Abstract
The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B. subtilis, P. aeruginosa and Salmonella enterica. Using the detailed information about the molecular interactions in two filament states, we successfully predict point mutations that shift the equilibrium between those two states. Further, we observe the dimerization of P. aeruginosa outer domains without any perturbation of the conserved interior of the filament. Our results give new insights into how the flagellin sequence has been “tuned” over evolution.
Highlights
The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity
It has served as an enlightening system for understanding how a protein polymer composed of a single protein, flagellin switches among different states to supercoil
Flagellin protein in Bacillus subtilis is expressed from the hag gene[29], which is homologous to fliC in Salmonella enterica
Summary
The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. The bacterial flagellar filament has been intensively studied for many years[2,3,4] It has served as an enlightening system for understanding how a protein polymer composed of a single protein, flagellin (except for the cap protein at the end that acts as an assembling chaperone5, 6) switches among different states to supercoil. Based upon extensive work from the Namba laboratory[23,24,25,26] using X-ray fiber diffraction, X-ray crystallography, and cryo-EM, atomic models have been proposed for straight Salmonella enterica filaments with all protofilaments in either the R-state[25] or the L-state[24] While these two atomic models represent a significant advance in understanding polymorphic switching of bacterial flagellar filaments, they do not provide sufficient mechanistic understanding of how switching occurs. We attempt to answer these questions and others using a direct electron detector by generating and studying locked, straight flagellar filaments from the Gram-positive bacterium B. subtilis and the Gram-negative P. aeruginosa with high resolution cryo-EM
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