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J Bacteriol, March 1998, p. 1030-1036, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Importance of RpoS and Dps in Survival of Exposure
of Both Exponential- and Stationary-Phase Escherichia coli
Cells to the Electrophile N-Ethylmaleimide
G. P.
Ferguson,*
R. I.
Creighton,
Y.
Nikolaev, and
I. R.
Booth
Department of Molecular and Cell Biology,
Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, AB25 2ZD, United Kingdom
Received 3 September 1997/Accepted 20 December 1997
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ABSTRACT |
The mechanisms by which Escherichia coli cells survive
exposure to the toxic electrophile N-ethylmaleimide (NEM)
have been investigated. Stationary-phase E. coli cells were
more resistant to NEM than exponential-phase cells. The KefB and KefC
systems were found to play an important role in protecting both
exponential- and stationary-phase cells against NEM. Additionally, RpoS
and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both
RpoS and Dps and triple mutants deficient in KefB and KefC and either
RpoS or Dps had an increased sensitivity to NEM in both exponential-
and stationary-phase cells compared to mutants missing only one of
these protective mechanisms. Stationary- and exponential-phase cells of
a quadruple mutant lacking all four protective systems displayed even
greater sensitivity to NEM. These results indicated that protection by
the KefB and KefC systems, RpoS and Dps can each occur independently of
the other systems. Alterations in the level of RpoS in exponentially
growing cells correlated with the degree of NEM sensitivity. Decreasing
the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and
gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.
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INTRODUCTION |
Within their natural environment,
bacteria go through periods of rapid growth when nutrients are
plentiful, but slow growth occurs as nutrients become limited and when
waste products accumulate. The transitions between these two states can
be mimicked in the laboratory by growth of bacteria in batch culture;
early exponential phase represents when nutrients are plentiful, and
stationary phase represents the nongrowing state. In addition,
alterations in the genotype of cells and changes to the nutrient
composition of the medium will affect the growth rate. Bacteria are
subject to an array of stresses within their natural environment, and it has been demonstrated previously that stationary-phase cells survive
these insults better than their exponential-phase counterparts (19). In Escherichia coli cells, this is, at
least in part, due to the alternative sigma factor, RpoS, which
accumulates in stationary-phase cells (23). RpoS is
responsible for the activation of transcription of at least 30 genes,
many of whose products encode proteins involved in protecting bacteria
against stress (17). Regulation of RpoS levels in E. coli cells is complex and occurs at many levels (17, 24,
37). In rapidly growing exponential-phase cells, RpoS is
maintained at a basal level both by low rates of synthesis and by a
reduction in its half-life by severalfold compared with
stationary-phase cells (24, 37). The short half-life of RpoS
in rapidly growing exponential-phase cells is due to the degradation of
RpoS by the ClpP protease system (33). However, it has been
shown that RpoS can accumulate in exponential-phase cells when the
growth rate is substantially reduced by either osmotic stress, glucose
limitation, growth on a poor carbon source such as succinate, or growth
at temperatures below 30°C (17, 23, 28, 30, 34).
Bacteria are exposed to toxic electrophiles both from within the cell
and from their environment. In E. coli cells, the endogenous electrophile methylglyoxal is produced when bacteria are grown on a
poor carbon source such as D-xylose in the presence of cAMP (1, 14, 18). Under these conditions, bacteria produce so much methylglyoxal that it is excreted into the environment. In addition, bacteria in the gut are likely to be exposed to electrophilic compounds from the diet. Methylglyoxal has also been found to accumulate during the cooking of food and is present in beverages such
as coffee and wine (29). Electrophiles can be generated during the chlorination of poultry and are used as herbicides (36,
42). To understand the mechanisms by which E. coli
cells defend themselves against toxic electrophiles, our research has focused on the electrophilic reagent N-ethylmaleimide (NEM).
Survival of E. coli cells upon exposure to electrophiles
such as methylglyoxal and NEM requires protective mechanisms. In
exponential-phase cells, the tripeptide glutathione is central to these
protective mechanism (4, 9, 14, 15, 27). Conjugation of the
electrophile to glutathione is the first step in detoxification, and
the resultant glutathione conjugate(s) activates either the KefB or
KefC potassium channel or both (9, 14, 27). The nature of
the glutathione adduct determines which potassium channel is activated
(14). For example, KefB is activated by glutathione adducts
formed from NEM and methylglyoxal, whereas KefC is activated by the NEM
adduct but is only poorly activated by the adduct of methylglyoxal. The activation of KefB and KefC results in the rapid loss of potassium from
the bacterial cell, via these channels, and this is balanced by an
influx of sodium ions and protons (5, 13, 15). The influx of
protons rapidly lowers the cytoplasmic pH of the E. coli
cell, which protects the cell against the toxic effects of electrophiles (12, 13, 15). Exponential-phase cells of
mutants lacking KefB and KefC are unable to acidify their cytoplasm
upon electrophile addition and consequently lose viability.
We suggested previously that interference with the KefB and KefC
systems could provide a novel antibacterial strategy since these
systems play an important role in the protection of exponential-phase bacterial cells against stress (12-15). However, the most
valuable targets for antibacterial therapy should be important for
survival in both growing and stationary-phase bacteria. Previously we
performed extensive studies of actively growing bacteria. Here we
present data demonstrating that the KefB and KefC systems also play an important role in the protection of stationary-phase E. coli
cells against the electrophile NEM. We also show that RpoS and the
DNA-binding protein Dps provide additional protection against NEM to
E. coli cells in both the exponential and stationary phases
of growth. These data demonstrate the importance of the KefB and KefC
systems, RpoS, and Dps in protecting actively growing and
stationary-phase E. coli cells against stress.
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MATERIALS AND METHODS |
Bacterial strains and growth.
