Download Swimming behavior of the monotrichous bacterium Pseudomonas

RESEARCH ARTICLE
Swimming behavior of the monotrichous bacterium
Pseudomonas fluorescens SBW25
Liyan Ping1, Jan Birkenbeil2 & Shamci Monajembashi3
1
Max-Planck-Institute for Chemical Ecology, Jena, Germany; 2Carl Zeiss Microscopy GmbH, Jena, Germany; and 3Imaging Facility and Nuclear
Organization Group, Leibniz Institute for Age Research (Fritz Lipman Institute), Jena, Germany
Correspondence: Liyan Ping, Max-PlanckInstitute for Chemical Ecology, Hans-KnoellStr. 8, 07745 Jena, Germany. Tel.:
+49 3641 57 1214; fax: +49 3641 57 1202;
e-mail: [email protected]
Received 28 November 2012; revised 20
January 2013; accepted 21 January 2013.
Final version published online 12 February
2013.
DOI: 10.1111/1574-6941.12076
MICROBIOLOGY ECOLOGY
Editor: Christoph Tebbe
Keywords
flagellation; free swim; circular motion; wall
effect; viscosity.
Abstract
Motility is an important trait for some bacteria living in nature and the analyses of it can provide important information on bacterial ecology. While the
swimming behavior of peritrichous bacteria such as Escherichia coli has been
extensively studied, the monotrichous bacteria such as the soil inhabiting and
plant growth promoting bacterium Pseudmonas fluorescens is not very well
characterized. Unlike E. coli that is propelled by a left-handed flagella bundle,
P. fluorescens SBW25 swims several times faster by rotating a right-handed flagellum. Its swimming pattern is the most sophisticated known so far: it swims
forward (run) and backward (backup); it can swiftly ‘turn’ the run directions
or ‘reorient’ at run-backup transitions; it can ‘flip’ the cell body continuously
or ‘hover’ in the milieu without translocation. The bacteria swam in circles
near flat surfaces with reduced velocity and increased turn frequency. The viscous drag load due to wall effect potentially accounts for the circular motion
and velocity change, but not the turn frequency. The flagellation and swimming behavior of P. fluorescens SBW25 show some similarity to Caulobacter, a
fresh-water inhabitant, while the complex swimming pattern might be an adaptation to the geometrically restricted rhizo- and phyllospheres.
Introduction
Flagella-mediated motion is indispensable in microbial
ecology, but was historically underdeveloped partially due
to the fact that the physics governing microbial behavior
is different from the macroscopic ecological phenomena
(Berg, 1990). Motility promotes bacterial fitness through
relocation to an optimal site in the ever-changing environment (Berg, 1975; Fenchel, 2002). As a consequence,
different cell shapes and flagellations have been evolved in
bacteria (Young, 2006). The swimming behavior of the
peritrichous rods such as Escherichia coli and Salmonella
enterica serovar Typhimurium has been extensively studied (Berg, 2003; Ping, 2012). Their lateral flagella form a
left-handed helical bundle that rotates counterclockwise
(when viewed from behind the cell) to propel the cell forward (Termed run; Darnton et al., 2007). The nearly
straight runs are punctuated by erratic reorientation every
1 s, when flagella reverse rotation direction (Termed
tumbles; Ping, 2012).
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Unlike enteric bacteria, pseudomonads are traditionally
regarded as monotrichous, i.e. they are propelled by a single flagellum at one of the cell poles (Lugtenberg et al.,
2001). This is a highly diverse bacterial genus, including
plant growth promoting organisms (e.g. Pseudomonas fluorescens), bioremediation agents (e.g. Pseudomonas putida),
and opportunistic pathogens (e.g. Pseudomonas aeruginosa;
Holloway & Morgan, 1986). The available information on
their swimming behavior, though sparse in literature,
clearly indicates that they adopt completely different strategies from the peritrichous ones (Ping, 2012). For example, P. aeruginosa swims much faster than E. coli (Vaituzis
& Doetsch, 1969; Conrad et al., 2011); Pseudomonas citronellolis swims backward (Termed backup) rather than
tumble when its flagellum reverses rotation (Taylor &
Koshland, 1974); P. putida can swiftly change the direction while swim forward (Termed turn; Harwood et al.,
1989; Duffy & Ford, 1997; Davis et al., 2011).
