Tumble Suppression Is a Conserved Feature of Swarming Motility

Bacteria within a swarm move characteristically in packs, displaying an intricate swirling motion in which hundreds of dynamic rafts continuously form and dissociate as the swarm colonizes an increasing expanse of territory. The demonstrated property of E. coli to reduce its tumble bias and hence increase its run duration during swarming is expected to maintain and promote side-by-side alignment and cohesion within the bacterial packs. In this study, we observed a similar low tumble bias in five different bacterial species, both Gram positive and Gram negative, each inhabiting a unique habitat and posing unique problems to our health. The unanimous display of an altered run-tumble bias in swarms of all species examined in this investigation suggests that this behavioral adaptation is crucial for swarming.

S warming is defined as a rapid collective migration of bacteria across a surface, powered by flagella and assisted by a wide array of phenotypic adaptations (1)(2)(3). A common attribute of all swarms is a pattern of ceaseless circling motion, in which packs of cells all traveling in the same directions split and merge, with continuous exchange of bacteria between the packs (3)(4)(5). This behavior differs from movement of the bacteria in bulk liquid, where they swim individually (6). In Escherichia coli, the mechanics of flagella are similar during both swimming and swarming in that peritrichous flagella driven by bidirectional rotary motors switch between counterclockwise (CCW) and clockwise (CW) directions. However, while CCW rotation promotes formation of a coherent flagellar bundle that propels the cell forward (run) during both swimming and swarming, a transient switch in rotational direction (CW) causes the cell to tumble while swimming but reverse direction while swarming (7,8). There are variations on this theme. For example, the motility of Pseudomonas aeruginosa is characterized as a run-reverse-turn, where prolonged runs are interrupted by a reversal and "flick" to cause a change in direction (9).
The switching frequency of the flagellar motor is controlled by the chemotaxis system, best studied in E. coli, where transmembrane receptors detect extracellular signals and transmit them via phosphorelay to the motor, to promote migration to favorable locales during swimming (10). The ability to perform chemotaxis is not essential for swarming, but a basal tumble bias (TB) is important (11). We recently reported that E. coli cells taken from a swarm exhibit more highly extended runs and higher speeds than planktonic cells and that this low tumble bias is the optimal bias for maximizing swarm expansion (12). Posttranscriptional changes that alter the levels of a key signaling protein suggested that the chemotaxis signaling pathway is reprogrammed for swarming. A low tumble bias is consistent with the superdiffusive Lévy walk run trajectories observed in swarms of Serratia marcescens and Bacillus subtilis (13) and could improve swarming performance at the minimum by favoring the alignment of cells all travelling in the same direction in a pack. Whether bacteria still perform chemotaxis during swarming is not known, but an avoidance response was observed when antibiotics were added to the swarm media; this response was not to the antibiotics per se (14). Swarming allows bacteria opportunities for dispersal in ecological niches and contributes to pathogenicity in many species (15), notably in conferring enhanced resistance to antibiotics (14).
In this study, we examined TB and speeds during swarming in a selected mix of swarmer species, united only in their macroscopic display of swirling packs. The disparities between these bacteria relating to swarming behavior are many. To begin with, the bacteria are fastidious with respect to the consistency of the agar on which they swarm, and accordingly, they display different phenotypes. For example, Proteus mirabilis elongates substantially (10 to 80 m) on hard agar (1.5% and above) (16) but not on softer agar. The other four bacteria swarm only on softer agar (0.5% to 0.8% agar). Flagellum arrangements in these bacteria also vary: P. aeruginosa has a polar flagellum (17), while the other bacteria are peritrichously flagellated. P. mirabilis is substantially hyperflagellated on hard agar (16) and B. subtilis and P. aeruginosa double their flagellum numbers (17,18), while Salmonella enterica and S. marcescens do not substantially change these numbers (3). S. enterica does not secrete surfactants or polysaccharides that lubricate the surface; the others all do. Despite these various swarming adaptations, we found that these bacteria all share the same low TB and higher run speeds as reported for E. coli, suggesting that this behavior is a universal adaptation for successful migration on a surface.
