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Journal of Bacteriology, February 1999, p. 981-990, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cell Density-Dependent Starvation Survival of
Rhizobium leguminosarum bv. phaseoli: Identification of the
Role of an N-Acyl Homoserine Lactone in Adaptation
to Stationary-Phase Survival
Stephen H.
Thorne
and
Huw D.
Williams*
Department of Biology, Imperial College of
Science, Technology and Medicine, London SW7 2AZ, United Kingdom
Received 23 March 1998/Accepted 11 November 1998
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ABSTRACT |
The cell density dependence of stationary-phase survival of
Rhizobium leguminosarum has been investigated. Following
starvation by exhaustion of carbon or nitrogen, but not of phosphorus,
the survival of cultures was dependent on the cell density at entry into stationary phase. High-density cultures survived with little or no
loss of viability over a 20-day period in stationary phase. In
contrast, low-density cultures lost viability rapidly but consisted of
a heterogeneous population, a small fraction of which successfully adapted and eventually formed a stable, surviving population. The
threshold density above which the cultures survived successfully in
stationary phase was dependent on the growth conditions and the strain
used. We took advantage of the fact that R. leguminosarum survives poorly following starvation by resuspension in carbon-free medium to demonstrate that cell density-dependent survival was mediated
by a component accumulating in the growth medium. The effects of this
medium component on survival in resuspension assays could be mimicked
by an N-acyl homoserine lactone,
N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone, previously demonstrated to have a role in controlling cell
density-dependent phenomena in R. leguminosarum. The Sym plasmids pRP2JI and pRL1JI were found to be essential for the production of the extracellular factor, which could also be made in
Escherichia coli carrying the cosmid clone pIJ1086
containing a specific region of pRL1JI.
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INTRODUCTION |
Nutrient starvation is a condition
in which bacteria in many environments, including soil, regularly find
themselves (9, 20, 31, 37). However, little is known about
the physiological processes involved in the starvation survival of soil
bacteria. We are investigating the starvation survival response of
Rhizobium leguminosarum. We have previously reported on the
changes in the physiology and metabolism of R. leguminosarum
bv. phaseoli in response to nutrient starvation (36). In
contrast to the starvation survival kinetics of enteric bacteria, no
reduction in the viability of R. leguminosarum cultures was
observed during more than 2 months in carbon-starved stationary phase
(36). A further distinguishing feature is the poor survival
of R. leguminosarum following nutrient starvation by
resuspension in minimal medium without carbon, indicating that this
bacterium cannot respond to such a rapid deprivation of nutrients
(36).
It has been known for over 30 years that a population effect is
observed when a culture of bacteria is starved (16, 28, 29).
In those early studies the specific death rates of high-density bacterial populations were lower than those of low-density populations. It is also well established that Myxococcus xanthus and
Bacillus subtilis show developmental responses to nutrient
deprivation, i.e., sporulation and fruiting body formation,
respectively, that are dependent on population cell density (7,
13, 18, 19). The effect of cell density on survival has not been
thoroughly addressed in recent studies of the starvation survival of
bacteria (9, 11, 21, 26, 27).
In this study we demonstrate the cell density-dependent
stationary-phase survival of R. leguminosarum following
starvation by nutrient exhaustion of carbon or nitrogen. We show that
this response is controlled by the accumulation of an extracellular factor(s) in the growth medium and that the effect is mimicked by the
N-acyl homoserine lactone (AHL)
N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone (7cisHtDHL).
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MATERIALS AND METHODS |
Strains and plasmids.
R. leguminosarum bv. phaseoli
4292 is a rifampin-resistant strain that carries the pRP2JI Sym plasmid
and nodulates beans (23). This strain was used in our
earlier study of starvation survival of R. leguminosarum
(36). R. leguminosarum 8401 is a derivative of a
biovar phaseoli isolate (strain 8002), and it is streptomycin resistant
and has been cured of the Sym plasmid pRP2JI. R. leguminosarum 8401/pRL1JI carries the biovar viciae Sym plasmid.
The four pLAFR1-derived cosmids pIJ1527, pIJ1529, pIJ1085, and pIJ1086
contain DNA cloned from the R. leguminosarum Sym plasmid
pRL1JI. pIJ1085 and pIJ1086 are two overlapping cosmids carrying about
60 kb of DNA from the nif and nod gene-carrying regions of pRL1JI (8). pIJ1527 and pIJ1529 are poorly
characterized, overlapping cosmids carrying about 45 kb of DNA from a
different region of pRL1JI, which does not overlap with pIJ1085 and
pIJ1086 (7a). Together the four cosmids carry DNA covering
about half of the Sym plasmid pRL1JI.
