Dynamic motility selection drives population segregation in a bacterial swarm

Abstract

Population expansion in space, or range expansion, is widespread in nature and in clinical settings. Space competition among heterogeneous subpopulations during range expansion is essential to population ecology, and it may involve the interplay of multiple factors, primarily growth and motility of individuals. Structured microbial communities provide model systems to study space competition during range expansion. Here we use bacterial swarms to investigate how single-cell motility contributes to space competition among heterogeneous bacterial populations during range expansion. Our results revealed that motility heterogeneity can promote the spatial segregation of subpopulations via a dynamic motility selection process. The dynamic motility selection is enabled by speed-dependent persistence time bias of single-cell motion, which presumably arises from physical interaction between cells in a densely packed swarm. We further showed that the dynamic motility selection may contribute to collective drug tolerance of swarming colonies by segregating subpopulations with transient drug tolerance to the colony edge. Our results illustrate that motility heterogeneity, or "motility fitness," can play a greater role than growth rate fitness in determining the short-term spatial structure of expanding populations.

Keywords: adaptive stress response; antibiotic tolerance; bacterial swarming; collective motion; flagellar motility.

Fig. 1.

Spatial segregation of subpopulations with motility heterogeneity in E. coli swarms. (A) Illustration of the protocol to induce motility heterogeneity in E. coli swarms on an antibiotic gradient plate. E. coli YW191 cells (KAN-resistant, labeled by GFP) and YW263 (KAN-sensitive, labeled by Katushka2S) were mixed and inoculated onto the drug-free side of a KAN gradient plate (SI Appendix, Fig. S2 and Methods). The dashed line on the plate marks the boundary between drug-free and drug-infused regions on the plate, and the color scale indicates relative KAN concentration. The spatial distribution of both subpopulations was measured by fluorescence microcopy along the swarm expansion direction (indicated by the black straight arrow) when the swarm had entered the KAN gradient for ∼25 mm. (B) Representative fluorescent image sequence showing the enrichment of the higher-speed subpopulation (YW191, green) near the swarm edge. Red fluorescence was from YW263 cells that had a smaller average speed than YW191 in the drug-infused region of KAN-gradient swarm plates. (C) Representative fluorescent image sequence showing the spatial distribution of YW191 (green) and YW263 (red) cells grown on nonswarming hard agar plates with the same KAN gradient as in B. The image sequences in B and C were taken at different locations whose relative distance to the starting position of the KAN gradient is specified by the ruler below panel C (unit: millimeters; KAN concentration increases from left to right). (Scale bars, 0.1 mm.) (D) Proportion of YW191 cells in swarms on KAN-gradient plates (Left) and in colonies on nonswarming hard agar plates (Middle) plotted against distance to the starting point of the KAN gradient. The population proportion (i.e., ratio between YW191 cell number and total cell number) was measured based on the fluorescence microscopy images as shown in B or C (Methods). The proportion of YW191 cells in swarms on antibiotic-free plates is shown for comparison (Right; distance = 0 mm is located at the plate center). Each line in the plots represents data from an independent colony.

Fig. 2.

Motion pattern of E. coli swarm cells during the spatial segregation of subpopulations with motility heterogeneity. (A) Representative trajectories of the higher-speed subpopulation (YW191) at ∼5 mm from the swarm edge. The portions of the trajectories moving toward and away from the swarm edge are colored in blue and brown, respectively. (B) Speed distribution of the faster (YW191, green, n = 94) and the slower (YW263, red, n = 314) subpopulations. Lines are Gaussian fits to the speed distributions to obtain the mean and SD of population speed used in main text. (C and D) Angular probability distribution of single-cell velocity directions for the faster (C) and the slower (D) subpopulations, respectively. To generate these plots, single-cell trajectories were divided into 1-s segments and the average velocity direction of these segments was computed as an angle ranging from 0° to 360°, with the swarm expansion direction set as degree 0. The obtained velocity directions were then grouped into 80 polar angle bins of a full circle (360°), with each bin covering an angle of 4.5°. The radii of colored circular sectors in C and D are proportional to the normalized count in the corresponding angle bin and thus represent the probability of single-cell velocity directions falling within the bin. The radius of the dashed circle in each plot indicates a probability of 0.015. (E and F) Average speed of cells plotted against velocity direction for the faster (E) and the slower (F) subpopulations, respectively. In the plots of E and F the polar angle was divided into 80 bins in a way similar to C and D. Single-cell trajectories were divided into 1-s segments and for a specific polar angle bin the average speed of all trajectory segments whose velocity direction fell within this bin was computed. The radii of colored circular sectors in E and F are proportional to the average speed of cells computed for the corresponding polar angle bin, with the radius of the dashed circle indicating a speed of 30 μm/s. Blue and brown colors in C–F indicate moving toward and away from the swarm edge, respectively.

