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Journal of Bacteriology, June 2001, p. 3365-3371, Vol. 183, No. 11
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.11.3365-3371.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Trehalose in Growth at High Temperature of
Salmonella enterica Serovar Typhimurium
David
Cánovas,1,2,
Susanne A.
Fletcher,1
Mikachi
Hayashi,1 and
Laszlo N.
Csonka1,*
Department of Biological Sciences, Purdue
University, West Lafayette, Indiana 47907-1392,1
and Departamento de Microbiologia y Parasitologia, Facultad de
Farmacia, Universidad de Sevilla, Seville,
Spain2
Received 2 January 2001/Accepted 5 March 2001
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ABSTRACT |
Moderate osmolality can stimulate bacterial growth at
temperatures near the upper limit for growth. We investigated the
mechanism by which high osmolality enhances the thermotolerance of
Salmonella enterica serovar Typhimurium, by isolating
bacteriophage MudI1734-induced insertion mutations that blocked
the growth-stimulatory effect of 0.2 M NaCl at 45°C. One of these
mutations proved to be in the seqA gene (a regulator of
initiation of DNA synthesis). Because this gene is cotranscribed with
pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm
gene. Pgm catalyzes the conversion of glucose-6-phosphate to
glucose-1-phosphate during growth on glucose, and therefore loss of Pgm
results in a deficiency in a variety of cellular constituents derived
from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734
mutant at high temperature in medium of high osmolality is due to the
block in trehalose synthesis, we determined the effect of an
otsA mutation, which inactivates the first step of the
trehalose biosynthetic pathway. The otsA mutation caused
a growth defect at 45°C in minimal medium containing 0.2 M
NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this
medium at 37°C. These results suggest that the growth defect of the
seqA-pgm mutant at high
temperature could be a consequence of the block in trehalose synthesis.
We found that, in addition to the well-known osmotic control, there is
a temperature-dependent control of trehalose synthesis such that, in
medium containing 0.2 M NaCl, cells grown at 45°C had a fivefold
higher trehalose pool size than cells grown at 30°C. Our observations
that trehalose accumulation is thermoregulated and that mutations that
block trehalose synthesis cause a growth defect at high temperature in
media of high osmolality suggested that this disaccharide is crucial
for growth at high temperature either for turgor maintenance or for
protein stabilization.
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INTRODUCTION |
Although high osmolality is
generally regarded as a source of "stress" (3), this
is not necessarily always the case, because raising the osmotic
strength of the medium can increase the thermotolerance of bacteria
(5, 16, 19, 32).
In bacteria, thermotolerance can be quantified by assays of at least
two responses: viability at lethal high temperatures and growth
rate at nonlethal but inhibitory high temperatures. Moderate or high
osmolality enhances both of these aspects of thermotolerance in
Escherichia coli and Salmonella enterica serovar Typhimurium. It is not clear whether these two osmoregulated responses, namely enhancement of viability at lethal high temperatures and stimulation of growth near the upper limit of nonlethal temperatures, are different manifestations of a single osmotically controlled thermotolerance mechanism or they represent two independent responses. Hengge-Aronis et al. (19) observed that the
high-osmolality-dependent acquisition of increased viability at high
temperatures in exponentially growing cells was abolished by an
rpoS::Tn10 mutation, indicating that
the stationary-phase factor 
S is required for
this response. However, there have been no studies of the mechanism
that regulates the second response, the stimulation of growth by high
osmolality at high but nonlethal temperatures. We investigated this
issue by isolating MudI1734-induced mutations in serovar Typhimurium,
which blocked this response. Our analysis of mutants revealed that
trehalose
(O-
-D-glucosyl-[1
1]--D-glucoside) is crucial for the cells to grow in media of moderate osmolality at
limiting high temperature.
This work was supported by the U.S. Department of Agriculture
grant 98-35201-6219. David Cánovas was supported by a fellowship from the Ministerio de Educación y Cultura of Spain.
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MATERIALS AND METHODS |
Media.
The rich medium was Luria-Bertani medium (LB)
(11), and the minimal medium was M63
(8), containing the indicated concentrations of
D-glucose or D-galactose as carbon sources.
Because the biosynthesis of methionine is impaired at temperatures
above 42.5°C (29), this amino acid was added at 0.5 mM
to M63. When used, glycine betaine · HCl was added at 1 mM. The
osmotic strength of M63 was increased with 0 to 0.2 M NaCl, and the pH
of the medium was adjusted with NaOH to 7.2. The osmolality of M63 and
M63 plus 0.2 M NaCl is 0.2 and 0.6 osm, respectively (12).
When used, sodium ampicillin was at 25 µg/ml and kanamycin sulfate
was at 75 µg/ml. Solid media contained 20 g of agar per liter
(Difco). For the determination of trehalose levels and growth rates at
different temperatures, cultures were grown with aeration in a Julabo
shaking water bath, model SW-21C (guaranteed temperature stability of
±0.1°C), with a shaking frequency of 150 oscillations/min. Unless
stated otherwise, all chemicals were purchased from Sigma (St. Louis,
Mo.).
Bacterial strains.
