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.

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.

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ(B) stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a σ(B)-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.