Growth and starvation of bacteria.
Strains were grown and
starved for carbon (mannitol), nitrogen (NH4Cl), and
phosphorus (K2HPO4 and
KH2PO4) in MOPS (morpholinepropanesulfonic acid) minimal medium as described previously (36). For
routine culture maintenance, growth of starter cultures, and plating
for viable-cell counting, rifampin was added at 25 µg
ml
1. YEM medium was also used at pH 7.5 (38)
and consisted of (per liter) yeast extract (0.4 g) (Oxoid),
K2HPO4 (0.5 g), NaCl (0.1 g),
MgSO4 · 7H2O (0.2 g), and mannitol (10 g).
Starter cultures were prepared by inoculating a single colony from a
plate stock into 5 ml of medium and incubating overnight at 30°C in a
shaking incubator (200 rpm). A 0.5-ml portion of this exponentially
growing culture was then subcultured into a second 5 ml of medium and
again incubated overnight to ensure that cells were fully adapted to
exponential growth. About 5 ml of this starter culture was then
inoculated into 50 ml of medium in a 250-ml conical flask to give an
initial optical density at 600 nm of 0.02. This method of inoculation
was used throughout this study unless otherwise stated. Growth was
measured spectrophotometrically as optical density at 600 nm (with
1-cm-path-length cuvettes in a Shimadzu MPS2000 spectrophotometer) and
by viable-cell counting, following plating onto solid minimal medium
after appropriate dilution in minimal medium. It was found that
although all of the strains had similar growth kinetics in the liquid
MOPS minimal medium (36), 8401/pRL1JI and 8401 did not grow
well on MOPS minimal medium agar. Therefore, the viability of these
strains was determined by diluting the samples in TY medium and plating on TY agar. TY medium consisted of (per liter) Bacto tryptone (5 g),
yeast extract (3 g), and CaCl2 · 6H2O
(1.3 g). Cells were osmotically stressed by adding NaCl to a final
concentration of 2.5 M as described previously (36).
Escherichia coli 803 strains were also grown in MOPS minimal
medium at 30°C.
Determination of affects of spent culture medium. (i) Preparation
of aseptic samples of spent culture medium.
To obtain spent
medium, cells were removed from a carbon-starved culture by
centrifugation at 4,000 × g for 45 min. The
supernatant fraction was then collected and sterilized by filtration
through 0.2-µm-pore-size cellulose acetate filters. The aseptic spent medium was then used to resuspend cells collected by centrifugation (see below). Spent medium was usually prepared from 6-day-old, high-density (1.5 × 109 CFU ml1),
carbon-starved stationary-phase cultures and is referred to as
high-density spent medium (HDSM). For some experiments the spent medium
was pretreated before being added to cells. Heat treatment involved
5.0-ml samples of spent medium being placed in tubes in a water bath at
100°C for 15 min. For protease treatment, spent medium was treated
with pronase attached to agarose beads (1 U ml1; Sigma
P4531) for 60 min at 30°C. The beads were then removed by filtering
the sample through a 25-mm glass fiber filter (pore size, 1 µm). To
approximately determine the size of the spent-medium component, samples
of stationary-phase spent medium from carbon-starved R. leguminosarum cultures were ultrafiltrated in a stirred cell by
using an Amicon YM3 ultraflow membrane with a molecular mass cutoff of
3 kDa.
(ii) Collection and resuspension of cells.
Fifty-milliliter
cultures of R. leguminosarum were harvested by
centrifugation at 4,000 × g for 20 min. The
supernatant fraction was then removed, and the cells were washed twice
by resuspending them in 50 ml of fresh MOPS minimal medium with no
carbon source (MM-C). This was done in order to make sure that any
residual carbon source or molecules secreted into the medium by the
cells were completely washed from the cells. The bacteria were then pelleted by centrifugation one final time, and the pellet was resuspended by vortexing in 5.0 ml of MM-C. A 0.5-ml portion of the
resuspended cells was then added to 5.0 ml of MM-C or spent culture
medium. Cultures were then kept at 30°C in a shaking incubator (200 rpm), and survival was monitored for several days by viable-cell counting.
Use of R. leguminosarum autoinducer.
A synthetic
preparation of the R. leguminosarum autoinducer
7cisHtDHL, containing a mixture of L and
D stereoisomers that differ in the orientation of the
3-hydroxy group, was kindly provided by Kendall Gray (12,
34). For use in bioassays, a sample of the autoinducer (in ethyl
acetate) was added to an empty culture vessel and dried down in a
stream of sterile air. The assay culture was then added to the vessel
to resuspend the autoinducer.
