Genetic Evidence for a Phosphorylation-Independent Signal Transduction Mechanism within the Bacillus subtilis Stressosome

Abstract

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 role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus

“…Therefore, whatever the transduced signal may be, the system seems unrelated to classical bacterial phosphorylation relays. Thus, a phosphorylation‐independent conformational change or binding/unbinding to the membrane itself may propagate the transduced signal as in other systems (Gaidenko and Price, ). These putative two forms (DrpB On and DrpB Off ) are represented in the figure as quadrangular and oval forms.…”

Mechanism and Role of Globin-Coupled Sensor Signalling

“…As phosphorylation of both RsbR and RsbS was absolutely required for activation of the B. subtilis Rsb signaling pathway (Gaidenko & Price, 2014; Kim et al, 2004a), these studies suggest that Vb RsbR controls O 2 -dependent stressosome signaling in V. brasiliensis (Figure 13). These data also suggest that the conformational changes that occur in Vb RsbR upon O 2 association/dissociation are sufficient for signal propagation to multiple binding partners (Jia et al, 2016).…”

“…The non-heme globin maintains the sensor globin structure, but mutations within the globin heme pocket (including mutation of the proximal histidine) yielded a protein with a sensor globin fold that is unable to bind heme (Murray et al, 2005). The stressosome was demonstrated to be an ~1.8 MDa complex in B. subtilis formed by RsbR, RsbS (STAS domain that makes protein-protein interactions with the RsbR STAS domain), and RsbT (kinase, phosphorylates RsbR/RsbS) that senses environmental stress and, through a phosphorylation cascade involving additional rsb proteins, eventually resulting in activation of σ B and transcription of over 150 genes (Eymann et al, 2011; Gaidenko & Price, 2014; Kim, Gaidenko, & Price, 2004a, 2004b; Marles-Wright et al, 2008; Price et al, 2001). However, the ligand(s) that bind and activate the B. subtilis non-heme globin-containing RsbR are unknown.…”

“…However, the ligand(s) that bind and activate the B. subtilis non-heme globin-containing RsbR are unknown. Therefore, bioinformatic searches that predicted the occurrence of GCS proteins with STAS output domains suggested that these proteins could provide insights into both stressosome signaling and function of additional GCS proteins (Chen et al, 2004; Gaidenko & Price, 2014; Murray et al, 2005). …”

Activation of the General Stress Response of Bacillus subtilis by Visible Light

“…Phosphorylation of stressosome proteins is critical for the release of (the kinase) RsbT and the initiation of the GSR , although there may also be a phosphorylation‐independent way to release RsbT . RsbS contains a single serine residue that can be phosphorylated by RsbT: Ser59 .…”