From the Department of Cell and Molecular Biology-Microbiology, Göteborg University, Box 462, 405 30 Göteborg, Sweden
Received for publication, September 26, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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When Escherichia coli cells enter
stationary phase due to carbon starvation the synthesis of ribosomal
proteins is rapidly repressed. In a The alarmone guanosine tetraphosphate
(ppGpp)1 of the stringent
response network in Escherichia coli affects ribosome
production by specifically lowering the transcription of ribosomal RNA
(rRNA) (1) and some of the genes encoding ribosomal proteins (2-4). The production of ribosomal proteins is also post-transcriptionally feedback-regulated to match the rRNA production (reviewed in 5). Two
different ppGpp synthetases (PS) exist in E. coli, the
ribosome-associated PS I, encoded by relA, and the
cytoplasmic PS II (6), encoded by spoT (7, 8). The protein
PS II is also responsible for ppGpp hydrolysis (1).
Increased levels of ppGpp not only down-regulate ribosome
production but also induce transcription from several promoters (9-14). As suggested by Schreiber et al. (9), the E. coli promoters can be divided into three groups depending on their
response to ppGpp: promoters specifically induced or repressed by the
stringent response and promoters that are unaffected by the rise in
ppGpp levels. Promoters dependent on the housekeeping Whether or not RNAP availability is involved in the actual mechanism of
the stringent response has been discussed, and several models for this
have been suggested (13-16, 20). Arguments have been raised both for
an increased and diminished availability of RNAP during entry into
stationary phase. In addition, different models exist on how changes in
the availability of RNAP would affect the stringent promoters. Barker
et al. (13, 14) and Zhou and Jin (15) suggest an increased
availability of free RNAP in stationary phase since ppGpp destabilizes
the open complex resulting in RNAP drop off at stable RNA promoters,
which form intrinsically unstable open complexes. As a consequence of
the increased availability of RNAP, positively regulated promoters are
then induced since they are relatively poor at recruiting RNAP and are
subsaturated during normal growth according to this model. Thus, this
model encompasses a direct and active role for ppGpp on stringently
controlled promoters (e.g. rrn) and a passive role on the positively regulated promoters (through RNAP availability). Jensen and Pedersen (16), on the other hand, have argued for a
diminished availability of RNAP during stringency. The "stringent" promoters, e.g. stable RNA promoters, are dependent on high
concentration of RNAP to transcribe at their maximal rate and are
therefore argued to be repressed when the RNAP availability is
diminished. Krohn and Wagner (21) showed that ppGpp increases pausing
of RNAP during transcription in general but more at the stringently controlled genes, which could be another reason for inhibition of
expression of these genes. Jensen and Pedersen (16) suggested that one
way through which the RNAP availability could be diminished during
stringency is through ppGpp-dependent pausing and therefore sequestering of RNAP in transcription. However, Vogel and Jensen (22)
have demonstrated that ppGpp-induced pausing is not required for the
stringent response since the stringent response was still observed when
ppGpp-induced pausing was abolished and the transcriptional elongation
speed was made constant by the introduction of the nusAcs10
allele. Thus, it is presently unclear if and how the availability of
RNAP changes during the stringent response.
There are also models for how the stringent response works that do not
involve RNAP availability. For example Barrachini and Bremer (23)
suggested that RNAP can exist in two forms, one with and one without
ppGpp bound to it. The RNAP without ppGpp can only transcribe genes
encoding stable RNA, while RNAP with ppGpp bound can transcribe from
both stable RNA and mRNA promoters but prefers mRNA promoters.
The fact that cells totally deficient in making ppGpp can still grow
shows that there is no absolute requirement for ppGpp for the
transcription of mRNA promoters, but no evidence presented so far
discredits the idea that there are direct effects of ppGpp on promoter selection.
The questions that need to be solved are whether variations in the
level of free RNAP play a physiologically relevant role in gene
regulation and if this mechanism is involved in the stringent response.
The specific question we set out to answer in this work was whether
variation in the levels of free Bacterial Strains and Growth Conditions--
The E. coli strains used in this work are listed in Table
I. The markerless
Cultures were grown in liquid M9-defined medium (27) aerobically in
Erlenmeyer flasks in a rotary shaker at 37 °C. The M9 medium was
supplemented with a limiting concentration of glucose (0.08%), all the
amino acids in excess (28) and thiamine (10 mM). IAA (0.2 or 0.02 mM) and chloramphenicol (30 µg/ml) were added
when appropriate.
