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Journal of Bacteriology, April 1999, p. 2593-2601, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gliding Mutants of Myxococcus xanthus
with High Reversal Frequencies and Small Displacements
Alfred M.
Spormann1,2,* and
Dale
Kaiser3
Max-Planck-Institut für Terrestrische
Mikrobiologie, 35043 Marburg, Germany,1 and
Environmental Engineering and Science, Department of Civil
and Environmental Engineering,2 and
Departments of Biochemistry and Developmental
Biology,3 Stanford University, Stanford,
California 94305
Received 10 November 1998/Accepted 5 February 1998
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ABSTRACT |
Myxococcus xanthus cells move on a solid surface by
gliding motility. Several genes required for gliding motility
have been identified, including those of the A- and S-motility systems
as well as the mgl and frz genes.
However, the cellular defects in gliding movement in many of these
mutants were unknown. We conducted quantitative, high-resolution
single-cell motility assays and found that mutants defective in
mglAB or in cglB, an A-motility gene, reversed
the direction of gliding at frequencies which were more than 1 order of
magnitude higher than that of wild type cells (2.9 min
1
for 
mglAB mutants and 2.7 min1 for
cglB mutants, compared to 0.17 min1 for
wild-type cells). The average gliding speed of mglAB
mutant cells was 40% of that of wild-type cells (on average 1.9 µm/min for mglAB mutants, compared to 4.4 µm/min for
wild-type cells). The mglA-dependent reversals and
gliding speeds were dependent on the level of intracellular MglA
protein: mglB mutant cells, which contain only 15 to 20%
of the wild-type level of MglA protein, glided with an average
reversal frequency of about 1.8 min1 and an average
speed of 2.6 µm/min. These values range between those exhibited by
wild-type cells and by mglAB mutant cells. Epistasis
analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation,
but not a frzE mutation, partially suppressed the
mglA phenotype. In contrast to mgl mutants,
cglB mutant cells were able to move with wild-type speeds
only when in close proximity to each other. However, under those
conditions, these mutant cells were found to glide less often with
those speeds. By analyzing double mutants, the high reversing
movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of
mglAB cells was only partially reduced by a
pilR mutation. These results suggest that the MglA protein
is required for both control of reversal frequency and gliding speed
and that in the absence of A motility, type IV
pilus-dependent cell movement includes reversals at high frequency.
Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.
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INTRODUCTION |
Many bacterial species spread over
surfaces as expanding swarms (1, 3, 9, 18). The swarming
behavior of Myxococcus xanthus is due to gliding motility of
individual cells; M. xanthus cells carry no flagella and
cannot swim. Gliding movement consists of translocation along the
cell's long axis on a solid surface with one or the other pole leading
the way. Swarms of M. xanthus expand by coordinating
the gliding movements of individual cells (15, 18). Two
multigene systems, the A- and S-motility systems, and the
mgl and frz genes have been identified as
affecting the ability of vegetative colonies to swarm on agar plates
(2, 12, 13, 27).
Among the M. xanthus motility mutants isolated so far,
mglA mutants have the strongest defect in that colonies are
rendered nonswarming by a mutation in a single gene (11,
12); their colonies are heaped in the center and have a sharp
edge that expands outward due only to cell growth and division. Because
the colony shape of mgl mutants is indistinguishable from
that of A S double mutants, the
mgl gene is considered to be required for both A motility
and S motility (13). These observations had initially
suggested that the MglA protein might be part of the gliding motor
(22), but the protein was localized to the cytoplasm and the
predicted amino acid sequence resembles those of GDP/GTP binding
proteins of the p21Ras type (6, 7). Therefore,
it appears to be more likely that the MglA protein is involved in
regulating the gliding motor. The mglA gene is cotranscribed
with an adjacent gene called mglB. Deletion of the
mglB gene reduces the level of mglA protein to about 15% of the wild-type level (8). The swarming behavior of mglB mutants is intermediate between those of
mglA mutants and the wild type; mglB mutants
spread at about 25% of the rate of wild-type swarms and 10 times
faster than mglA mutant swarms (8).
Wild-type swarming of M. xanthus requires both A
motility and S motility (13, 15). Previous studies showed
that mutants defective in the A-motility system were impaired in
swarming, and single cells were found to be unable to glide as
individual isolated cells (12, 19b). Molecular analysis of
one of the A-motility genes, the cglB gene, showed that this
gene encodes a lipoprotein with an unusually high cysteine content
(19b). All motility retained in this and other
A mutants is considered to be S motility, because
A S double mutants do not swarm at all
(12). Recently, Wu and Kaiser (23-26) discovered
that all of the S-motility genes in the sglI region of
M. xanthus encode proteins required for the synthesis, export, assembly, and regulation of type IV pili. How type IV pili
produce S motility is unknown; however, these pili are also required
for twitching motility (3, 10).