All bacterial strains used in
this study were derivatives of E. coli K-12 and are
described in Table 1. Exponential- and stationary-phase cultures were prepared as stated in the text in either
K0 supplemented with 10 mM KCl (K10)
(10) or M9 minimal medium. Unless stated otherwise, 0.2%
(wt/vol) glucose was included as the carbon source. The medium was also
supplemented with 0.4% (wt/vol) casein hydrolysate (CAS) and bases
(adenine, cytosine, guanine, thymidine, and uracil) at 50 µg
ml
1 as defined in the text. Cultures in K10
were prepared as follows. Stationary-phase cultures were prepared by
growth from a single colony at 37°C and 300 rpm for ca. 16 h.
Exponential-phase cultures were prepared by diluting an overnight
culture 15-fold into fresh growth medium to give a starting optical
density at 650 nm (OD650) of 0.05 to 0.1. The culture was
then grown to an OD650 of 0.4. The stationary- and
exponential-phase cultures were then diluted into fresh prewarmed
medium (37°C) to give a starting OD650 of 0.04, and then
either NEM or sodium acetate was added to the concentration defined in
the text. For the viability experiments, 50-µl samples were removed
from the cultures at the times defined in the text. Samples for the
Western blots were prepared by filtering (4.5 cm, 0.45-µm pore size;
Whatman) 2 × 20 ml of culture at defined times. For protein
measurements, the samples were resuspended in 0.1 M NaOH.
Exponential-phase cultures in M9 were prepared from a growth-arrested
overnight culture (0.04% [wt/vol] glucose) as follows.
The overnight
culture was diluted into fresh prewarmed medium
(37°C) to give an
OD
650 of 0.05 and then grown up to an OD
650 of
0.3. Samples for the Western blots were harvested by centrifugation
(4,500 ×
g, 15 min). An identical sample was filtered
(4.5 cm,
0.45-µm pore size; Whatman) to determine the protein
concentration,
using the trichloroacetic acid (TCA) method (see below).
The exponential-phase
cells were then diluted into fresh prewarmed
medium (37°C) to
give a starting OD
650 of 0.05, and NEM
was added. All NEM additions
were made from a freshly prepared 50 mM
stock in 50% (vol/vol)
ethanol. Viability experiments were conducted
exactly as described
previously (
13-15). For viability
experiments with cells grown
in K
10 medium, cells were
diluted into K
0 lacking all supplements,
and recovery was
conducted on K
10 plates. Cells from M9 viability
experiments were diluted into 0.9% (wt/vol) NaCl, and recovery
was
performed on LB plates. In both cases, samples had to be diluted
at
least 10-fold prior to spotting, as the neat sample did not
recover due
to the presence of NEM.
Western blots.
Western blotting was performed by a standard
method (39). The cell pellets were resuspended in 100 µl
of sodium dodecyl sulfate solubilization buffer (20% [vol/vol]
glycerol, 2% [wt/vol] sodium dodecyl sulfate, 0.5 g of
bromophenol blue liter1, and 0.75 M 
-mercaptoethanol
in 0.125 M Tris-HCl [pH 6.8]), and incubated at 100°C for 10 min,
and 30 µg of protein was loaded per well. The RpoS mouse monoclonal
antibody was diluted 40,000-fold in 0.5% Marvel dissolved in PBS-T (15 mM phosphate buffer, 150 mM NaCl, 0.05% [vol/vol] Tween 20). The
binding of the RpoS antibody was detected by using the SuperSignal
ULTRA chemiluminescent substrate (Pierce, Rockford, Ill.). To eliminate
problems with background, the blot was washed once with PBS-T prior to
exposure.
Protein determinations.
For the M9-grown cultures, the
samples were resuspended in ice-cold TCA to a final concentration of
0.5 M. The TCA-treated cultures were then filtered (4.5 cm; Whatman
GF/F), washed with an equal volume of M9 medium (CAS-derived peptides
that might interfere with the protein assay are soluble in the TCA and
are therefore removed at this step), resuspended in 0.2 M NaOH, and left overnight at room temperature. Distilled water (2 ml) was added,
and then the extract was filtered (4.5 cm, 0.45-µm pore size;
Whatman) to remove debris. The total protein concentration of the
extract was determined by using the Lowry method adapted for microtiter
plates (25).
Reproducibility.
NEM is highly toxic, and variations in the
actual survival data were observed between different days, although the
trends were always the same. Therefore, it was important to test all strains for comparison, including the appropriate controls on the same
day. Each graph represents data obtained from experiments conducted on
the same day only and is representative of at least two experiments.
Error bars represent the standard deviation from the mean for one
experiment. At higher concentrations of NEM (above 0.3 mM), experiments
were very reproducible and therefore results from different days could
be averaged.
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RESULTS |
KefB and KefC play an important role in protecting stationary-phase
cells against NEM.
We have shown previously that the KefB and KefC
potassium channels protect exponential-phase cells against the toxic
effects of NEM (15). However, the role of these potassium
channels in stationary-phase cells had not been established.
Stationary-phase cells of MJF274 (KefB+ KefC+)
and MJF276 (kefB kefC::Tn10) were
diluted to the same starting cell density and then exposed to 0.2 mM
NEM (Fig. 1a). The viability of
stationary-phase cells of MJF274 remained constant throughout the
75-min exposure to 0.2 mM NEM. In contrast, the viability of
stationary-phase cells lacking KefB and KefC was retained for up to 15 min and then declined. These results demonstrate that KefB and KefC
have an important role in protection against NEM in stationary-phase
cells. To investigate whether stationary-phase cells had increased
resistance to NEM, exponential- and stationary-phase cells of MJF274
(KefB+ KefC+) were exposed to 0.2 mM NEM and
cell viability was determined (data not shown). Under these conditions,
both exponential- and stationary-phase cells of MJF274 remained viable
for the 75-min exposure. However, when a higher concentration of NEM
(0.4 mM) was used, both exponential- and stationary-phase cells of
MJF274 retained almost complete viability for up to 35 min, after which their viability steadily declined (Fig. 1b). The decline in viability was more rapid for the exponential-phase cells of MJF274, and after 80 min of incubation with NEM, there were no surviving cells. In contrast,
3 × 105 stationary-phase cells survived 80 min of
exposure to 0.4 mM NEM. It has also been shown that stationary-phase
cells of MJF274 are more resistant than exponential-phase cells to the
endogenous electrophile methylglyoxal (10a). These data
demonstrate that stationary-phase cells are more resistant than
exponential-phase cells to electrophiles.