The experiments described in this
paper were carried out with derivatives of Salmonella
enterica serovar Typhimurium strain TL1, a line of wild-type
strain LT2 propagated in this laboratory (10). Phage P22
HT105/1 int201 (designated P22 hereafter) was used as
the generalized transducing phage (11). Strains SF1005 (rpoS::amp) (15)
and XF373 (otsA::MudI1734) (14)
were obtained from F. Fang. Strains TL3131
(rpoS::amp) and TL3354
(otsA::MudI1734) were derived by transduction
of TL1 to Ampr and Kanr by P22 grown on SF1005
and XF373, respectively. Strain TL3356 (rpoS::amp
otsA::MudI1734) is a Kanr transductant of
TL3131 by P22 grown on TL3354. The presence of the rpoS
mutation in strains TL3131 and TL3356 was verified by reaction with
H2O2. Treatment of colonies of the wild-type
strain on LB plates with a drop of 30% H2O2
results in vigorous bubbling, due to the generation of O2
by catalases encoded by the katE and katG
genes. Because the transcription of these genes is dependent on RpoS,
mutants lacking this transcriptional activator have reduced catalase
activity (20). As expected, the rpoS
mutants TL3131 and TL3356 showed a greatly reduced efficiency of bubble
formation upon treatment with H2O2 compared to
that of control rpoS+ strains.
The seqA101::MudI1734 insertion was isolated as a
mutation that impaired growth stimulation by high osmolality at high
temperature. Approximately 105 derivatives of TL1
carrying random insertions of MudI1734 (24), obtained on
LB plus kanamycin plates, were replica plated to two M63 plates
containing 0.2 M NaCl and methionine, one of which was incubated at
48°C and the other at 30°C. Derivatives which could not grow at
48°C but could grow at 30°C were collected. (48°C was used for
the screening of the mutants, because we found that the wild type was
able to form colonies in replica plates at this high temperature.) The
MudI1734 insertion from potential mutants was transduced with P22 into
strain TL1, and the transductants were tested for their thermotolerance
at 45°C in liquid M63-0.2 M NaCl-glucose-methionine medium. We
identified 12 strains that showed various gradations of impaired growth
at 45°C but grew normally at 37°C in the same medium. Strain TL3283
(seqA101::MudI1734) was obtained as one of these transductants.
The location of this MudI1734 insertion was determined by cloning and
DNA sequencing. Cloning steps were carried out according
to procedures
described by Sambrook et al. (
30). In phage MudI1734,
the
Kan
r gene is located between
HindIII and
BamHI sites that are 1 and
2.8 kbp from the "left" (or
c) end of the phage
(
6). To clone
the DNA flanking the MudI1734 insertions,
total DNA was isolated
from strain TL3283 and was subjected to a
partial digestion with
Sau3AI (New England Biolabs, Beverly,
Mass.). The fragments were
ligated into the
BamHI site of
pBluescript IIKS (Stratagene, La
Jolla, Calif.) and introduced into
strain DH5
(Gibco-BRL, Grand
Island, N.Y.) by electroporation, and
then Kan
r derivatives were selected. In phage
MudI1734, there is an
NsiI
site 0.5 kbp from the above
HindIII site. As an initial screening
for constructs
that were likely to contain the left end of MudI1734
and hence, the
flanking host DNA, the plasmids were digested with
NsiI and
HindIII, and plasmids that contained both of these sites
were used for DNA sequence analysis. The sequences of the inserts
were
determined at the Iowa State University DNA sequencing facility,
Ames,
Iowa. The sequencing reactions employed the primer
5'-CCAATGTCCTCCCGGTTTTT-3',
which directs the sequence
determination outward from phage MudI1734,
starting at nucleotide 25 from the left end of the phage. Sequence
analysis revealed that the
left end of the phage in TL3283 is
immediately upstream of nucleotide
358 of the
seqA gene of serovar
Typhimurium (see
http://genome.wustl.edu/gsc/Blast/client.pl for
the
S. enterica serovar Typhimurium genomic sequence) and is thus
likely
to be polar on the expression of the
pgm gene
(
23). As
expected (
2), the
seqA101::MudI1734 insertion was cotransducible
with the
kdp locus in P22 transduction (~50% linkage).
The
seqA101::MudI1734
mutation resulted in a
defect in galactose utilization in M63
and in MacConkey galactose
medium, similar to that described in
E. coli by Lu and
Kleckner (
23). This insertion also conferred
resistance to
phage P22, which could be reversed by galactose,
similar to mutations
in other genes encoding enzymes of the pathway
between UDP-galactose
and glucose-6-phosphate (Fig.
1)
(
13).
These observations, which we confirmed further by
trehalose measurements
(see below), indicated that the
seqA101::MudI1734 insertion diminishes
the
expression of the
pgm gene by transcriptional polarity.
Determination of growth rates at 45°C.
Cells from single
colonies grown on LB plates at 37°C were inoculated into LB and grown
to saturation at 30°C. The cells were then subcultured at a dilution
of 1:100 into M63-10 mM glucose-methionine and the indicated
concentrations of NaCl, with or without glycine betaine or 5 mM
galactose, and were grown to saturation overnight at 30°C. The cells
were then subcultured again at a 1:8 dilution into 8 ml of medium of an
identical composition to that used for the overnight growth, except the
glucose concentration was increased to 20 mM and galactose to 10 mM,
and then these cultures were grown for 120 min at 30°C (to an optical
density at 600 nm [OD600] of 0.4 to 0.6). At this point,
the cells were diluted to an OD600 of 0.10 into 20 ml of
medium which had the same composition as was used for the previous step
and which had been prewarmed to 45°C. The cells were grown for the
growth curve measurements at this temperature in the Julabo water bath.