The M. xanthus frz genes were first identified in
mutants with an altered aggregation behavior during fruiting-body
development (27). The predicted amino acid sequences of the
Frz proteins exhibit extensive homology to those of proteins of
two-component signal transduction systems, specifically those involved
in chemotaxis (che genes) in a variety of prokaryotes and in
control of twitching motility in Pseudomonas aeruginosa
(5, 19). Single isolated cells of frz mutants are
defective in reversing the direction of gliding. With the exception of
a frzD mutant, frz mutants
(frzA, -B, -C, -E, and
-F) reverse less frequently than wild-type cells (2). A transposon insertion in the frzD region of
the frzCD gene generated a mutant where the cells reverse
more often than wild-type cells (2).
During our motility studies of mgl and cglB
mutants, we recognized that both motility mutants reverse the direction
of movement more often than the wild type. We then investigated
systematically the movement of individual cells with high optical and
temporal resolutions (21). With the assay employed,
displacements of as low as 0.03 µm and translocation speeds of as low
as 1 µm/min were detectable. Movements of single cells of
mgl, cglB, frz, and various double
mutants were quantified to examine the relationship between the A- and
S-motility genes and the mgl and frz genes in
controlling single-cell motility.
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MATERIALS AND METHODS |
Bacteria and growth.
M. xanthus (14)
DK1622 and its motility mutant strains were routinely grown on CTT agar
plates (11). To assay gliding motility, single colonies were
picked, transferred to 5 ml of CTT broth in 50-ml flasks, and grown
at 32°C. Cultures were aerated by rotary shaking at 200 rpm.
Strains DK9711 (frzE226::Tn5) and DK9713 (mglAB frzE226::Tn5) were
constructed by Mx8 transduction of kanamycin resistance from
strain DZ3377 (frzE226::Tn5) to
DK1622 (frz+) and to DK6204
(frz+ mglAB), respectively.
Strains DK9712 (frzCD224::Tn5) and
DK9714 (mglAB frzCD224::Tn5) were
constructed by Mx8 transduction of kanamycin resistance from strain
DZ4033 (frzCD224::Tn5) to DK1622 and to
DK6204, respectively. Strain DK9715 was constructed by Mx8 transduction
of kanamycin resistance from strain DK3163
[pilR::Tn5(
3163)] to DK6204.
Strains DK9716 and DK9717 were constructed by Mx8 transduction of
tetracycline resistance from strains DK3164
[pilR::Tn5-132(3164)] and DZ4040
[frzE::Tn5(234)], respectively, to
strain JZ315. Strains DZ3377, DZ4033, and DZ4040 were kindly provided
by D. Zusman, and strain JZ315 was kindly provided by J. Zissler
(2, 16, 19). The M. xanthus strains used and
their relevant genotypes are summarized in Table
1.
Recording and quantification of cell movement by video
microscopy.
From cultures grown to 80 to 100 Klett density units
(4 × 108 to 5 × 108 cells/ml),
10-µl aliquots were withdrawn and spotted on CTT plates (1.5% agar)
which had been prepared the day prior to use (21). The
droplets were allowed to dry onto the agar for 10 to 15 min and were
immediately examined by microscopy. Recording and analyzing of cell
movement were conducted by methods described previously (21).
Swarming motility under starvation conditions.
M.
xanthus DK1622, DK9712, DK6204, and DK9714 were grown in CTT broth
to a density of 100 Klett units (5 × 108 cells/ml)
and concentrated 10 times. Ten-microliter aliquots were spotted on TPM
plates (17) containing 0.6% agar and incubated for 24 h at 32°C in the dark. Plates were prepared the day before use.
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RESULTS |
M. xanthus cells move on solid surfaces by gliding
(4, 18, 21). Occasionally, cells reverse the direction of
movement, so the leading end of the cell becomes the lagging end.
During our video microscopic studies of M. xanthus,
we noticed that cells of mglAB mutants and of A-motility
(cglB) mutants reversed their direction of gliding more
often than wild-type cells. We therefore first studied gliding of
M. xanthus wild-type cells with respect to
reversals and gliding speed in both directions.
Reversals in wild-type cells.
The gliding speed of
M. xanthus wild-type cells was measured, using a
motility assay with high optical and temporal resolutions (21). M. xanthus wild-type cells
(DK1622) that executed at least one reversal during the period of
observation were clocked in both their forward and backward directions,
and gliding speeds were measured for each. Figure
1 displays the average speed in each
direction for 10 cells. No significant difference in gliding speed was
evident despite the fourfold variation in average speed among these
cells, implying that individual M. xanthus cells
have no kinetically preferred gliding direction. Speed variations
between M. xanthus cells are common, and the
observed speeds fall within the normal range (21).