Many of the fluorescent pseudomonads are plant commensals living in rhizosphere and phyllosphere (Loper
FEMS Microbiol Ecol 86 (2013) 36–44
37
Swimming behavior of P. fluorescens SBW25
et al., 2012). Pseudmonas fluorescens SBW25 is a widelyused microorganism in ecological research due to its
potential applications in agriculture (J€aderlund et al.,
2008; Silby et al., 2009). It was originally isolated from
the leave of sugar beet (Thompson et al., 1995). When
inoculated in soil or on seeds, it colonizes the root
(Maraha et al., 2004) and other plant surfaces (Thompson
et al., 1995; Unge & Jansson, 2001; Humphris et al.,
2005). Motility plays a key role in the dispersion and surface colonization of soil inhabiting bacteria (Barahona
et al., 2010). When P. fluorescens SBW25 colonizes rhizosphere, one of the induced genes is fliF, encoding a component of bacterial flagella (Gal et al., 2003; Silby et al.,
2009). Motile P. fluorescens SBW25 cells are more efficient on attaching to the plant root compared to the isogenic nonmotile cells (Turnbull et al., 2001). Hypermotile
variants of P. fluorescens F113 arise spontaneously when
they grow in rhizosphere (Navazo et al., 2009). However,
the motility of P. fluorescens SBW25 and other strains
were merely studied en masse that based on agar diffusion
and capillary assays thus far (Faust & Doetsch, 1969;
Turnbull et al., 2001; Barahona et al., 2010). In this communication, we report the first systematic analysis of single-cell behavior of P. fluorescens SBW25 and the
comparison with what is known for the peritrichous bacteria and other Pseudomonas. The ecological implications
of the sophisticated swimming pattern were also discussed.
Materials and methods
Fluorescence labeling
Fluorescence staining of bacterial flagella was performed
with a modified procedure that was originally developed
for staining the flagella of E. coli by Turner et al. (2000).
Briefly, a single colony of P. fluorescens SBW25 was inoculated in LB medium and grew to the early stationary
phase (OD600 = 0.4) at 23 °C with shaking at 160 rpm.
Cells from 10 mL culture were washed three times with
the chemotaxis medium designed for E. coli motility study
(Hedblom & Adler, 1980) and re-suspended in 500 lL flagella staining buffer (Turner et al., 2000) containing
0.6 mg mL1 Alexa Fluor 594 (Sigma) or 0.5 mg mL1
fluorescein-5-isothiocyanate (AAT Bioquest, Inc., Sunnyvale, CA). Twenty-five microlitre of 1 M Sodium bicarbonate solution was added to adjust the pH to 7.8. The
cell suspension was shaken for 1 h in dark at 23 °C. Free
dye was removed by three exchanges of 10 mL chemotaxis
medium after shaking for 5 min in dark. The cells were
finally suspended in 1 mL staining buffer for observation.
Five microlitre cell suspension was loaded on a Superfrost Ultra Plus slide (Thermo Scientific, Gerhard Menzel
FEMS Microbiol Ecol 86 (2013) 36–44
GmbH, Braunschweig, Germany) and observed with an
Axio Imager Z1 microscope (Carl Zeiss Microscopy
GmbH, Jena, Germany) equipped with a 63 9 oil objective lens. Images were taken within 5 min as described
previously (Ping, 2010). Non-flagellated cells were not
counted in data collection. The parameters of flagella
were measured on images of stationary flagella either
detached or on immobilized cells as previously described
(Wang et al., 2012). The contour length (L) of flagella
was calculated using formula
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
L ¼ N P2 þ ðpDÞ2 ;
where N is the number of turns per filament, P is the
pitch size, and D is the diameter. The sizes of cell bodies
were determined on bright-field images taken on the
same samples. The lower quartile of the collected data
were regarded as the size of normal cells as published
before (Ping et al., 2012).
Motility study
Bacteria were cultivated in tryptone broth (Armstrong
et al., 1967) to the early exponential phase (OD600 = 0.2)
with shaking at 23 °C. Unstained and stained cells were
concentrated five times in chemotaxis medium. After
standing still on bench for 30 min with air exposure,
2 lL cell suspension were taken from the surface layer
and filled into a sample well of a diagnostic slide
(Thermo Scientific, Gerhard Menzel GmbH). Because
oxygen depletion would handicap the bacterial motility,
all movie clips were recorded within 5 min after putting
on the coverslip. Movies were recorded at 18 frames per
second using the Axio Imager Z1 microscope with 40 9
oil objective lens and an AxioCam HSm high speed
monochrome camera (Carl Zeiss Microscopy GmbH,
G€
ottingen, Germany) within 5 min. Movies were analyzed
with the AxioVision software provided by the manufacturer and ImageJ 1.37 (National Institutes of Health,
Bethesda, MD). To calculate the full swimming speed,
only runs longer than 50 lm and backups longer than
10 lm were analyzed. Cells spontaneously tethered to the
slide surface by their flagella were recorded under same
condition. Rotation events with an angular displacement
less than p were omitted in analysis.