Resolution of Proteins by Two-dimensional Polyacrylamide Gel
Electrophoresis--
After growth for at least five generations,
samples were taken in exponential phase, and 1 ml of the culture was
mixed with 5 µl of [35S]methionine (10 mCi/ml, 1000 Ci/mmol, Amersham Biosciences) for pulse labeling. Incorporation
was allowed to proceed for 5 min at 37 °C, then non-radioactive
methionine (50 µl, 0.2 M) was added and a chase was
allowed for 3 min. The samples were processed for resolution on
two-dimensional polyacrylamide gels according to O'Farrell (29) with
modifications (30). Electrophoresis in the first dimension was carried
out between pH 3.5 and pH 10, and the gels were run according to the
NEPHGE protocol (Non Equilibrium pH Gel Electrophoresis, (31, 32)). The
NEPHGE protocol was used since it results in two-dimensional gels where
the ribosomal proteins (most with very basic pI) are visible. The
second dimension was run on 11.5% polyacrylamide gels. Radiolabeled
proteins were detected in a phosphorimaging device (Personal FX,
Bio-Rad), and the images were analyzed using the PDQuest 6.2 software
(Bio-Rad). Alphanumeric designations and/or protein names were assigned
to protein spots after matching them to the reference two-dimensional images of the gene-protein data base of E. coli (33) or
after performance of mass spectrometry.
Measurement of Cellular Components--
Western blot analysis
was performed as described (20). Cell sampling and measurement of the
cellular level of ppGpp with high pressure liquid chromatography
was done according to Neubauer et al. (34). For RNA and
protein measurements, bacteria were first incubated in 5%
trichloroacetic acid on ice and subsequently washed once with
5% trichloroacetic acid and lysed with 1 M NaOH (30 min at
40 °C). Protein content in the cell extracts was analyzed using the
BCA protein assay kit (Pierce). RNA concentration was measured using
the UV absorption assay as described (35). Based on the values obtained
(normalized to optical density of the culture at the time of sampling),
the RNA/protein ratio was calculated for cells underproducing
A ppGpp0 Mutant Fails to Respond to Carbon
Starvation--
A proteomic analysis demonstrated that the ribosomal
proteins are immediately down-regulated 4- to 64-fold when wild type cells enter stationary phase due to carbon starvation (Fig.
1, B and D). The
fold reduction in the synthesis rate is different for each of the
ribosomal proteins but eventually the production of all the ribosomal
proteins falls below the detection limits (data not shown). As
demonstrated previously a set of proteins is induced during entry into
stationary phase (Fig. 2A).
Among those are a set of
Apart from differences between the rate of production of specific
proteins in the wild type and relaxed strain, it is clear that the
magnitude of the proteome response is much reduced in the
The ppGpp levels were not affected by the
Lowering the levels of E In this work we demonstrate that a strain unable to produce ppGpp
is largely decontrolled upon the entry to stationary phase and its
proteome appears "locked" in a growth mode. By comparing the
effects of a wild type and a ppGpp0 strain going into
stationary phase (Fig. 1) we can define the significant changes in the
proteome mediated by the stringent response under these conditions. The
ribosomal proteins are, as expected, down-regulated by the entry into
stationary phase, and this effect is dependent on ppGpp (Fig. 1,
C and D). Some proteins, e.g. UspA,
are induced in a ppGpp-dependent manner as has been shown
previously (10, 11). A few amino acid biosynthesis proteins (e.g. the proteins encoded by carB,
gltD, ilvE, metC, metE,
metH, and tyrB) are identified on NEPHGE
two-dimensional gels (33), but those were not among the proteins
significantly affected by the stringent response to carbon starvation.
In a recently published study using macroarrays, the only genes
encoding amino acid biosynthesis proteins that were induced by the
stringent response were the ones involved in the histidine and arginine
biosynthetic pathways (37). Thus, it appears that promoters requiring
ppGpp do not necessarily behave identically during a stringent response
as demonstrated by the fact that the production of amino acid
biosynthetic gene products, in contrast to the Usp family proteins, are
not induced during carbon starvation induced stringency.