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FIG. 1.
Forward (solid bars) and backward (open bars)
translocation velocities of individual DK1622 cells. Ten cells were
measured when moving in the forward and backward directions. For each
cell all speed values from each direction were pooled, and the average
speed and standard deviation were calculated.
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A reversal of gliding in a single
M. xanthus cell
is characterized as the following sequence: movement in the forward
direction,
stop, and movement in the backward direction. The transition
from
gliding in one direction to gliding in the reverse direction is
observed as a transient decrease in gliding speed to less than
1 µm/min (the minimum speed that is measurable [
21])
before
a cell resumes movement in the opposite direction. During our
studies on the reversal behavior, we noticed that gliding
movements
which are interrupted by stops can be grouped into
three classes:
(i) forward movement, stop for less than 9 s, and
backward movement
(we considered this sequence a reversal); (ii)
forward movement,
stop for more than 9 s, and forward movement (we
called this sequence
a pause); and (iii) forward movement, stop for
more than 9 s,
and backward movement. In this study, we analyzed
only the first
type of
reversals.
How often do wild-type cells reverse the direction of gliding? The time
interval between two reversals in DK1622 cells was
found to be
variable. Of 250 cells that all moved during the observation
period
(Table
2), 13 were found to reverse once
or more than
once within a period of 20 min. Those cells that reversed
performed
on average 0.17 reversal per min (Table
2). In an independent
experiment, 5 of 90 wild-type cells reversed once or more than
once
within 10 min. Monitoring cells for longer than 20 min proved
to be
difficult because cells would often associate with other
cells in a
group and the cell being tracked could not be identified
within the
group. Since the longer reversal times were difficult
to measure, the
true average reversal frequency for wild-type
cells may be less than
the 0.17 reversal per min recorded.
Reversals in mgl mutant cells.
To investigate the
function of the MglA protein in gliding, movement of individual
mglAB motility mutant cells was quantified. During a
20-min interval, 696 of 792 mglAB cells moved: only 12%
failed to move. Figure 2 displays the
velocity profile of one cell. As illustrated, the movement pattern of
this particular mglAB cell was characterized by frequent
reversals and abrupt, often jerky changes in gliding speed (at
t = 1 to 140 s and 200 to 310 s) which
alternated with intervals where no significant movement was detectable
(t = 140 to 160 s and 310 to 400 s). During the total observation time of about 474 s, the cell shown in Fig. 2 completed 14 reversals. The net displacements performed were less
than 1 µm in 9 min. Figure 3 summarizes
the speed measurements for 50 mglAB cells. The average
speed of an actively gliding mglAB mutant cell was
1.9 ± 1.1µm/min. Figure 3 also indicates that 43% of the 2,927 recorded speed values were below 1.0 µm/min, which is the lower limit
of reliable measurement (21). Reversal frequencies for
50 mglAB mutant cells were quantified. An average reversal frequency of 2.9 min1 (standard deviation, 1.4 min1) was calculated from 439 reversals (Table 2),
indicating that the reversal frequency of mglAB mutant
cells is at least 1 order of magnitude higher than that of DK1622 cells
(Table 2). Cells of mglAB mutants traveled on average
0.3 ± 0.4 µm before reversing their directions. These data are
compatible with the time-lapse photographs of mglA mutant
cells published by Hodgkin and Kaiser (13), who reported no
net movement over a period of 3 h.
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FIG. 2.
Velocity profile of an M. xanthus
mglAB cell. Strain DK6204 was grown to a density of 5 × 108 cells/ml in CTT broth. Ten microliters was spotted on a
1-day-old CTT plate (1.5% agar). The lagging end of the cell was
tracked for 474 s. Velocity (solid line) and net displacement
(dashed line) are displayed. The cell reversed the direction of
movement 14 times, at t = 15, 30, 63, 75, 96, 102, 192, 222, 261, 288, 363, 399, 444, and 453 s. Reversal frequencies were
calculated from intervals t = 15 to 30, 63 to 75, 96 to
102, 222 to 261, and 261 to 288 s.
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FIG. 3.
Distribution of gliding speeds of mglAB
(DK6204) and mglB (DK6206) cells. Gliding speeds from 50 mglAB cells (open bars) and 50 mglB cells
(solid bars) were measured. The speed values for each strain were
grouped, and the number of speed values qualifying for each category
was determined. For mglAB cells 2,927 values were
collected, and for mglB cells 11,169 values were
collected.
