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Appl Environ Microbiol, May 1998, p. 1708-1714, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Starvation-Induced Changes in Motility, Chemotaxis,
and Flagellation of Rhizobium meliloti
Xueming
Wei1 and
Wolfgang D.
Bauer2,*
Department of Plant
Biology1 and
Department of Horticulture
and Crop Science,2 Ohio State University,
Columbus, Ohio 43210
Received 12 January 1998/Accepted 9 March 1998
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ABSTRACT |
The changes in motility, chemotactic responsiveness, and
flagellation of Rhizobium meliloti RMB7201, L5-30, and
JJ1c10 were analyzed after transfer of the bacteria to buffer with no
available C, N, or phosphate. Cells of these three strains remained
viable for weeks after transfer to starvation buffer (SB) but lost all motility within just 8 to 72 h after transfer to SB. The rates of
motility loss differed by severalfold among the strains. Each strain
showed a transient, two- to sixfold increase in chemotactic responsiveness toward glutamine within a few hours after transfer to
SB, even though motility dropped substantially during the same period.
Strains L5-30 and JJ1c10 also showed increased responsiveness to the
nonmetabolizable chemoattractant cycloleucine. Cycloleucine partially
restored the motility of starving cells when added after transfer and
prevented the loss of motility when included in the SB used for initial
suspension of the cells. Thus, interactions between chemoattractants
and their receptors appear to affect the regulation of motility in
response to starvation independently of nutrient or energy source
availability. Electron microscopic observations revealed that R. meliloti cells lost flagella and flagellar integrity during
starvation, but not as fast, nor to such a great extent, as the cells
lost motility. Even after prolonged starvation, when none of the cells
were actively motile, about one-third to one-half of the initially
flagellated cells retained some flagella. Inactivation of flagellar
motors therefore appears to be a rapid and important response of
R. meliloti to starvation conditions. Flagellar-motor
inactivation was at least partially reversible by addition of either
cycloleucine or glucose. During starvation, some cells appeared to
retain normal flagellation, normal motor activity, or both for
relatively long periods while other cells rapidly lost flagella, motor
activity, or both, indicating that starvation-induced regulation of
motility may proceed differently in various cell subpopulations.
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INTRODUCTION |
As nutrient availability approaches
zero in natural environments, populations of motile bacteria face a
potentially crucial choice between searching actively for additional
nutrients or shutting off motility to conserve energy and substrates.
On the one hand, continued motility and chemotaxis may offer a
population its best chance to find new nutrient supplies, avoid stress,
mate, and reproduce. On the other hand, motility and taxis are
inherently costly cellular processes, requiring the synthesis of
perhaps 50 gene products and the performance of considerable mechanical work, with flagella rotating up to 15,000 rpm and requiring about 1,000 protons per revolution (19, 26, 28, 29). Thus, survival and
competitive success in natural environments may require sophisticated, perhaps subpopulation-level regulation of motility and chemotaxis in
response to low-level and fluctuating nutrient availability. Relatively
few studies have examined the changes in behavior of flagellated
bacteria in response to starvation or reduced nutrient availability,
and none so far has involved soil or rhizosphere bacteria. Terracciano
and Canale-Parola (30) reported that carbon-limited growth
of a Spirochaeta species in chemostat cultures resulted in a
10- to 1,000-fold enhancement of chemotactic responsiveness to the
specific sugars used to support growth, indicating that this bacterium
selectively enhanced its behavioral sensitivity to low concentrations
of growth-limiting nutrients. Certain marine vibrios became
considerably more motile and chemotactically responsive between 15 and
72 h after transfer to starvation medium and also shifted to the
use of high-affinity transporters (3, 11, 31, 32). However,
another marine vibrio lost motility and responsiveness within 10 h
after transfer to starvation conditions (20), while a marine
Pseudomonas strain increased its motility after starvation
for 27 h (35). It is not clear from these limited studies whether there are common patterns of behavioral regulation for
flagellated bacteria in response to starvation conditions.