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RESULTS |
Effects of culture cell density on stationary-phase survival of
R. leguminosarum.
By varying the mannitol concentration in
MOPS minimal medium, R. leguminosarum bv. phaseoli cultures
were grown and allowed to enter stationary phase at cell densities
ranging from ~1.3 × 109 to 1 × 108 CFU ml1. It was found that the survival
of cultures in the 10 to 14 days following entry into carbon-starved
stationary phase was dependent on the cell density at the time of entry
(Fig. 1A). Cultures entering stationary
phase at a density of ~1.3 × 109 CFU
ml1 had 108% viability after 14 days of stationary
phase, whereas cultures that had entered stationary phase at ~1 × 108 CFU ml1 retained only 15% viability
after the same time period. Cultures with intermediate cell densities
at entry into stationary phase showed intermediate survival (Fig. 1A).
This experiment was repeated with YEM medium, in which mannitol was the
major but not the sole carbon source. Changing the concentration of
mannitol in YEM allowed the cell density at which cultures entered
stationary phase to be altered. It was found that stationary-phase
survival in YEM was dependent on the culture density (Fig. 1B). The
survival of nitrogen-starved stationary-phase cultures was also
dependent on the culture cell density, with a higher cell density at
the entry into stationary phase resulting in better survival (Fig. 1C).
However, cell density-dependent survival was not seen in phosphorus-starved cultures; phosphorus-starved cultures entering stationary phase at densities of between 7 × 107 and
8 × 105 CFU ml1 all survived well
during the first 14 days of stationary phase (Fig. 1D).
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FIG. 1.
Effect of culture cell density on the stationary-phase
survival of nutrient-starved R. leguminosarum bv. phaseoli
4292 cultures. Cultures were grown in minimal medium (A, C, and D) or
YEM (B) with various limiting amounts of carbon source (A and B),
nitrogen (C), or phosphorus (D) so that the cell density upon
nutrient-limited entry into stationary phase was varied. (A) In
carbon-limited minimal medium, the mannitol concentrations in grams
liter1 (cell densities entering stationary phase in CFU
milliliter1) were 10 ( ) (1.3 × 109),
5 ( ) (5 × 108), 2 ( ) (1.5 × 108), and 0.3 ( ) (1 × 108). (B) In
YEM, the mannitol concentrations in grams liter1 (cell
densities entering stationary phase) were 10 () (9.8 × 109), 5 () (4.7 × 109), 2 ()
(1.9 × 109), and 0.3 () (1.1 × 109). (C) In nitrogen-limited minimal medium, the
NH4Cl concentrations in grams liter1 (cell
densities entering stationary phase) were 0.25 () (9.5 × 108), 0.1 () (3.1 × 108), 0.05 ()
(1.7 × 108), and 0.025 () (1.1 × 108). (D) In phosphorus-limited minimal medium, the
phosphate concentrations in grams liter1 (cell densities
entering stationary phase) were 0.05 () (7 × 107),
0.025 () (8.5 × 106), 0.01 () (3 × 106), and 0.005 () (9 × 105). Error
bars represent standard deviations.
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A heterogeneous population enters stationary phase in low-density
cultures.
An experiment similar to that of Fig. 1A was set up, but
stationary-phase survival was monitored for a much longer time period. As expected, the high-density cultures survived long-term starvation with little loss of viability (Fig. 2)
(36). In the low-density culture, there was an initial loss
of viability over the first 16 days in stationary phase (up to 20 days
after inoculation), and then the proportion of viable cells stabilized
at about 20% of the starting population. There was no further loss of
viability for 15 days (Fig. 2). However, this was followed by a further reduction of viability from day 35 onwards that continued until the end
of the experiment at day 60. The survival kinetics of low-density
cultures suggests that a heterogeneous population enters stationary
phase or is present immediately after entry, comprising a fraction
(~20%) that goes on to survive long-term stationary phase and those
cells that will die during the first 15 days (Fig. 2). We investigated
whether the population was also heterogeneous with respect to another
property of stationary-phase-adapted cells, resistance to osmotic
stress (36). The osmotic stress resistance of high- and
low-density cultures at 5 and 25 days into stationary phase was
determined (Fig. 3). The
high-cell-density, 5-day stationary-phase culture was resistant to
challenge by 2.5 M NaCl, while the equivalent low-density culture was
clearly sensitive (Fig. 3A). At 150 min after the exposure to osmotic
stress, the viability of the low-density culture had been reduced by
70%, but the surviving 30% of the population was resistant to 2.5 M NaCl and there was no further reduction in viability during the remaining 250 min of the experiment (Fig. 3A). These data are consistent with the idea that there is a fraction of the population that has failed to successfully adapt to stationary-phase survival and
consequently retains physiological traits of exponentially growing
cultures. In support of this idea, the decimal reduction times
(D values) (1) for osmotically stressed,
exponential-phase cultures (calculated from the data in Fig. 7 of
reference 36) and for the osmotically sensitive
fraction of the population in low-density cultures (Fig. 3A) were
similar, being approximately 150 and 200 min, respectively.