Influence of viscosity
Bacteria from an early exponential culture were resuspended in chemotaxis medium containing 0.1–1.0%
methyl cellulose (M7140, M6385, and M0262; Sigma). It
has been demonstrated that at the low concentrations, the
solutions of these un-branched polymers are Newtonian
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38
(Berg & Turner, 1979). Viscosity of the media was measured at 23 °C with an AVS 350 capillary viscometer
(Schott Ger€ate GmbH, Ludwigshafen, Germany). To
study the swimming behavior, the microscope was
focused to a position where uniform curvature of swimming paths was unobservable (> 100 lm away from the
surfaces). Incomplete runs and backups were analyzed
only if they were longer than 50 lm. When several runs
and backups of the same bacteria were recorded, only the
first and last runs and backups were measured to reduce
bias. Those runs shorter than 10 lm and backups shorter
than 5 lm were omitted. Chemotaxis medium without
methylcellulose was used as control.
Results and discussion
L. Ping et al.
(b)
(a)
(c)
(d)
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.).
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 lm. 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).
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Fig. 1. Free-swimming behavior of Pseudmonas fluorescens SBW25.
(a) The trajectory of a swimming bacterium. Scale bars equal 10 lm.
The inset shows a fluorescence stained bacterium. The segments of
the trajectory are labeled with Roman numbers. The backup and run
speeds (lm s1) 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.
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 lm and the diameter is
0.9 0.1 lm. 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).
FEMS Microbiol Ecol 86 (2013) 36–44
39
Swimming behavior of P. fluorescens SBW25
Table 1. Flagellar characters of Pseudmonas fluorescens SBW25 and other monotrichous bacteria
Flagellar parameters
Bacteria
Handedness
Pitch (lm)
Diameter (lm)
References
P. fluorescens SBW25
C. crescentus CB15
P. aeruginosa strain PAK
P. syringae pv glycinea
P. syringae pv tabaci 6605
Right
Right
Left
Left
Left
1.76
1.08
1.38
1.59
1.59
0.79
0.27
0.39
0.43
0.18
This study
Koyasu & Shirakihara (1984)
Fujii et al. (2008)
Fujii et al. (2008)
Taguchi et al. (2008)
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 lm s1 and
the maximum speed 102.0 lm s1; the average backup
speed is 18.0 lm s1 and the maximum 22.4 lm s1
(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 lm s1 (Kanda et al.,
2011) or 83 lm s1 (Taguchi et al., 2008). Pseudomonas
aeruginosa ATCC 15692 swim at c. 60 lm s1 (Conrad
et al., 2011). The average swimming speed of P. putida
was reported to be 44 lm s1 with maximum at
75 lm s1 (Harwood et al., 1989). Pseudomonas putida
strain KT2440 swims even slower, with an averaged c.
20.9 lm s1 and a maximum at 51.2 lm s1 (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
FEMS Microbiol Ecol 86 (2013) 36–44
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 righthanded 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).
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,
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40
L. Ping et al.
(a)
(b)
(c)
Fig. 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).
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 s1; 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
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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 lm 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 lm s1 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 lm and the average 14.6 lm
(Fig. 3c). The radius of the circular path of P. aeruginosa
ATCC 15692 was reported to be c. 12 lm, 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.
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
FEMS Microbiol Ecol 86 (2013) 36–44
41
Swimming behavior of P. fluorescens SBW25
down. We attribute this to the suppressed turns and
backups.
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.
(a)
(b)
(c)
Ecological implications
Fig. 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 lm.
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).