We also show that it is possible to mimic the down-regulation of
ribosomal proteins seen in the stringent response by underproducing the
housekeeping Our results of The fact that lowering the availability of Lowering the levels of In this article we show that the ppGpp0 mutant
appears locked in a growth state even after entering stationary phase
due to carbon starvation. We also show that it is possible to mimic a stringent down-regulation of ribosome production by underproducing relA
spoT mutant, defective in the production of the alarmone
guanosine tetraphosphate (ppGpp), this regulation of the levels of the
protein synthesizing system is abolished. Using a proteomic approach we
demonstrate that the production of the vast majority of detected
E. coli proteins are decontrolled during carbon starvation
in the
relA
spoT strain and that the starved cells behave as if they were growing exponentially. In addition
we show that the inhibition of ribosome synthesis by the stringent
response can be qualitatively mimicked by artificially lowering the
levels of the housekeeping
factor,
70. In other
words, genes encoding the protein-synthesizing system are especially
sensitive to reduced availability of
70 programmed RNA
polymerase. This effect is not dependent on ppGpp since lowering the
levels of
70 gives a similar but less pronounced effect
in a ppGpp0 strain. The data is discussed in view of the
models advocating for a passive control of gene expression during
stringency based on alterations in RNA polymerase availability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor,
70, exist in at least two of these groups; one group is
positively regulated by ppGpp, e.g. PuspA (11)
and Phis (8), whereas the other group is repressed during
stringency, e.g. rrnP1. Attempts to explain this
dual effect of ppGpp have involved considerations of RNAP (RNA
polymerase) availability and intrinsic differences in the kinetic
properties of the promoters affected (15). Several lines of evidence
suggest that the availability of transcriptional and/or translational
machinery play a role in global gene regulation in concert with
classical activators and repressors (13-17). One well known example of
this type of regulation, where the level of RNAP is involved, is
factor competition in both Bacillus subtilis (18) and
E. coli (19, 20).
70-programmed RNAP does
affect gene expression and, if so, what genes are sensitive to the
altered RNAP availability? We used two-dimensional gel analysis to
elucidate the global effects of underproduction of the housekeeping
factor,
70, during exponential growth and to study the
role of ppGpp in this effect. We found that lowering the amounts of
70-programmed RNAP mimics the proteome of stringent
cells and that this effect is not ppGpp-dependent. We
speculate on the physiological relevance of these results with respect
to the availability of RNAP during the stringent response.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
relA
spoT strain was constructed by K. Kvint and co-workers
(20) by the method of Datsenko and Wanner (24).
70
levels were manipulated by using a system where
70
(RpoD) is under control of the trp-promoter,
Ptrp, (25). Levels of
70 were
controlled with IAA (Indole-3-acrylic acid, an antagonist of the Trp
repressor). The (CamR) Ptrp-rpoD allele was
transduced into the different strains by standard P1 transduction (26). The IAA concentration giving wild type levels of
70 was
determined previously (0.2 mM IAA, (20)), and a 2-fold underproduction of
70 was accomplished by diluting to a
final concentration of 0.02 mM IAA.
E. coli strains
70 and compared with the control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70-dependent
proteins, e.g. UspA and GlyA (Fig. 1D). In a
ppGpp0 strain the ribosomal proteins are not down-regulated
when the cells enter stationary phase (Fig. 1E), and the
protein production pattern, as seen on the two-dimensional gels, looks
rather like a growing cell even after the entry into stationary phase
(Fig. 1C). It should be noted that
relA+ and relA1 strains behave
indistinguishably during the conditions analyzed. The SpoT protein
(PS II) is responsible for ppGpp production during the glucose
starvation conditions employed (8), and the relA1 allele did
not affect the proteome during these conditions.
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Fig. 1.
Effects of carbon starvation in a wild type
strain and in a ppGpp0 strain. Cells were
grown into starvation in defined media, and samples were labeled in
exponential phase (A420 = 0.5) and stationary
phase (20 min after transition). A, location on the NEPHGE
reference two-dimensional gel of the proteins analyzed in this work.
The production of all these proteins depends on the housekeeping factor,
70. B, two-dimensional gels of
proteins produced in the wild type strain during growth
(Exp.) and in stationary phase (Stat.).
C, two-dimensional gels of proteins produced in the
ppGpp0 strain during growth (Exp.) and in
stationary phase (Stat.). D, the effects on a
subset of proteins in response to carbon starvation in a wild type
strain. The dark-gray bars indicate the ribosomal proteins, and
the light-gray bars indicate a set of proteins that are
non-ribosomal but under control of
70. The
y-axis shows the fold change in a 2Log scale
which means that 1 is a 2-fold induction, while
1 is a 2-fold
repression of the production of a protein. The fold change is
calculated by dividing the quantity of the specific protein produced
during 5 min in stationary phase (normalized to total protein
production) by the quantity of the specific protein produced during 5 min in exponential phase (normalized to total protein production).
E, the effect on the proteins shown in C) in a
ppGpp0 strain entering stationary phase.
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Fig. 2.
The global effects on protein production by
entering stationary phase in a wild type strain (A)
and in a ppGpp0 strain
(B). The graph was made the same way as in Fig.