In this paper, we examine the starvation-induced changes in the
motility behavior of a common soil and rhizosphere bacterium, Rhizobium meliloti. R. meliloti is an agriculturally
important species that symbiotically nodulates alfalfa and fixes
atmospheric nitrogen. It typically has five to eight peritrichous
complex flagella. Complex flagella require divalent cations for
functional integrity (24), and they are more rigid and
appear to be more efficient in propulsion through viscous media than
plain flagella (12, 13, 17, 21). R. meliloti is
reported to swim by exclusive clockwise rotations of its flagella, with
brief pauses or changes in speed for redirection, and it exhibits
chemokinesis, swimming at higher speeds when exposed to higher
attractant concentrations (13, 28). This bacterium is
positively chemotactic toward a variety of amino acids, dicarboxylic
acids, and sugars (2, 4, 12, 14), toward the nodulation
gene-inducing flavonoids secreted by the roots of its host (8,
9), and toward unknown attractants secreted at localized sites on
these roots (14). Mutants defective in motility or
chemotaxis are impaired in their ability to compete for sites of nodule
initiation on the host root (1, 7). The present article
describes some of the changes in motility, chemotaxis, and flagellation
seen in three strains of R. meliloti after transfer to
starvation conditions and seeks to characterize the behavioral
strategies used by this soil-rhizosphere species in response to
starvation and the variability among strains of this species in their
responses.
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MATERIALS AND METHODS |
Bacterial strains, media, and culture.
R. meliloti
RMB7201 (15), L5-30 (7), and JJ1c10
(34) were maintained in 15% glycerol stock cultures at
80°C. The strains were routinely cultured at 28°C in
1/10-strength TY medium (0.6 g of tryptone, 0.3 g of yeast
extract, and 0.5 g of CaCl2 · 2H2O per liter). In some experiments, NM minimal salts medium
(24) was used, with 20 mM sodium succinate as the carbon
source and 5 mM KNO3 as the nitrogen source. After
autoclaving of the NM medium, and immediately before its use, 5 mg of
CaCl2 · 2H2O and 1 ml of Gotz vitamin
stock solution (12) were added per liter. Escherichia
coli c118 (16) and HB101 (6) were cultured
on Luria-Bertani medium at 37°C. All liquid cultures were grown on a
rotary shaker at 175 to 200 rpm. Solid media were prepared by addition
of 1.5% agar (Difco). All chemicals used for these media were
analytical or reagent grade (Baker Chemical Co. or Sigma Chemical Co.).
Cells were routinely harvested for transfer to starvation medium during
early exponential phase (A590, ca. 0.15), the
period during which the R. meliloti strains exhibit the
greatest motility.
SB.
The chemotaxis buffer of Robinson et al. (24)
was used as a starvation medium for these studies. Starvation buffer
(SB) contains 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0.1 mM EDTA-Na, and 0.2 mM CaCl2, in
high-performance liquid chromatography-grade (Baker Chemical Co.) or
nanopure water, pH 7.0. This buffer was found to be optimal for
maintaining the motility of R. meliloti and the integrity of
its complex flagella (24). It is also suitable as a
starvation medium since it contains no utilizable carbon, nitrogen, or
energy source.
Motility and chemotaxis assays.
For routine analysis of
motility after transfer of cells to starvation medium,
early-exponential-phase cultures on 1/10-strength TY or NM-succinate
medium were harvested by centrifugation at 1,800 × g
for 10 min. The pellet was resuspended by vortexing in an equal volume
of SB; a micropipet was carefully used to remove droplets of liquid
from the tube after decanting and prior to cell resuspension. To
determine the percentage of motile cells, an aliquot of a bacterial
suspension was placed in a Petroff-Hauser counting chamber and observed
at a magnification of ×860 under phase-contrast optics (Zeiss model
I-35 inverted microscope). Only those cells in the focal plane of the
grid surface were counted, and counting was performed rapidly (1 to 2 min) to minimize the settling of nonmotile cells. Reported percentages
are averages of counts from two independent replicate suspensions for
all experiments. A total of 60 to 100 cells from two or three different
fields were counted per determination. The percentage of motile cells determined in this way was normally reproducible within ±10% for replicate samples. In some cases, video-recorded data were analyzed to
obtain motile-cell percentages and to characterize motility behavior.
