From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
Received for publication, January 8, 2003, and in revised form, February 10, 2003
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ABSTRACT |
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Previously we found that a mutation in
either pgi or pfkA, encoding phosphoglucose
isomerase or phosphofructokinase A, respectively, facilitates
degradation of the ptsG mRNA in an RNase
E-dependent manner in Escherichia coli (1). In
this study, we examined the effects of a series of glycolytic genes on
the degradation of ptsG mRNA and how the mutations
destabilize the ptsG mRNA. The conditional lethal
mutation ts8 in fda, encoding
fructose-1,6-P2 aldolase just downstream of
pfkA in the glycolytic pathway, caused the destabilization
of ptsG mRNA at the nonpermissive temperature. Mutations in any other gene did not destabilize the ptsG
mRNA; rather, they reduced the ptsG transcription
mainly by affecting the cAMP level. The rapid degradation of
ptsG mRNA in mutant strains was completely dependent
upon the presence of glucose or any one of its compounds, which enter
the Embden-Meyerhof glycolytic pathway before the block points. A
significant increase in the intracellular glucose-6-P level was
observed in the presence of glucose in the pgi strain. An
overexpression of glucose-6-phosphate dehydrogenase eliminated
both the accumulation and the degradation of ptsG mRNA in the pgi strain. In addition, accumulation of
fructose-6-P led to the rapid degradation of ptsG mRNA
in a pgi pfkA mutant strain lacking glucose-6-P. We
conclude that the RNase E-dependent destabilization of
ptsG mRNA occurs in response to accumulation of
glucose-6-P or fructose-6-P.
In bacteria, a number of sugars represented by glucose are
transported into the cells coupled with their phosphorylation by the
phosphoenolpyruvate-dependent sugar phosphotransferase
system (PTS)1 (2-4), whereas
the translocation of some other sugars such as lactose is
catalyzed by non-PTS transport systems. In either case, the
incorporated sugars are metabolized primarily by the Embden-Meyerhof glycolytic pathway and by the pentose phosphate pathway to produce numerous intermediary metabolites as well as energy in cells (5). The
PTS in Escherichia coli consists of two common cytoplasmic proteins, enzyme I and HPr (histidine-containing protein of the PTS),
as well as an array of sugar-specific enzyme II complexes (EIIs). The
glucose-specific EII (glucose transporter) consists of cytoplasmic
protein IIAGlc and membrane receptor IICBGlc
encoded by crr and ptsG, respectively. The
phosphoryl group from phosphoenolpyruvate is transferred sequentially
to enzyme I, HPr, the EIIs, and finally glucose as it is translocated
across the membrane.
In addition to sugar transport and phosphorylation, the PTS plays
important regulatory roles in a variety of cellular activities. This is
particularly evident for the glucose-specific PTS. For example,
IIAGlc regulates both the transport of non-PTS sugars and
the activity of adenylate cyclase depending on its phosphorylation
state (2-4). The former process, called inducer exclusion, is fully
responsible for the glucose-lactose diauxie that is a prototype
of catabolite repression (6, 7). A striking recent discovery regarding the regulatory function of PTS is that IICBGlc, depending
on its phosphorylation state, interacts with Mlc to modulate the
cellular localization and activity of this global repressor protein
(8-10).
The expression of the ptsG gene encoding IICBGlc
is regulated by two global control systems at the level of
transcription initiation (7, 11, 12). First, it is under positive
control by CRP-cAMP; hence, the ptsG expression is
absolutely dependent on this complex. Second, the transcription of the
ptsG gene is negatively regulated by a global repressor,
Mlc. Recent studies have established that external glucose induces
ptsG transcription by modulating the Mlc-mediated regulatory
pathway (7, 11, 12). When glucose is transported into the cell,
IICBGlc is dephosphorylated and binds Mlc, resulting in the
sequestration of Mlc at the membrane (8-10). Another unexpected
discovery regarding the regulation of ptsG expression is
that a mutation in either pgi or pfkA, encoding
glycolytic enzyme phosphoglucose isomerase or phosphofructokinase A,
respectively, leads to a rapid degradation of ptsG mRNA
and that RNase E is responsible for the degradation of ptsG
mRNA (1). Thus, ptsG expression also is regulated at the
level of mRNA degradation, presumably in response to the glycolytic flux. However, it remains unclear how the glycolytic flux is involved in the destabilization of ptsG mRNA, and little is known
about the mechanism of the stimulation of ptsG mRNA
degradation except that RNase E is a major player in this process.
