The flagellum is a rotary motor with a filament several times longer than the cell. Most of its subunits are exported by a type-III secretion system and assemble outside the cytoplasm.
Flagella enable bacteria to swim towards nutrients and away from harmful substances. Many pathogens, including Salmonella enterica, require motility to reach and colonise the host. The filament consists of several thousand flagellin subunits. Each subunit is exported in an unfolded state by the flagellar type-III secretion system (fT3SS), travels through the 2 nm channel of the growing structure and folds at the tip.
100 nm/mininitial elongation rate
We labelled successive filament fragments with maleimide dyes and measured filament growth in single cells. The elongation rate decreased with filament length, from about 100 nm/min initially to about 20 nm/min. A model in which the proton motive force injects subunits into the channel and the subunits then diffuse to the tip describes these kinetics.
2.5 µmminimal filament length
We synchronised flagellar assembly in Salmonella and related filament length to swimming speed. Cells with filaments shorter than about 2.5 µm did not swim. Biophysical modelling attributes this threshold to an elasto-hydrodynamic instability of the swimming cell.
10,000 aa/smaximal secretion rate
Stepwise filament labelling combined with electron microscopy confirmed flagellin secretion rates of up to 10,000 amino acids per second. In a model of filament growth, this rate balances the elongation rate against the energy cost per subunit. A higher injection rate would shorten the time to motility only slightly.
3.7 Åresolution of the FliD cap
Together with the Bergeron group, we determined cryo-EM structures of the complete extracellular flagellum, including the FliD cap at 3.7 Å and the hook-filament junction at 2.9 Å. The structures and structure-guided mutagenesis show that movement of the FliD terminal domains and rotation of the cap allow each flagellin subunit to fold into the filament.
5:2MotA:MotB stoichiometry
Together with the Taylor group, we showed that the stator unit consists of a MotA pentamer around a MotB dimer. Structures in different functional states support a model in which proton flux drives rotation of MotA around MotB. MotA in turn rotates the rotor of the flagellum.
Salmonella mutants that lack the periplasmic protein FlhE assembled flagella in the periplasm. These flagella disrupted cell-wall synthesis and caused cell lysis. We conclude that FlhE acts as a chaperone during rod assembly.
We analysed Salmonella strains with mutations in the catalytic domain of the export ATPase FliI. Minimal ATPase activity was sufficient for early substrate export and hook-basal-body assembly. Efficient substrate-specificity switching and export of late substrates required near-wild-type activity.
We investigate the regulation of the flagellar genes and the heterogeneity of flagellation in single cells, the type-III export apparatus, the filament cap and the recruitment of stator units. We measure the unfolding energy that the fT3SS applies during early and late secretion, and the kinetics of hook assembly. We also compare external and periplasmic flagella.
Contact: Marc Erhardt, E-mail
ANR-DFG project BacCellFactory
The fT3SS secretes proteins into the culture medium at a high rate. Together with the Université de Bordeaux, we determine which factors limit the secretion of recombinant proteins. We engineer strains for the production of peptides and proteins for diagnostics and biomedicine.
Contact: Min Meijer, E-mail