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The
mglA gene is cotranscribed with another gene, called
mglB. Cells of a
mglB mutant strain contain
only 15% of the wild-type
level of the MglA protein (
8).
This reduction in MglA protein
level is not due to transcriptional
control. In contrast to
mglA point mutants and
mglAB deletion mutants, some swarming of
mglB colonies is observed on a 1.5% agar surface. However, the swarm
expansion rate is one-quarter of that of the wild type (
8).
We examined 412 individual
mglB mutant cells by video
microscopy.
More than 98% of these cells moved during a 20-min time
interval.
The gliding profile of a single
mglB mutant
cell is displayed
in Fig.
4. In contrast
to the
mglAB mutant cell (Fig.
2), this
mglB cell moved during the entire period of observation.
The
cell maintained movement in one direction for up to 160 s
before
reversing. Gliding speeds for 50
mglB mutant cells
were determined
and are represented in Fig.
3. Their average speed is
2.6 ± 1.5
µm/min. Of the 11,169 speed values obtained, 6% were
below the
minimal detectable speed, indicating that a
mglB cell is actively
gliding for 94% of the time. A
particular cell (Fig.
4) accomplished
four reversals in 474 s.
Reversals for 50
mglB mutant cells were
quantified, and
of 393 reversals counted, the average value was
calculated to be 1.8 reversals per min with a standard deviation
of 1.3 (Table
2). To test
whether the difference in the average
reversal frequencies of
mglAB and
mglB mutant cells is statistically
significant, the two distributions were subjected to a paired
Student
t test. Taking each measurement as being independent,
the
probability that the two distributions were derived from one
and the
same distribution and appeared to be different by chance
was found to
be less than 10
17. Although the mean values are close
(2.9 and 1.8 min
1, respectively), the average reversal
frequencies are significantly
different. Thus, a decrease in the level
of
mglA protein is reflected
by an increase in the frequency
of reversal.
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FIG. 4.
Velocity profile of an M. xanthus
mglB cell. Strain DK6206 was grown to a density of 5 × 108 cells/ml in CTT broth. Ten microliters was spotted on a
1-day-old CTT plate (1.5% agar). The lagging end of the cell was
followed for 474 s. The solid line indicates velocity, and the
dashed line indicates net displacement. The cell conducted four
reversals (at t = 150, 195, 255, and 303 s).
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Reversals in A mutant cells.
Colonies of mutants
defective in the A-motility system exhibit sharp edges, and no single
cells are detectable at the perimeter (12, 19b). Because no
agl null mutant is available, single-cell analyses were
conducted with strain JZ315, which carries a Tn5phoA insertion in the cglB gene (16). Cells of this
strain and agl mutant strains are nonmotile when separated
from each other by more than 2 µm (12, 19b). However, when
in close proximity to each other (<2 µm), cglB mutant
cells showed active movements, presumably reflecting their S motility.
These movements included an increased and highly variable pattern of
cell reversals. The patterns are illustrated in the velocity profiles
of three individual cells (Fig. 5).
During the approximately 200-s observation period, the cell whose
pattern is shown in Fig. 5A performed abrupt movements that included 15 reversals. If movement of cglB cells included such frequent
reversals and if most of the runs in one direction lasted for 30 s
or less, then we considered these cells to be moving in a high-reversal
mode. More than half (54%) of 50 cells that were analyzed in detail
moved in the high-reversal mode as illustrated in Fig. 5A. Considering
reversals of 50 cells in a high-reversal mode, the average number of
reversals was 2.7 (±1.4) min1 (Table
3). Another one-third of the
cglB cells investigated moved in a pattern that is
characterized by the absence of reversals, such as is shown in Fig. 5B.
This cell translocated at high speed for a period of up to 150 s
without a reversal. This fast forward movement was not smooth but was
subject to abrupt changes in translocation speed; after the first
reversal, the speed fluctuated between the following approximated
values (micrometers per minute): 8, 2, 6, 4, 11, 8, 12, and 8. The
apparent bimodal movement pattern (high reversal [Fig. 5A] or low
reversal [Fig. 5B]) of cglB cells is not due to two
subpopulations in the culture, because the same cell can move in both
modes, as shown in Fig. 5C. About 16% of the cells investigated were
able to move in a pattern where periods of high reversals (Fig. 5C, 30 to 100 s) alternated with intervals of unidirectional, relatively
fast movement (Fig. 5C, 100 to 170 s). Thus, for cglB
mutant cells in close proximity to each other, each cell is able to
move in either a high-reversal or a low-reversal mode, with a higher
probability of moving in the former mode. A correlation between a
particular reversal mode and the cell-to-cell distance or the cell
orientation could not be addressed, because these movements were
observed for cells in contact with each other.