In this study, we first investigated whether mutations in glycolytic
genes other than pgi and pfkA affect the
stability of ptsG mRNA. We found that the conditional
lethal mutation ts8 in fda, encoding
fructose-1,6-P2 aldolase (Fda) (13), stimulates degradation
of ptsG mRNA but that mutations in any other glycolytic gene did not affect ptsG mRNA degradation. Then we
addressed the question of how the mutations destabilize ptsG
mRNA. The degradation of ptsG mRNA in mutant cells
occurred only in the presence of glucose or any one of the
glycolytic intermediates upstream of the block. More specifically, the
degradation of ptsG mRNA was associated with elevated
levels of glucose-6-P and/or fructose-6-P. We propose that accumulation
of glucose-6-P or fructose-6-P is responsible for ptsG
mRNA degradation. The physiological relevance of this novel
feedback regulatory system at the post-transcriptional step is
discussed below.
Strains, Plasmids, and Growth Conditions--
The bacterial
strains used in this study are listed in Table
I. A series of disruption mutants were
constructed by the one-step gene inactivation protocol that is based on
the high efficiency of the phage Northern Blot Analysis--
Total cellular RNAs were isolated
from exponentially growing cells as described (16). The RNAs were
resolved by 1.2% agarose gel electrophoresis in the presence of
formaldehyde and blotted onto a Hybond-N+ membrane
(Amersham Biosciences) as described (17). The mRNAs were
visualized using digoxigenin reagents and kits for nonradioactive nucleic acid labeling and detection systems (Roche Molecular
Biochemicals) according to the procedure specified by the manufacturer.
The 305-bp digoxigenin-labeled DNA probe corresponding to the
5'-ptsG region was used.
Determination of Intracellular cAMP--
Bacterial cells were
grown in LB medium to A600 = 0.6. Two
milliliters of the culture were taken and centrifuged at 12,000 × g for 20 s at 4 °C. The pellet was suspended in 40 µl of 5 mM NaCl and immediately heated at 100 °C for 5 min. After adding 160 µl of ethanol, the mixture was chilled at
Determination of Intracellular
Glucose-6-P--
Bacterial cells were grown in LB medium to
A600 = 0.6 unless otherwise specified. One
milliliter of the culture was centrifuged at 10,000 × g for 2 min at room temperature. The pellet was
suspended in 100 µl of H2O, immediately added to 50 µl
of 5 M HClO4, and chilled on ice. After adding
100 µl of 2.5 M K2CO3, the
mixture was centrifuged at 14,000 × g for 10 min at
4 °C. The supernatant was used for the glucose-6-P assay. The assay
was performed according to the method of Hogema et al. (20).
The intracellular concentration of glucose-6-P was calculated as in the
case of the cAMP assay.
The ts8 fda Mutation Stimulates the Degradation of ptsG mRNA at
the Nonpermissive Temperature--
Among glycolytic genes other than
pgi and pfkA, we first focused on fda,
which encodes Fda just downstream of pfkA in the Embden-Meyerhof glycolytic pathway (Fig.
1). It is known that the Fda activity in
a strain carrying a temperature-sensitive fda allele
(ts8 or h8) was essentially undetectable at
42 °C (13). The ts8 mutation was shown to inhibit stable
RNA synthesis and cell growth depending on glucose metabolism at the
nonpermissive temperature (21). To examine whether the ts8
mutation affects the expression and/or degradation of ptsG
mRNA, the ts8 allele was transferred to a W3110
background, and the expression of ptsG mRNA in the
presence of glucose was analyzed by Northern blotting using a
ptsG DNA probe. The full-length ptsG mRNA was
expressed stably in the ts8 fda strain at 30 °C as in the
wild-type strain, whereas the pgi mutation dramatically
reduced the full-length ptsG mRNA, resulting in an
extensive smear because of the stimulation of RNase E-mediated mRNA
degradation (Fig. 2, lanes
1-3). When the temperature was shifted to 42 °C, the ts8
fda strain gave a smear of degradation intermediates of
ptsG mRNA (Fig. 2, lane 4), whereas the
temperature upshift to 42 °C did not affect the Northern pattern in
the wild-type and pgi strains (data not shown). This implies
that the loss of Fda activity leads to the rapid degradation of
ptsG mRNA in the presence of glucose. The introduction of an rne allele, ams1 (22, 23), encoding a
temperature-sensitive RNase E into the ts8 fda mutant
prevented the rapid degradation of ptsG mRNA at the
nonpermissive temperature (Fig. 2, lane 5). Thus, the
ptsG mRNA is destabilized in an RNase
E-dependent manner in the ts8 fda strain at
42 °C as in the case of the pgi and pfkA strains (1).
