The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria

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 σ(B) 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

Selective Heterogeneity in Exoprotease Production by <em>Bacillus subtilis</em>

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the Gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical “bet-hedging” behaviour