Bacteria use type-IV pili to slingshot on surfaces

Abstract

Bacteria optimize the use of their motility appendages to move efficiently on a wide range of surfaces prior to forming multicellular bacterial biofilms. The "twitching" motility mode employed by many bacterial species for surface exploration uses type-IV pili (TFP) as linear actuators to enable directional crawling. In addition to linear motion, however, motility requires turns and changes of direction. Moreover, the motility mechanism must be adaptable to the continually changing surface conditions encountered during biofilm formation. Here, we develop a novel two-point tracking algorithm to dissect twitching motility in this context. We show that TFP-mediated crawling in Pseudomonas aeruginosa consistently alternates between two distinct actions: a translation of constant velocity and a combined translation-rotation that is approximately 20× faster in instantaneous velocity. Orientational distributions of these actions suggest that the former is due to pulling by multiple TFP, whereas the latter is due to release by single TFP. The release action leads to a fast "slingshot" motion that can turn the cell body efficiently by oversteering. Furthermore, the large velocity of the slingshot motion enables bacteria to move efficiently through environments that contain shear-thinning viscoelastic fluids, such as the extracellular polymeric substances (EPS) that bacteria secrete on surfaces during biofilm formation.

Fig. 1.

Analysis of the two-point velocity profiles of the ΔfliM mutant. (A) x and y position of one focus (p_x(t) and p_y(t)) as a function of time. Yellow symbols show raw data and cyan lines indicate the denoising regression (threshold = 2 pixels). (B) Velocity amplitudes (|v_pLead(t)| and |v_pTrail(t)|) of foci p_Lead and p_Trail as a function of time. Velocity amplitudes are calculated from the regression data.

Fig. 2.

Identical ΔfliM bacteria exhibit individuation of motion preferences. (A) 2D histogram of |v_pLead(t)| versus |v_pTrail(t)|, from datasets of two different ΔfliM mutant bacteria. The dashed line (slope = 1.0) is a guide to the eye to indicate translational motion (|v_pLead(t)| = |v_pTrail(t)|); the dashed circle is a guide to the eye to indicate rotational motion (|v_pLead(t)| ≠ |v_pTrail(t)|); the dotted green lines indicate the velocity threshold (|v_pLead,pTrail| = 0.3 μm/s) separating release actions and pull actions. Genetically identical bacteria exhibit distinct individual motion preferences: (i) rotation; (ii) translation. (B) Portion of trajectories from (top) bacterium (i), with schematic illustrating rotational motion, and (bottom) bacterium (ii), with schematic illustrating translational motion. In (B) the blue and red circles indicate positions of p_Lead and p_Trail, respectively.

Fig. 3.

Analysis of the velocity of leading pole p_Lead reveals distinct pulls and releases. (A) Schematic definition of an action in the velocity profile: For the ith action A_i, |v_pLead(A_i)| is the velocity amplitude, τ(A_i) is the duration time, and D(A_i) is the total displacement. (B) 2D histogram of the velocity amplitude of connected actions (A_i and A_i+1) for leading pole p_Lead. (C) Velocity amplitude |v_pLead(A)| as a function of duration τ(A) for the leading pole p_Lead in the trajectory of a single ΔfliM bacterium (N = 12,000 points); the color scale indicates the total displacement D(A). The two dashed lines (slope = -1.0) are guides to the eye indicating total displacements between 0.01 μm and 0.1 μm. (D) Histogram of the duration τ(A). The dashed line indicates the time threshold (τ_c = 0.2 s) separating release actions (red bars) from pull actions (blue bars). (E) Histogram of the velocity amplitude |v_pLead(A)|. The dashed line indicates the velocity threshold (|v_pLead,c| = 0.3 μm/s) separating release actions (red bars) from pull actions (blue bars). (F) Histogram of the displacement D(A) of release actions (red bars) and of pull actions (blue bars). In D–F the line is the Gaussian fit to the distribution.

Fig. 4.

Identical ΔfliM bacteria exhibit distinct orientation preferences. (Inset A) Schematic plot of the p_Lead action velocity vector; θ is the deviation angle between the p_Lead velocity vector and the body axis of the bacterium, with clockwise (counterclockwise) motion defined as positive (negative). (A) Normalized distribution of the deviation angle (θ), p(θ) calculated from 10 trajectories of different single ΔfliM bacteria, including approximately 180,000 images. (B) Distribution of deviation angle for a single bacterium, showing that single cells also exhibit orientational preference. In A and B distributions for release actions (red line) and pull actions (blue line) are decomposed from the entire trajectory.

Fig. 5.

Model for twitching motility. Schematic illustrating the mechanisms for the alternating pull (left) and release (right) actions of a crawling bacterium.