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FIG. 5.
Movement patterns of cglB cells (JZ315)
[A S+]) when in close proximity to each
other. Panels A, B, and C show results for three different cells. For
details, see the text.
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We examined the average translocation speed of
cglB cells
when in close proximity. For 2,621 speed measurements obtained from
50 cells, the average speed of high-reversal cells was determined
to be
2.5 (±1.7) µm/min (Table
3). This value is below the average
speed
of 5.0 µm/min for wild-type cells when close to each other
(
21). However, for low-reversal cells the average velocity
was
4.7 (±3.0) µm/min, and those
cglB cells were able to
glide with
the same high velocities as wild-type cells (Fig.
6). Both wild-type
and
cglB
cells utilize the same high speeds at approximately the
same frequency
(Fig.
6, speed ranges of >4 µm/min). One noticeable
difference is
that
cglB cells moved significantly more often at
speeds of
1 to 2 µm/min. Because only a small fraction of these
cells were
conducting unidirectional, high-speed movements and
because the average
speed value integrates all speed measurements,
this average value does
not reflect adequately the ability of
A
cells to move
with wild-type speeds in close proximity.
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FIG. 6.
Distribution of gliding speeds of wild-type (solid bars)
and cglB (open bars) cells when in close proximity
(cell-cell distance of  1 µm). The gliding speeds of 50 cells were
measured for each strain. Distributions were calculated from 3,446 speed measurements for DK1622 cells and 2,621 measurements for JZ315
cells.
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Reversals in double-mutant cells containing frz
mutations.
Individual cells of M. xanthus
mutants defective in the frz genes had previously been
described to be altered in control of reversal frequency
(2). However, the phenotype of a frz deletion mutant is opposite from that of a mgl mutant; most
frz mutants have a decreased rather than increased reversal
frequency (2) (Table 2). Under our experimental conditions,
a frzE mutant (DK9711) moved with an average speed of
4.3 ± 2.0 µm/min and an average reversal frequency of <0.02
reversal per min (Table 2).
One exception to the pattern of decreased reversal frequency of
frz mutants is a Tn
5 insertion in the 3' end of
the
frzCD gene. Such a
frzD mutant cell (strain
DK9712) glides on average
with a speed of 4.0 ± 2.3 µm/min
and performs reversals at a rate
of 1.5 ± 1.1 per min. This
average reversal frequency is comparable
to the value determined
earlier by Blackhart and Zusman (
2).
To investigate the
possibility of a connection between the
frz-dependent
and
the
mgl-dependent control of reversal frequency, two
double-mutant
strains, a
mglAB frzE mutant (DK9713) and a
mglAB frzD mutant
(DK9714), were constructed, and the
behavior of individual double-mutant
cells was examined by
high-resolution video microscopy. As shown
in Table
2, cells of the
mglAB frzE double mutant reversed the
direction of
movement with an average frequency of about 2.7 ±
1.4 min
1. This frequency was very similar to the 2.9 ± 1.4 min
1 which was measured for
mglAB
mutants and was clearly different
from the frequency found for
frzE mutant cells (<0.02 min
1) (Table
2). In
addition, the average gliding speed of 2.1 ±
1.4 µm/min for
mglAB frzE double mutants was similar to that
for
mglAB mutants (1.9 ± 1.1 µm/min) but markedly
different from
that for
frzE mutant cells (4.3 ± 2.0 µm/min) (Table
2). Single
cells of the
mglAB frzD
strain (DK9714) also showed a reversal
frequency and average gliding
speed that are very similar to those
of
mglAB (Table
2).
We also examined the effect of a
frzE mutation on the
high-reversal phenotype of
cglB cells by analyzing the
single-cell movement
of a
cglB frzE double mutant (Table
3).
About 98% of the 240
cells examined were actively moving. Detailed
analysis of 50
cglB frzE cells revealed that the
high-reversal phenotype of
cglB cells
was retained (Table
3). The average number of reversals for the
double-mutant cells moving
in a high-reversal mode was 2.6 ± 1.4
min
1. Similar
to the
cglB strain, cells were observed to move also
in a
low-reversal mode with increased speed (3.4 ± 2.8 µm/min
[data
not shown]). Thus, a
frzE mutation does not affect the
high-reversal
behavior of
cglB cells.
mglA (
17) and
frz (
27) are
also important for cell movement under starvation conditions that
induce fruiting-body development.
