Functional studies of the stressosome

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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.