Twenty-five-day-old high- and low-density stationary-phase
cultures were equally resistant to osmotic stress (Fig. 3B),
confirming that 25 days into stationary phase, the surviving bacteria
in the low-density culture are fully adapted for stationary-phase
survival. It seems likely, but is not proven, that this surviving
population is the fraction of the low-density culture that was
resistant to osmotic stress after 5 days into stationary phase.
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FIG. 2.
Growth and stationary-phase survival of R. leguminosarum bv. phaseoli 4292 cultures entering stationary phase
at high and low cell densities. Cultures were grown in minimal medium
with limiting amounts of carbon source. , growth with 10 g of
mannitol liter1, entering stationary phase at ~1 × 109 CFU ml1, , growth with 0.3 g
of mannitol liter1, entering stationary phase at 2.6 × 108 CFU ml1. Growth was monitored by
viable-cell counting. Arrows 1 and 2 correspond to the times that
samples were taken for osmotic stress tests (see Fig. 3). Error bars
represent standard deviations.
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FIG. 3.
Osmotic stress resistance of high- and low-density
stationary-phase cultures of R. leguminosarum bv. phaseoli
4292. , growth in minimal medium with 10 g of mannitol
liter1, entering stationary phase at ~1 × 109 CFU ml1; , growth in minimal medium
with 0.3 g of mannitol liter1, entering stationary
phase at 2.6 × 108 CFU ml1. Cultures
were osmotically stressed by the addition of sterile NaCl to a final
concentration of 2.5 M. The stress was applied to cultures 10 days
after inoculation, when they had been in stationary phase for 5 days
(arrow 1 in Fig. 2) (A), or 30 days after inoculation, when they had
been in stationary phase for 25 days (arrow 2 in Fig. 2) (B). Survival
after the osmotic stress was monitored by viable-cell counting. Error
bars represent standard deviations.
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High-density stationary-phase cultures contain an extracellular
component that promotes adaptation to starvation survival.
We
investigated whether an extracellular compound(s) accumulates that is
able to promote the adaptation to stationary-phase survival. We took
advantage of the fact that R. leguminosarum survives poorly
following carbon starvation by resuspension in MM-C (36).
The effect of filter-sterilized, spent culture medium from a
high-density culture on the survival of a low-density culture was
investigated. A low-density culture, entering stationary phase at
108 CFU ml1, was harvested and resuspended in
MM-C containing spent medium from a high-density stationary-phase
culture, and the viability was monitored. The data in Fig.
4 clearly show that the HDSM promotes survival of the low-density culture. At 96 h after resuspension, 98% of the cells resuspended in HDSM were viable, with only 21% of
those resuspended in MM-C retaining viability. HDSM could also promote
survival or adaptation of exponentially growing
cells to stationary phase (data not shown). This indicates that the action of the HDSM component is not limited to cells in the transition phase between exponential and stationary phases. Preliminary
characterization indicated that the active component(s) of HDSM was
small (<3 kDa), stable, and heat resistant (surviving 100°C for 15 min), and it was protease resistant, suggesting that it was not a
protein (data not shown).
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FIG. 4.
Effect of HDSM and an AHL on stationary-phase survival
of R. leguminosarum bv. phaseoli 4292. Late-exponential-phase, low-density cultures (density, ~1 × 108 CFU ml1) were harvested and resuspended
to the same density in spent medium from high density cultures
(density, ~1.5 × 109 CFU ml1) of
strain 4292 (), in MM-C ( ), and in MM-C containing 200 ng of
7cisHtDHL ml1 ( ). Survival was monitored by
viable-cell counting. Data are the mean values from four experiments,
with error bars representing standard deviations.
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The accumulation of an extracellular component may trigger the
adaptation to stationary-phase survival in high-density cultures,
with
the failure of low-density cultures to adapt being a consequence
of the
compound(s) not reaching a threshold level. This was demonstrated
by
growing a culture to carbon-starved stationary phase while
taking
samples of spent medium, at cell densities above and below
the
threshold necessary for successful stationary-phase survival,
and
testing their ability to promote survival in resuspension
assays (Fig.
5). The spent medium from an
exponentially growing
culture that had reached a density of 5 × 10
7 CFU ml
1 (Fig.