Mutations in Glycolytic Genes Downstream of fda Affect the
Expression but Not the Degradation of ptsG mRNA--
To examine
the effects of mutations in other glycolytic genes on the degradation
of ptsG mRNA, a series of deletion mutants of glycolytic
genes downstream of fda were constructed. We successfully disrupted tpiA, pgk, gpm, and
pyk encoding triose-phosphate isomerase, phosphoglycerate
kinase, phosphoglycerate mutase, and pyruvate kinase,
respectively, although we failed to disrupt gap and
eno encoding glyceraldehyde-3-phosphate dehydrogenase and
enolase, respectively. The ptsG mRNA expression in each
mutant was analyzed by Northern blotting. As shown in Fig. 2, each of
these mutations affected the expression of ptsG mRNA to
various extents. However, the Northern patterns of the ptsG
mRNA in these mutant strains were essentially the same as those of
the wild-type, indicating that these mutations do not affect
significantly the degradation of ptsG mRNA (Fig. 2,
lanes 6-9). We conclude that mutational blocks only in
early stages of the glycolytic pathway produce a signal that leads to
the rapid degradation of ptsG mRNA. The effects of
mutations in the glycolytic genes on ptsG expression also
were analyzed by Western blotting. The level of IICBGlc was
correlated with that of the full-length ptsG mRNA in
each strain (data not shown). It is likely that the mutations in the glycolytic genes downstream of fda reduce the
ptsG expression at the level of transcription initiation
presumably by affecting the CRP-cAMP and/or Mlc pathways because
ptsG transcription is regulated by these two global systems
(7, 11, 12). However, the introduction of the mlc mutation
did not affect the ptsG mRNA level in each mutant strain
in the presence of glucose, suggesting that the Mlc pathway is not
involved in the reduction of ptsG expression (data not
shown). We then determined the intracellular cAMP levels in each
mutant strain both in the presence and absence of glucose. We observed
that the reduction in the intracellular cAMP level by glucose was more
significant in the tpiA, gpm, and pyk
strains than in the wild-type strain (Table
II). Thus, the reduced expression of
ptsG mRNA in the tpiA, gpm, and
pyk strains particularly in the presence of glucose appears
to be at least in part due to the reduction of the intracellular
cAMP level. It remains to be studied why ptsG expression is
reduced in the pgk strain, in which no significant reduction
of cAMP was observed.
The Destabilization of ptsG mRNA in Mutant Strains Is Dependent
upon Glucose Uptake and Metabolism--
The ptsG mRNA
is highly expressed in the mlc strain without the addition
of glucose to the medium, and the ptsG mRNA expression is moderately reduced in the presence of external glucose because of
the reduction in cAMP and CRP levels (11). When the ptsG mRNA was analyzed in the pgi mlc double mutant growing
in LB medium, rapid degradation of ptsG mRNA occurred in
the presence of external glucose as expected (Fig.
3, lane 4). However, a
significant degradation of ptsG mRNA also was observed
even without the addition of external glucose (Fig. 3, lane
3). This raises a question as to whether the rapid degradation of
ptsG mRNA is dependent upon glucose metabolism. To test
this, we introduced the ptsI mutation into the
pgi mlc strain and examined the expression of
ptsG mRNA by Northern blotting. The ptsG
mRNA was stabilized in the pgi mlc ptsI triple mutant where the uptake of glucose is prevented (Fig. 3, lanes 5 and 6). It is apparent that the degradation of
ptsG mRNA observed in the pgi mlc double
mutant growing in LB medium without external glucose was due to trace
amounts of endogenous glucose in the LB medium. In fact, the rapid
degradation of ptsG mRNA no longer occurred in the
mlc pgi and mlc pfkA double mutant strains
without the addition of glucose when TB medium was used (Fig.
