Direct upstream motility in Escherichia coli

Abstract

We provide an experimental demonstration of positive rheotaxis (rapid and continuous upstream motility) in wild-type Escherichia coli freely swimming over a surface. This hydrodynamic phenomenon is dominant below a critical shear rate and robust against Brownian motion and cell tumbling. We deduce that individual bacteria entering a flow system can rapidly migrate upstream (>20 μm/s) much faster than a gradually advancing biofilm. Given a bacterial population with a distribution of sizes and swim speeds, local shear rate near the surface determines the dominant hydrodynamic mode for motility, i.e., circular or random trajectories for low shear rates, positive rheotaxis for moderate flow, and sideways swimming at higher shear rates. Faster swimmers can move upstream more rapidly and at higher shear rates, as expected. Interestingly, we also find on average that both swim speed and upstream motility are independent of cell aspect ratio.

Figure 1

Experimental setup. (a) WT E. coli were subjected to various shear flows within a microfluidic channel and imaged from below in an inverted microscope setup. (b) Cells were observed at the center of the channel ceiling as they swam over its PDMS surface. In choosing a coordinate system for data analysis, we follow the convention of Kaya and Koser (19), where the +y axis points upstream and the z axis denotes the surface normal (for channel ceiling) pointing into the flow channel. In this convention, a positive y velocity component corresponds to upstream migration. The cell body orientation angle (ϕ) is measured relative to the +x axis. The microscope-camera system flips the images along the vertical image axis, such that the z axis points into the page in recorded pictures (see Fig. 3).

Figure 2

Bacteria orientation angles (ϕ) and swim speed components under no-flow conditions. (a) The simulated PDF of ϕ is uniform under circular or random swimming assumptions. (b) Assuming a constant swim speed (v = 22.3 μm/s), the corresponding velocity components along the x or y axes of the surface (v_x or v_y) have a characteristic U-shaped distribution, with their PDF peaking at ±v. (c) In reality, the measured swim speed of the bacteria is normally distributed (μ = 22.3 μm/s; σ = 4.6 μm/s at the beginning of the experiment). (d) The corresponding PDFs for v_x and v_y are M-shaped and symmetric about the origin.

Figure 3

Sample bacterial trajectories. (a) Under quiescent conditions, bacteria swam in circular trajectories, occasionally tumbling or colliding, and randomly changing direction. (b) Once moderate laminar flow was introduced, most bacteria rapidly turned to face and swim upstream (i.e., positive rheotaxis; trajectories shown for 5.9 s⁻¹ shear). The overall population displayed positive rheotaxis until 6.4 s⁻¹. (c) At higher shear rates, an increasing percentage of bacteria succumbed to the drag from the flow and swam sideways to eventually get out of the fast shear (trajectories shown for 35 s⁻¹ shear). In a–c, images are contrast-enhanced composites comprising minimum pixels of every fifth sequential frame (20). Original frame rates: 30 frames/s for a and b, and 240 frames/s for c. See Movie S1, Movie S2, and Movie S3 for original image sequences.

Figure 4

Three hydrodynamic modes of E. coli swimming over a surface. (a) Without flow, there is no net rheotaxis (i.e., v_x and v_y distributions are symmetric and zero mean) and all cell body orientation angles (ϕ) are equally likely. Here, we depict the PDFs for instantaneous v_x, v_y, and ϕ values at a shear rate just above 0 (∼0.1 s⁻¹) to elucidate the beginning of upstream orientation. (b) Positive rheotaxis is dominant up to a critical shear rate (6.4 s⁻¹), with the majority of the population swimming upstream (up to a mean v_y of 4 μm/s). Here, a peak in ϕ distribution just under 90° is clearly visible at a shear rate of 5.9 s⁻¹. (c) As the shear rate is increased beyond the critical, the bacteria begin to turn toward the +x axis and swim sideways as they get dragged by the flow. The ϕ peak correspondingly shifts to lower angles and approaches 0° with increasing flow. Here, the shear rate is 35 s⁻¹.