At different time intervals, samples were withdrawn, and the cell
density was monitored by determining the OD600.
Because the light scattering was not proportional to cell density above
an OD600 of >1, at high cell densities, the
OD600 was determined for a 1:10 dilution of the cultures. Specific growth rates were calculated from the best exponential fit of
the corrected OD600 versus time for the exponential portion of the growth curves, using the Cricket Graph III, version 1.01 Macintosh application. We used 30°C as the growth temperature prior
to the final growth phase at 45°C as a precaution to minimize possible protective effects that might arise from the induction of the
heat shock system (35). However, pilot experiments
indicated that cultures that were grown first at 42°C and shifted to
45°C had growth rates at the latter temperature that were within
experimental error equal to those of cultures that were grown at 30°C
and shifted to 45°C (data not shown). For determination of the growth
rates of control cultures at 37°C and 42°C, the same pregrowth
steps were used, except that the final cultures were incubated at these latter temperatures.
Determination of trehalose levels.
Cells were grown in
liquid LB, and then two successive subcultures were grown in M63 plus
glucose plus methionine plus the indicated concentrations of NaCl,
glycine betaine, or galactose, as described above for the determination
of the growth rates at 45°C, except that these cultures were grown at
30, 37, or 42°C, depending on the desired temperature at which the
cellular trehalose level was to be determined. After growth for 120 min
and verification that the cultures were in exponential growth, the
cells were diluted to an OD600 of 0.10 in 20 ml of medium
of the same composition used for the previous step (M63-20 mM
glucose-methionine plus the indicated concentrations of NaCl,
galactose, or glycine betaine). These last cultures were incubated at
the same temperature used for the pregrowth, with the exception of the
experiment conducted at 45°C, for which the inocula were taken from
cultures grown at 42°C. This temperature shift in the 45°C
experiment was performed because the latter temperature would not have
supported the growth of control cultures with 
0.10 M NaCl, whereas
42°C was permissive for growth, regardless of the NaCl concentration.
When the cell density in the final cultures reached an
OD
600 of 0.4 to 0.6 (2 to 2.6 doublings), 10-ml
samples were removed,
and the cells were sedimented by centrifugation
at 10,000 ×
g for 15 min at 4°C. In order to reduce
the carryover of glucose
from culture media that would interfere with
the trehalose determination
(see below), the cells were resuspended in
10 ml of glucose-free
M63 plus methionine plus the same concentration
of NaCl used for
the growth and were sedimented by centrifugation, as
above. The
cells were suspended in 1 ml of 80% (vol/vol) ethanol and
heated
to 65°C for 15 min. The cell debris was removed by
centrifugation
in a microfuge, and the ethanol extract was transferred
to new
tubes and dried under vacuum at 37°C in a SpeedVac (Savant).
The
residue was dissolved in 0.75 ml of H
2O.
The trehalose contents of the above aqueous extracts were determined by
a modification of the procedure recommended by the
manufacturer (Sigma,
St. Louis, Mo.) for the assay of trehalase.
In this procedure,
trehalose is hydrolyzed to two molecules of
glucose, which are
converted to glucose-6-phosphate and oxidized
by NADP, producing two
molecules of NADPH per molecule of trehalose
input. Samples of 0.05, 0.10, and 0.15 ml of the aqueous cell
extracts were brought to 0.20 ml
with H
2O, and 0.0054 U of trehalase
was added in
0.02 ml of 25 mM potassium phosphate, pH 6.5. The
mixtures were
incubated for 4 h at 37°C, during which the trehalose
was
hydrolyzed to glucose. Then 1 ml of a mixture of 0.25 M
triethanolamine
· HCl (pH 7.6), 10 mM
MgCl
2 · 6H
2O, 0.95 mM Na-ATP, 1.4 mM NADP,
0.32 U of hexokinase, and 0.18 U of
glucose-6-phosphate dehydrogenase
was added to each sample and
incubated at 37°C for 60 min. The
NADPH produced was measured
by its
A340.
The extracts could contain a number of compounds that would interfere
with the measurement of trehalose, such as glucose,
glucose-6-phosphate, NADH, or NADPH. In order to nullify the
effects
of any contribution from these sources, we carried out control
assays, from which trehalase was omitted, and the
A340 of these
controls was subtracted
from the
A340 of the assays with
trehalase.
The factor for conversion of
A340 to trehalose concentration was
determined from standard curves generated with various concentrations
of trehalose, subjected to identical assays as described above.
In the
standard curves,
A340 was linear with
the input concentration
of trehalose in the range of 0 to 160 mM
(
r2 > 0.98). The trehalose levels
were expressed as nanomoles per
milligram of protein, with the latter
calculated from the OD
600 of the cultures at the
time of harvest and standard curves relating
OD
600 to milligrams of cellular protein per
milliliter, determined
with the Bradford assay for cells grown in media
of appropriate
NaCl concentrations. It has been reported in
E. coli that if the
periplasmic TreA trehalase is not inactivated,
measurements of
trehalose levels might be artifactually low because of
the hydrolysis
of the solute in the cell extracts (
22). To
test whether this
is a problem in our procedure in which the cell
extracts were
prepared by ethanol lysis, we compared the trehalose
levels in
a
treA152::MudQ insertion mutant with those in
the wild type.