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 pathlengths 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 slow-
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 runbackup 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
Table 2. Influence of viscosity on the free-swimming behavior of Pseudmonas fluorescens SBW25
Sample sizes
Velocity (lm s1)
Relative amount of run (%)
Viscosity (cP)
Cell
Run
Backup
10–30 lm
30–50 lm
> 50 lm
Length of backup (lm)
1
2
4
7
19
178
200
204
179
221
239
248
256
249
278
68
57
30
14
5
21.8
17.3
33.3
42.5
38.1
24.4
24.2
23.1
29.2
25.1
53.8
58.5
43.8
28.3
36.7
15.9
18.3
11.1
13.4
17.8
FEMS Microbiol Ecol 86 (2013) 36–44
11.1
10.7
6.8
6.4
11.7
Run
45.9
44.3
27.6
27.9
17.1
Backup
12.2
12.1
9.7
9.3
7.6
18.5
19.3
8.3
9.6
7.5
Duration of run (s)
7.0
6.6
4.9
4.4
2.1
1.2
1.2
1.6
1.6
2.4
0.6
0.5
0.9
0.7
1.3
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42
L. Ping et al.
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 (G€
otz & 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 nutrientscarce 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:
F ¼ 6pgrv;
here v is swimming velocity and r is the radius of a
spherical cell. A typical E. coli cell is 2.5 lm long and
0.8 lm in diameter (Berg, 2003). The radius of a sphere
with equal volume is c. 0.62 lm. Escherichia coli generally
swims at 25 lm s1 (Berg, 2003). The P. fluorescens
SBW25 cells are slightly larger and the radius of the
equivalent sphere is c. 0.75 lm. If we take the viscosity of
the medium g 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
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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.
Acknowledgements
This research is supported by the Max-Planck Society.
We thank Christian Kost of MPI-CE Jena for providing
the bacterial strain, who obtained the strain from Paul
Rainey at New Zealand Institute for Advanced Study. We
also thank Ewald Grosse-Wilde of MPI-CE Jena for assistance on microscopy, Marcus Franke and Yanze Ren of
FSU Jena for assistance on viscosity measurements. We
also thank E. Peter Greenberg of University of Washington and Reuben Ramphal of University of Florida for
sharing their opinions about the flagellation of P. aeruginosa.
References
Armitage JP & Schmitt R (1997) Bacterial chemotaxis:
Rhodobacter sphaeroide and Sinorhizobium meliloti –
variations on a theme? Microbiology 143: 3671–3682.
Armstrong JB, Adler J & Dahl MM (1967) Nonchemotactic
mutants of Escherichia coli. J Bacteriol 93: 390–398.
Atsumi T, Maekawa Y, Yamada T, Kawagishi I, Imae Y &
Homma M (1996) Effect of viscosity on swimming by the
lateral and polar flagella of Vibrio alginolyticus. J Bacteriol
178: 5024–5026.
Barahona E, Navazo A, Yousef-Coronado F, Aguirre de Carcer
D, Martınez-Granero F, Espinosa-Urgel M, Martın M &
Rivilla R (2010) Efficient rhizosphere colonization by
Pseudomonas fluorescens f113 mutants unable to form
biofilms on abiotic surfaces. Environ Microbiol 12:
3185–3195.
Berg HC (1975) Bacterial behaviour. Nature 254: 389–392.
Berg HC (1990) Physical constraints on microbial
behavior How you act if you are very small. J Chem Ecol 16:
119–120.
Berg HC (2003) E. coli in Motion. Springer-Verlag, New York,
NY.
FEMS Microbiol Ecol 86 (2013) 36–44
Swimming behavior of P. fluorescens SBW25
Berg HC & Turner L (1979) Movement of microorganisms in
viscous environments. Nature 278: 349–351.
Berg HC & Turner L (1993) Torque generated by the flagellar
motor of Escherichia coli. Biophys J 65: 2201–2216.
Block SM, Blair DF & Berg HC (1989) Compliance of bacterial
flagella measured with optical tweezers. Nature 338: 514–518.
Chen J & Jin Y (2011) Motility of Pseudomonas aeruginosa in
saturated granular media as affected by chemoattractant.
J Contam Hydrol 126: 113–120.
Conrad JC, Gibiansky ML, Jin F, Gordon VD, Motto DA,
Mathewson MA, Stopka WG, Zelasko DC, Shrout JD &
Wong GCL (2011) Flagella and pili-mediated near-surface
single-cell motility mechanisms in P. aeruginosa. Biophys
J 100: 1608–1616.
Darnton NC, Turner L, Rojevsky S & Berg HC (2007) On
torque and tumbling in swimming Escherichia coli.
J Bacteriol 189: 1756–1764.
Davis ML, Mounteer LC, Stevens LK, Miller CD & Zhou A
(2011) 2D motility tracking of Pseudomonas putida KT2440
in growth phases using video microscopy. J Biosci Bioeng
111: 605–611.
