AbstractPathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.
Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS