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FIG. 1.
Stationary-phase resistance against NEM. Cells were
grown in K10 medium, and cell viability was determined
exactly as described in Materials and Methods. (a) Stationary-phase
cells of MJF274 ( ) and MJF276 (kefB
kefC::Tn10;  ) were treated with 0.2 mM NEM
at time zero. (b) Exponential ( )- and stationary ()-phase cells
of MJF274 were treated with 0.4 mM NEM at time zero. The arrow
represents no viable cells by the next time point; error bars represent
the standard deviation from the mean for one experiment.
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RpoS and Dps protect exponential- and stationary-phase E. coli cells against NEM.
To investigate additional components
involved in the protection of E. coli cells against NEM, we
sought to determine the roles of the alternative sigma factor RpoS and
of Dps, a DNA-binding protein first identified in starved bacterial
cells (2, 17). We proposed previously that NEM damages
bacterial cells by interacting with the nucleophilic centers of
cellular macromolecules such as DNA (15). RpoS is known to
regulate the expression of DNA repair enzymes; Dps is believed to bind
to the DNA of the bacterial cell, with a lack of sequence specificity,
and it has been found to protect cells against oxidative damage
(2, 17, 26, 31, 38, 40). Strain MJF274 was transduced to
Tetr and Kanr by using strains RH90
(rpoS::Tn10) and ZK1058
(dps::kan), respectively, to create
MJF378 (MJF274, rpoS::Tn10) and MJF371
(MJF274, dps::kan) (Table 1).
Exponential- and stationary-phase cells of MJF274, MJF378, and MJF371
were treated with 0.2 mM NEM, and cell viability was determined (Fig.
2a). Loss of either RpoS or Dps increased the sensitivity of both exponential- and stationary-phase E. coli cells to NEM, with a greater effect due to the loss of Dps
than to that of RpoS. Under these conditions, exponential-phase cells of the mutant strains appeared to be slightly more sensitive to NEM
than their stationary-phase counterparts (Fig. 2a). These data provided
evidence that both RpoS and Dps play important roles in the protection
of exponential- and stationary-phase E. coli cells against
NEM. The greater sensitivity of the Dps-deficient mutant than of the
strain lacking RpoS suggested that an RpoS-independent factor could
regulate the level of Dps in both stationary- and exponential-phase
cells.
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FIG. 2.
RpoS and Dps protect exponential- and stationary-phase
cells against NEM. Cells were grown in K10 medium, and cell
viability of exponential (open symbols)- and stationary (closed
symbols)-phase cells was determined exactly as described in Materials
and Methods. (a) NEM was added at 0.2 mM to cells of MJF274 (, ),
MJF378 (rpoS::Tn10; ,  ), and
MJF371 (dps::kan;  ,  ) at time
zero; (b) 0.1 mM NEM was added to cells of MJF381
(rpoS::Tn10,
dps::kan; , ), MJF371 (, ),
and MJF378 (, ) at time zero. The arrows represent no viable
cells by the next time point; error bars represent the standard
deviation from the mean for one experiment.
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To confirm that protection by RpoS and that by Dps could occur
independently, a double mutant lacking both RpoS and Dps was
created by
transduction of strain MJF378 to Kan
r with strain ZK1058 as
the donor to create strain MJF381 (MJF274,
rpoS::Tn
10
dps::
kan; Table
1). Exponential- and
stationary-phase
cells of the double mutant lacking both RpoS and Dps
were highly
sensitive to 0.2 mM NEM and rapidly lost viability (data
not shown).
To monitor the loss of viability of cells of the double
mutant,
the concentration of NEM was reduced to 0.1 mM. At the lower
NEM
concentration (0.1 mM), stationary- and exponential-phase cells
of
the double mutant lacking RpoS and Dps displayed an increased
sensitivity compared with cells of the single mutants lacking
either
RpoS or Dps alone (Fig.
2b). These data demonstrate that
RpoS and Dps
can provide protection to both exponential- and stationary-phase
E. coli cells in the absence of each other.
Exponential-phase
cells of the double mutant lacking RpoS and Dps were
more resistant
to 0.1 mM NEM than stationary-phase cells of this
strain, suggesting
that these systems could have a greater contribution
to survival
against stress in cells of the latter condition (Fig.
2b).
Relationship between the KefB and KefC systems, RpoS, and Dps in
protecting exponential- and stationary-phase cells against NEM.
Having determined a role for RpoS and Dps in the protection of E. coli cells against NEM, we sought to investigate the relationship between these systems and the KefB and KefC potassium channels. Isogenic strains carrying mutations in KefB and KefC and either RpoS or
Dps were created by transduction (Table 1). Stationary-phase cells of
MJF274, MJF358 (MJF274, rpoS::kan),
MJF371 (MJF274, dps::kan), MJF276
(kefB, kefC::Tn10), MJF359
(MJF276, rpoS::kan), and MJF376 (MJF276, dps::kan) were diluted to the
same starting cell density and then exposed to 0.1 mM NEM (Fig.