Fig. 3.

Persistence time analysis for single-cell motion in E. coli swarms during the spatial segregation of subpopulations with motility heterogeneity. (A and B) Probability distribution of the duration of outward-moving traces (empty blue columns) and inward-moving traces (filled brown columns) for the faster (YW191; A) and the slower (YW263; B) subpopulations, respectively. Lines represent plots of exponential fit of trace duration distribution in the form of f(t)= (1/τ)*exp(−t/τ), with the persistence time τ being either τ_out or τ_in (i.e., the outward or the inward persistence time). τ was obtained by fitting the corresponding cumulative probability distributions to F(t) = 1 − exp(−t/τ) (Methods), with the values given as follows: For YW191, τ_out = 0.45 s, τ_in = 0.36 s; for YW263, τ_out = 0.43 s, τ_in = 0.39 s. (C) Overall persistence time (τ_all) plotted as a function of cell speed for the faster (YW191, green square) and the slower (YW263, red circle) subpopulations. Lines are linear fits to the data, with R² being 0.95 and 0.99 for YW191 and YW263 cells, respectively. (D) The outward bias of persistence time (β) plotted as a function of cell speed for the faster (YW191, green square) and the slower (YW263, red circle) subpopulations. Lines are linear fits to the data, with R² being 0.71 and 0.91 for YW191 and YW263 cells, respectively. Error bars in C and D represent the error introduced by temporal uncertainty of single-cell tracking (Methods).

Fig. 4.

Motion pattern and persistence time analysis for wild-type E. coli swarm cells on drug-free agar plates. (A) Angular probability distribution of single-cell velocity directions. The plot was generated in the same manner as done for Fig. 2 C and D. The polar angle (as a measure of single-cell velocity direction) was divided into 80 bins, and the radii of colored circular sectors in the polar plot are proportional to the probability of single-cell velocity directions falling within the corresponding polar angle bin. The radius of the dashed circle corresponds to a probability of 0.015. The swarm expansion direction is set as degree 0. (B) Average speed of cells plotted against velocity direction. The plot was generated in the same manner as done for Fig. 2 E and F. The radii of colored circular sectors in the polar plot are proportional to the average speed of cells computed for the corresponding polar angle bin, with the radius of the dashed circle indicating a speed of 40 μm/s. Blue and brown colors in A and B indicate moving toward and away from swarm edge, respectively. (C) Probability distribution of the duration of outward-moving traces (empty blue columns) and inward-moving traces (filled brown columns). Lines represent exponential fit of trace duration distribution in the form of f(t) = (1/τ)*exp(−t/τ), with the persistence time τ being either τ_out or τ_in and obtained in the same way as in Fig. 3: τ_out = 0.59 ± 0.01 s, τ_in = 0.39 ± 0.01 s. (Inset) Trace-duration distributions for noninteracting cells swimming near the diluted swarm edge do not show persistence time bias (both τ_out and τ_in are 0.52 s). (D) The overall persistence time (τ_all, circle) and the outward bias of persistence time (β, square) plotted as a function of cell speed. Error bars represent the error introduced by temporal uncertainty of single-cell tracking (Methods). Lines are linear fits to the data, with R² for τ_all and β being 0.98 and 0.84, respectively.

Fig. 5.

Cells near the edge of E. coli swarms that encountered KAN stress displayed KAN tolerance. (A) Workflow of cell survival rate measurement. Wild-type cells were inoculated on the drug-free region of the KAN-gradient swarm plates and of the hard agar plates (that do not support swarming) (SI Appendix, Fig. S2 and Methods). Cells located in between ∼15 mm and ∼25 mm inside the KAN gradient were harvested either from the swarm plates at 3 h after the swarm had ceased expansion or from the hard agar plates after 3 h of incubation. The harvested cells were then subjected to CFU and OD₆₀₀ measurements, and the ratio of the deduced cell numbers was taken as cell survival rate (Methods). (B) Results of cell survival rate measurement. The survival rate of cells harvested from KAN-gradient swarm plates was $0.16\pm 0.09$ (blue column, Inside Gradient), with the survival rate of cells harvested from drug-free swarm plates as the control (blue column, Control). For comparison, the survival rate of cells harvested from KAN-gradient hard agar plates was $\left(4\pm 5\right)\times {10}^{-4}$ (orange column, Inside Gradient), with the survival rate of cells harvested from drug-free hard agar plates as the control (orange column, Control). Each column presents data of 6 to 21 measurements from three or more independent experiments, and error bars represent SD. Also see SI Appendix, Fig. S8.