Chemotactic responsiveness toward glutamine (10 mM in SB) or other
attractants was determined by capillary assay in Palleroni chambers
(22). Responsiveness is expressed as a chemotaxis ratio (the
average number of cells entering attractant-filled capillaries divided
by the average number of cells entering buffer-filled capillaries).
Relative motility was also measured by entry into SB-filled capillary
tubes (27). In some experiments, responsiveness was assayed
with the attractant present in both the bacterial suspension and
buffer-filled capillaries as a control. Entry into capillary tubes for
motility and chemotaxis assays was determined after a 30-min incubation
at room temperature. The average CFU per capillary tube was determined
by plating aliquots of suspensions from four replicate tubes per data
point.
Transfer to starvation conditions by membrane filtration.
Ten-milliliter aliquots of a mid-exponential-phase 1/10-strength TY or
NM culture were filtered through micropore membranes of various types
(0.02-µm-pore-size Anodisc [Alltech Associates, Inc., Deerfield,
Ill.], 0.45-µm-pore-size cellulose acetate and nylon [Magna,
Honeye, N.Y.], 0.22-µm-pore-size mixed cellulose esters [Poretics,
Livermore, Calif.], or 0.4-µm-pore-size polycarbonate [Nucleopore
Co., Pleasanton, Calif.]). After filtration, the filters were washed
twice with SB (20 ml each), and the cells were subsequently resuspended
by gentle agitation of the membrane in 10 ml of SB.
Flagellar staining and electron microscopy.
The percentage
of flagellated cells was determined at various times after transfer to
SB. A 3-µl droplet of the suspension was placed on a piece of
Parafilm, and 1 µl of 2% uranyl acetate stain was added. After a few
seconds of mixing, 2 µl of the droplet was transferred to a
Formvar-coated 300-mesh copper grid. The cells were allowed to react
with the stain and to settle onto the surface of the film for 5 min
prior to being air dried in a laminar-flow hood. Grids were observed
through a Phillips model 201 transmission electron microscope. Air
drying of the grid surface left areas of rather dense stain, but other
areas had a light background and were suitable for observation of
bacteria and flagella. The percentage of cells with flagella was
determined by observation of 75 to 100 cells per grid on at least two
duplicate grids per sample.
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RESULTS |
Survival of R. meliloti in starvation medium.
Viable-cell counts of all three R. meliloti strains remained
high after transfer to SB. The number of viable cells of strain JJ1c10
gradually diminished about 50-fold over a period of 120 days, but
counts of strains L5-30 and RMB7201 remained almost unchanged, at about
108 CFU/ml, during this period.
Changes in motility in response to starvation conditions.
The
visible motility of all three strains of R. meliloti
diminished to almost zero within 6 to 72 h after transfer to
starvation medium (Fig. 1). The length of
time required for each of the three strains to reach either
half-maximal or essentially zero motility was quite reproducible and
also considerably different for each strain. These times were not
significantly affected by the growth phase prior to harvest. The loss
of motility seen in Fig. 1 was not affected by addition of trace metals
or vitamins.
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FIG. 1.
Changes in motility of R. meliloti L5-30
( ), RMB7201 ( ), and JJ1c10 ( ) at various times after transfer
to SB. Cells were transferred to SB and, at intervals, assayed
microscopically to determine the percentage of motile cells as
described in Materials and Methods. The data are from a single
experiment. The curves shown are representative of those obtained in at
least four other experiments. Replicate samples normally showed less
than 10% variation from the mean.
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The bacteria lost motility after transfer to SB regardless of whether
they were transferred by centrifugal washing (Fig.