3a). Cells of the triple mutant lacking
KefB and KefC and either RpoS or Dps were more sensitive to NEM in the
stationary phase of growth than cells lacking either the potassium
channels, RpoS, or Dps alone. This was also the case for
exponential-phase cells of the triple mutants lacking KefB and KefC and
either RpoS or Dps (data not shown). These data provided strong
evidence that the KefB and KefC systems, RpoS, and Dps play important
roles in the protection of both exponential- and stationary-phase cells
against NEM. However, they also suggest that each of these protective
mechanisms can function independently. Cells of the triple mutant
lacking KefB and KefC and Dps were slightly more sensitive to 0.1 mM
NEM than the triple mutant lacking RpoS (Fig. 3a). This was even more
apparent when the NEM concentration was increased to 0.2 mM (data not
shown). These results were consistent with the data for the single
mutants, where Dps appears to play a more dominant role in protection
in the presence of 0.2 mM NEM (Fig. 2a), suggesting that the
contribution of the protective system is determined by the
concentration of NEM in the medium. We also observed that stationary-
and exponential-phase cells of MJF358
(rpoS::kan) were always slightly more
resistant to 0.1 mM NEM than cells of MJF378
(rpoS::Tn10) when the experiments were
conducted on the same days (data not shown). However, the reason(s)
behind this is not known, although the same observation was made when
the role of RpoS in protection against the electrophilic anticancer
drug mechloroethamine was analyzed (12a).
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FIG. 3.
The KefB and KefC systems, RpoS, and Dps can provide
protection against NEM independently. Cells were grown in
K10 medium, and cell viability and Western blot assays were
conducted with stationary-phase cells exactly as described in Materials
and Methods. (a) NEM was added at 0.1 mM to cells of MJF274 (),
MJF276 (kefB kefC::Tn10; ), MJF358
(rpoS::kan;  ), MJF371
(dps::kan; ), MJF359 (kefB
kefC::Tn10 rpoS::kan;
), and MJF376 (kefB kefC::Tn10
dps::kan; ) at time zero, and cell
viability was determined. (b) Western blot analysis using an RpoS
monoclonal antibody. Lane 1, MJF274, no addition; lane 2, MJF274, 0.2 mM NEM (10 min); lane 3, MJF274, 0.2 mM NEM (30 min); lane 4, MJF276,
no addition; lane 5, MJF276, 0.2 mM NEM (10 min); lane 6, MJF276, 0.2 mM NEM (30 min). Lanes 7 to 12 were the same except that sodium acetate
was added to a final concentration of 50 mM instead of NEM. (c) NEM was
added at 0.1 mM to cells of MJF359 (kefB
kefC::Tn10 rpoS::kan;
), MJF413 (kefB kefC::Tn10
dps::cam; ), and MJF411 (kefB
kefC::Tn10 rpoS::kan
dps::cam; ) at time zero, and cell
viability was determined. The arrows represent no viable cells by the
next time point; error bars represent the standard deviation from the
mean for one experiment.
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We have previously shown that activation of the KefB and KefC systems
results in acidification of the cytoplasm and that this
protects
E. coli cells against the toxic effects of electrophiles
such as NEM (
15). It has also been proposed that
acidification
of the cytoplasm by weak acid addition to
E. coli cells induces
RpoS accumulation (
32). To
investigate whether RpoS was induced
by the activation of the KefB and
KefC systems under our experimental
conditions, stationary-phase cells
of MJF274 (KefB
+ KefC
+) and MJF276 (
kefB
kefC::Tn
10) were treated with 0.2 mM NEM, and
the level of RpoS was monitored by Western blotting using an RpoS
monoclonal antibody (Fig.
3b). The amount of RpoS remained constant
over a 30-min incubation in the presence of NEM irrespective of
whether
cells possessed KefB and KefC. This was also found to
be the case for
exponential-phase cells (data not shown). These
data showed that under
our experimental conditions, activation
of the KefB and KefC systems
did not protect by induction of RpoS.
To confirm that the addition of
weak acids could induce RpoS under
our conditions, stationary-phase
cells of MJF274 and MJF276 were
exposed to 50 mM sodium acetate and the
effect on RpoS levels
was detected by Western blotting (Fig.
3b).
Sodium acetate induced
RpoS in both strains within 30 min of addition,
demonstrating
that weak acids can result in RpoS accumulation in our
strains
and growth medium.
The separate nature of the KefB and KefC systems, RpoS, and Dps was
further supported by the creation of a quadruple mutant,
MJF411,
lacking KefB, KefC, RpoS, and Dps. Strain MJF411 was created
by
transduction of strain MJF359 (
kefB
kefC::Tn
10
rpoS::
kan) by
using strain ZK1146
(
dps::
cam) as a donor (Table
1). To
allow
comparison with the appropriate triple mutants, strain MJF276
(
kefB kefC::Tn
10) was also transduced
to Cm
r by using ZK1146 as the donor, to give strain MJF413
(
kefB kefC::Tn
10 dps::
cam; Table
1). Stationary-phase cells of
the quadruple mutant
were found to be highly sensitive to 0.1 mM NEM
compared with
cells of MJF413 and MJF359 (Fig.
3b). This was also found
to be
the case for exponential-phase cells of the quadruple mutant
(data
not shown). These data support the view that the KefB and KefC
systems, RpoS, and Dps are important systems in NEM protection
that can
act independently of each other to protect cells against
stress.
Alterations in the RpoS level of exponential-phase cells affects
NEM sensitivity.
It has been demonstrated previously that RpoS
levels are low in rapidly growing exponential-phase cells due to a
reduction in its half-life compared to stationary-phase cells (23,
24, 33, 37). However, RpoS has been found to accumulate in
exponential-phase cells when the growth rate has been substantially
reduced (17, 23, 28, 30, 34). The data presented so far in
this report clearly show a role for RpoS in protecting
exponential-phase E. coli cells against electrophile damage.
