Flagellation and cell size
Bacteria swim in the low Reynolds number regime of fluid dynamics (Berg, 1975; Ping, 2012). The cell dimension and flagellation are key parameters that determine output. The cells of P. fluorescens SBW25 are monotrichous as expected, but not in sensu stricto. More than 60% flagellated cells are with one flagellum (Fig. 1a inset). Among the 254 fluorescence-labeled cells examined, c. 38% carry more than two flagella. The average number of flagella per cell is 1.5 ± 1.1, lower than those of the other reported Pseudomonas species. The average number of flagella on Pseudomonas syringae pv. tabaci cells is 2.7 (Sigee & El-Masry, 1989; Kanda et al., 2011). Pseudomonas putida PRS2000 generally have five to seven flagella (Harwood et al., 1989). The number of flagella on P. fluorescens SBW25 cells is probably similar to that of P. aeruginosa, which often carry a single flagellum (E.P. Greenberg and R. Ramphal, pers. commun.).
Figure 1. Free-swimming behavior of Pseudmonas fluorescens SBW25. (a) The trajectory of a swimming bacterium. Scale bars equal 10 μm. The inset shows a fluorescence stained bacterium. The segments of the trajectory are labeled with Roman numbers. The backup and run speeds (μm s−1) are shown beside segment II and III. In segment I, the bacterium first performed a run within the focal plane, and then swam towards the bottom after a turn (arrow). Segment II corresponds to a backup. A second run followed (segment III). This run ended with a flip following by a hover. The cell eventually dashed away from the focal plane (segment IV). (b) The average speeds of runs (n = 27) and backups (n = 9). (c) The run speed was plotted against the backup speed of the same bacterium with a linear fit. (d) The flip event boxed in (a). Seven cell positions captured every 55 ms was superimposed in 1; 2–9 show three dimensional reconstructions of the cell in each frame. The upper ends correspond to the flagellated poles.
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The flagella of P. fluorescens SBW25 are right-handed helices with averagely 2.5 turns per filament. The flagellar contour length is 8.4 ± 1.3 μm. The pitch size and diameter are much larger than those reported for P. aeruginosa strain PAK, P. syringae pv glycinea, and P. syringae pv tabaci 6605 (Table 1). At physiological pH, the normal flagellar form of P. aeruginosa and P. syringae have been reported as left-handed (Fujii et al., 2008; Taguchi et al., 2008). However the left-handed curly flagella on E. coli mutants cannot generate enough swimming force (Wang et al., 2012). Whether the previously studied P. aeruginosa and P. syringae pathovars are motile needs further verification. On the other hand, the flagella of the swarmer cells of Caulobacter crescentus, another fresh water monotrichous bacterium, are right-handed (Koyasu & Shirakihara, 1984). The Caulobacter flagellum has even smaller pitch and diameter, comparing to P. fluorescens SBW25 (Table 1).
Table 1. Flagellar characters of Pseudmonas fluorescens SBW25 and other monotrichous bacteria
|Handedness||Pitch (μm)||Diameter (μm)|
|P. fluorescens SBW25||Right||1.76||0.79||This study|
|C. crescentus CB15||Right||1.08||0.27||Koyasu & Shirakihara (1984)|
|P. aeruginosa strain PAK||Left||1.38||0.39||Fujii et al. (2008)|
|P. syringae pv glycinea||Left||1.59||0.43||Fujii et al. (2008)|
|P. syringae pv tabaci 6605||Left||1.59||0.18||Taguchi et al. (2008)|
The cell body of P. fluorescens SBW25 is larger than that of E. coli. The average length of cell bodies of P. fluorescens SBW25 is 3.1 ± 0.8 μm and the diameter is 0.9 ± 0.1 μm. These results were in good agreement with the previous observations on other pseudomonads (Harwood et al., 1989; Sigee & El-Masry, 1989). The flagellar filaments of P. putida PRS2000 usually have two to three wavelength and are about two body lengths long (Harwood et al., 1989). Similar observation was obtained with transmission electron microscopy on P. syringae pv. tabaci strain Wolf and Foster 1917 (Sigee & El-Masry, 1989).