1),
by membrane
filtration, or by dialysis (data not shown). Centrifugal
washing and
resuspension was adopted for routine use in subsequent
starvation
response studies. Dialysis was considered unsuitable
due to the
relatively long time interval (>8 h) between the initiation
of
dialysis and essentially complete removal of nutrients from
the
bacterial suspensions. Membrane filtration through micropore
membrane
filters of various types allowed for rapid transfer to
starvation
conditions but resulted in large (>50%) reductions
in the percentage
of motile cells after resuspension in SB, presumably
due to flagellar
adhesion or damage (data not shown). Centrifugal
washing was rapid and
resulted in relatively small (5 to 10%)
reductions in the percentage
of motile cells and the percentage
of flagellated cells during the
transition from exponential-phase
1/10-strength TY cultures to fresh SB
suspensions. The number
of detached flagella present in the
supernatants of the SB suspensions
was found to be low, indicating that
centrifugation and resuspension
caused relatively little damage to
flagella. Centrifugal washing,
however, did not give complete recovery
of the motile cells in
the original cultures. At the relatively low
centrifugation speeds
that were used to pellet the cells as gently as
possible, 10 to
15% of the motile cells remained in the supernatant.
Changes in chemotactic responsiveness upon exposure to starvation
conditions.
All three strains showed significant (two to sixfold)
but transiently enhanced responsiveness to 10 mM glutamine within a few
hours after transfer to starvation medium (Fig.
2). The enhanced responses occurred
despite the fact that the overall motile activity diminished rapidly
during the same time period. As shown in Fig. 3, L5-30 cells suspended in SB showed a
transiently increased responsiveness to the nonmetabolizable attractant
cycloleucine, similar to that obtained with glutamine. Cycloleucine is
a potent attractant for R. meliloti (25) but does
not support its growth and is not toxic (data not shown). The entry of
cells into buffer-filled capillaries was not appreciably affected by
the addition of cycloleucine to the bacterial suspension. Thus,
enhanced entry of starving R. meliloti cells into
cycloleucine-filled capillaries is not due to localized activation of
motility in cells exposed to the attractant.
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FIG. 2.
Changes in motility and chemotaxis of R. meliloti L5-30 (a), RMB7201 (b), and JJ1c10 (c) after transfer to
SB. Cultures were transferred to SB and assayed at various times after
transfer for percentage of motile cells and for chemotactic
responsiveness as described in Materials and Methods. Graphed are the
percentage of motile cells (), the number of cells entering
SB-filled capillary tubes (), and the number of cells entering
capillary tubes containing 5 mM glutamine (). The chemotaxis ratio
( ) was calculated by dividing the number of cells in
attractant-filled capillaries by the number in SB-filled capillaries.
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FIG. 3.
Chemotactic responsiveness of R. meliloti
L5-30 to different concentrations of cycloleucine (CL) before and after
starvation. L5-30 cells freshly transferred from 1/10-strength TY
cultures to SB () or starved in SB for 2 h () were assayed
for entry into capillary tubes filled with 5, 1, 0.2, 0.04, or 0.008 mM
cycloleucine as described in Materials and Methods. Data points
represent averages of values from four replicate capillaries. The
chemotaxis ratio was calculated by dividing the number of cells in
attractant-filled capillaries by the number in SB-filled capillaries.
Curves are from a single experiment that was repeated with similar
results.
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Swimming behavior of cells exposed to starvation conditions.
Light and videomicroscopy revealed clear changes in the character of
swimming behavior after transfer of R. meliloti cells to
starvation medium. Immediately after transfer to SB, when 40 to 60% of
the cells were motile, the cells were very active, swimming quite
continuously with almost no discernible pauses. The swimming was
relatively fast (ca. 20 to 30 µm/s) and straight, the bacteria often
covering distances of 100 µm between abrupt changes of direction. The
proportion of cells of all three strains that exhibited this normal
swimming behavior diminished steadily after transfer to SB. By the time
the percentages of motile cells had dropped to 15 to 25% of the
initial levels, very few cells exhibited any smooth forward swimming.
The average rate of forward movement of these starved cells was slower,
perhaps one-third of the speed seen in fresh suspensions, and few cells
moved more than 50 µm from their starting point within 1 min. Motile
cells in starved suspensions seemed to change direction more frequently
than those in fresh SB suspensions, and many of the swimming cells were
observed to come to a complete stop, with no visible motion. Sometimes these stops lasted for a fraction of a second, while at other times
they lasted 1 s or more before swimming was resumed. Near the grid
surface, where motion is constrained, many motile cells in these
starved suspensions were observed to move in circles 3 to 10 cell
lengths in diameter. With longer periods of starvation, an increasing
proportion of the motile cells appeared unable to swim at all and just
spun or tumbled weakly in one location.