Our data suggested that growth in glucose-replete minimal medium must
pose enough stress to cells to allow RpoS accumulation. To investigate
this further, we analyzed the effect of enriching minimal medium with
CAS on the level of RpoS and NEM sensitivity. Exponential-phase cells of Frag1 (RpoS+) were grown in M9 minimal medium in either
the absence or presence of CAS and diluted to the same cell density in
medium lacking CAS, and viability was determined in the presence of 0.3 mM NEM (Fig. 4a and b; the growth rates
prior to NEM treatment were 0.7 and 1.1 h1,
respectively). Cells of Frag1 grown in the presence of CAS rapidly lost
viability upon exposure to NEM, and by 30 min there were no viable
cells remaining (Fig. 4b). In contrast, there were still 106 cells of Frag1 grown in the absence of CAS remaining
after 30 min of exposure to NEM (Fig. 4a). A low level of RpoS could be detected in cells of Frag1 (Fig. 5a) upon
Western blotting using an RpoS monoclonal antibody; however, due to the
weak signal, no significant difference was observed when cells were
grown in M9 supplemented with or without CAS (data not shown).
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FIG. 4.
RpoS protects rapidly growing exponential-phase cells
against NEM. Exponential-phase cells were grown in M9 medium, and cell
viability was determined exactly as described in Materials and Methods.
(a) NEM was added at 0.3 mM to cells of Frag1 (RpoS+; ).
MJF405 (Frag1, clpP::Tn9; ), and
MJF372 (Frag1, rpoS::Tn10; ) at time
zero. (b) Cells were grown to exponential phase in M9 supplemented with
0.4% (wt/vol) CAS and then treated with 0.3 mM NEM at time zero
(symbols same as panel a except open). The arrows represent no viable
cells by the next time point; error bars represent the standard
deviation from the mean for one experiment.
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FIG. 5.
Effects of growth conditions and strain differences on
RpoS levels. Exponential-phase cells were grown in M9 medium with the
defined supplements, and the Western blot analyses were conducted
exactly as described in Materials and Methods (where stated, CAS was
added to 0.4% [wt/vol]). (a) Lane 1, Frag1; lane 2, MSD462 (exposure
time, 10 times longer than in panel b). (b) Lane 1, Frag1; lane 2, MJF405 (Frag1, clpP::Tn9) with CAS;
lane 3, MJF405; lane 4, MSD462 with CAS; lane 5, MSD462; lane 6, MSD462
with bases (adenine, cytosine, guanine, thymine and uracil; 50 µg.ml1) and CAS; lane 7, MJF385 (MSD462,
clpP::Tn9) with CAS; lane 8, MJF385;
lane 9, stationary-phase MSD462 (positive control); lane 11, stationary-phase MJF402 (MSD462,
rpoS::Tn10; negative control).
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To confirm that the difference in sensitivity to NEM between Frag1
growing in the presence or absence of CAS was due to changes
in RpoS,
strain Frag1 was transduced to Tet
r by using strain RH90
(
rpoS::Tn
10) as the donor to create
strain
MJF372 (Frag1,
rpoS::Tn
10)
(Table
1). Cells of MJF372 were grown
to exponential phase in M9 in
either the absence or presence of
CAS and then diluted into medium
without CAS and exposed to 0.3
mM NEM (Fig.
4a and b; the growth rates
of the RpoS mutant were
unaltered compared with the parent strain under
the same conditions,
1.1 and 0.7 h
1, respectively). Cells
lacking RpoS were highly sensitive to NEM
compared with cells of the
parent strain (Fig.
4b). However, the
addition of CAS did not increase
the sensitivity of the RpoS-deficient
cells to NEM but instead slightly
enhanced survival compared to
cells of the same strain grown at the
slower growth rate (Fig.
4). These data confirmed the importance of
RpoS in protecting
relatively rapidly growing (growth rates of 0.7 and
1.1 h
1) exponential-phase cells of
E. coli
against NEM and also supported
the view that changes in RpoS levels are
responsible for the large
increase in NEM sensitivity caused by
supplementing the growth
medium with CAS.
It has been shown previously that the ClpP protease is responsible for
maintaining low levels of RpoS in rapidly growing exponential-phase
cells (
33). The importance of RpoS in the protection of
exponential-phase
cells against NEM was also further supported by the
creation of
a ClpP-deficient mutant of Frag1 by transduction (Table
1).
Exponential-phase
cells of MJF405 (Frag1,
clpP::Tn
9), which lacked the ability to
produce the ClpP protease, accumulated high levels of RpoS (Fig.
5b)
and were highly resistant to NEM (Fig.
4). However, increasing
the
growth rate of MJF405 from 0.7 to 1.0 h
1 by the addition
of CAS slightly decreased the level of resistance
to NEM (Fig.
4),
although no difference in the RpoS level was
detected (Fig.
5b). These
data suggested that in this ClpP-deficient
strain, an RpoS-independent
factor can have an effect on NEM sensitivity.
To investigate further the link between RpoS and protection against
NEM, we sought to analyze cells of another
E. coli strain,
MSD462 (RpoS
+). The growth rate of cells of MSD462 in
minimal medium is substantially
lower and the level of RpoS is
significantly higher than that
of cells of Frag1 (Table
1 and Fig.
5,
respectively). As predicted
from the high RpoS levels, cells of MSD462
were more resistant
to NEM than cells of Frag1 under the same growth
conditions (Table
2). In addition, the
RpoS levels of cells of MSD462 were virtually
the same whether cells
had been grown in the presence or absence
of CAS (Fig.
5b), and this
correlated with the very similar NEM
sensitivity data (Table
2).
However, growth of MSD462 cells in
medium enriched further by the
addition of both CAS and bases
significantly reduced RpoS levels (Fig.
5b) and drastically increased
sensitivity to NEM (Table
2). As observed
with the RpoS
and ClpP-deficient mutants of Frag1, cells
of MJF402 (MSD462,
rpoS::Tn
10) had an
increased sensitivity to NEM, whereas cells
of MJF385 (MSD462,
clpP::Tn
9) accumulated RpoS (Fig.
