Water reservoir maintained by cell growth fuels the spreading of a bacterial swarm

Abstract

Flagellated bacteria can swim across moist surfaces within a thin layer of fluid, a means for surface colonization known as swarming. This fluid spreads with the swarm, but how it does so is unclear. We used micron-sized air bubbles to study the motion of this fluid within swarms of Escherichia coli. The bubbles moved diffusively, with drift. Bubbles starting at the swarm edge drifted inward for the first 5 s and then moved outward. Bubbles starting 30 μm from the swarm edge moved inward for the first 20 s, wandered around in place for the next 40 s, and then moved outward. Bubbles starting at 200 or 300 μm from the edge moved outward or wandered around in place, respectively. So the general trend was inward near the outer edge of the swarm and outward farther inside, with flows converging on a region about 100 μm from the swarm edge. We measured cellular metabolic activities with cells expressing a short-lived GFP and cell densities with cells labeled with a membrane fluorescent dye. The fluorescence plots were similar, with peaks about 80 μm from the swarm edge and slopes that mimicked the particle drift rates. These plots suggest that net fluid flow is driven by cell growth. Fluid depth is largest in the multilayered region between approximately 30 and 200 μm from the swarm edge, where fluid agitation is more vigorous. This water reservoir travels with the swarm, fueling its spreading. Intercellular communication is not required; cells need only grow.

Fig. 1.

(A) A phase-contrast image of the region near the edge of a typical E. coli swarm grown on 0.6% Eiken agar (see Materials and Methods). Microbubbles appear as bright spots. Some move in the river that flows clockwise in front of the swarm (e.g., black arrow), and others move within the body of the swarm (e.g., white arrow). A monolayer of cells appears at the swarm edge (to the right of the second vertical dashed line). The area bounded by the first and second dashed lines looks more porous, but the cells are multilayered (stacked on top of one another). The swarm is expanding to the right, as shown by the arrow x. See Movie S1. (B) Normalized brightness fluctuation of pixels, P, in a direction parallel to the arrow y averaged over 900 consecutive frames of Movie S1, plotted as a function of the distance from the edge of the swarm. See Materials and Methods.

Fig. 2.

Net radial displacement in the laboratory frame [, black solid line] of bubble trajectories at different regions inside the swarm, shown as a function of time. Linear fits to , indicating radial drift velocities, v_x, are shown by red dashed lines. The gray areas indicate standard errors in the mean. The insets show typical bubble tracks in the laboratory frame measured in μm, beginning at + and ending at x. Tracking began at distances from the swarm edge shown on the ordinates at t = 0 and continued over the time span shown on the abscissas. The numbers of tracks analyzed were (A) 29, (B) 35, (C) 43, and (D) 22. Data analysis was continued until about half of the trajectories extended beyond the region of interest; e.g., the cell monolayer, A; the multilayered region, B; and the regions beyond the multilayered region, C and D.

Fig. 3.

Mean-squared displacement (MSD) of microbubbles as a function of time, corrected for drift. Four datasets of bubble trajectories (the same as those used in the panels of Fig. 2, truncated at t = 5 s) were computed and plotted with different symbols. Each solid line is the best linear fit of log[MSD(t)] versus log(t), which yields the effective self-diffusion coefficient D_eff and the anomalous diffusion exponent α, as the y-intercept and the slope, respectively. The datasets are representative of the bubble motion in the following regions of a swarm: the swarm-edge monolayer (D_eff = 31 ± 1 μm²/s, α = 1.11 ± 0.04, squares); the multilayered region (D_eff = 64 ± 2 μm²/s, α = 1.20 ± 0.03, circles); the region between approximately 200 and 300 μm from the swarm edge (D_eff = 26 ± 1 μm²/s, α = 1.27 ± 0.03, triangles); and the region between approximately 300 and 350 μm from the swarm edge (D_eff = 26 ± 1 μm²/s, α = 1.15 ± 0.04, upside-down triangles).

Fig. 4.

Average radial cell speed as a function of the distance from the swarm edge. Particle image velocimetry was performed on a phase-contrast movie of a typical swarm lasting approximately 33 s, and the radial components of the velocity vectors in each velocity field were averaged (see Materials and Methods). The gray area indicates standard error of the mean.

Fig. 5.

Cell-density profiles of swarms of cells of E. coli strain HCB1668 shown as a function of the distance from the swarm edge. The agar contained a membrane-specific fluorescent dye, FM 4-64. The solid curve is the average fluorescence intensity profile (n = 5), with the gray area indicating the standard error of the mean. The dashed lines are best linear fits of the black solid curve ranging from -200 to -150 μm and from -50 to -30 μm, with slopes 0.0039 ± 0.0001 μm^-1 and -0.0092 ± 0.0004 μm^-1, respectively.

Fig. 6.

A model of E. coli swarm expansion. (A) The predicted flows in and out of the agar substrate (v_o) computed with Eq. 1. (B) An illustration of fluid balance of a swarm traveling from left to right. As the swarm fluid spreads, the height profile of swarm fluid shifts outward (changing from the black solid line to the red dashed line). Along the direction of swarm expansion, successive gray dots at the swarm/agar interface denote distances from the swarm edge of 300, 200, 100, 30, and 0 μm, respectively. Note that the length scales in the horizontal and vertical directions are different. The observed drift of swarm fluid in different regions is depicted by the solid arrows, with the relative length of arrows roughly corresponding to the magnitude of the measured flow speeds (Fig. 2). The predicted flows in and out of the agar are illustrated by the open arrows with a dashed boundary, with the relative height of arrows roughly corresponding to the magnitude of the predicted flow speeds in A.