5A) was unable to promote
survival of low-density cultures
following resuspension (Fig.
5B). In
contrast, spent medium from
an exponential-phase culture at
approximately 2 × 10
9 CFU ml
1 (Fig.
5A), was able to promote starvation survival following
resuspension
(Fig.
5B). Interestingly, spent medium from high-density
cultures at
~32 days into stationary phase was also able to promote
starvation
survival in resuspension assays, indicating that the
spent-medium
component is still present and active during long-term
stationary phase
(Fig.
5B). Therefore, a molecule(s) secreted
into the medium has a role
in the adaptation of
R. leguminosarum cultures to stationary
phase.
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FIG. 5.
Effect of spent medium from different stages of the
growth curve on stationary-phase survival of R. leguminosarum 4292 in resuspension assays. (A) R. leguminosarum 4292 was grown in minimal medium (5 g of mannitol
liter1), entering stationary phase at a high cell density
(3 × 109 CFU ml1). During growth,
samples were taken at 24 h (5 × 107 CFU
ml1), 96 h (2 × 109 CFU
ml1), and 30 days (3 × 109 CFU
ml1) for the preparation of spent medium. (B) Cells
obtained from a low-density (~108 CFU ml1),
late-exponential-phase culture, grown in minimal medium with 0.3 g
of mannitol liter1, were resuspended to
~108 CFU ml1 in MM-C () or in
spent-medium samples taken at the different time points as described
for panel A, and the viability was monitored. Spent medium was from
24 h (), 96 h (), and 30 days () after inoculation.
Error bars represent standard deviations.
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An AHL promotes cell density-dependent survival.
The AHL
7cisHtDHL has been shown to induce a cell
density-dependent response in R. leguminosarum (12,
34). It activates the RhiR protein to induce expression of the
rhiABC operon and to inhibit growth (12). We
tested whether 7cisHtDHL could also protect low-density
cultures during starvation survival. Cells from a low-density,
late-exponential-phase culture were resuspended in MM-C with
7cisHtDHL added, and survival was monitored. Ninety-six percent of the cells remained viable 4 days after resuspension in the
7cisHtDHL-containing medium, compared to 98% of cells
resuspended in HDSM and only 21% of the cells in MM-C (Fig. 4).
7cisHtDHL is clearly able to promote the survival of
low-density cultures. As 7cisHtDHL has been demonstrated to
be present in spent culture medium of R. leguminosarum 4292, it is possible that the HDSM component promoting stationary-phase
survival is an AHL similar to or the same as 7cisHtDHL,
although this is not directly demonstrated by the present data. The
7cisHtDHL used was a racemic mixture of L and
D stereoisomers that differ in the orientation of the 3-hydroxy group, although the biological importance of chirality at the
hydroxylated C-3 position has yet to be established (12, 34). The experiment was repeated with various concentrations of
7cisHtDHL in order to find the threshold value at which it is effective at promoting survival. A concentration of 200 ng of
7cisHtDHL ml1 conferred full protection on
low-density cultures in stationary phase (Fig.
6) while less than 100 ng
ml1 caused only a small difference in starvation
survival. These data support the idea that a quorum-sensing effect,
involving AHLs, is functioning to improve the stationary-phase survival of R. leguminosarum. It seems reasonable to conclude that
AHLs have a role in the survival of high-density cultures and that the
absence of a quorum leads to loss of viability in low-density cultures.
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FIG. 6.
Concentration of 7cisHtDHL needed to produce
a stationary-phase response. Cells from late-exponential-phase,
low-density cultures (density, ~108 CFU
ml1) were resuspended in MM-C or in MM-C containing
various concentrations of 7cisHtDHL. The percent survival of
the cultures was determined 4 days after resuspension by viable-cell
counting. Data are the mean values from four experiments, with error
bars representing standard deviations.
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Role of the Sym plasmids pRL1JI and pRP2JI in cell
density-dependent survival.
The R. leguminosarum 4292 strain used so far in this study contains the biovar phaseoli Sym
plasmid pRP2JI. In order to investigate the role of Sym plasmids in
cell density-dependent starvation survival, the related strains
R. leguminosarum 8401 (no Sym plasmid) and R. leguminosarum 8401/pRL1JI (strain 8401 with the biovar viciae Sym
plasmid pRL1JI) were also used. Growth of 8401/pRL1JI to stationary
phase in carbon-limited minimal medium indicated that changing the Sym
plasmid from pRP2JI to pRL1JI had no effect on the cell
density-dependent stationary-phase survival (Fig. 7). R. leguminosarum
8401/pRL1JI grown to high density survived subsequent starvation well,
while cultures grown to low density showed a loss of viability in
stationary phase similar to that of R. leguminosarum
4292/pRP2JI (Fig. 1 and 7). R. leguminosarum 8401 cultures
(no Sym plasmid) showed a loss and subsequent stabilization of
viability similar to those of the strains with a Sym plasmid. Also, the
R. leguminosarum 8401 cultures grown to high-cell-density stationary phase did not show a significant difference in survival compared to strains with a Sym plasmid.