5b)
and gained
resistance to NEM compared with cells of the parent strain
(Table
2). These findings confirmed the interrelationship of RpoS
accumulation
and sensitivity to NEM in exponentially growing cells.
| |
DISCUSSION |
The data presented here demonstrate that stationary-phase E. coli cells are more resistant than exponential-phase cells to electrophiles. Resistance to NEM was found to be dependent on the KefB
and KefC potassium channels, RpoS, and Dps. All four systems were
required for protection against stress both in actively growing and in
stationary-phase cells. By the analysis of single, double, triple, and
quadruple mutants, we provide strong evidence that the individual
protective mechanisms can function in the absence of each other.
However, it is likely that these systems are not completely independent
and that Dps is still responsible for some of the RpoS-regulated
protection, since the expression of Dps is known to be affected by RpoS
(3, 17). The greater sensitivity of the Dps mutant than of
the RpoS-deficient strains in the presence of 0.2 mM NEM suggests the
strategic importance of the Dps protein for cell survival. NEM is
thought to damage bacterial cells by interacting with the nucleophilic
centers of macromolecules such as DNA, and hence protection by Dps is
likely to involve binding to the DNA to shield it from NEM attack
(2, 26). However, it is also possible that Dps is an
activator of gene expression and is responsible for the induction of
other proteins involved in protection against NEM (2). The
finding that Dps can still provide some protection to cells against NEM even in the absence of RpoS in both stationary- and exponential-phase cells provides evidence that some other factor(s) in the cell must be
able to regulate dps expression. It has been shown
previously that the dps promoter can be activated by OxyR in
growing cells and by integration host factor in stationary-phase cells
(3). Recently, it has been demonstrated that OxyR is
produced in exponential-phase cells of E. coli during
aerobic growth (16). Consistent with this finding,
exponential-phase cells of an OxyR-deficient mutant of Salmonella
typhimurium were found to be highly sensitive to NEM, and an
OxyR(Con) mutant that expresses the OxyR regulon constitutively was
highly resistant to NEM (7). It is also possible that OxyR regulates Dps in RpoS-deficient stationary-phase cells, since it has
been demonstrated that in the absence of RpoS, OxyR accumulates in
E. coli cells in this phase of growth (16).
RpoS is clearly important for survival of E. coli cells upon
exposure to NEM. While the role of RpoS in the expression of Dps can in
part account for this, other gene products of the RpoS regulon must
also be important since protection is observed even in the absence of
Dps. Three candidates known to be regulated by RpoS are the DNA repair
proteins Ada, AidB, and exonuclease III (22, 31, 35, 38,
40). Preliminary data suggest that exonuclease III could play a
role, since mutants unable to make this protein were sensitive to both
NEM and another electrophile, methylglyoxal (15a). The role
of Ada is less clear, since we found that Ada was not induced by NEM
but preinduction of this system by RpoS could protect cells against NEM
(15a, 38). Alternatively, NEM may interact with the cell to
produce oxidative damage, and therefore the RpoS-regulated
katE gene, encoding hydroperoxidase HPII, may play an
important role (31). RpoS has also been found to regulate
the accumulation of certain fatty acids, and hence membrane
permeability could be altered by the presence or absence of RpoS
(41). However, RpoS was not found to influence the entry of
NEM into E. coli cells (26a).
The finding that cells of a triple mutant lacking both KefB and KefC
and either RpoS or Dps exhibited an enhanced sensitivity, and cells of
the quadruple mutant lacking all four systems displayed an even greater
sensitivity to NEM, provided evidence for the separate nature of these
protective mechanisms. The activation of KefB and KefC results in
acidification of the cytoplasm, and it has been demonstrated previously
that RpoS is induced by the addition of weak acids that lower the
intracellular pH (32). However, we have shown that
protection by KefB and KefC can occur in the absence of RpoS,
suggesting that if RpoS is induced by the activation of these channels,
it is not responsible for all of the KefB-KefC-mediated protection. We
also found that under the conditions of our experiments, RpoS did not
accumulate after activation of the KefB and KefC systems by NEM,
although induction of RpoS did occur after weak acid addition. These
data provide evidence that the protection by the KefB and KefC systems
does not result from an increase in the level of RpoS.
The data presented in this report also highlight the fact that clearly
targeting one or more of these stress protective mechanisms would
create a very effective antibacterial strategy. It would also be
difficult for bacterial cells to develop resistance, since these
mechanisms can act independently of each other. The KefB-KefC-like systems have been detected in all gram-negative bacteria tested to
date, and Dps homologs have also been detected in both gram-negative and gram-positive bacteria (2, 6, 8). Although RpoS homologs have not been identified in gram-positive bacteria, changes associated with stationary phase have been observed. For example, in cells of the
human pathogen Staphylococcus aureus, an alternative sigma factor, 
B, is believed to be regulated in response to
growth phase, and in cells of Bacillus subtilis, a similar
sigma factor is responsible for the expression of a wide array of
stress genes (21). It has been demonstrated by other workers
that in S. typhimurium, RpoS regulates virulence genes and
mutations in RpoS lead to avirulence (11, 20). From our
experiments, interference with both RpoS and either the potassium
channels or Dps would greatly sensitize bacteria to the environment of
the host and lead not only to the inability to cause disease but also
to bacterial cell death.
It had been shown previously that RpoS plays an important role in
protecting stationary-phase cells against certain types of stress
(17, 19). RpoS has also been found to play a role in
exponential-phase cells where the growth rate is substantially reduced
(17, 23, 28, 30, 34). The data presented here point to the
importance of RpoS in relatively rapidly growing exponential-phase
cells (growth rate ranging from 0.5 to 1.1 h1) compared
to previous studies where the growth rates were much lower. Even in
cells of Frag1 where the RpoS level is barely detectable by Western
blot analysis, deletion of RpoS dramatically sensitizes cells to NEM.