Duffy K & Ford R (1997) Turn angle and run time
distributions characterize swimming behavior for
Pseudomonas putida. J Bacteriol 179: 1428–1430.
Faust MA & Doetsch RN (1969) Effect of respiratory
inhibitors on the motility of Pseudomonas fluorescens.
J Bacteriol 97: 806–811.
Fenchel T (2002) Microbial behavior in a heterogeneous
world. Science 296: 1068–1071.
Frymier PD, Ford RM, Berg HC & Cummings PT (1995)
Three-dimensional tracking of motile bacteria near a solid
planar surface. P Natl Acad Sci USA 92: 6195–6199.
Fujii M, Shibata S & Aizawa S-I (2008) Polar, peritrichous,
and lateral flagella belong to three distinguishable flagellar
families. J Mol Biol 379: 273–283.
Gal M, Preston GM, Massey RC, Spiers AJ & Rainey PB
(2003) Genes encoding a cellulosic polymer contribute
toward the ecological success of Pseudomonas fluorescens
SBW25 on plant surfaces. Mol Ecol 12: 3109–3121.
G€
otz R & Schmitt R (1987) Rhizobium meliloti swims by
unidirectional, intermittent rotation of right-handed
flagellar helices. J Bacteriol 169: 3146–3150.
Greenberg EP & Canale-Parola E (1977) Motility of flagellated
bacteria in viscous environments. J Bacteriol 132: 356–358.
Harwood CS, Fosnaugh K & Dispensa M (1989) Flagellation
of Pseudomonas putida and analysis of its motile behavior.
J Bacteriol 171: 4063–4066.
Hedblom ML & Adler J (1980) Genetic and biochemical
properties of Escherichia coli mutants with defects in serine
chemotaxis. J Bacteriol 144: 1048–1060.
Holloway BW & Morgan AF (1986) Genome organization in
Pseudomonas. Annu Rev Microb 40: 79–105.
Humphris SN, Bengough AG, Griffiths BS, Kilham K, Rodger
S, Stubbs V, Valentine TA & Young IM (2005) Root cap
influences root colonisation by Pseudomonas fluorescens
SBW25 on maize. FEMS Microbiol Ecol 54: 123–130.
FEMS Microbiol Ecol 86 (2013) 36–44
43
J€aderlund L, Arthurson V, Granhall U & Jansson JK (2008)
Specific interactions between arbuscular mycorrhizal
fungi and plant growth-promoting bacteria: as revealed
by different combinations. FEMS Microbiol Lett 287:
174–180.
Kanda E, Tatsuta T, Suzuki T, Taguchi F, Naito K, Inagaki Y,
Toyoda K, Shiraishi T & Ichinose Y (2011) Two flagellar
stators and their roles in motility and virulence in
Pseudomonas syringae pv. tabaci 6605. Mol Genet Genomics
285: 163–174.
Kato J, Kim H-E, Takiguchi N, Kuroda A & Ohtake H (2008)
Pseudomonas aeruginosa as a model microorganism for
investigation of chemotactic behaviors in ecosystem. J Biosci
Bioeng 106: 1–7.
Keller JB (1974) Effect of viscosity on swimming velocity of
bacteria. P Natl Acad Sci USA 71: 3253–3254.
Koyasu S & Shirakihara Y (1984) Caulobacter crescentus
flagellar filament has a right-handed helical form. J Mol Biol
173: 125–130.
Lauga E, DiLuzio WR, Whitesides GM & Stone HA (2006)
Swimming in circles: motion of bacteria near solid
boundaries. Biophys J 90: 400–412.
Li G, Tam L-K & Tang JX (2008) Amplified effect of
Brownian motion in bacterial near-surface swimming.
P Natl Acad Sci USA 105: 18355–18359.
Loper JE, Hassan KA, Mavrodi DV et al. (2012) Comparative
genomics of plant-associated Pseudomonas spp.: insights into
diversity and inheritance of traits involved in multitrophic
interactions. PLoS Genet 8: e1002784.
Lugtenberg BJJ, Dekkers L & Bloemberg GV (2001) Molecullar
determinants of rhizosphere colonization by Pseudomonas.
Annu Rev Phytopathol 39: 461–490.
Magariyama Y, Masuda S-y, Takano Y, Ohtani T & Kudo S
(2001) Difference between forward and backward swimming
speeds of the single polar-flagellated bacterium, Vibrio
alginolyticus. FEMS Microbiol Lett 205: 343–347.