Within experimental error, the
treA mutation
did not have an effect
on the trehalose levels measured in cells grown
in M63 or in M63-0.2
M NaCl at 37 or 42°C (data not shown);
therefore, we concluded
that the trehalase activity did not cause a
significant interference
with our
measurements.
-Galactosidase assays.
For -galactosidase
measurements, strains carrying rpoS-lacZ fusions were
grown at the indicated temperatures in M63-20 mM glucose-methionine,
with or without 0.2 M NaCl and glycine betaine, as described above for
growth rate measurements. The -galactosidase activities were
determined at two points in the growth cycle: in exponential phase
(OD600, 0.2 to 0.25) and in stationary phase, after 24 h of incubation (corrected OD600, 3.9 to 4.2). The
-galactosidase activities were assayed as described by Miller
(25), with the specific activity expressed as nanomoles of
product formed per minute per milligram of protein, with the protein
concentration determined as described above for the trehalose level measurements.
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RESULTS |
Trehalose synthesis was required for high-osmolality-dependent
growth stimulation at 45°C.
As described above, our search for
mutations that abolish the osmotic stimulation of growth at high
temperature yielded the seqA101::MudI1734
insertion. The seqA gene, which encodes a negative regulator of initiation of chromosomal DNA replication, is upstream of
the pgm gene in a single operon (23). The
product of the latter gene, phosphoglucomutase, catalyzes the formation
of glucose-1-phosphate from glucose-6-phosphate during growth on
glucose (Fig. 1). Glucose-1-phosphate is the precursor of a variety of
saccharide moieties in a number of cellular constituents (Fig. 1). In
principle, the growth defect of the
seqA::MudI1734 mutant at 45°C in the
presence of high salt might be due to the loss of function of the SeqA
protein or to reduced expression of Pgm because of transcriptional
polarity. One of the metabolic consequences of the lack of Pgm is the
inability to make trehalose. Because this disaccharide has been
suggested to have thermoprotective functions (9, 33) and
because its cellular levels increase in response to high osmolality
(17), we investigated the possibility that it is essential
for growth at very high temperatures in media of moderate osmolality.
Trehalose is derived from UDP-glucose and glucose-6-phosphate by two
reactions catalyzed by the
otsA and
otsB gene
products
(Fig.
1). In order to test the possibility that the growth
defect
of the
seqA mutant at high temperature might be
connected to trehalose
deficiency, we compared the growth rates of this
mutant with that
of an
otsA::MudI1734 mutant at
45°C (Fig.
2). Typical results,
obtained with wild-type serovar Typhimurium, are shown in Fig.
2A. This
strain was unable to grow in M63 plus methionine at 45°C,
but
supplementation with 0.2 M NaCl stimulated it to grow at a
specific
growth rate of 0.38 generation/h. As reported previously
(
16), glycine betaine did not have an effect on the growth
rate
of the wild type in the presence of 0.2 M NaCl. The
seqA::MudI1734
mutant had a severe growth defect
in M63-methionine-0.2 M NaCl
(Fig.
2B). The
otsA mutant
showed a similar growth defect to that
of the
seqA mutant at
45°C (Fig.
2C). These results demonstrated
that trehalose is
necessary for the cells to respond to growth
stimulation by increased
osmolality at high temperature.
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FIG. 2.
seqA::MudI1734 and
otsA mutations block the high-osmolality-dependent
growth stimulation at 45°C. The growth rates of the wild-type strain
TL1 (A), the seqA::MudI1734 mutant TL3283 (B),
and the otsA::MudI1734 mutant TL3354 were
determined at 45°C, as described in Materials and Methods. Symbols:
 , M63-20 mM glucose-0.5 mM methionine;  , medium supplemented
with 0.2 M NaCl;  , medium supplemented with 0.2 M NaCl and 1 mM
glycine betaine (GB);  , medium supplemented with 10 mM galactose.
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The effects of the
seqA::MudI1734 mutation might
be complicated, not only because it could impair the expression of the
pgm gene but conceivably because the lack of the
seqA gene product
could have pleiotropic consequences.
Supplementation of the growth
medium with galactose or glycine betaine
restored growth to this
mutant at 45°C (Fig.
2B). Because galactose
is metabolized to
glucose-1-phosphate (Fig.
1), it is possible to
supply this intermediate
in strains lacking Pgm by providing them with
galactose during
growth on glucose (
13). The result, that
galactose improved
the growth of the
seqA::MudI1734 mutant in M63-0.2 M NaCl at
45°C,
indicated that the high-temperature growth defect of this
mutant
could be ascribed to the loss of metabolic product(s) derived
from glucose-1-phosphate, rather than to the loss of the SeqA
protein.
The fact that glycine betaine could also alleviate the
growth defect at
high temperature in this mutant suggested that
the problem arose from
the lack of trehalose, whose function as
a compatible solute could be
supplanted by glycine betaine. These
conclusions were supported by the
observations (Fig.