Swarming motility integrates the
gliding movements of individual
cells and can be easily detected with
the unaided eye. To further
investigate the connection between the
frz-dependent and
mglA-dependent
control of cell
movement, the macroscopic swarming behaviors of
the
mglA frz
double mutants under growth and starvation conditions
were compared. To
examine swarming edges under vegetative conditions,
CTT plates (1.5%
agar) were inoculated with single cells of strains
DK1622, DK6204,
DK9712, and DK9714 and incubated at 32°C. Swarming
edges of single
colonies were recorded after 5 days (Fig.
7).
To study swarming edges under
starvation conditions, cells of
these strains were grown in CTT medium,
concentrated 10 times,
and spotted on petri plates containing TPM
starvation medium with
0.6% agar. Edges of swarms were examined after
incubation at 32°C
for 24 h (Fig.
8). As expected,
mglAB
mutants exhibited a sharp
swarming edge (Fig.
7C and
8C) compared to
the wild type (Fig.
7A and
8A). Swarms of
frzD mutants
showed an edge containing numerous
pronounced flares and projections
(Fig.
7B and
8B).
mglAB frzD double-mutant cells had
projections (Fig.
7D and
8D) like those
of
frzD mutant cells
(Fig.
7B and
8B), which was qualitatively
different from the
mglAB mutant (Fig.
7C and
8C). The wave-shaped
swarming
edge of strain DK9714 (
mglAB
frzCD224::Tn
5), which is
clearly different
from that of DK6204 (
mglAB) cells, implies
active net
cell movement. The swarming pattern of
mglAB frzE mutant
cells under the same conditions was indistinguishable from
that of
mglAB mutant cells; i.e., there was a sharp edge like
that in Fig.
7C and
8B (data not shown). The swarming behavior
of the
wild type and the
mglAB and
mglAB frz
mutants on TPM
starvation plates containing 1.5% (data not shown) or
0.6% (Fig.
8) agar was observed. The swarming effect of
mglAB
frzD was most
pronounced on the support with the lower percentage
of agar.
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FIG. 7.
Swarming patterns of M. xanthus
motility strains under vegetative conditions on CTT plates (1.5%
agar). Cells were streaked on CTT agar plates, which were
prepared 1 day before use. The plates were incubated at 32°C for 5 days in the dark. (A) DK1622; (B) DK9712; (C) DK6204; (D) DK9714. Bar,
100 µm.
|
|
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|
FIG. 8.
Swarming patterns of M. xanthus
motility strains on TPM starvation agar. The agar plates contained 10 mM Tris-HCl (pH 7.6), 1 mM potassium phosphate, 8 mM magnesium sulfate,
and 0.6% agar. The plates were prepared on the day prior to use. Cells
were grown in CTT broth to a density of 5 × 108
cells/ml and concentrated 10 times, and 10-µl drops were spotted on
the agar plates. The plates were incubated at 32°C for 24 h in
the dark. (A) DK1622; (B) DK9712; (C) DK6204; (D) DK9714. Bar, 100 µm.
|
|
Dependence of cell reversals on S motility.
In M. xanthus, movement of cells in close proximity is controlled by
the S-motility system (12, 15). Recently, Wu and Kaiser
(23-26) showed that the S-motility genes in the
sglI region encode type IV pili. To examine the effect of S
motility on the high-reversal phenotype of mglAB mutants, we
analyzed 310 cells of an mglAB pilR double mutant. In
general, the motility of this strain was strongly impaired. Of those
cells, only about 30% showed visible movement. In contrast to DK6204,
the movement consisted of short translocations, and only 11 of the 20 actively moving cells that were analyzed in detail retained a mode of
frequent reversals. In those cells, the average number of reversals was reduced to 2.0 (±1.4) per min (Table 3). This observation indicates that the S-motility defect reduced the extent of motility significantly but only partially affected the high-reversal mode of mglAB
mutant cells. Interestingly, the average speed of mglAB pilR
double-mutant cells was 1.9 (±1.0) µm/min, as determined from 1,694 measurements, and thus was similar to speeds of mglAB cells.
In order to examine the effect of S motility on the high-reversal
pattern of
cglB mutant cells, we constructed a
cglB
pilR double mutant and analyzed the movement of individual cells
by
video microscopy. The high-reversal phenotype in these
A
S
double-mutant cells was completely
abolished (Table
3). During
the 20-min observation period, 18 of the
164 cells conducted only
a short, one-stroke displacement of about
1.2 (±0.9) µm and then
stopped. During these short movements, the
average speed was 2.0
(±1.0) µm/min (determined from 904 speed
measurements). In contrast
to the
mglAB pilR double-mutant
phenotype, no reversals were observed.