These data demonstrate that low levels of RpoS that accumulate in
rapidly growing exponential-phase cells can have a profound effect on
cell survival in the presence of stress. Thus, for many studies in the
laboratory that use growing cells, and in the environment where cells
achieve submaximum growth rates, RpoS will be a major determinant of
the survival potential of the cell. In conclusion, these data
demonstrate the complexity of protective mechanisms against the
electrophile NEM in both actively growing and stationary-phase E. coli cells. The importance of these systems in both phases of
growth for survival against stress suggests that one or more of these
mechanisms could be targeted in the design of novel antibiotics.
| |
ACKNOWLEDGMENTS |
G.P.F. is a Wellcome Trust Toxicology Fellow, Y.N. was supported
by a Wellcome Trust Visiting Fellowship, R.I.C. is supported by the
BBSRC, I.R.B. is a Wellcome Trust Research Leave Fellow, and the group
is supported by a Wellcome Trust Programme grant.
We acknowledge Neil Hunter for preliminary experiments on
stationary-phase resistance and thank Karen Sutherland and Vanessa Santana for technical assistance. Thanks also go to Susan Gottesman, Regina Hengge-Aronis, Roberto Kolter, and Asuncion Martinez for the
provision of donor strains and to Nancy Thompson for the RpoS monoclonal antibody.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom. Phone:
44 1224 273151. Fax: 44 1224 273144. E-mail:
g.p.ferguson{at}abdn.ac.uk.
| |
REFERENCES |
| 1.
|
Ackerman, R. S.,
N. R. Cozzarelli, and W. Epstein.
1974.
Accumulation of toxic concentrations of methylglyoxal by wild-type Escherichia coli K-12.
J. Bacteriol.
119:357-362[Abstract/Free Full Text].
|
| 2.
|
Almirón, M.,
A. J. Link,
D. Furlong, and R. Kolter.
1992.
A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli cells.
Genes Dev.
6:2646-2654[Abstract/Free Full Text].
|
| 3.
|
Altuvia, S.,
M. Almiron,
G. Huisman,
R. Kolter, and G. Storz.
1994.
The dps promoter is activated by OxyR during growth and by IHF and s in stationary phase.
Mol. Microbiol.
13:265-272[Medline].
|
| 4.
|
Apontoweil, P., and W. Berends.
1975.
Isolation and characterization of glutathione-deficient mutants of Escherichia coli K12.
Biochim. Biophys. Acta
399:10-22[Medline].
|
| 5.
|
Bakker, E. P., and W. E. Mangerich.
1982.
N-ethylmaleimide induces K+-H+ antiport activity in Escherichia coli K-12.
FEBS Lett.
140:177-180[Medline].
|
| 6.
|
Chen, L., and J. D. Helmann.
1995.
Bacillus subtilis MrgA is a Dps (PexB) homologue: evidence for metalloregulation of an oxidative-stress gene.
Mol. Microbiol.
18:295-300[Medline].
|
| 7.
|
Christman, M. F.,
R. W. Morgan,
F. S. Jacobson, and B. N. Ames.
1985.
Positive control of a regulon for defences against oxidative stress and some heat-shock proteins in Salmonella typhimurium.
Cell
41:753-762[Medline].
|
| 8.
|
Douglas, R. M.,
J. A. Roberts,
A. W. Munro,
G. Y. Richie,
A. J. Lamb, and I. R. Booth.
1991.
The distribution of homologues of the Escherichia coli KefC K+-efflux system in other bacterial species.
J. Gen. Microbiol.
137:1999-2005[Abstract/Free Full Text].
|
| 9.
|
Elmore, M. J.,
A. J. Lamb,
G. Y. Ritchie,
R. M. Douglas,
A. Munro,
A. Gajewska, and I. R. Booth.
1990.
Activation of potassium efflux from Escherichia coli by glutathione metabolites.
Mol. Microbiol.
4:405-412[Medline].
|
| 10.
|
Epstein, W., and B. S. Kim.
1971.
Potassium transport loci in Escherichia coli K-12.
J. Bacteriol.
108:639-644[Abstract/Free Full Text].
|
| 10a.
| Evans, G., G. P. Ferguson, and I. R. Booth. Unpublished data.
|
| 11.
|
Fang, C. F.,
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].
|
| 12.
|
Ferguson, G. P.,
A. D. Chacko,
C. Lee, and I. R. Booth.
1996.
The activity of the high-affinity potassium uptake system Kdp sensitizes cells of Escherichia coli towards methylglyoxal.
J. Bacteriol.
178:3957-3961[Abstract/Free Full Text].
|
| 12a.
| Ferguson, G. P., A. Larrson, and B. Mannervik.
Unpublished data.
|
| 13.
|
Ferguson, G. P.,
D. Mclaggan, and I. R. Booth.
1995.
Potassium channel activation by glutathione-S-conjugates in Escherichia coli: protection against methylglyoxal is mediated by cytoplasmic acidification.
Mol. Microbiol.
17:1025-1033[Medline].
|
| 14.
|
Ferguson, G. P.,
A. W. Munro,
R. M. Douglas,
D. Mclaggan, and I. R. Booth.
1993.
Activation of potassium channels during metabolite detoxification in Escherichia coli.
Mol. Microbiol.
9:1297-1303[Medline].
|
| 15.
|
Ferguson, G. P.,
Y. Nikolaev,
D. Mclaggan,
M. Maclean, and I. R. Booth.
1997.
Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels.
J. Bacteriol.
179:1007-1012[Abstract/Free Full Text].
|
| 15a.
| Ferguson, G. P., Y. Nikolaev, B. Sedgewick, and
I. R. Booth. Unpublished data.
|
| 16.
|
Gonzalez-Flencha, B., and B. Demple.
1997.
Transcriptional regulation of the Escherichia coli oxyR gene as a function of cell growth.
J. Bacteriol.
179:6181-6186[Abstract/Free Full Text].
|
| 17.
|
Hengge-Aronis, R.
1993.
Survival of hunger and stress: the role of RpoS in early stationary phase gene regulation in E. coli.