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FIG. 7.
Effect of the Sym plasmid pRL1JI on stationary-phase
survival at high and low culture cell densities. Cultures of R. leguminosarum 8401/pRL1JI ( and ) and R. leguminosarum 8401 ( and ) were grown under carbon-starved
conditions at either a high (open symbols) (5 g of mannitol
liter1, entering stationary phase at 2 × 108 CFU ml1) or low (closed symbols (0.3 g of
mannitol liter1, entering stationary phase at 1 × 107 CFU ml1) cell density. Growth was
monitored by viable-cell counting, and data are the mean values from
four experiments, with error bars representing standard deviations.
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The Sym plasmids pRL1JI and pRP2JI play an essential role in the
production of the extracellular stationary-phase survival factor.
We examined whether Sym plasmids have a role in the production of or
response to the spent-medium component. Cells from low-density, late-exponential-phase cultures were resuspended in HDSM, MM-C, or MM-C
containing 200 ng of 7cisHtDHL per ml. Both cells and HDSM
from three strains of R. leguminosarum (4292/pRP2JI,
8401/pRL1JI, and 8401) were used, and the results are summarized in
Table 1. As expected, none of the strains
could survive well in MM-C. Also, as shown in Fig. 4, R. leguminosarum 4292/pRP2JI cells could survive in MM-C if
7cisHtDHL was added. However, neither R. leguminosarum 8401/pRL1JI nor R. leguminosarum 8401 showed significantly increased survival in the presence of
7cisHtDHL. This leads to two important conclusions. First,
the ability of R. leguminosarum 4292 to respond to
7cisHtDHL is encoded on the Sym plasmid pRP2JI, as strain
8401 is cured of this plasmid and can no longer respond to
7cisHtDHL. Second, the fact that 7cisHtDHL does
not support survival of R. leguminosarum 8401/pRL1JI
suggests that its cell density-dependent starvation survival must be
mediated by a second spent-medium component. Consistent with this
conclusion, we found that when cells from each of the three strains
were resuspended in HDSM from strain 8401, which contains no Sym
plasmid but is known to produce 7cisHtDHL (12,
34), only strain 4292/pRP2JI showed improved survival (Table 1).
However, the cell density-dependent survival of R. leguminosarum 8401/pRL1JI does require a spent-medium component.
Although this strain is unable to produce 7cisHtDHL (12), it can produce a spent-medium component, which we
refer to as SMC8401, capable of promoting starvation survival of itself and of R. leguminosarum 4292/pRP2JI. The strain with pRP2JI
could respond to both 7cisHtDHL and SMC8401. Additionally,
HDSM from R. leguminosarum 4292/pRP2JI promotes survival of
R. leguminosarum 4292/pRP2JI and 8401/pRL1JI, but not 8401. This implies that 4292/pRP2JI produces a molecule (which we call
SMC4292) that is able to promote survival of strain 8401/pRL1JI and
that is chemically distinct from 7cisHtDHL. Whether SMC8401
and SMC4292 are chemically identical remains to be established, but it
is clear from a recent study that Rhizobium strains can
produce a significant number of different AHLs (30).
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TABLE 1.
Role of the Sym plasmids pRP2JI and pRL1JI in production
of or response to the HDSM components conferring stationary-phase
survival and the AHL 7cisHtDHLa
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Which region of the Sym plasmid is essential for the production of
the spent-medium component?
To locate which region of the Sym
plasmid pRL1JI is required for production of AHL8401, we used four
cosmid clones in E. coli, each containing a different region
of pRL1JI (see Materials and Methods). HDSM was obtained from cultures
of E. coli carrying one of each of these clones, and its
effect was compared to those of HDSM from R. leguminosarum
4292/pRP2JI and of MM-C. HDSM from E. coli strains carrying
pIJ1085, pIJ1527, and pIJ1529 proved unable to support starvation
survival of R. leguminosarum 4292/pRP2JI cultures (Fig.
8). In contrast, 81% of the cells
resuspended in spent medium from E. coli/pIJ1086 were still
viable 4 days after resuspension (Fig. 8). This finding indicates that
the pRL1JI-derived DNA in cosmid pIJ1086 contains genes that have a
role in the production of the starvation survival factor produced
by pRL1JI. Interestingly, pIJ1086 contains the rhiABCR
genes. RhiR activates the rhiABC genes in the presence of
7cisHtDHL (3, 8).
