Firmicutes form long-lived, resistant spores. In response to germinants, they metamorphose into active vegetative cells. The demands of resistance/dormancy versus rapid germination must be reconciled, but how? The radical structural changes taking place in germination will be our focus, specifically the spore-cellular envelope transition. These proteinaceous envelopes are resistant assemblies, so how are they breached to allow the cell to emerge? If we can explain in structural terms how the spore-vegetative cell transition takes place we can exploit vulnerabilities. We will use CryoEM, AFM, fluorescence microscopy & molecular biology to understand how this remarkable metamorphosis takes place. We will test our ability to perform structural work on an anaerobe- C. sporogenes. We will develop a workflow of analysis from germination to the final mature vegetative state. We will then apply our gained expertise to C. difficile. (1) We will determine 'baseline' structures from which we can follow changes & test hypotheses. We will reveal for the first time the 3D structure of a Clostridial spore in molecular detail. (2) We will follow germinating spores in a time-resolved manner initially as snapshots by CryoEM but then dynamically by AFM. We will establish early surface changes that occur in germination/outgrowth. (3) We will map envelope domains involved in cell exit, by CryoEM, AFM, fluorescence microscopy and mutagenesis. AFM will map mechanical properties. We will generate the first 3D picture simultaneously revealing the architectural, mechanical & chemical properties of the spore envelope. (4) We will test whether the envelope breach is enzymatic or mechanical. We will knock out structural proteins & proteases. We will complement these studies by time-resolved AFM. (5) By fluorescence microscopy, CryoEM and AFM we will visualise the timing & localisation of appearance of new protein subunits on the emerging vegetative cell surface.