4, A and B,
lane 1). The addition of glucose caused a dramatic
degradation of ptsG mRNA in both strains growing on TB
medium (Fig. 4, A and B, lane 2).
These results clearly indicate that the destabilization of
ptsG mRNA in mutant strains occurs depending on the
glucose uptake and metabolism.
The Destabilization of ptsG mRNA Occurs Only in the Presence of
Glycolytic Intermediates Upstream of the Block--
To test whether
glucose generated internally also could lead to the destabilization of
ptsG mRNA in the mutant strains, the effect of the
addition of lactose on the degradation of ptsG mRNA was
examined. The addition of lactose in the culture medium stimulated the
degradation of ptsG mRNA in both the pgi mlc
and pfkA mlc strains (Fig. 4, A and B,
lane 3). Thus, internally produced glucose, which is
believed to be phosphorylated by glucokinase encoded by glk
(5), also leads to the destabilization of ptsG mRNA in
the mutant strains. We also examined the effect of the addition of
several glycolytic intermediates and/or carbon sources on the ptsG mRNA degradation in pgi mlc and
pfkA mlc double mutant strains. The addition of glucose-6-P,
which is taken up by UhpT (24), caused the rapid degradation of
ptsG mRNA in both strains (Fig. 4, A and
B, lane 8). On the other hand, the addition of
fructose-6-P, which also is taken up by UhpT, failed to cause the rapid
degradation of ptsG mRNA in the pgi mlc
strain (Fig. 4A, lane 9), although it caused the
degradation of ptsG mRNA in the pfkA mlc
strain (Fig. 4B, lane 9). The addition of
mannitol and xylose, which are taken up by mannitol-specific PTS and
non-PTS xylose permease, respectively (25), and then converted to
fructose-6-P, also caused the rapid degradation of ptsG
mRNA in the pfkA mlc (Fig. 4B, lanes
4 and 5) but not in the pgi mlc (Fig.
4A, lanes 4 and 5) strains. However,
the addition of fructose, which enters the Embden-Meyerhof pathway past
fructose-6-P, did not affect the stability of ptsG mRNA
in either strain (Fig. 4, A and B, lane 6). Likewise, the stability of ptsG mRNA in mutant
strains was not affected by the addition of glycerol (Fig. 4,
A and B, lane 7), which also enters
the central carbon metabolism past fructose-1,6-P2. We
conclude that the mutational block in the glycolytic pathway destabilizes ptsG mRNA depending on the accumulation of
glycolytic intermediates up to the block points.
Elevated Glucose-6-P Level Is Associated with the Rapid Degradation
of ptsG--
Among glycolytic intermediates, glucose-6-P would
be the most likely participant in the destabilization of
ptsG mRNA because this intermediate is expected to
accumulate at least in the pgi strain growing in the glucose
medium. In fact, it is known that the glucose-6-P level in the
pgi strain markedly increases when grown in the presence of
glucose (26, 27). To examine a link between the glucose-6-P level and
the destabilization of ptsG mRNA, we determined the
intracellular levels of glucose-6-P in various strains growing in LB
medium containing exogenous glucose. The glucose-6-P level in the
pgi strain was about 8 times higher than that in the
wild-type strain (Table III, lines 1 and
2). A moderate but still significant accumulation of glucose-6-P was observed in the pfkA strain (Table III, line 3). On the
other hand, little or no increase in the glucose-6-P level was observed
in other mutant strains (Table III, lines 4-7). A large increase in the glucose-6-P level was observed in the mlc pgi but not in
the mlc and the mlc pgi ptsI strains when cells
were grown in LB medium with exogenous glucose (Table
IV, lines 2, 4, and 8). As expected, the
accumulation of glucose-6-P in the mlc pgi strain occurred even when cells were grown in LB medium without exogenous glucose because of trace amounts of glucose in the medium (Table IV, line 3).
However, no increase in the glucose-6-P level was observed in the
pgi strain when cells were grown in TB medium without
exogenous glucose (Table IV, line 5). All of these results are entirely consistent with a view that accumulation of glucose-6-P is a
degradation signal for the ptsG mRNA at least in the
pgi strain.
