt stress. However, details of the signaling mechanism within the stressosome remain uncertain. The core of the complex comprises one or more members of the RsbR co-antagonist family together with the RsbS antagonist protein, which binds the RsbT kinase in the absence of stress. As part of the response, RsbT first phosphorylates the RsbRA co-antagonist on T171 and then RsbS on S59; this latter event correlates with the stress-induced release of RsbT to activate downstream signaling. Here we examine the in vivo consequence of S59 phosphorylation in a model strain whose stressosome core is formed solely with the RsbRA co-antagonist and RsbS. A phosphorylation-deficient S59A substitution in RsbS blocked response to mild stress but had declining impact as stress increased: with strong ethanol challenge response with S59A was 60% as robust as with wild type RsbS. Genetic analysis narrowed this S59-independent activation to the stressosome and established that significant signaling still occurred in a strain bearing both the T171A and S59A substitutions. We infer that S59 phosphorylation increases signaling efficiency but is not essential, and that a second (or underlying) mechanism of signal transduction prevails in its absence. This interpretation nullifies models in which stressosome signaling is solely mediated by control of RsbT kinase activity toward S59

Strains bearing the S59A substitution retain significant stress activation.

<p>β-galactosidase accumulation from a σ^B-dependent <i>ctc-lacZ</i> fusion, assayed in logarithmically growing cells either wild type for RsbS (open circles) or with the S59A substitution (closed circles), before and after 4% ethanol addition. In all three panels an RsbU null strain defective for the response served as the negative control (PB495, open triangles). (A) Wild type stressosome with all four co-antagonists and wild type RsbS (PB198, open circles); RsbS-S59A (PB470, closed circles); or RsbU null with RsbS-S59A (PB1274, closed triangles). (B) Minimal stressosome containing RsbRA as sole co-antagonist and wild type RsbS (PB1078, open circles); RsbS-S59A (PB1161, closed circles); or RsbU null with RsbS-S59A (PB1273, closed triangles). (C) Minimal stressosome containing RsbRB as sole co-antagonist and wild type RsbS (PB1255, open circles); RsbS-S59A (PB1256, closed circles); or RsbU null with RsbS-S59A (PB1275, closed triangles). Representative results are shown; in independent experiments S59A supported a response 13% as robust as wild type RsbS in the strain with all four co-antagonists (+/−2.0% SEM, n = 4); 63% in the strain with RsbRA alone (+/−2.1%, n = 7); and 33% in the strain with RsbRB alone (+/−1.3%, n = 3).</p

S59-independent activation does not require the YtvA blue-light sensor.

<p>Peak β-galactosidase accumulation from a <i>ctc-lacZ</i> fusion after 4% ethanol stress, in the presence or absence of YtvA (+YtvA or ΔYtvA). Strains encoded a minimal stressosome containing RsbRA as sole co-antagonist together with either wild type RsbS (PB1078 for +YtvA or PB1085 for ΔYtvA; open bars) or RsbS bearing the S59A substitution (PB1161 or PB1272; shaded bars). Error bars represent range in two independent assays.</p

Positive epistasis between S59A and T171A in a model stressosome.

<p>β-galactosidase accumulation from a <i>ctc-lacZ</i> fusion in logarithmically growing cells, before and after 4% ethanol addition. (A) Wild type stressosome with all four co-antagonists and wild type RsbS and RsbRA (PB198, open circles); RsbS-S59A (PB470, closed circles); RsbRA-T171A (PB830, open squares); or RsbS-S59A together with RsbRA-T171A (PB1219, closed squares). (B) Minimal stressosome containing RsbRA as sole co-antagonist and wild type RsbS and RsbRA (PB1078, open circles); RsbS-S59A (PB1161, closed circles); RsbRA-T171A (PB1205, open squares); or RsbS-S59A with RsbRA-T171A (PB1190, closed squares). Representative results are shown; in independent experiments the T171A-S59A mutant manifested a response 0.2% that of wild type RsbRA and RsbS in the strain with all four co-antagonists (+/−0.03% SEM, n = 3); and 21% in the strain with RsbRA alone (+/−5.8%, n = 3).</p

The PrpC Serine-Threonine Phosphatase and PrkC Kinase Have Opposing Physiological Roles in Stationary-Phase Bacillus subtilis Cells

Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of β-galactosidase from a general stress reporter fusion. These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells. Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro

Plasmids for strain construction.

<p>Plasmids for strain construction.</p

Stressosome and model of RsbRA-RsbS-RsbT activation.

<p>(A) Cytoplasmic stressosome complex (RsbR-RsbS-RsbT) controls activation of the environmental phosphatase (RsbU) in response to diverse physical and chemical signals. STAS domains of RsbR and RsbS form the stressosome core (red); N-terminal domains of RsbR are hypothetical sensors (red crosshatch); dissociable RsbT kinase (green) is positive activator of RsbU (yellow). RsbU removes the phosphoryl group (orange) from S56 on the RsbV anti-anti-σ, ultimately activating the σ^B stress factor. The energy phosphatase (RsbP) and the phosphatase-independent cold stress pathways are shown in dotted outline. Arrowheads indicate activation of protein targets or enzymatic reactions; T-headed lines indicate inhibition. (B) Model of stressosome control of RsbU activity. Stressosome core comprises partially redundant RsbRA, RB, RC, and RD co-antagonists (represented here as RsbRA) and the RsbS antagonist, which binds the RsbT kinase. In unstressed cells RsbT phosphorylates RsbRA on T171, facilitating subsequent activation of RsbT kinase activity toward RsbS (+ arrow). During the stress response RsbT phosphorylates RsbS on S59; RsbT is released to bind and activate RsbU. The RsbX feedback phosphatase (dotted outline) dephosphorylates RsbS-P. (C) RsbS comprises a single STAS domain (red rectangle), whereas the larger RsbRA has an N-terminal non-heme globin domain (red crosshatching) and C-terminal STAS domain (red). Phosphorylated S59 and T171 residues lie in the STAS domains; T205 (light grey) is phosphorylated only under extreme stress (see text). RsbRB, RC and RD are similar to RsbRA, with corresponding phosphorylated residues (not shown); these three paralogs were removed in some strains. YtvA is an RsbR family member that increases stressosome output in response to blue light, sensed by its N-terminal LOV (Light-Oxygen-Voltage) domain (blue). Figure modified from ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090741#pone.0090741-Gaidenko3" target="_blank">[20]</a>.</p