To ensure that light microscopic determinations of the percentage of
motile cells provided an accurate measure of the changes
in motility
induced by starvation, and to see whether the reduced
forward swimming
behavior exhibited by starving cells might affect
entry into pores
(such as those found in soil), motility was measured
by capillary assay
as described by Segal et al. (
27). Motilities
measured by
light microscopy and capillary assay did not always
change in simple
parallel to each other with time after cell transfer
to starvation
medium (Fig.
2). However, both measures of motility
seem valid and
useful. They provide averages over different time
scales, with the
microscopic assay giving an average over a short
(roughly 1-min) time
period and the capillary assay giving an
average motility over a longer
(30-min) time period.
Changes in flagellation of cells exposed to starvation
conditions.
Initially, it seemed possible that the loss and
disintegration of flagella could explain the observed losses of
motility and normal swimming behavior following transfer to starvation
conditions. To test this possibility, a simple method for determining
the percentage of cells that retained flagella was devised. Grids for
electron microscopy were prepared by mixing an SB suspension of
bacteria with the uranyl acetate stain and drying this mixture directly
on the grid film, without washing or blotting. This procedure prevented
the potentially selective loss of actively motile cells during blotting
as well as the potentially selective retention of any cells that might
adhere more strongly to the grid film during blotting or washing.
Addition of the uranyl acetate stain was observed to stop all motility
within seconds, making it likely that the motile, flagellated cells
settled on the surface of the film in much the same manner as
nonflagellated cells. When freshly transferred suspensions of bacteria
were examined, relatively few loose flagella or fragments of flagella
were observed on the grids. Cells from growing cultures and those from
freshly transferred suspensions were similar with regard to the number
and length of flagella associated with the bacteria. These observations
provide evidence that low-speed centrifugal transfer of cells to SB,
exposure to uranyl acetate, and subsequent drying normally resulted in relatively little breakage of flagella or large-distance separation of
flagella from their cell of origin. About 10 to 15% of the cells on
the grids were present in clumps of five or more cells. Abundant
flagella were usually seen in association with the clumped cells,
possibly indicating a tendency for flagellated cells to adhere to each
other. The cells present in such clumps were not included in counts of
flagellated cells since it was not easy to distinguish between
individual cells with and without flagella in such clumps.
The reliability of this direct counting method for determining
flagellated-cell percentages was tested by mixing a suspension
of L5-30
containing a high percentage of flagellated cells (i.e.,
freshly
transferred to SB) with a suspension containing a low
percentage of
flagellated cells (i.e., 20 h after transfer to
SB). The
percentage of flagellated cells counted in the mixture
was in close
(±3%) agreement with the percentage predicted from
counts of the two
initial suspensions. Flagellated-cell percentages
determined from
duplicate grids were usually found to agree within
5%, indicating that
the method is quantitatively reproducible.
After suspension of L5-30 cells in starvation medium, the percentage of
cells with flagella decreased gradually from about
45% to about 32%
over an 8-h period (Fig.
4). The
percentage of
visibly motile cells, however, decreased from about 40%
to essentially
zero during this same period. Most of the L5-30 cells
that retained
flagella after several hours of starvation did not have
the normal
number of full-length flagella. Indeed, many retained only a
short
(0.5- to 1.0-µm) remnant of a single flagellum. About one-third
of the flagellated cells in 8-h, nonmotile suspensions of L5-30,
however, retained two or more full-length flagella. A similar
proportion of flagellated cells in suspensions of L5-30 starved
for
20 h or more had two or more full-length flagella, even though
the
overall percentage of flagellated cells dropped to 15 to 20%.
Strains
RMB7201 and JJ1c10 behaved similarly: a relatively high
proportion
(30%) of the cells from nonmotile suspensions of these
strains,
starved for 24 to 48 h, still retained flagella, and
a
considerable fraction (approximately one-third) of these flagellated
cells had two or more long flagella.
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FIG. 4.