Cell
72:165-168[Medline].
|
| 18.
|
Hopper, D. J., and R. A. Cooper.
1971.
The regulation of Escherichia coli methylglyoxal synthase: a new control site in glycolysis?
FEBS Lett.
13:213-216[Medline].
|
| 19.
|
Jenkins, D. E.,
J. E. Schultz, and A. Matin.
1988.
Starvation induced cross protection against heat or H2O2 challenge in Escherichia coli.
J. Bacteriol.
170:3910-3914[Abstract/Free Full Text].
|
| 20.
|
Kowarz, L.,
C. Coynault,
V. Robbe-Saule, and F. Norel.
1994.
The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.
J. Bacteriol.
176:6852-6860[Abstract/Free Full Text].
|
| 21.
|
Kullik, I., and P. Gianchino.
1997.
The alternative sigma factor B in Staphylococcus aureus: regulation of the sigB operon in response to growth phase and heat shock.
Arch. Microbiol.
167:151-159[Medline].
|
| 22.
|
Landini, P.,
L. I. Hajec, and M. R. Volkert.
1994.
Structure and transcriptional regulation of Escherichia coli adaptive response gene aidB.
J. Bacteriol.
176:6583-6589[Abstract/Free Full Text].
|
| 23.
|
Lange, R., and R. Hengge-Aronis.
1991.
Identification of a central regulator of stationary-phase gene expression in Escherichia coli.
Mol. Microbiol.
5:49-59[Medline].
|
| 24.
|
Lange, R., and R. Hengee-Aronis.
1994.
The cellular concentration of the S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability.
Genes Dev.
8:1600-1612[Abstract/Free Full Text].
|
| 25.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurements with Folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 26.
|
Martinez, A., and R. Kolter.
1997.
Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.
J. Bacteriol.
179:5188-5194[Abstract/Free Full Text].
|
| 26a.
| Mclaggan, D., and I. R. Booth. Unpublished
data.
|
| 27.
|
Meury, J., and A. Kepes.
1982.
Glutathione and the gated potassium channels of E. coli.
EMBO J.
1:339-343[Medline].
|
| 28.
|
Muffler, A.,
D. D. Traulsen,
R. Lange, and R. Hengge-Aronis.
1996.
Posttranscriptional osmotic regulation of the S subunit of RNA polymerase in Escherichia coli.
J. Bacteriol.
178:1607-1613[Abstract/Free Full Text].
|
| 29.
|
Nagoa, M.,
Y. Fujita, and T. Sugimura.
1984.
.
Methylglyoxal in beverages and foods: its mutagenicity and carcinogenicity.
Presented at the IARC Meeting, Lyon, France.
|
| 30.
|
Notely, L., and T. Ferenci.
1996.
Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli?
J. Bacteriol.
178:1465-1468[Abstract/Free Full Text].
|
| 31.
|
Sak, B. D.,
A. Eisenstark, and C. Touati.
1989.
Exonuclease III and the catalase hydroperoxidase II in E. coli are both regulated by the katF gene product.
Proc. Natl. Acad. Sci. USA
86:3271-3275[Abstract/Free Full Text].
|
| 32.
|
Schellhorn, H. E., and V. L. Stones.
1992.
Regulation of katF and katE in Escherichia coli K-12 by weak acids.
J. Bacteriol.
174:4769-4776[Abstract/Free Full Text].
|
| 33.
|
Scheweder, T.,
K. Lee,
O. Lomovskaya, and A. Matin.
1996.
Regulation of Escherichia coli starvation sigma factor (s) by ClpXP protease.
J. Bacteriol.
178:470-476[Abstract/Free Full Text].
|
| 34.
|
Sledjeski, D. D.,
A. Gupta, and S. Gottesman.
1996.
The small RNA, DsrA, is essential for the low-temperature expression of RpoS during exponential growth in Escherichia coli.
EMBO J.
15:3993-4000[Medline].
|
| 35.
|
Smirnova, G. V.,
O. N. Oktyabrsky,
E. V. Moshonkina, and N. V. Zakirova.
1994.
Induction of the alkylation-inducible aidB gene of Escherichia coli by cytoplasmic acidification and N-ethylmaleimide.
Mutat. Res.
314:51-56[Medline].
|
| 36.
|
Stevens, K. L.,
R. E. Wilson, and M. Friedman.
1995.
Inactivation of a tetrachloride mutagen from stimulated processing water.
J. Agric. Food Chem.
43:2424-2427.
|
| 37.
|
Takayanagi, Y.,
K. Tanaka, and H. Takahashi.
1994.
Structure of the 5' upstream region and the regulation of the rpoS gene of Escherichia coli.
Mol. Gen. Genet.
243:525-531[Medline].
|
| 38.
|
Taverna, P., and B. Sedgwick.
1996.
Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli.
J. Bacteriol.
178:5105-5111[Abstract/Free Full Text].
|
| 39.
|
Towbin, H.,
T. Staehelin, and G. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 40.
|
Volkert, M. R.,
L. I. Hajec,
Z. Matijasevic,
F. C. Fang, and R. Prince.
1994.
Induction of the Escherichia coli aidB gene under oxygen limited conditions requires a functional rpoS (katF) gene.
J. Bacteriol.
176:7638-7645[Abstract/Free Full Text].
|
| 41.
|
Wang, A. Y., and J. E. Cronan.
1994.
The growth-phase dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS(KatF)-dependent promoter plus enzyme stability.
Mol. Microbiol.
11:1009-1017[Medline].
|
| 42.
|
Zablotowicz, R. M.,
R. E. Hoagland,
M. A. Locke, and W. J. Hickey.
1995.
Glutathione S-transferase and metabolism of glutathione conjugates by rhizosphere bacteria.
Appl. Environ. Microbiol.
61:1054-1060[Abstract].
|
J Bacteriol, March 1998, p. 1030-1036, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