Sophisticated free-swimming behavior
Pseudmonas fluorescens SBW25 exhibited a sophisticated swimming behavior unknown to any other bacteria. Figure 1a shows the trajectory of a free-swimming cell. It can swiftly adjust the direction when swimming forward (runs) without retardation on speed. This behavior has been called ‘turns’ by Harwood et al. (1989). When they swim backward (backups), they sometimes follow the same path of the immediately preceding run (Between segment II and III), but often change the direction to an acute angle at transitions; with the next run deviated again from the backup path, a zig-zag trajectory was hence generated. Turn has only been observed in P. putida previously (Harwood et al., 1989; Duffy & Ford, 1997; Davis et al., 2011), and the zig-zag trajectory in P. citronellolis (Taylor & Koshland, 1974). In previous computer-aided analyses on P. putida swim, a bimodal distribution of the changing angle was observed, with frequency peaking at about 40° and 160°, respectively (Duffy & Ford, 1997; Davis et al., 2011). The obtuse angle would be resulted from turns, and the acute angle from reorientation at run-backup transitions.
Pseudmonas fluorescens SBW25 is one of the fastest swimmers in Pseudomonas. The average run speed of P. fluorescens SBW25 was observed as 77.6 μm s−1 and the maximum speed 102.0 μm s−1; the average backup speed is 18.0 μm s−1 and the maximum 22.4 μm s−1 (Fig. 1b). Backups are often short. Cells that run fast tend to backup fast (Fig. 1c). Pseudomonas syringae pv. tabaci 6605 was observed to swim at c. 50 μm s−1 (Kanda et al., 2011) or 83 μm s−1 (Taguchi et al., 2008). Pseudomonas aeruginosa ATCC 15692 swim at c. 60 μm s−1 (Conrad et al., 2011). The average swimming speed of P. putida was reported to be 44 μm s−1 with maximum at 75 μm s−1 (Harwood et al., 1989). Pseudomonas putida strain KT2440 swims even slower, with an averaged c. 20.9 μm s−1 and a maximum at 51.2 μm s−1 (Davis et al., 2011). None of the backup speed of other Pseudomonas species has been reported.
The swimming behavior of P. fluorescens SBW25 is much more sophisticated than the peritrichous rods. Besides the above mentioned runs, backups, turns, and run-backup reorientations, they can make a series of continuous rocking (Fig. 1d), or standstill for a while. In both cases, there is no net displacement. These two kinds of behavior will be referred as ‘flip’ and ‘hover’ respectively for convenience. The boxed area in Supporting Information, Movie S1 highlights a prolonged flip, while normal flips of P. fluorescens SBW25 were shorter and simpler (Fig. 1d). The bacteria must control their flagellar rotation very precisely to perform this kind of movement. When hovering still, the cells still rotated. Movie S2 showed a congenitally curved cell. The cell body rotated counterclockwise at 6.7 Hz. The flagellar propulsion force and the translational viscous drag load must be precisely balanced in this process.
Flagella and cell body rotation
The flagellum and cell body must rotate against each other to generate propulsion force (Berg, 2003). A right-handed flagellum pushes the cell forward when it rotates clockwise, and pull the cell backward when rotates counterclockwise. When fluorescence-labeled cells swam into the inspection field relatively slowly, this could be clearly observed. We also examined bacteria that spontaneously tethered to the glass slide surface (Fig. 2). The cells rotate alternatively to both directions equally well with a slight preference to clockwise direction on speed (P = 0.38, two-tailed paired Student's t-test) and duration (P = 0.09), confirming that flagella rotate clockwise during the runs. It has been reported that the cell body of P. citronellolis rotates chiefly in counterclockwise direction, and change to a clockwise rotation periodically in an isotropic medium (Taylor & Koshland, 1974). Our observation on P. fluorescens SBW25 is more similar to the observation on E. coli. When E. coli cells were tethered by a single flagellum, the cells rotate alternatively to both directions randomly at similar angular speed (Block et al., 1989).