Figure 5

Evolution of E. coli hydrodynamic characteristics over the course of our experiment. (a) The (Gaussian) swim speed (v) distributions for the bacteria under no-flow conditions were virtually the same before (μ = 22.3 μm/s; σ = 4.6 μm/s; dashed curve) and after (μ = 22.5 μm/s; σ = 5.0 μm/s; solid curve) the experiment (>7 h long). (b) The PDF for cell length (log-normal; μ = 4.9 μm; σ = 1.3 μm) was also unchanged during this period. Results in A and B imply that the hydrodynamic characteristics of E. coli remained constant throughout the experiment. (c) The (Gaussian) PDF for deduced velocity (v_ded; μ = 22.2 μm/s; σ = 5.1 μm/s; solid curve) matches that for v (shown here for the beginning of the experiment; histogram) under quiescent conditions, as expected. (d) Variation of mean v_ded with shear rate. Each data point was extracted from the center location of a Gaussian fit to the distribution of v_ded at the given shear rate (representing thousands of bacteria trajectories). The 95% confidence interval in the mean location for each fit is typically below ±0.05 μm/s. The vertical dashed line depicts the critical shear rate (6.4 s⁻¹); the horizontal dashed lines mark v_ded extrema for shear rates below the critical.

Figure 6

Peak orientation and average swim speed components of E. coli at various shear flows. (a) The peak in ϕ distribution remains at ∼90° until the critical shear rate (γ_c), beyond which the bacteria gradually turn sideways with increasing flow. (b) Mean v_x steadily increases with shear flow and saturates at the average swim speed of the overall observed cells (solid line), as expected. (c) On average, bacteria swim upstream for γ < γ_c and get dragged with faster flow. (d) We estimate the critical shear rate for any subpopulation of E. coli by fitting a second-order polynomial to the average v_y data in the vicinity of the transition from upstream swimming to downstream drag. Dashed lines in c and d indicate the point of zero net v_y.

Figure 7

Relative ratios of circular/random swimmers (circles), hydrodynamically aligned bacteria (squares), and bacteria that display Jeffery orbits (triangles) as a function of shear rate. Hydrodynamic alignment steadily rises within the population with increasing shear rate, whereas the incidence of circular/random swimming decreases. Solid lines are fits that guide the eye; they bear a resemblance to first-order enzyme binding kinetics. For circular/random swimmers, ratio = 0.9/(1+(γ/7.2)^1.5); for hydrodynamically aligned cells, ratio = 0.7 × γ/(4.3 + γ); and for cells with Jeffery orbits, ratio = 0.5 × (γ − 2.9)/(17.1 + γ) for γ > 2.9.

Figure 8

Relationships among γ_c, v_y, bacteria length, and deduced swim speed (v_ded). Swim rates and cell lengths are obtained from average values within each tracked bacterium trajectory. (a) γ_c as a function of cell length remains within ± 1 (s⁻¹) of the mean value for the entire population (6.4 s⁻¹). (b) Mean v_ded (at all shear rates) does not appear to change with bacteria length either, implying that longer cells, on average, may have more flagella that compensate for the increased hydrodynamic drag of their bodies. Solid lines in a and b indicate average values for γ_c and v_ded for the entire population, respectively. (c) γ_c is higher for faster bacteria, which are more likely to swim upstream at a given shear rate (d). Here, squares and circles stand for 5.9 s⁻¹ and 12 s⁻¹ shear, respectively, and the dotted line marks zero mean v_y.

Figure 9

Distribution of v_y among a subpopulation of 1035 bacteria that swim at 24 μm/s (deduced) on average (range: 22.5–25.5 μm/s) at a shear rate of 2.9 s⁻¹. While the mean v_y is 2.4 μm/s, 7.2% of the cells swim upstream much faster (> 20 μm/s). Others get dragged downstream, either because they happen to face away from the +y direction as they swim on the surface, or simply because they are somewhat farther away from the surface itself and the local flow they experience is faster.

Figure 10

Steady-state lateral angle (α) can be estimated by balancing the –x directed torque (L_f) on the cell body that arises from increased drag over the surface with the hydrodynamic torque that tends to orient the cell parallel to the surface as it swims.