The Addition of Glucose Rapidly Leads to the Accumulation of
Glucose-6-P and the Degradation of ptsG mRNA in the pgi
Strain--
To examine how rapidly glucose-6-P accumulates to
stimulate the degradation of ptsG mRNA after the
addition of glucose, both mlc and mlc pgi cells
were grown to early log phase in TB medium, glucose was added at a
final concentration of 1%, and the incubation was continued. The
glucose-6-P level was monitored at various times after the addition of
glucose. The glucose-6-P level started to increase immediately after
the addition of glucose, reaching the plateau level (about a 2-fold
increase) characteristic of cells grown in the presence of glucose
after less than 3 min in the mlc cells (Fig.
5A). On the other hand, the
glucose-6-P level continuously increased up to 9-fold for 10 min in the
mlc pgi cells (Fig. 5A). The expression of
ptsG mRNA also was examined at various times after the
addition of glucose by Northern blotting. It was expected that the
addition of glucose would affect the ptsG expression at the
levels of both transcription and mRNA degradation. In fact, the
ptsG mRNA level was moderately reduced by the addition of glucose (Fig. 5B, lanes 1-5). This effect of
glucose, called catabolite repression, is apparently a result of the
reduction of both cAMP and CRP levels (11, 28, 29). Remarkably, the full-length ptsG mRNA was eliminated almost completely
within 5 min after the addition of glucose in the mlc pgi
cells (Fig. 5B, lanes 6-10). It is apparent that
the dramatic decrease of ptsG mRNA in the mlc
pgi cells is because of the degradation of the full-length
ptsG mRNA level. The data clearly indicate that the
addition of glucose rapidly leads to the accumulation of glucose-6-P, resulting in the stimulation of the degradation of ptsG
mRNA.
Accumulation of Fructose-6-P Can Be an Additional Metabolic
Signal for the Degradation of ptsG--
It is rather complicated to
specify the degradation signal in the pfkA strain, in which
not only glucose-6-P but also fructose-6-P is expected to accumulate.
To access a possible role of accumulation of fructose-6-P in the
degradation of ptsG mRNA in the pfkA strain, an mlc pgi pfkA triple mutant was constructed. The effects
of glucose, glucose-6-P, and fructose-6-P on the degradation of
ptsG mRNA were examined in cells growing in TB medium.
As expected, the addition of glucose (Fig.
6, lane 2) or glucose-6-P
(Fig. 6, lane 3) caused a rapid degradation of
ptsG mRNA because of the accumulation of glucose-6-P in
the triple mutant strain. Interestingly, the destabilization of
ptsG mRNA also occurred when fructose-6-P was added
(Fig. 6, lane 4). Under this condition, cells should be
virtually devoid of glucose-6-P because the interconversion from
fructose-6-P to glucose-6-P is prevented. In fact, we observed that the
glucose-6-P level was not elevated in the presence of fructose-6-P
(Table IV, line 11), whereas it dramatically increased in the presence
of glucose (Table IV, line 10). This implies that accumulation of
fructose-6-P could act as an additional signal to stimulate the
degradation of ptsG mRNA. On the other hand, the
addition of fructose or fructose-1,6-P2 did not cause the rapid degradation of ptsG mRNA in the ts8 fda
strain at the nonpermissive temperature, suggesting that the increased
fructose-6-P rather than the accumulation of
fructose-1,6-P2 itself is responsible for the
destabilization of ptsG mRNA in the fda
strain (data not shown).
Overexpression of Glucose-6-P Dehydrogenase Prevents the Rapid
Degradation of ptsG mRNA--
If the increased intracellular pool
of glucose-6-P is responsible for the rapid degradation of
ptsG mRNA, an overexpression of glucose-6-P
dehydrogenase (Zwf) is expected to affect the degradation of
ptsG mRNA because it may stimulate the flow from
glucose-6-P to 6-P-gluconolactone resulting in a reduction in the
glucose-6-P level. To test this idea, a multicopy plasmid (pTM6)
carrying the zwf gene was constructed and introduced into
the pgi and pfkA strains. As expected, the
glucose-6-P level was reduced dramatically in both strains growing in
glucose medium when Zwf was overexpressed by introducing pTM6 (Table
IV, lines 13 and 14). Interestingly, the rapid degradation of
ptsG mRNA in both strains was prevented completely under
the same conditions (Fig. 7, lanes
2 and 4), whereas the introduction of a control plasmid
did not affect the degradation (Fig. 7, lanes 1 and
3). This strongly supports the argument that the
increased level of glucose-6-P is surely responsible for the rapid
degradation of ptsG mRNA in the pgi strain.