1. The set of proteins in A and B were sorted
depending on the magnitude of the response not on the protein identity.
Therefore the proteins are not necessarily in the same order in
A and B.
relA
spoT mutant strain (Figs. 1 and 2).
The proteome of the
relA
spoT mutant strain
appears to be locked in a growth mode.
70 Underproduction Qualitatively Mimics a Stringent
Response--
When
70 was underproduced in a wild type
strain the ribosomal proteins were found to be specifically affected.
When the
70 levels were lowered to half the level of a
normal strain, the ribosomal proteins were down-regulated 2- to 3-fold
(Fig. 3A). This effect was not
as great as the effect of carbon starvation, but this was expected
since the cells are still growing in the
70
underproduction experiment. The effect of
70
underproduction on ribosomal proteins has been confirmed on the transcriptional level by the use of
macroarrays.2
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Fig. 3.
Effects of 70
underproduction in growing wild type cells. A,
the effects of
70 underproduction on the subset of
proteins seen in Fig. 1C. B, measurement of ppGpp
levels during growth with normal levels of
70 (0.2 mM IAA) and lower levels of
70 (0.02 mM IAA). C, total RNA/total protein during
growth with normal levels of
70 (0.2 mM IAA)
and lower levels of
70 (0.02 mM IAA)
expressed relative to the control (0.2 mM IAA).
70
underproduction (Fig. 3B), which means that the
down-regulation of ribosomal protein production is not caused by
elevated levels of ppGpp in this experiment. In agreement with the
effect on ribosomal proteins the RNA/protein ratio was reduced (Fig.
3C). This measurement can be used as a rough measure of
stable RNA since total RNA consists of 98% stable RNA (36). In
addition, the levels of RNAP
-subunit were not affected by the
reduced
70 levels (data not shown).
70 also affected the growth rate
(Fig. 4A), as has previously
been shown for ppGpp overproduction (9, 10). In the case of
70 underproduction no steady state of growth could be
obtained, but rather the growth rate decreased gradually over time
(Fig. 4A). The
70 underproduction levels were
therefore checked at later times in exponential phase and showed that
the level of underproduction did not change within the range of the
experiment (data not shown). The effects of
70
underproduction were studied at two different absorbance values during growth (A420 = 0.5 and 0.7), and no
significant differences in the protein expression between the two
absorbance values were found (data not shown). Finally, the
70 underproduction was performed in both a
relA1 and a relA+ strain with the
same result (data not shown).
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Fig. 4.
A, effects of 70
underproduction on the growth rate in a wild type strain. Growth with
normal levels of
70 (0.2 mM IAA) is depicted
with filled circles and lower levels of
70
(0.02 mM IAA) with open circles. The
arrow indicates the point of labeling for Fig. 3.
B, effects of
70 underproduction on the
growth rate in a ppGpp0 strain. Growth with normal levels
of
70 (0.2 mM IAA) is depicted with
filled squares, and lower levels of
70 (0.02 mM IAA) with open squares. The arrow
indicates the point of labeling for Fig. 5.
70 Underproduction in a ppGpp0
Gives Repression of Stringently Controlled Proteins--
The specific
effect on ribosomal proteins is not ppGpp-dependent since
the down-regulation of ribosomal proteins was obtained also during
70 underproduction in a ppGpp0 strain (Fig.
5A). The global effects of
70 underproduction were less pronounced, but the same
trend was seen as in the wild type strain (comparison of Fig.
5A with 3A). In addition, the effect of
70 underproduction on growth rate (Fig. 4B)
was not ppGpp-dependent. Moreover, the level of
underproduction of
70 was the same in the
ppGpp0 as in the wild type (data not shown), and the levels
of RNAP
-subunit were not affected in the ppGpp0 strain.
In agreement with the effect on ribosomal proteins the RNA/protein
ratio was reduced (Fig. 5B).
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Fig. 5.
Effects of 70
underproduction in growing ppGpp0
cells. A, the effects of
70
underproduction on the subset of proteins seen in Fig. 1C.