2C) that
in the
otsA mutant, the growth
defect at high temperature was
not repaired by galactose, which could
not be converted in this
mutant to trehalose, but it was corrected by
glycine betaine,
which could replace trehalose as a compatible
solute.
Mutations blocking the synthesis of trehalose (
otsA,
otsB, and
galU) (see Fig.
1) have been isolated
previously by their phenotype
of sensitivity to high osmolality (
0.45
M NaCl) (
17,
28).
Although these previous experiments
indicated that the loss of
the ability to accumulate trehalose does not
cause a significant
growth defect at 37°C at NaCl concentrations
below 0.45 M, conceivably
there might be an increased need for this
disaccharide at higher
temperatures in media of moderate osmolality.
Therefore, we determined
the growth rates of the relevant strains in
media containing 0.2
M NaCl at various temperatures (Table
1). At 37°C, the growth
rate of the
otsA mutant in the presence of 0.2 M NaCl was only
2% lower
than that of the wild-type strain, in accord with the
earlier
conclusions in
E. coli (
17,
28) that at this
temperature,
trehalose is not required for cytoplasmic osmoregulation
at moderate
osmolalities. However, the
otsA mutation caused
a more substantial
growth defect in the presence of 0.2 M NaCl as the
temperature
was increased. At 42°C, this mutation caused an ~25%
decrease
in the growth rate, and at 45°C, it resulted in a >90%
reduction
in growth rate in M63-0.2 M NaCl, compared to the wild type.
The
growth rate defect caused by the loss of trehalose synthesis at
these higher temperatures could be corrected by glycine betaine.
These
results suggested that trehalose becomes more crucial for
growth at
moderate osmolality with increasing temperature.
Like the
otsA mutant, the
seqA mutant also
exhibited a growth defect in M63-0.2 M NaCl at 42°C and especially
at 45°C. However,
the
seqA mutant had a lower growth rate
than the wild type even
in M63 without NaCl at 37°C and 42°C, and
the growth rate of this
strain was not completely restored by glycine
betaine in M63-0.2
M NaCl to wild-type values at any temperature. The
latter result
suggested that, while the growth defect in the
seqA mutant could
at least in part be due to the block in
trehalose synthesis, the
Pgm or SeqA deficiency could also cause a more
pleiotropic growth
reduction. Loss of the ability to grow at 45°C in
M63-methionine-0.2
M NaCl in the SeqA/Pgm mutant was not due to the
defect in the
normal composition of the outer membrane, because a
galE mutation,
which also interferes with the synthesis of
the sugar moieties
in the lipopolysaccharide (Fig.
1), did not cause a
detectable
defect in the osmotically induced growth stimulation at high
temperature
(data not
shown).
The seqA::MudI1734 mutation impairs the
synthesis of trehalose.
In order to verify that the
seqA::MudI1734 mutation blocks the synthesis of
trehalose, we measured the levels of this disaccharide in various
strains at low and high osmolality (Table
2). Our results confirmed observations in
E. coli (31) that high osmolality stimulated
the accumulation of trehalose in wild-type serovar Typhimurium and that
this response was blocked by glycine betaine. As expected, the
otsA mutation prevented the high-osmolality-dependent accumulation of trehalose. The seqA mutation had a similar
consequence, demonstrating that indeed this insertion is polar on the
expression of pgm. The results shown in Table 2 were
obtained at 37°C, but we found that the seqA insertion
mutation eliminated trehalose synthesis at 42°C also (data not
shown). Supplementation with galactose restored the synthesis of
trehalose in the seqA mutant in M63-glucose-0.2 M NaCl
(Table 2), in accord with the notion that the metabolic defect in this
Pgm-deficient mutant can be circumvented by galactose.
High-osmolality-dependent accumulation of trehalose was enhanced at
high temperature.
Because of our finding that the synthesis of
trehalose was critical for growth at high temperature in the presence
of 0.2 M NaCl, we examined whether the levels of trehalose were
regulated by temperature. This experiment (Fig.
3) revealed that the accumulation of
trehalose was subject to a temperature-dependent control in media of
moderate osmolality; in M63-0.2 M NaCl, cells that were grown at
45°C had ~fivefold higher trehalose levels than cells that were
grown at 30°C. Because there was a requirement for at least 0.075 M
NaCl for growth at 45°C, we could not determine whether this high
temperature would be sufficient signal by itself to elevate the
trehalose pool size, but at lower temperatures, high osmolality was
required to observe trehalose accumulation. The effect of increasing
temperature was to lower the threshold osmolality that was required to
elevate the trehalose pool size; at 45°C, 0.075 M NaCl was sufficient
to induce trehalose accumulation, whereas at 30°C, 0.2 M NaCl was
required.
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FIG. 3.
Regulation of trehalose levels by osmolality and
temperature in the wild-type serovar Typhimurium. The trehalose levels
were determined in serovar Typhimurium strain TL1 in M63 plus glucose
plus methionine, containing the indicated concentrations of NaCl at the
indicated temperatures, as described in Materials and Methods. The data
represent the averages and standard errors of results obtained in at
least two independent measurements for all conditions, except for the
results obtained with 0.05 and 0.075 M NaCl at 42 and 45°C, which
were obtained in single measurements.
