A huge but elusive macromolecular cage in enterobacterial stress response as seen by cryoEM and X-ray crystallography

Orateur : Irina GUTSCHE (IBS/Groupe Microscopie Electronique et Méthodes)

The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close biosynthetic paralogue. We showed that a unique macromolecular cage formed by two decamers of LdcI and five hexamers of the AAA+ ATPase RavA could counteract acid stress under starvation in E. coli (El Bakkouri et al., 2010 ; Kanjee et al., 2011). We constructed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer (Malet et al., 2014). We also performed cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC at their active pH. Comparison with each other and with available structures, coupled to a phylogenetic analysis of enterobacterial lysine decarboxylases, uncovered differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA (Kandiah et al., 2016). The core genome of an important pathogen of plants, animals and humans, Pseudomonas aeruginosa, contains a gene that codes for a PLP-dependent lysine decarboxylase, whereas the ravA gene appears to be present only in the accessory genome of some virulent P. aeruginosa strains. Thus, in order to study the structure-function relationships of these proteins in P. aeruginosa in comparison to E. coli we analyzed the function of P. aeruginosa Ldc in vivo and produced its structure at 4.2 A resolution by cryoEM. The resulting pseudoatomic model of the P. aeruginosa Ldc allows to determine if it is closer to the E. coli LdcI or LdcC, to predict if it has a potential of forming a cage with RavA, and to get more insights into the role of this system in different bacteria. Further research by a combination of cryoEM, X-ray crystallographym super-resolution fluorescence microscopy and other structural and biochemical, biophysical and cell biology techniques is on-going.

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