Retention of motility and flagella by strain L5-30 after
transfer to SB. Cells were transferred to SB and then assayed at
various times for the percentage of motile cells () and the
percentage of flagellated cells () as described in Materials and
Methods. The results are representative of two independent
experiments.
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Effects of glucose, glutamine, culture filtrates, and
nonmetabolizable chemoattractants on retention of motility and reversal
of motility loss.
As shown in Table
1, the addition of 5 mM glucose,
glutamine, or cycloleucine to the SB used to wash and resuspend the
initial cultures of L5-30 resulted in an almost complete retention of motility for a period of several hours. The addition of 0.05 mM cycloleucine was just as effective as 5 mM cycloleucine in blocking motility loss. The addition of 5 mM itaconic acid, another
nonmetabolizable (but weaker) chemoattractant, was also effective in
blocking the loss of motility during starvation but did so only about
half as well as cycloleucine. L5-30 cells washed and resuspended in SB
containing either glucose or one of the nonmetabolizable attractants did not grow, and they gradually lost motility after the first few
hours. Cells washed and resuspended in SB containing glutamine, which
provides both N and C for growth, were able to multiply and maintained
motility for longer periods of time. L5-30 cells resuspended in SB
containing one-fifth-strength culture filtrate from 3-day-old
stationary-phase cultures of L5-30, RMB7201, or JJ1c10 grown in
NM-succinate defined medium also retained essentially full motility for
several hours.
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TABLE 1.
Motility of R. meliloti L5-30 after transfer
to SB containing an added nutrient, culture filtrate, or
nonmetabolizable attractant
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Because the loss of motility induced by starvation could be prevented
by the addition of either a nonmetabolizable attractant
or an energy-C
source such as glucose, it was of interest to determine
if these
substances could reverse the loss of motility after a
period of
starvation. Cells of L5-30 were washed and resuspended
in SB and
incubated for either 1.5 or 8 h, so that the visible
motility of
the suspensions was reduced by 50 or 98%, respectively.
Then either
glucose, cycloleucine, or both were added to a concentration
of 5 mM.
As seen in Table
2, delayed additions of
glucose and
cycloleucine were able to restore the motility of starving
L5-30
cells to some extent. Both the glucose and glucose-cycloleucine
additions quickly restored the motility of 1.5-h-starved cells
to full,
prestarvation levels of normal swimming activity (Table
2). The
addition of cycloleucine alone appeared to activate a
comparable number
of nonmotile cells, but their motility was largely
restricted to
circling and tumbling activity, with little evidence
of straight
swimming. When glucose and cycloleucine were added
to L5-30 cells which
had been starved for 8 h and had lost ca.
98% of their motility,
it took 1 h or more for the added compounds
to have any
appreciable effect on motility. Restoration of motility,
even after 6 to 12 h, was only partial, particularly in terms
of straight
swimming activity (Table
2). In similar experiments
with strain JJ1c10,
the addition of cycloleucine to suspensions
which had been starved for
24 h in SB and had lost about 50% of
their motility was found to
immediately, though modestly, increase
the percentage of motile cells
and the number that entered cycloleucine-filled
capillaries. The
addition of cycloleucine 24 h after transfer
did not prevent the
cell motility from gradually decreasing over
the next 24 h of
starvation. When glucose was added to these 24-h-starved
JJ1c10
suspensions, the cells were found to clump in large aggregates
within a
few hours, so motility could not be assessed.
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DISCUSSION |
Starvation-induced changes in chemotactic responsiveness.
All
three strains of R. meliloti studied here showed increased
but transiently expressed chemotactic responsiveness during starvation.
Enhanced chemotactic responsiveness may represent a general starvation
response by which motile bacteria increase their probability of finding
new nutrient supplies without committing internal reserves to continued
flagellar synthesis or motor activity. The molecular mechanisms
involved in the chemotactic-sensitivity facet of R. meliloti's starvation response are unknown. In starving R. meliloti cells, enhanced responsiveness does not appear to involve
significant changes in either the threshold of or the optimal
concentration for responses to attractants (Fig. 3). Since starving
R. meliloti cells exhibit increased responsiveness to cycloleucine, a nonmetabolizable attractant, we conclude that this
organism's enhanced responsiveness to attractants does not require
exposure to new nutrient or energy supplies. It is not clear why, or
even whether, the increased responsiveness of R. meliloti is
transient. It is quite possible that responsiveness per se remains at a
high level during prolonged starvation and that the reduction in entry
into attractant-filled capillaries simply reflects reduced motility.