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Thermoregulation of expression of otsA.
In
E. coli K-12 derivatives, the otsAB operon is
induced by high osmolality or by stationary phase under the control of
S (18, 21). In addition, it has
been shown that the otsAB operon exhibits a three- to
fourfold "heat shock" induction upon shift of the temperature from
30 to 42.5°C (26). Because we found that the trehalose
levels increased as a function of increasing temperature in cells that
were grown at constant temperature and salinity for more than nine
generations (Fig. 3), we determined whether there is a thermoregulation
of expression of the otsA-lacZ fusion in serovar Typhimurium
grown continuously at 30 or 42°C. As can be seen in Table
3, the expression of the
otsA-lacZ fusion was five- to sixfold higher at 42 than at
30°C, both in the absence and in the presence of 0.2 M NaCl. Thus,
the otsA operon remained induced continuously at high
temperature, unlike the typical genes in the heat shock regulon, which
are transcribed maximally only for a short time after a temperature
upshift (35). As has been seen in E. coli
(18, 21), the expression of the otsA-lacZ fusion in serovar Typhimurium was also induced in exponential phase
about threefold by 0.2 M NaCl. Glycine betaine reversed the
high-osmolality-dependent induction at both temperatures in exponentially growing cells. We observed stationary-phase-dependent induction of the fusion at 30 but not at 42°C. Induction of the otsA gene by high temperature in midexponential phase was
eliminated by the rpoS::amp insertion,
as was the induction by high osmolality in stationary phase.
This result, which was observed earlier in E. coli,
indicated that the S factor is required for
the thermoregulation of expression of otsA (18,
26).
| |
DISCUSSION |
Moderate or high osmolality generated by 0.2 M NaCl elevates the
upper limit of growth for serovar Typhimurium to 45°C
(16). In this work, we found that the synthesis of
trehalose was necessary for this high-osmolality-dependent growth
stimulation. It has been documented that trehalose is one of the
compatible solutes that is synthesized by bacteria in response to
osmotic stress, but we discovered that high temperature greatly
enhanced the high-osmolality-dependent accumulation of this disaccharide.
As discussed in the introduction, moderate osmolality imparts increased
thermotolerance to serovar Typhimurium and E. coli by two
different mechanisms: enhancement of resistance to lethal high
temperatures (19) and elevation of the upper limit of
temperature for growth (32). Hengge-Aronis et al.
(19) reported that otsAB mutations do not
impair the high-osmolality-dependent induction of increased
thermotolerance in exponentially growing E. coli cells,
which we confirmed in serovar Typhimurium with the trehalose-deficient seqA and otsA mutants (S. A. Fletcher and
L. N. Csonka, unpublished data). In contrast, the ability to
respond to growth stimulation by high osmolality at elevated
temperature was abolished by mutations that blocked the synthesis of
trehalose (Fig. 2 and Table 1).
With our enzymatic assay, we found that the trehalose level in
wild-type serovar Typhimurium grown in M63-0.2 M NaCl at 37°C was 20 nmol/mg of protein (Fig. 3 and Table 2). Laboratory lines of E. coli K-12 showed large variation in their trehalose levels at low
osmolalities. Wild-type K-12 carries an in-frame amber codon in the
rpoS gene. In this strain and in related amber
suppressor-free derivatives, trehalose was undetectable at 37°C in
M63-0.2 M NaCl (21). In strains that contain an amber
suppressor tRNA, the trehalose pool size was ~200 nmol/mg of protein
at 37°C in medium containing 0.2 M NaCl. Welsh et al.
(34) reported that the trehalose content of E. coli NCIB9484 was ~5 nmol/mg of protein at 0.2 M NaCl. Thus, the
trehalose level we measured with the enzymatic assay in serovar
Typhimurium at 37°C in M63-0.2 M NaCl was within the range reported
for wild-type E. coli strains grown at the low osmolalities
(0.2 M NaCl).
Previously, we found that a minimum 0.15 M NaCl is required to induce
growth stimulation at 45°C (16). This response can be
elicited by NaCl concentrations up to 0.6 M, but above 0.3 M, the
stimulatory effects of high osmolality at high temperature are
counteracted by its adverse effect due to osmotic inhibition that are
seen at all temperatures. In the present study, we used 0.2 M NaCl
because it is nearly optimal for stimulating the growth of serovar
Typhimurium at 45°C (16). At this temperature, the trehalose content of cells grown with this concentration of NaCl was 64 nmol/mg of protein (Fig. 3). The water-accessible cytoplasmic volume of
E. coli grown at an osmolality comparable to that of M63-0.2 M NaCl was reported to be ~1.7 µl/mg dry weight or ~3.4 µl/mg of protein (7). Using this value as the
cytoplasmic volume of serovar Typhimurium, we calculated that the
trehalose concentration at 45°C and 0.2 M NaCl was approximately 0.02 M.
Trehalose has been suggested to have a number of functions, which are
not mutually exclusive. By accumulating this disaccharide to high
levels, cells can generate the necessary turgor pressure in media of
high osmolality. Solutes that are accumulated at high concentrations in
intracellular compartments for turgor maintenance are "compatible"
because they do not interfere with cellular processes (4).