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|
FIG. 8.
Identification of regions of the Sym plasmid pRL1JI
required for the production of the spent-medium component. Survival of
R. leguminosarum 4292 cells harvested from
late-exponential-phase, low-cell-density cultures was monitored after
resuspension in R. leguminosarum HDSM (), MM-C (), or
spent medium from late-exponential-phase E. coli cultures
carrying cosmids containing different regions of the Sym plasmid pRL1JI
(, pIJ1085;  , pIJ1086;  , pIJ1527;  , pIJ1529). For all
experiments, the data are the means from four experiments and the
standard deviation was never more than 6%.
|
|
| |
DISCUSSION |
In this paper we have demonstrated the cell density-dependent
starvation survival of R. leguminosarum. This phenomenon was found to occur in both carbon- and nitrogen-starved cultures but not
phosphorus-starved cultures. It is possible that the accumulation of
large amounts of phosphorus storage compounds, such as polyphosphates, during exponential growth (4, 5) allows the cells to survive phosphorus starvation more effectively and disguises any cell density effects.
Quantification of an absolute threshold cell density, above which cells
are able to survive in stationary phase, was not possible, as the
density threshold was dependent on both the growth conditions (Fig. 1)
and the strains starved (compare survival of strains 4292 and
8401/pRL1JI in Fig. 1A and 7, respectively). R. leguminosarum 4292/pRP2JI cultures survived carbon starvation when
they entered stationary phase at densities greater than 1.5 × 109 CFU ml1. However, in YEM medium, cultures
needed to enter stationary phase at densities at or above
1010 CFU ml1 to survive subsequent
starvation. YEM cultures have a significantly higher growth rate than
minimal medium cultures, and the growth rate prior to starvation will
affect the physiology of the cell in numerous ways: the numbers of
ribosomes, DNA and protein levels, and number of active replication
forks are all altered (2, 15). Consequently, the growth rate
may affect the adaptation to starvation survival and the long-term
survival outcome. A link between growth rate and starvation survival is
suggested by the finding that bacterial populations grown for many
generations in chemostat culture are taken over by mutants which have
faster doubling times but which are less able to survive starvation
(22).
When fully adapted to stationary-phase survival, R. leguminosarum is cross-protected against several environmental
stresses (36). However, the osmotic stress resistance of
stationary-phase cultures (Fig. 3) indicated that at 5 days into
stationary phase, only a small proportion of cells in low-density
cultures was protected against osmotic stress and consequently fully
adapted to survival. Therefore, it is likely that at the onset of
starvation, low-density cultures consist of a heterogeneous population.
Approximately 20% of the cells either are able to successfully adapt
themselves to stationary phase or possess the ability to subsequently
adapt by utilizing metabolites leaked into the growth medium by dying cells. The reason for the further loss of viability of low-density cultures after 30 days in stationary phase (Fig. 2) is unclear.
We clearly demonstrate that an extracellular compound that is able to
promote stationary-phase survival accumulates in high-density cultures.
Resuspension assays in the presence of spent medium from high-density
cultures (HDSM) demonstrated that the compound active in promoting
survival accumulates to effective concentrations at high but not low
cell densities. Evidence is provided for the role of AHLs in
promoting the starvation survival of R. leguminosarum. 7cisHtDHL, a previously identified autoinducer from R. leguminosarum (12, 34), promotes survival of R. leguminosarum 4292/pRP2JI in resuspension assays (Fig. 4 and 6).
The ability of strain 4292/pRP2JI to respond to the starvation
survival-promoting function of 7cisHtDHL is encoded by
pRP2JI, since strain 8401, which is cured of this plasmid, was unable
to respond to 7cisHtDHL. R. leguminosarum 8401 can produce 7cisHtDHL but does not respond to it, while
R. leguminosarum 8401/pRL1JI does not produce
7cisHtDHL (12). Consistent with this idea is that
following resuspension of the three strains in HDSM from strain 8401, only strain 4292/pRP2JI showed improved starvation survival (Table 1).
7cisHtDHL promotes transcription of an rhiA-lacZ
gene fusion and growth inhibition as long as the RhiR protein, encoded
by the Sym plasmid pRL1JI but not by pRP2JI, is present (6,
12). Therefore, the 7cisHtDHL-promoted starvation survival of strain 4292/pRP2JI cannot involve the transcriptional regulator RhiR but must require an unidentified regulator encoded by
pRP2JI. This suggests that the cell density-dependent starvation survival response and the cell density-dependent induction of the
rhiABC operon in R. leguminosarum, although both
mediated by 7cisHtDHL, represent separate regulatory
pathways. Also, the amount of 7cisHtDHL required to promote
starvation survival (Fig. 6) was substantially higher (~200 ng
ml1) than that required to induce rhiA-lacZ
fusions (~10 ng ml1 [12]). Gray et al.