Regulation of motility in response to starvation.
In general,
the motility of R. meliloti cells was down regulated during
starvation. Both flagellar maintenance and motor activity seemed to be
affected. The bacteria lost flagella as a result of starvation, but
this loss of flagella could not fully account for the extensive loss of
motility during the initial phases of the starvation response (Fig. 4),
and many of the nonmotile cells in starving cultures retained flagella.
We conclude that one of the first responses of R. meliloti
to starvation is to turn off the rotation of flagellar motors in
certain cells. Flagellar-motor activity in R. meliloti is
reported to alternate between an "on" state and an "off" state
during normal swimming behavior (13). Similar behavior, with
considerably longer pauses between swims, is seen in the related,
uniflagellate species Rhodobacter sphaeroides (33). We speculate that starvation may progressively
increase the proportion of time that the individual flagellar motors of a cell spend in the off state. If so, then one would predict that the
time intervals that cells spend in straight swimming would gradually
decrease, that the average swimming speed would also gradually
decrease, and that the frequency of directional changes would
correspondingly increase. Later during starvation, the probability that
all flagellar motors on a given cell are off at the same time would
become appreciable and the cells would stop moving altogether for brief
periods. Finally, the proportion of flagellated cells that stay at rest
for extended time periods would increase to 100%. Our videomicroscopic
observations of motility are consistent with this starvation response
model. After a period of starvation sufficient to reduce the percentage
of motile cells to ca. 20% of their initial level, those cells that
remained motile often stopped moving altogether for periods of time
ranging from about 0.5 s to several seconds. Unstarved cells
rarely, if ever, stopped moving for more than a small fraction of a
second. The possibility that an increased amount of time in the off
position is a starvation-induced response seems in accord with the
increased proportion of time that E. coli flagellar motors
were reported to spend in the inactive, pausing mode in suspensions
without nutrients as opposed to suspensions with added glycerol
(18).
The switching off of flagellar-motor activity in response to starvation
seems to be at least partially reversible in all three
strains tested.
The reactivation of motor activity in starved
L5-30 cells by added
attractants was effectively instantaneous
during the first 1.5 h
after transfer to SB but required 1 h or
more when glucose or
cycloleucine was added after 8 h of starvation
(Table
2). It
remains to be determined how the cells changed
between 1.5 and 8 h
of starvation to prevent the full and rapid
reactivation of
flagellar-motor activity. Access to a carbon or
energy source was the
main limiting factor in reactivation of
motility in cells starved for
an extended period. However, the
appreciable reactivation of motor
activity by cycloleucine alone
(Table
2) indicates that the binding of
a receptor to a nonmetabolite
can be an important contributor to
reactivation, even if it is
not sufficient to restore normal swimming
behavior. It will be
of interest to learn whether one of the recently
described MotC
or MotD proteins (
23), which have no
counterparts in
E. coli,
might serve as a receptor for
signals corresponding to energy
sources or attractants that would
subsequently regulate flagellar-motor
activity.
In contrast to L5-30 and RMB7201, strain JJ1c10 remained fairly
responsive to reactivation by chemoattractants even after
48 to
100 h of starvation (Fig.
2; Table
2). Such differences
between
strain JJ1c10 and the other two strains in the reactivatability
of
flagellar motors, as well as the different periods of starvation
required to reduce motility to half-maximal or zero levels for
the
three strains (Fig.
1), suggest that all three strains of
R. meliloti have adopted somewhat different, genetically programmed
patterns of behavioral response to starvation. Strain JJ1c10 appears
to
be significantly more committed to sustained motility and chemotactic
responsiveness than either L5-30 or RMB7201. Perhaps as a consequence,
strain JJ1c10 loses viability (or enters a viable but nonculturable
state) significantly faster than the other two strains under conditions
of prolonged starvation, at least in vitro.
Loss of flagella in response to starvation.