The methodology and growth conditions used to monitor TB and speed in this study were similar to those used for E. coli (12) and were consistently applied across all swarming species. Eiken agar was used to solidify swarm media, because this agar facilitates swarming in non-surfactant producers (19). By using the same agar concentration for all species (0.5%), we kept P. mirabilis from elongating; long cells do not tumble. Under these conditions, the cell length of this bacterium during swarming was similar to that of all the other species (2.5 Ϯ 0.7 m; n ϭ 50), unchanged from that observed in liquid (2.1 Ϯ 0.5 m; n ϭ 50). Preliminary tracking experiments with S. marcescens cells taken from liquid showed large circular trajectories (Fig. S1, left). Such trajectories have been observed with E. coli and Caulobacter crescentus swimming close to a glass surface (20). We suspected that the surfactant serrawettin, a cyclic lipopeptide secreted by S. marcescens (3), might be responsible for this behavior by suppressing tumbles. A S. marcescens mutant deficient in serrawettin production abolished the circular motion (Fig. S1, right), so this strain was used for tracking. B. subtilis makes a surfactant similar to serrawettin (2) and also displayed circular trajectories, so we used a srfA mutant deficient in surfactin synthesis. P. aeruginosa surfactant has a different structure (rhamnolipid) (2) and did not show such trajectories, so we worked with the wild-type strain. We discuss our findings in the order of discovery of swarming in the bacterial species studied in this investigation (21)(22)(23)(24)(25).
Representative cell trajectories in liquid and swarm media for all bacterial species tested are shown in Fig. 1. All show a distinct shift in motion paths under the two conditions, becoming smoother (long run trajectories) during swarming. Quantitative analyses of these trajectories are shown in Fig. 2. While technically P. aeruginosa does not tumble, in our analysis, the run-reverse and reverse-flick were both identified as tumbles. The tumble angle distribution plots generated were consistent with runreverse-flick. The changes in median TB values from liquid to swarm were as follows: P. mirabilis, 0.27 to 0.14; S. marcescens, 0.23 to 0.037; S. enterica, 0.07 to 0.05; B. subtilis, 0.24 to 0.048; and P. aeruginosa, 0.53 to 0.31 (statistics are detailed in Table S1). While the overall pattern was that TBs shifted to lower values during swarming, we note that TB values for S. enterica are lower than for E. coli in liquid to begin with, as reported in single motor assays (26). For comparison, TB values for E. coli decreased from a median of 0.12 in liquid to 0.04 in swarmers (12).
The low TB displayed by E. coli swarmers was observed to be stable for up to 45 min and persisted through one cell division at room temperature (ϳ120 min); this physiological adaptation is different from the adaptation of the chemotaxis pathway through methylation (12). We therefore also included a 45-min time point (after lifting cells from the swarm) for tracking all five swarmers. At 45 min after removal from the swarm, most bacteria maintained their low TB values (statistics in Table S1).
As observed for E. coli, running speeds (micrometers per second) for a majority of the bacterial species increased significantly between liquid and swarm as follows: P. mirabilis, 9.01 to 13.3; S. enterica, 23.1 to 30.7; B. subtilis, 18.6 to 31; and P. aeruginosa, 21.9 to 41.6 (statistics in Table S1). These values for E. coli were 21 m/s in liquid and 25 m/s in swarmers (12).
In summary, keeping swarming conditions the same, we demonstrated that despite different natural habitats and widely different swarming adaptations discovered in the laboratory, the swarmers studied all modify their TB, and a majority modify run speeds during swarming, similar to what was reported for E. coli (12). This apparently common behavior suggests that it represents a successful strategy for collective migration across a surface. There are many ways to reduce TB and increase speed, and the precise mechanisms of altering these parameters may vary in the different species. In E. coli, elevation or stabilization of the chemotaxis component CheZ is responsible for the low TB (12). Other components in the chemotaxis pathway can potentially be altered to achieve the same outcome. As demonstrated for S. marcescens, surfactants themselves contribute to lowering TB (Fig. S1), although our experiments bypassed the surfactant. Swarming speed can be increased by hyperflagellation, torque-enhancing proteins, special stators, and increased proton motive force (3,27). Future work will reveal the mechanisms used by each of these bacteria to arrive at what is apparently a common solution for maximizing collective motion.
Methods. Cell culture, swarm setup, tracking experiments, and analysis were largely carried out as described previously (12).