Maraha N, Backman A & Jansson JK (2004) Monitoring
physiological status of GFP-tagged Pseudomonas fluorescens
SBW25 under different nutrient conditions and in soil by
flow cytometry. FEMS Microbiol Ecol 51: 123–132.
McCarter L, Hilmen M & Silverman M (1988) Flagellar
dynamometer controls swarmer cell differentiation of
V. parahaemolyticus. Cell 54: 345–351.
Navazo A, Barahona E, Redondo-Nieto M, Martınez-Granero
F, Rivilla R & Martın M (2009) Three independent
signalling pathways repress motility in Pseudomonas
fluorescens F113. Microb Biotechnol 2: 489–498.
Ping L (2010) The asymmetric flagellar distribution and
motility of Escherichia coli. J Mol Biol 397: 906–916.
Ping L (2012) Cell orientation of swimming bacteria: from
theoretical simulation to experimental evaluation. Sci China
Life Sci 55: 202–209.
Ping L, Mavridou DAI, Emberly E, Westermann M &
Ferguson SJ (2012) Vital dye reaction and granule
localization in periplasm of Escherichia coli. PLoS ONE 7:
e38427.
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
44
Ramia M, Tullock DL & Phan-Thien N (1993) The role of
hydrodynamic interaction in the locomotion of
microorganisms. Biophys J 65: 755–778.
Schneider WR & Doetsch RN (1974) Effect of viscosity on
bacterial motility. J Bacteriol 117: 696–701.
Sigee DC & El-Masry MH (1989) Changes in cell size and
flagellation in the phytopathogen Pseudomonas syringae pv.
tabaci cultured in vitro and in planta: a comparative
electron microscope study. J Phytopathol 125: 217–230.
Silby M, Cerdeno-Tarraga A, Vernikos G et al. (2009)
Genomic and genetic analyses of diversity and plant
interactions of Pseudomonas fluorescens. Genome Biol 10:
R51.
Silverman M & Simon M (1974) Flagellar rotation and the
mechanism of bacterial motility. Nature 249: 73–74.
Taguchi F, Shibata S, Suzuki T, Ogawa Y, Aizawa S-I,
Takeuchi K & Ichinose Y (2008) Effects of glycosylation on
swimming ability and flagellar polymorphic transformation
in Pseudomonas syringae pv. tabaci 6605. J Bacteriol 190:
764–768.
Taylor BL & Koshland DE Jr (1974) Reversal of flagellar
rotation in monotrichous and peritrichous bacteria:
generation of changes in direction. J Bacteriol 119: 640–642.
Thompson IP, Lilley AK, Ellis RJ, Bramwell PA & Bailey MJ
(1995) Survival, colonization and dispersal of genetically
modified Pseudomonas fluorescens SBW25 in the
phytosphere of field grown sugar beet. Nat Biotech 13:
1493–1497.
Turnbull GA, Morgan JAW, Whipps JM & Saunders JR (2001)
The role of bacterial motility in the survival and spread of
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
L. Ping et al.
Pseudomonas fluorescens in soil and in the attachment and
colonisation of wheat roots. FEMS Microbiol Ecol 36: 21–31.
Turner L, Ryu WS & Berg HC (2000) Real-time imaging of
fluorescent flagellar filaments. J Bacteriol 182: 2793–2801.
Unge A & Jansson J (2001) Monitoring population size,
activity, and distribution of gfp-luxAB -tagged Pseudomonas
fluorescens SBW25 during colonization of wheat. Microb Ecol
41: 290–300.
Vaituzis Z & Doetsch RN (1969) Motility tracks: technique for
quantitative study of bacterial movement. Appl Microbiol 17:
584–588.
Wang W, Jiang Z, Westermann M & Ping L (2012) Three
mutations in Escherichia coli that generate transformable
functional flagella. J Bacteriol 194: 5856–5863.
Xie L, Altindal T, Chattopadhyay S & Wu X-L (2011) Bacterial
flagellum as a propeller and as a rudder for efficient
chemotaxis. P Natl Acad Sci USA 108: 2246–2251.
Young KD (2006) The selective value of bacterial shape.
Microbiol Mol Biol Rev 70: 660–703.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Movie S1. A video clip showing the flip behaviour of
Pseudomonas fluorescens SBW25.
Movie S2. A movie shows the hover behaviour of Pseudomonas fluorescens SBW25.
FEMS Microbiol Ecol 86 (2013) 36–44