In general, when
R. meliloti cells were starved long enough to bring visible
motility to essentially zero, roughly half of the flagellated cells
retained only short lengths of just one or two flagella. It is likely
that cells with only one or two truncated flagella are incapable of
normal, smooth forward swimming. Thus, the progressive loss and
disintegration of flagella on many of the cells may help to explain why
most of those cells which remained visibly motile only circled,
twitched, or tumbled slowly. Frequent or extended pausing by some
individual flagella on a cell with several flagella might also result
in such aberrant swimming behavior (13, 28).
However, even in extensively starved suspensions in which no swimming
or even slowly spinning cells were detected, about one-third
of the
flagellated cells of all three strains appeared to retain
a normal
number of full-length flagella. In electron micrographs,
such cells
were indistinguishable from flagellated cells taken
from actively
growing cultures. We conclude that the loss and
disintegration of
flagella occur in just a selected subpopulation
of the cells. The
molecular mechanisms behind this selective loss
or retention of
flagella in certain subpopulations of this bacterium
remain to be
determined. The cell-specific, starvation-induced
regulation of
flagellar loss on the one hand and of flagellar-motor
activity on the
other hand appears to result in the production
of five distinct
subpopulations: (i) cells that have lost all
of their flagella and are
nonmotile, (ii) cells that retain normal
numbers of intact flagella and
have active flagellar motors, (iii)
cells that retain normal numbers of
intact flagella but have inactive
flagellar motors, (iv) cells that
have lost most of their flagella
but have active flagellar motors, and
(v) cells that have lost
most of their flagella and have inactive
flagellar motors. After
prolonged starvation, only the first, third,
and fifth subpopulations
of cells remain. It would be of considerable
interest to know
whether the different subpopulations generated by
these two kinds
of starvation response each have some kind of selective
advantage
in surviving starvation under natural environmental
circumstances
(
5).
Retention of motility during starvation.
The loss of motility
in L5-30 cells following transfer to starvation medium could be
prevented or minimized for an extended period by the addition of an
attractant to the starvation medium during transfer (Table 1). The
extent to which this loss of motility was prevented seemed to be
determined by how strongly the attractant elicited tactic responses and
not by whether the attractant was metabolizable or by its concentration
per se. Substances present in the filtrates from stationary-phase
cultures of R. meliloti had similar abilities to prevent
motility loss during starvation. Presumably the active substances in
these culture filtrates were nonmetabolizable attractants, but other
possibilities, including quorum-sensing autoinducers (10),
remain open. From these observations, we conclude that the presence of
chemoattractants can at least temporarily override the normal down
regulation of behavioral activity in R. meliloti. Based on
the effects of nonmetabolizable attractants like cycloleucine and
itaconic acid, the ability of chemoattractants to override
starvation-induced down regulation of motility seems to be independent
of any energy or nutrient input from the attractant. Thus, the
interaction of such chemoattractants with their receptors would seem to
provide one important input to the regulation of behavioral activity
under starvation conditions. A second, independent part of such
regulation undoubtedly involves nutrient-energy sensing and its
connections to flagellar maintenance and motor activity. Further
studies at both the molecular genetic and ecological levels are needed
to understand how bacteria regulate their patterns of behavioral
activity when faced with starvation.
| |
ACKNOWLEDGMENTS |
The assistance of Elke Kretschmar and Robert Whitmoyer in the use
of the electron microscope is much appreciated, as is the advice of
Catherine Wolkin regarding methods for flagellar staining. We thank
John Parkinson for valuable suggestions and John Streeter, Michael
Boehm, and Jayne Robinson for critical reading of the manuscript and
helpful comments.
Partial support for salaries, supplies, and publication costs was
provided by state and federal funds appropriated to the Ohio
Agricultural Research and Development Center, Ohio State University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Horticulture and Crop Science, 2021 Coffey Rd., Ohio State University, Columbus, OH 43210. Phone: (614) 292-9035. Fax: (614) 292-7162. E-mail:
bauer.7{at}osu.edu.
Ohio Agricultural Research and Development Center manuscript number
136-97.
| |
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Top
Abstract
Introduction
