Flagellar hook flexibility is essential for bundle formation in swimming Escherichia coli cells

Abstract

Swimming Escherichia coli cells are propelled by the rotary motion of their flagellar filaments. In the normal swimming pattern, filaments positioned randomly over the cell form a bundle at the posterior pole. It has long been assumed that the hook functions as a universal joint, transmitting rotation on the motor axis through up to ∼90° to the filament in the bundle. Structural models of the hook have revealed how its flexibility is expected to arise from dynamic changes in the distance between monomers in the helical lattice. In particular, each of the 11 protofilaments that comprise the hook is predicted to cycle between short and long forms, corresponding to the inside and outside of the curved hook, once each revolution of the motor when the hook is acting as a universal joint. To test this, we genetically modified the hook so that it could be stiffened by binding streptavidin to biotinylated monomers, impeding their motion relative to each other. We found that impeding the action of the universal joint resulted in atypical swimming behavior as a consequence of disrupted bundle formation, in agreement with the universal joint model.

Fig 1

Design and functionality of engineered strains. (a) Three-dimensional density maps of the hook, adapted from Structure (17) with permission from Elsevier. Shown is the structure of three FlgE units with the insertion sites for the AviTag marked in red and labeled A through E (Protein Data Bank [PDB] identifier 3A69). Scale bar, 50 Å. (b) Swim plate analysis of strains relative to the WT and a nonmotile control (ΔfliC). (c) Fluorescent images of streptavidin-Alexa Fluor 532 attached to exogenously biotinylated hooks; exposure, 300 ms; scale bar, 3 μm.

Fig 2

Biophysical characterization of site A and site C AviTag mutants in response to streptavidin. (a) Hook stiffness of single tethered cells over time calculated using the equipartition theorem (see the text for details). Streptavidin (100 μM) was added at t = 5 min; stiffnesses are means ± standard errors of 28, 31, and 27 hooks from WT, site A, and site C strains, respectively. The inset shows the variance in body angle of a single cell from the site A data set versus time. (b) Analysis of free-swimming cells (see Videos S1, S2, and S3 in the supplemental material); histograms of the difference in angle between the cells' swimming trajectory and the angle of the cell body (illustrated by the inset). A cell swimming along its long axis gives an angle of 0°. (c) Selected video frames (3-ms exposure) of swimming cells dyed with Cy3 monofunctional succinimidyl ester showing the position of filaments. Scale bar, 3 μm (see Videos S4, S5, and S6).

Fig 3

Quantification of the number of streptavidin molecules on the hook. (a) Stoichiometry of streptavidin-Alexa Fluor 532-labeled hooks via single molecule stepwise photobleaching. The top panel shows TIRF (false-color) images taken every 100 ms; the first image has the corresponding bright-field image superimposed. Scale bar, 1 μm. At the bottom is a histogram of data collected at saturating levels of streptavidin-Alexa Fluor 532 (≥10 μM; number of cells = 50 and number of spots = 114). Inset, stoichiometry at different concentrations of streptavidin-Alexa Fluor 532 fit with a Michaelis-Menten curve (K_m = 2 μM; V_max = 36). Measurements were made at concentrations of 0.1 μM (28, 50), 1 μM (19, 45), 5 μM (5, 5), 10 μM (15, 36), 50 μM (4, 17), 75 μM (13, 26), and 100 μM (18, 35); numbers in parentheses are numbers of cells and spots. (b) Schematic of streptavidin bound to the hook, based on the model of Samatey and coworkers (adapted by permission from Macmillan Publishers Ltd.: Nature [30]) (PDB, 1WLG). The α-carbon atoms of the curved hook are displayed as a solvent-accessible surface, with the D1 domains shown in white and the D2 domains shown in blue. Streptavidin molecules (PDB, 1SWE [15]) are shown as gray surfaces. The top panel shows how streptavidin binding along the 11-start helix may lead to steric clashes (red). The bottom panel shows an alternative packing strategy that avoids steric clashes, where the streptavidins are positioned in alternating arrangements along the 11-start helices. Images were generated using PyMOL.