Therefore, type IV pili are
required for
cglB mutant cells to
exhibit both the
high-reversal mode and the fast movements when
in close
proximity.
| |
DISCUSSION |
A high-resolution motility assay (21) was used to
examined the motility behavior of mglAB and cglB
mutant cells. Quantification of the gliding movement of both strains
revealed that individual cells moved in a jerky fashion with a high
reversal frequency and a reduced speed compared to DK1622 wild-type
cells (Fig. 2 and 5; Table 2). Thus, a decrease in continuous gliding
activity, a high reversal frequency, and a reduced speed can explain
the absence of net movement observed as the nonswarming phenotype of
growing mgl colonies (11), as well as the absence
of net movement after 3 h (12). We will first elaborate
on the phenotype of a cglB strain (JZ315) in order to
discuss the behavior of mgl strains.
S motility in single cells.
Recently, it was shown that the
sglI locus in M. xanthus encodes genes
necessary for the structure, export, assembly, function, and regulation
of type IV pili (15, 23-26). These extracellular appendages
are known to be involved in two types of bacterial surface
translocation mechanisms: (i) twitching motility, where cells
translocate for a few micrometers by sudden, jerky movements (3,
10), and (ii) S-motility gliding. Currently, details of twitching
movement are unknown, and the mechanistic basis of twitching is
obscure. Wild-type cells in close proximity to each other move with
high speeds (5.0 ± 2.6 µm/min) compared to speeds of isolated
cells (3.8 ± 1.4 µm/min) (21). In the
cglB strain JZ315 (A S+), movement
is detectable only when cells are in close proximity to each
other. Cells were observed to translocate either by jerky movement in a high-reversal mode or by unidirectional movement at
high, variable velocities. The latter feature is also observed in
wild-type cells (Fig. 6; Table 3). Since the cglB cells are defective in A motility, both movement patterns are solely due to S
motility. Single-cell studies on other A S+
strains, including agl mutants, should reveal whether
these motility patterns are representative of all A-motility mutants.
As determined in preliminary experiments, cglC mutant cells
(DK1219) exhibited a phenotype similar to the one observed for
cglB mutants (data not shown).
Considering that movement by S motility depends on type IV pili, it
seems plausible that S-motility in
M. xanthus
is related
to twitching; both motility forms require type IV pili
and are
comprised of jerky movements. However, in contrast to
twitching,
movement by S motility in
M. xanthus is
mostly in the direction
of a cell's long axis. S motility is observed
only when cells
are in close proximity to each other, i.e., when other
cells provide
part or all of the surface to move on (e.g., during
aggregation
and fruiting-body formation). It was previously shown that
the
A- and the S-motility systems provide advantages for
M. xanthus cells to translocate on different surfaces
(
20).
Motility defect of mgl mutants.
It was previously
shown that the cellular level of the MglA protein in an mglB
deletion mutant is reduced to about 15% of the wild-type level
(8). Here, we show that the average reversal frequency and
the gliding speed in mgl mutant cells also
correlate with the level of MglA protein; on average, wild-type cells
glide with a speed of 4.4 ± 2.2 µm/min and 0.17 reversal
per min, mglB mutant cells (containing 15% of the
wild-type MglA level) glide at 2.6 ± 1.5 µm/min and with 1.8 reversals per min, and mglAB mutant cells (containing no
MglA) glide at 1.9 ± 1.1 µm/min and with 2.9 reversals/min
(Table 2). An alteration in reversal frequency is not necessarily
coupled to a change in gliding speed, as is evident from the motility
phenotype of frzD mutant cells (Table 2). These cells move
as isolated cells with about wild-type speed (4.0 ± 2.3 µm/min)
but have an increased reversal frequency (1.5 reversals/min). In
contrast, mglB mutant cells reverse at a frequency (1.8 reversals/min) which is similar to that of frzD mutants
(1.5 reversals/min) but have a decreased gliding speed (2.6 ± 1.5 µm/min). Thus, mgl mutant cells carry two motility
defects: one in single-cell gliding speed and one in control of
reversal frequency.
In
M. xanthus, the reversal frequency is controlled
not only by
mglA but also by the
frz genes. Null
mutations in
frz and
mglAB have opposite effects;
single cells of
frzE::Tn
5 insertion
mutants showed a low number of reversals (<0.02 min
1),
whereas cells of
mglAB mutants exhibited a high number of
reversals (2.9 min
1) (Table
2). The single-cell motility
assay (Fig.
2,
3, and
4;
Table
2) and a swarming assay (Fig.
7 and
8)
were used to examine
the possible connection between
mgl-dependent and
frz-dependent
reversals of
gliding movements by analyzing
mglAB,
frz, and
mgl frz mutant cells. The observation that
mglAB
frzE double mutants
exhibit a high reversal frequency rules out
the possibility that
reversals in
M. xanthus are
generated independently by MglA and
FrzE.