B, total RNA/total protein production during growth with
normal levels of
70 (0.2 mM IAA) and lower
levels of
70 (0.02 mM IAA) expressed
relative to the control (0.2 mM IAA).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor,
70 (Fig. 3A). The
expression of ribosomal proteins has been shown to depend on the
availability of rRNA, and the ribosomal protein promoters have also
been shown to be under direct stringent control (2-4). Thus, the
effects seen on ribosomal proteins in the
70
underproduction experiment could be due to effects on rRNA production via transcription of rrn promoters causing a subsequent
feedback control of the expression of genes encoding ribosomal
proteins. However, we can not rule out the possibility of direct
effects on the transcription from ribosomal protein promoters. The
effects seen are most likely not post-transcriptional since the
ribosomal protein transcripts were similarly affected by a reduction in
70-programmed RNAP.2 It should also be noted
that the total RNA/total protein ratio decreased during
70 underproduction demonstrating that rRNA levels
decreased. The data indicate that among the genes expressed during
exponential growth of E. coli the expression of rRNA and
ribosomal protein genes is more sensitive than the average to a
reduction in RNAP availability. The results support the idea of
stringent promoters requiring high levels of free RNAP, as suggested by
Jensen and Pedersen (16). In fact, rrn promoters may not
work at their maximal capacity even during logarithmic growth since
data from Squires and co-workers (38) showed that a cell containing
only one rrn operon instead of seven was still able to
produce 56% of the wild type rRNA.
70 underproduction also go in line with
the model of Jishage et al. (20) who suggested that one
mechanism of ppGpp-dependent gene regulation is to affect
the relative competitiveness of
factors, possibly by affecting the
affinity of
70 to the core enzyme. The lowering of
70 interaction with RNAP increases the opportunities for
alternative
factors to bind RNAP core enzyme and redirect RNAP to
their respective promoters, and at the same time it lowers the
availability of RNAP holoenzyme for the
70-dependent promoters. If the mechanism
behind the stringent control is RNAP availability, either by changing
the pool of free RNAP or by affecting
factor competition or both,
the constitutively stringent mutations in RNAP (e.g. (15))
should also affect these parameters. One interesting feature of the
stringent rpoB mutants characterized by Zhou and Jin (15,
39) is that they express lower levels of
70. The
stringent rpoB alleles had effects on transcription in
vitro so the lower level of
70 expressed by these
cells is not the only explanation for their action. But, considering
the results presented here, it is possible that the lower
70 levels of the mutants fortify the stringent phenotype
in vivo.
70-programmed
RNAP has effects that mimic the stringent response does not, per
se, prove that a reduction of
70-programmed RNAP is
part of a bona fide stringent response, but it is possible
to speculate on such a mechanism. Even if changing the availability of
70-programmed RNAP is one effect of ppGpp in the cell,
direct positive effects of ppGpp on expression of specific
genes/proteins has been shown previously by Choy (40) using a coupled
in vitro transcription-translation system. It is possible
that the RNAP availability works together with more direct effects of
ppGpp on specific promoters.
70-programmed RNAP also affects
growth rate (Fig. 4, A and B), as has previously
been shown for cells overproducing RelA and consequently ppGpp (9, 10).
It could be argued that the effects seen on the protein synthesizing
system are merely an effect of the growth rate-dependent
regulation of these particular genes. But, then the question remains
how
70 underproduction mechanistically causes a reduced
growth rate, since most gene products, with the exception of for
example the ribosomal proteins, were unaffected by the reduction in
E
70. Maybe the lowered growth rate is a
result of the lower levels of the protein synthesizing
system due to lower availability of free
70-programmed
RNAP. The rrn promoters and ribosomal protein promoters are
affected before the other promoters because they are, in this model,
sensitive to changes in free RNAP. It has long been known that the
stringent response and growth rate-dependent control have
many effectors in common, but it is still controversial whether the one
system, ppGpp and stringent control, is involved in the other, growth
rate control. RNAP availability could account for growth
rate-dependent regulation of gene expression whether or not
the signal controlling RNAP levels is ppGpp.
70. We suggest that the effects on expression of
ribosomal proteins by lowering the levels of
70 indicate
that the ribosomal protein promoters are sensitive to the availability
of RNAP. Thus, it is formally possible that the stringent response
could encompass a mechanism of lowered availability of
70-programmed RNAP. Further experiments will address the
question of whether available, transcription-competent, RNAP levels
change significantly in response to ppGpp accumulation.
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ACKNOWLEDGEMENTS |
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We thank Carol Gross for supplying the Ptrp-rpoD construct. Peter Neubauer and Ha Le Thanh for help with the ppGpp measurements. Joakim Norbeck and Thomas Larsson for the help with mass spectrometry analysis of proteins. We also thank Kristian Kvint and Miki Jishage for sharing unpublished results and strains and for helpful discussions and suggestions on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-0-31-7732567;
Fax: 46-0-31-7732599; E-mail: anne.farewell@gmm.gu.se.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209881200
2 Miki Jishage, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ppGpp, guanosine 3', 5'-bis diphosphate; RNAP, RNA polymerase; IAA, indole-3-acrylic acid; NEPHGE, non-equilibrium pH gel electrophoresis.
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