Figure 2. Rotation of Pseudmonas fluorescens SBW25 cells tethered on glass slide surfaces by their flagella. (a) the angular velocities of two representative cells. Clockwise rotation was arbitrarily assigned as positive. (b) The rotation speed of six anchored cells (n = 93). (c) The duration of rotation events showing in (b).
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In our experiments, the rotation speed of tethered P. fluorescens SBW25 cells towards clockwise directions was c. 2.4 Hz. The tethered polyhook mutant of E. coli rotate at 2–9 Hz (Silverman & Simon, 1974), and a fully energized E. coli cell spins at 10 Hz (Berg & Turner, 1993). Unlike E. coli, whose flagellar motors sit on lateral surface, the polar filaments of Pseudomonas must bend when the cell bodies rotate in a horizontal plane. The disalignment of the cell bodies with the flagellar axis would account for, at least partially, the observed slow speed. The peaks appearing immediately after direction switching testified this assumption: that likely results from a sudden release of the accumulated tension (Fig. 2a). Flagellum(a) tethered P. aeruginosa ATCC 15692 cells also spin towards both directions equally well, but with a lower angular velocity of c. 0.8 Hz (5 rad s−1; Conrad et al., 2011), probably due to additional thrust/hindrance from surplus flagella.
Circular motion near flat surface
When living in rhizo- and phyllosphere, bacteria unavoidably encounter liquid-solid interface, of which a flat solid surface is the simplest form (Ping, 2012). The presence of a solid surface in solution increases the viscosity of nearby fluid. This is termed wall effect (Ramia et al., 1993; Lauga et al., 2006; Ping, 2012). It is negligible in macroscopic system, but significant at the microscopic scale. Pseudmonas fluorescens SBW25 cells performed circular motion in runs when they swam near the slide surface (< 50 μm above the surface). When viewed from above and the solid surface is below, they appear to be rolling to the left (Fig. 3a). They swam in right-hand direction in backups. Near the surface, their average run speed decreases to c. 65 μm s−1 in circular motion (Fig. 3b). The swarmer cells of C. crescentus perform similar circular motion. They swim clockwise near the bottom surface in run when observed from below (Li et al., 2008; Ping, 2012). It has been reported that P. citronellolis swims in clockwise circles near bottom when viewed from above like E. coli (Taylor & Koshland, 1974; Berg, 2003), and hereby different from of P. fluorescens SBW25. Pseudomonas putida PRS2000 swim in a counterclockwise direction near a bottom surface when viewed from below, but whether it was in run or in backup was not clarified (Harwood et al., 1989). The circular trajectories of P. aeruginosa ATCC 15692 was also observed, however, the direction was not determined (Conrad et al., 2011).
Interestingly, P. fluorescens SBW25 cells turn much more frequently in circular motion than in free-swim. Kinks were observed on the circular trajectories (Fig. 3a). However, between two kinks the curvatures were always constant for a given bacterium. The median of the radius of these curvatures was 12.5 μm and the average 14.6 μm (Fig. 3c). The radius of the circular path of P. aeruginosa ATCC 15692 was reported to be c. 12 μm, in good agreement with our data (Conrad et al., 2011). The heterogeneity of viscous drag load originated from wall effect that experienced by the rotating cell body and flagellar helix pushes the swimming cell constantly off track and slows it down (Lauga et al., 2006). The radius of the circular path is determined by the physical dimension of the organism, the swimming velocity and the out-of-plane rotation rate (Ramia et al., 1993; Lauga et al., 2006). When these parameters are fixed, the curvature should remain unchanged.
