Functional studies of the stressosome

For survival, all organisms must respond to changes in their environment. One of the most important ways by which all organisms signal to themselves that a response is required to a change in growth conditions is mediated by protein phosphorylation. Here, the covalent modification of regulatory proteins by phosphorylation changes the properties of the target protein and hence its functions. Phosphorylation is performed by enzymes called kinases, which require ATP as a co-factor for the phosphorylation reaction. The stress response of the bacterium Bacillus subtilis and its close relatives is also regulated by phosphorylation. The response to stress provides the cell with a protective mechanism against a wide range of chemical and physical insults. Ultimately the stress signaling pathway controls the activity of the molecular machine, RNA polymerase, which is required to initiate the first step in the synthesis of the proteins that together act to provide the cell with its resistance against the imposed stress. Key to the information-processing pathway that indicates to the cell that its environment is deteriorating is a large structure called the 'stressosome'. The stressosome is composed of several proteins that together assembles the stressosome into a small virus-like structure. The proteins in this 'mini-virus' act in concert to trap a key kinase prior to stress and then to release it on stress to act in the signaling system that ultimately activates RNA polymerase. We are currently determining the structure of the stressosome in a variety of functional states and these on-going studies are challenging us to answer questions that the structures pose. These answers will only be obtained by functional studies using classical biochemical approaches in combination with bacterial cell biology techniques. For instance, we need to establish why the stressosome exists in the first place - perhaps its purpose is to provide a response to stress that is much greater in magnitude than the stress signal to guarantee that the health of the cell is maintained. We will aim to destroy the formation of the stressosome complex to see the impact on this disruption on the ability of the bacterial cell to respond appropriately to stress. Furthermore, there are missing components in the stress-signalling pathway, for instance how is stress actually perceived by a single-celled organism? Clues to the cellular location and identity of stress-receptors may be found by tagging the key signalling kinase with a protein that will colour the kinase green and enable us to track its location using a microscope during the cell cycle and during the response to stress. Finally, stressosome proteins are also found in many other bacteria and they are proposed to, and we will demonstrate that they do play roles regulating other important cellular processes.

Macromolecular assemblies and post-translational modifications of proteins play pivotal roles in a vast array of biological processes including signal transduction cascades. The signaling system we study regulates the response of Bacillus subtilis and its relatives to stress in a phosphorylation-dependent fashion. The phosphorylation state of proteins in the signaling pathway regulates the availability of sigmaB for the formation of transcriptionally-competent complexes with RNA polymerase, activating the transcription of genes required for the response to stress. At the core of this stress signaling pathway is a large complex structure termed the stressosome. We are currently engaged in a study that focuses on the structural analysis of the stressosome, using a combination of X-ray crystallography with cryo-electron microscopy and single particle analysis. Our structural studies have revealed the exquisite beauty of the stressosome, which resembles a small icosahedral virus. The interpretation of our preliminary molecular envelopes from cryo-EM data is on-going and in this proposal we seek to obtain answers to questions of stressosome function that the structures pose. We will investigate for the presence of allostery in the signalling system using classical biochemistry and establish by genetics the fate of B. subtilis cells if stressosomes formation is disrupted. We will use microbial cell biology GFP-tagging techniques to study the localisation of stressosomes in real time during the response to stress. Finally, we will confirm the widespread importance of stressosome homologues in other bacteria that appear, but are not yet demonstrated, to regulate other important cellular processes such as the biosynthesis of secondary messenger signaling molecules.