(12) showed that 7cisHtDHL added to exponentially growing cultures of R. leguminosarum 8401/pRL1JI caused
cessation of growth. However, we did not find 7cisHtDHL to
be able to promote starvation survival of 8401/pRL1JI in our
experiments, perhaps suggesting that we are not observing the same
phenomenon as Gray et al. (12).
Previous studies have shown that 7cisHtDHL is produced by
strains of R. leguminosarum bv. phaseoli (12,
39). It seems possible, although it is not proven, that
7cisHtDHL is the compound that accumulates in high-density
R. leguminosarum 4292/pRP2JI cultures and promotes
adaptation to and survival in subsequent carbon-starved stationary
phase (Fig. 1). However, our data (Table 1) indicate that R. leguminosarum 4292/pRP2JI produces another survival-promoting
compound in addition to 7cisHtDHL and that strain
8401/pRL1JI produces a compound distinct from 7cisHtDHL. It
is clear from recent work that some Rhizobium species
produce a large number of AHLs (30).
The widespread role of cell density-mediated control of bacterial
physiology (quorum sensing) is now recognized (10, 32). How
widespread is AHL-mediated cell density-dependent stationary-phase survival? In E. coli and other gram negative bacteria, the
development of stationary-phase properties and subsequent
stationary-phase survival is coordinately regulated by the action of
the stationary-phase sigma factor RpoS (reference 40
and references therein). The cellular level of RpoS increases
significantly upon entry into stationary phase, and it is controlled at
the level of protein stability (40). Huisman and Kolter
(17) proposed a mechanism by which E. coli cells
sense starvation via a homoserine lactone (HSL)-dependent signalling
pathway which leads directly to increased cellular levels of RpoS. They
demonstrated that HSL, or a metabolite derived from it, induced
rpoS expression and increased cellular levels of RpoS. They
postulated that HSL constitutes an intracellular signal that
accumulates in starved cells regardless of cell density and that at
high cell densities this signal molecule can be acylated to allow
diffusion across membranes, forming an extracellular, cell
density-dependent signal. However, in recent studies AHL synthesis from
intracellular homoserine lactone pools has been demonstrated not to be
valid in studies in which LuxI-type AHL synthases have been expressed
in E. coli. Rather AHL are synthesized from
S-adenosylmethionine and acyl carrier protein conjugates (14, 25, 33). No further direct support for these ideas has
been forthcoming from experiments on E. coli. However,
recent work with Pseudomonas aeruginosa has shown that the
expression of rpoS is abolished in P. aeruginosa
lasR mutants, which are unable to respond to either of the two
AHLs [N-(3-oxododecanoyl)-L-homoserine lactone
and N-butyryl homoserine lactone] produced by this
bacterium (24). This provided direct evidence linking cell
density-dependent signalling systems with the regulation of the
stationary-phase sigma factor RpoS. Further experiments showed that the
regulator RhlR, when activated by N-butyryl homoserine
lactone, functions as a transcriptional activator of RpoS
(24). The full significance of these findings for the
stationary-phase or starvation survival of P. aeruginosa is
not clear. At present it is not known whether an RpoS homologue is
involved in stationary-phase survival of R. leguminosarum.
Recently it has been shown that an extracellular signal molecule(s) is
involved in the carbon starvation response of Vibrio
sp. strain S14 (35). When an extract of
stationary-phase medium was added to exponential-phase
Vibrio sp. strain S14, it resulted in the up-regulation of
carbon starvation-induced proteins, a process that was inhibited by
halogenated furanones, compounds that are putative antagonists of AHLs
(35).
| |
ACKNOWLEDGMENTS |
This work was funded by a grant from The Royal Society. S.H.T.
was supported by a BBSRC research studentship.
We are very grateful to Kendall Gray for providing us with samples of
R. leguminosarum autoinducer and to Allan Downie for strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Imperial College of Science, Technology and Medicine, Sir
Alexander Fleming Building, Imperial College Rd., London SW7 2AZ,
United Kingdom. Phone: 44(171) 594 5383. Fax: 44(171) 584 2056. E-mail: h.d.williams{at}ic.ac.uk.
Present address: School of Biological Sciences, University of
Surrey, Guildford, Surrey GU2 5XH, United Kingdom.
| |
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Abstract
Introduction