Figure 3. The motion of Pseudmonas fluorescens SBW25 cells near bottom surface. (a) The trajectory of a bacterium swimming near a bottom surface when viewed from above. Scale bar equals 10 μm. Beginning and end of the movie are indicated by open arrows. Run is depicted by black dots, and backup by grey open circles. Turns are pointed by black arrows. Curved arrows indicate the smooth segments in backup. The smooth segments in run are fitted with grey broken circles. The numbers beside the trajectories are averaged speeds in each segment. (b) The distribution of cell velocities in the circular runs (n = 106). (c) The distribution of curvature radius plotted in (b).
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Influence of isotropic viscosity
To test whether the high turn frequency in circular motion is triggered by the high viscous drag load on cells, chemotaxis media with different viscosities were used (Table 2). The backup frequency of P. fluorescens SBW25 was significantly reduced as viscosity increased (Table 2). At 19 centipoises (cP), backup was seldom observable. The run speed of P. fluorescens SBW25 did not show significant difference at 2 cP compared to the controls (Table 2), but the backup speed was slightly increased. On the other hand, the path-length of runs and backups at 2 cP were significantly longer than those at 1 cP (Table 2). As viscosity was further increased, the path-lengths of runs and backups decreased linearly until their minima were reached at 7 and 4 cP, respectively. The speed of runs and backups decreased linearly as well. At 19 cP, some runs and backups showed intermittent slowdown. We attribute this to the suppressed turns and backups.
Table 2. Influence of viscosity on the free-swimming behavior of Pseudmonas fluorescens SBW25
|Viscosity (cP)||Sample sizes||Relative amount of run (%)||Length of backup (μm)||Velocity (μm s−1)||Duration of run (s)|
|Cell||Run||Backup||10–30 μm||30–50 μm||> 50 μm||Run||Backup|
|1||178||239||68||21.8||24.4||53.8||15.9 ± 11.1||45.9 ± 12.2||18.5 ± 7.0||1.2 ± 0.6|
|2||200||248||57||17.3||24.2||58.5||18.3 ± 10.7||44.3 ± 12.1||19.3 ± 6.6||1.2 ± 0.5|
|4||204||256||30||33.3||23.1||43.8||11.1 ± 6.8||27.6 ± 9.7||8.3 ± 4.9||1.6 ± 0.9|
|7||179||249||14||42.5||29.2||28.3||13.4 ± 6.4||27.9 ± 9.3||9.6 ± 4.4||1.6 ± 0.7|
|19||221||278||5||38.1||25.1||36.7||17.8 ± 11.7||17.1 ± 7.6||7.5 ± 2.1||2.4 ± 1.3|
Low viscosity is known to bestead bacterial swim (Keller, 1974). Pseudomonas aeruginosa and E. coli swim fastest at 2 cP (Schneider & Doetsch, 1974; Atsumi et al., 1996), but the speed of P. fluorescens SBW25 was unchanged at this viscosity. It is worth noting that E. coli cells are multiply flagellated, while P. fluorescens SBW25 is, dominantly, monoflagellated. That of P. aeruginosa is uncertain (E.P. Greeberg, pers. commun.). A mutant of Vibrio alginolyticus that only produces a single polar flagellum swim fastest at 1 cP, while the isogenic strain that produce multiple lateral flagella swim at maximum speed at 5 cP (Atsumi et al., 1996). Above 2 cP, the swimming speeds of P. aeruginosa and E. coli correlate with medium viscosity inversely (Greenberg & Canale-Parola, 1977; Atsumi et al., 1996). Pseudmonas fluorescens SBW25 behaved the same. Because the path-length and the velocity decreased proportionally, the durations of each run and backup actually remained unchanged.