In general, compatible solutes are excluded from the water of hydration
surrounding the proteins, and therefore such compounds promote
macromolecular stability under conditions of low water activity
(1, 27). Trehalose also has been found at very high levels
(up to 20% of dry weight) in a number of desiccation-tolerant plants
under extreme dehydration (9). It has been suggested that
under extremely anhydrobiotic conditions, trehalose at very high
concentrations may promote the integrity of membranes and other
macromolecules by functioning as a water substitute (9). Although our results demonstrate that the synthesis of trehalose is
required in serovar Typhimurium for growth at high temperature in media
of moderate osmolality and that high temperature stimulates the
accumulation of trehalose, the basis for its thermoprotective function
is not clear in serovar Typhimurium. At 0.02 M internal concentration,
trehalose could balance only about 3% of the osmotic pressure
generated by M63-0.2 M NaCl. At such modest concentrations, it is not
plausible that trehalose could have the stabilizing effects on
membranes that have been proposed at trehalose contents approaching
20%. To our knowledge, there has been no investigation of the
temperature dependence of the turgor maintenance by compatible solutes
or their exclusion from the water of hydration around proteins, but our
observation that glycine betaine could compensate for the growth defect
at high temperature arising from the block in trehalose production
suggests that there is a greater need for compatible solutes at high
temperature even at moderate osmolality. While our results demonstrated
that trehalose was required for growth near the limiting high
temperatures in media of elevated osmolality, further experiments are
necessary to determine the role of this disaccharide.
| |
ACKNOWLEDGMENTS |
We thank K. O'Connor for critical comments on the
manuscript, D. S. Cayley, D. Fraenkel, and M. T. Record, Jr.,
for thought-provoking discussions, N. C. Carpita for suggesting to
us the method for measurement of the trehalose levels, and E. Groisman
for providing us with the sequence of the Mu sequencing primer.
| |
ADDENDUM IN PROOF |
After this paper was accepted, we became aware of a large body of
literature on the accumulation and function of trehalose in response to
heat stress in yeast. Examples are the work of DeVirgilio et al. (C. DeVirgilio, T. Hottinger, J. Dominguez, T. Boller, and A. Wiemken, Eur.
J. Biochem. 219:179-186, 1994) and Hottinger et al. (T. Hottinger, C. DeVirgilio, M. N. Hall, T. Boller, and A. Wiemken, Eur.
J. Biochem. 219:187-193, 1994). In addition, prior use of
trehalase to assay trehalose has been reported (I. Kienle, M. Burgert, and H. Holzer, Yeast 9:607-611, 1993).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Purdue University, West Lafayette, IN 47907-1392. Phone: (765) 494-4969. Fax: (765) 496-1496. E-mail:
lcsonka{at}bilbo.bio.purdue.edu.
Present address: Departamento Biotecnologia Microbiana, Centro
Nacional de Biotecnologia-CSIC, Campus UAM-Cantoblanco, 28049 Madrid, Spain.
| |
REFERENCES |
| 1.
|
Arakawa, T., and S. N. Timasheff.
1985.
The stabilization of proteins by osmolytes.
Biophys. J.
47:411-414[Medline].
|
| 2.
|
Berlyn, M.,
K. B. Low, and K. E. Rudd.
1996.
Linkage map of Escherichia coli K-12, edition 9, p. 1715-1902.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. ASM Press, Washington D.C.
|
| 3.
|
Bremer, E., and R. Krämer.
2000.
Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes in bacteria, p. 79-97.
In
G. Storz, and R. Hengge-Aronis (ed.), Bacterial stress. ASM Press, Washington D.C.
|
| 4.
|
Brown, A. D.
1990.
Microbial water stress physiology: principles and perspectives.
John Wiley & Sons, Ltd., Chichester, United Kingdom.
|
| 5.
|
Calhoun, C. L., and W. C. Frazier.
1966.
Effect of available water on thermal resistance of three nonsporeforming species of bacteria.
Appl. Microbiol.
14:416-420[Medline].
|
| 6.
|
Castilho, B.,
P. Olfson, and M. J. Casadaban.
1984.
Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposon.
J. Bacteriol.
158:488-495[Abstract/Free Full Text].
|
| 7.
|
Cayley, S.,
B. A. Lewis,
H. J. Guttman, and M. T. Record, Jr.
1991.
Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity.
J. Mol. Biol.
222:281-300[CrossRef][Medline].
|
| 8.
|
Cohen, G. N., and R. H. Rickenberg.
1956.
Concentration specifique reversible des amino acides chez E. coli.
Ann. Inst. Pasteur (Paris)
91:693-720[Medline].
|
| 9.
|
Crowe, J. H.,
F. A. Hoekstra, and L. M. Crowe.
1992.
Anhydrobiosis.
Annu. Rev. Physiol.
54:579-599[CrossRef][Medline].
|
| 10.
|
Csonka, L. N.
1981.
Proline over-production results in enhanced osmotolerance in Salmonella typhimurium.
Mol. Gen. Genet.
182:82-86[CrossRef][Medline].
|
| 11.
|
Davis, R. W.,
D. Botstein, and J. R. Roth.
1980.
Advanced bacterial genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 12.
|
Dunlap, V. J., and L. N. Csonka.
1985.