One important question is how a swarming pattern relates to the gliding
speed and reversal frequency of single cells. Wild-type
cells glide
with an average speed of 4.4 µm/min and an average
reversal frequency
of 0.17 reversal per min (
21) (Table
2).
The edge of a
swarming wild-type colony shows many single cells
and cells in loose
groups (Fig.
7A). Single cells of
frzD mutants
glide with an
average speed of 4.0 µm/min and an average of 1.5
reversals per min
(Table
2) (
2). This reversal frequency is
about 10-fold
higher than that of wild-type cells. Edges of growing
colonies of
frzD mutant cells are sharp and coherent, and no single
cells are visible (Fig.
7B). Finger-like projections or flares
can be
observed (Fig.
7B). Colonies of
mglAB mutants have a
similar
morphology except that they are smaller and do not have
finger-like
projections (Fig.
7C). Single-cell analysis of
mglAB mutant cells
revealed a reversal frequency of
2.9 reversals per min, which
is about 20-fold above the wild-type
reversal frequency. Additionally,
the gliding speed of
mglAB cells was dramatically reduced, to
1.9 µm/min
from the 4.4 µm/min for wild-type cells (Table
2).
Thus, it seems
that a 10-fold increase in reversal frequency to
above that found for
wild-type cells is sufficient for the formation
of a sharp edge of an
M. xanthus colony. It is also important
to notice
that reversals in
frz mutants have been observed for
isolated cells, whereas with
cglB and most
mglAB
mutant cells,
reversals were detected for cells in close proximity to
each
other.
Studies of single-cell movement and of swarming patterns under
vegetative and developmental conditions for
mgl,
frz, and
mgl frz mutants revealed an interesting
connection between the two
sets of genes: the average gliding speed
and reversal frequency
observed for
mglAB mutant cells
are epistatic in a
frzE mutant
background (Table
2). The
motility behavior of
mglAB frzD double-mutant
cells seems to
be quite different from that of
mglAB and
mglAB frzE mutant cells. Under vegetative (Fig.
7D) and developmental
(Fig.
8D) conditions, the flares at the edges of
mglAB frzD
double-mutant
swarms resemble those of
frzD mutants but not
those of
mglAB mutants.
Flare formation suggests that
movement occurred in
mglAB frzD cells to an extent that is
different from that in
mglAB cells.
Analysis of the average
gliding speed and reversal frequency of
single
mglAB and
mglAB frzD cells did not reveal a significant
difference
between the two mutant strains (Table
2). Small differences
in
gliding speed and reversal frequency between different mutant
strains
may not be detectable by quantitative video microscopy
under the
conditions employed and may be recognized as a difference
in swarming
behavior only after prolonged incubation. Flares observed
in
mglAB frzD swarms were noticeable only after incubation for
more than 4 days on CTT agar (Fig.
7A). Thus, a
frzD
mutation
can partially suppress the S-motility (swarming) defect of
mglAB mutant cells. Since
mglAB colonies do not
swarm, the suppression
of this defect by
frzD seems to be a
gain of function in S
motility.
Although colonies of both
mglAB and A
S
mutants exhibit round, sharp edges, the motility
patterns of single cells are clearly
different (Fig.
2,
5, and
6;
Tables
2 and
3). In contrast to
A
S
cells,
mglAB cells show a residual movement pattern of
high
reversals that requires the presence of type IV pili (encoded
by
S-system genes [Table
3]). Furthermore, a
frzD mutation
partially
suppresses the group swarming defect of
mglAB
cells. This effect
is more visible under conditions of low percent agar
concentration,
where
M. xanthus moves
mostly by S motility (
20). Comparing
the gliding
movements of
mglAB and A
S
cells, it seems that the
mglAB mutant
behaves as if it is a
strong A-motility system mutant with only
partially defective
S
motility.
| |
ACKNOWLEDGMENTS |
We thank Hans Warrick for many helpful discussions and advice on
using the optical and electronic equipment.
This work was supported by National Science Foundation grant MCB
9423182 to D.K. A.M.S. was a recipient of a postdoctoral fellowship from the Max-Planck-Gesellschaft.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Environmental
Engineering and Science, Department of Civil and Environmental
Engineering, Stanford University, Stanford, CA 94305-4020. Phone: (650)
723-3668. Fax: (650) 725-3164. E-mail:
spormann{at}ce.stanford.edu.
| |
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Journal of Bacteriology, April 1999, p. 2593-2601, Vol. 181, No. 8
0021-9193/99/$04.00+0
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Top
Abstract
Introduction