The sophisticated swimming behavior of P. fluorescens SBW25 has not been observed in any other well-studied bacteria so far. It is known that V. alginolyticus flicks their flagellum upon resuming runs to change body orientation (Xie et al., 2011). If the turns, flips, and run-backup reorientations of Pseudomonas were operated with similar mechanism, Pseudomonas would have a very precise control on flagella movements. Furthermore, the flagella of bacteria like Vibrio can sense the surrounding viscosity to initiate lateral flagella biosynthesis (McCarter et al., 1988). Whether the flagella of Pseudomonas can also serve as a dynamometer is unclear. However, the flagellar motors of Vibrio are sodium-driven (McCarter et al., 1988; Xie et al., 2011), while the Pseudomonas motors are proton-driven (Kanda et al., 2011). Vibrio swim faster in backups than in runs (Magariyama et al., 2001). They might employ very different mechanisms.
The precise movement control of P. fluorescens SBW25 certainly has ecological advantages in the restricted geometry of soil and plant cavities. Intuitively, backup is helpful to prevent jam and turn, flip and hover to avoid collision. It has been observed that the turn frequency is significantly increased when P. aeruginosa swim in glass-bead column (Chen & Jin, 2011). The cell body spinning has been proposed to facilitate the release of tethered cells (Conrad et al., 2011). Other soil bacteria such as Sinorhizobium meliloti are known to adopt different strategies for the same goal: they use a combination of peritrichous and lophotrichous flagella to swim (Götz & Schmitt, 1987; Armitage & Schmitt, 1997). Although the right-handed flagellar bundle only rotates clockwise, they can slowdown, turn, or stop through motor control.
Monotrichous flagellaion, rather than peritrichous flagellaion is believed to be an adaption to the nutrient-scarce aquatic environment for fast swim with low energy cost (Ping, 2012). On several aspects, P. fluorescens SBW25 resembles more to the fresh-water bacterium C. crescentus than the enteric bacteria (Li et al., 2008; Ping, 2012). For free-swimming bacteria, translation at low Reynolds number is torque free and equals the drag load on the cell body given by Stoke's law:
here v is swimming velocity and r is the radius of a spherical cell. A typical E. coli cell is 2.5 μm long and 0.8 μm in diameter (Berg, 2003). The radius of a sphere with equal volume is c. 0.62 μm. Escherichia coli generally swims at 25 μm s−1 (Berg, 2003). The P. fluorescens SBW25 cells are slightly larger and the radius of the equivalent sphere is c. 0.75 μm. If we take the viscosity of the medium η as 1 cP like water, the propulsion force generated by a typical E. coli swimming cell and a P. fluorescens SBW25 cell can be calculated as c. 0.29 and 1.11 pN, respectively.
In the circular motion of E. coli, frequent reorientation was not observed, except that they might leave the surface after tumble (Frymier et al., 1995). Since the turn frequency of P. fluorescens SBW25 was not influenced by the viscosity of bulk media, it is possible that the symmetry breaking of viscous drag load due to wall effect is responsible for the phenomenon. Nevertheless, the isotropic viscosity has its own significance in rhizo- and phyllospheres, because plant secretion and decomposed organic matters would increase viscosity locally. Speeding up at low cP values and slowing down at high cP values as well as the suppression of turn and backup at high viscosity might all influence nutrient uptake and chemotaxis efficiency. In water-saturated rhizo- and phyllosphere, submerged surface is very common. The circular motion with low swimming speed might also enhance chemotaxis sensitivity and nutrient gain as proposed for Caulobacter (Li et al., 2008). At mean time, it mimics the behavior of foraging animals, though operated passively in bacteria, and might enhance the host seeking and invasion.
In accordance with the sophisticated swimming behavior, the chemotactic sensors in Pseudomonas are highly diverse. There are 26 receptor genes belonging to the methyl-accepting-chemotaxis proteins (MCPs) family on the genome of P. aeruginosa PAO1, while only four in E. coli (Kato et al., 2008). On the genome of P. fluorescens SBW25 (Accession no. NC_012660), 52 putative MCPs have been annotated. Loper et al. (2012) compared this genome with nine other genomes and found a tremendous ecological and physiological diversity within the P. fluorescens group. When comparative genomic analysis is combined with single-cell motility study in the future the ecological importance of these detector-propeller networks will begin to emerge.