Osmotic regulation of L-proline transport in Salmonella typhimurium.
J. Bacteriol.
163:296-304[Abstract/Free Full Text].
|
| 13.
|
Enomoto, M., and B. A. D. Stocker.
1974.
Transduction by phage P1kc in Salmonella typhimurium.
Virology
60:503-514[CrossRef][Medline].
|
| 14.
|
Fang, F. C.,
C. Y. Chen,
D. G. Guiney, and Y. Xu.
1995.
Identification of s-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma s and cyclic AMP-receptor protein.
J. Bacteriol.
178:5112-5120[Abstract/Free Full Text].
|
| 15.
|
Fang, F. C.,
S. J. Libby,
N. A. Buchmeier,
P. C. Loewen,
J. Switala,
J. Harwood, and D. G. Guiney.
1992.
The alternative -factor KatF (RpoS) regulates Salmonella virulence.
Proc. Natl. Acad. Sci. USA
89:11978-11982[Abstract/Free Full Text].
|
| 16.
|
Fletcher, S. A., and L. N. Csonka.
1998.
Characterization of the induction of increased thermotolerance by high osmolality in Salmonella typhimurium.
Food Microbiol.
15:307-317[CrossRef].
|
| 17.
|
Giæver, H. M.,
O. B. Styrvold,
I. Kaasen, and A. R. Strøm.
1988.
Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.
J. Bacteriol.
170:2841-2849[Abstract/Free Full Text].
|
| 18.
|
Hengge-Aronis, R.,
W. Klein,
R. Lange,
M. Rimmele, and W. Boos.
1991.
Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli.
J. Bacteriol.
173:7918-7924[Abstract/Free Full Text].
|
| 19.
|
Hengge-Aronis, R.,
R. Lange,
N. Henneberg, and D. Fischer.
1993.
Osmotic regulation of rpoS-dependent genes in Escherichia coli.
J. Bacteriol.
175:259-265[Abstract/Free Full Text].
|
| 20.
|
Hengge-Aronis, R.
2000.
The general stress response of Escherichia coli, p. 161-178.
In
G. Storz, and R. Hengge-Aronis (ed.), Bacterial stress. ASM Press, Washington, D.C.
|
| 21.
|
Kaasen, I.,
P. Falkenberg,
O. B. Styrvold, and A. R. Strøm.
1992.
Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by KatF (AppR).
J. Bacteriol.
174:889-898[Abstract/Free Full Text].
|
| 22.
|
Larsen, P. I.,
L. K. Sydnes,
B. Landfald, and A. R. Strøm.
1987.
Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose.
Arch. Microbiol.
147:1-7[CrossRef][Medline].
|
| 23.
|
Lu, M., and N. Kleckner.
1994.
Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli.
J. Bacteriol.
176:5847-5851[Abstract/Free Full Text].
|
| 24.
|
Maloy, S. R.
1990.
Experimental techniques in bacterial genetics.
Jones and Bartlett, Boston, Mass.
|
| 25.
|
Miller, J. H.
1991.
A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 26.
|
Muffler, A.,
M. Barth,
C. Marschall, and R. Hengge-Aronis.
1997.
Heat shock regulation of s turnover: role for DnaK and relationship between stress responses mediated by s and 32 in Escherichia coli.
J. Bacteriol.
179:445-452[Abstract/Free Full Text].
|
| 27.
|
Record, T. M., Jr.,
E. S. Courtenay,
D. S. Cayley, and H. J. Guttman.
1998.
Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments.
Trends Biochem. Sci.
23:190-194[CrossRef][Medline].
|
| 28.
|
Rod, L. M.,
K. Y. Alam,
P. R. Cunningham, and D. P. Clark.
1988.
Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors.
J. Bacteriol.
170:3601-3610[Abstract/Free Full Text].
|
| 29.
|
Ron, E. Z., and M. Shani.
1971.
Growth rate of Escherichia coli at elevated temperatures: reversible inhibition of homoserine trans-succinylase.
J. Bacteriol.
107:397-400[Abstract/Free Full Text].
|
| 30.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 31.
|
Styrvold, O. B., and A. R. Strøm.
1991.
Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of periplasmic trehalase.
J. Bacteriol.
173:1187-1192[Abstract/Free Full Text].
|
| 32.
|
Tesone, S.,
A. Hughes, and A. Hurst.
1981.
Salt extends the upper temperature limit for growth of food-poisoning bacteria.
Can. J. Microbiol.
27:970-972[Medline].
|
| 33.
|
Van Laere, A.
1989.
Trehalose, reserve and/or stress metabolite?
FEMS Microbiol. Rev.
63:201-210[CrossRef].
|
| 34.
|
Welsh, D. T.,
R. H. Reed, and R. A. Herbert.
1991.
The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture.
J. Gen. Microbiol.
137:745-750[Medline].
|
| 35.
|
Yura, T.,
M. Kanemori, and M. T. Morita.
2000.
The heat shock response: regulation and function, p. 3-18.
In
G. Storz, and R. Hengge-Aronis (ed.), Bacterial stress. ASM Press, Washington, D.C.
|
Journal of Bacteriology, June 2001, p. 3365-3371, Vol. 183, No. 11
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.11.3365-3371.2001
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Top
Abstract
Introduction