The fact that the rapid degradation of ptsG mRNA was
prevented by the introduction of pTM6 suggests that overexpression of
Zwf may eliminate the accumulation of not only glucose-6-P but also
fructose-6-P. The introduction of pTM6 caused only a slight decrease in
the glucose-6-P level in the wild-type strain (Table IV, line 12).
One of the questions addressed in the present study is whether
mutations in other glycolytic genes affect the expression and/or degradation of ptsG mRNA. We demonstrate that the
ts8 mutation in fda, just downstream of
pfkA in the glycolytic pathway, destabilizes the
ptsG mRNA at the nonpermissive temperature in
glucose-grown cells as in the pgi and pfkA
mutations. On the other hand, mutations downstream of fda
did not affect significantly the stability of ptsG mRNA,
although these mutations somehow reduced ptsG expression at
the level of transcription initiation by affecting primarily the
intracellular cAMP level. Thus, we conclude that a mutational block in
only the early stages of the glycolytic pathway leads to the
destabilization of ptsG mRNA in glucose-grown cells.
An important conclusion of the present study is that the rapid
degradation of ptsG mRNA in the mutant strains is
completely dependent on the presence of glucose or any one of the
compounds that enters the Embden-Meyerhof glycolytic pathway before the block points. These results led us to propose that the accumulation of
early glycolytic intermediates due to the metabolic block triggers the
stimulation of RNase E-mediated degradation of ptsG
mRNA. Several lines of evidence indicate that accumulation of
glucose-6-P or fructose-6-P is responsible for the rapid degradation of
ptsG mRNA in the pgi strain. First, a large
increase in glucose-6-P levels was observed in the pgi
strain in the presence of glucose where the rapid degradation of
ptsG mRNA occurs. Second, overexpression of glucose-6-P
dehydrogenase eliminated both the accumulation of glucose-6-P and the
degradation of ptsG mRNA in the pgi strain. Third, the ptsG mRNA was destabilized in the pgi
pfkA mlc triple mutant strain lacking glucose-6-P when
fructose-6-P was supplied. In addition, we observed that the
degradation of ptsG mRNA was stimulated by the addition
of a nonmetabolizable glucose analog, The rapid degradation of ptsG mRNA in response to the
accumulation of glucose-6-P or fructose-6-P illuminates an elaborate feedback regulatory mechanism in carbon central metabolism. The physiological relevance of this feedback regulation at the mRNA degradation step is clear because accumulation of metabolic
intermediates sometimes would be deleterious to cells (21). It is known
that the accumulation of hexose phosphates such as glucose-6-P and fructose-6-P damages DNA resulting in a dramatic increase in mutation frequency (26, 27). The down-regulation of ptsG
expression through mRNA degradation certainly prevents glucose
uptake by avoiding too much accumulation of hexose phosphates in mutant cells. It would be interesting to know whether the expression and
stability of other sugar transport-related mRNAs also are affected
by the accumulation of early glycolytic intermediates. We tested the
effect of the pgi or pfkA mutation on the
expression of several other mRNAs. So far, no clear evidence for
the destabilization of other mRNAs by the pgi or
pfkA mutation has been obtained.
Are there any situations in which the metabolic perturbation in
the early stages of the Embden-Meyerhof glycolytic pathway occurs in
the wild-type cells? One clear case for this is when cells incorporate
nonmetabolizable glucose analogs such as Another important aspect of our study is that the metabolic
flow in the glycolytic pathway regulates the stability of a specific mRNA of a solute transporter gene. Although the degradation of mRNA is an important step for controlling gene expression (32-36), little is known about how a particular environmental and/or internal signal regulates the stability of a specific mRNA in E. coli cells. The acceleration of ptsG mRNA
degradation in response to the metabolic blocks in the early stages of
the Embden-Meyerhof pathway certainly offers a good opportunity to
investigate both the mechanism and the role of regulation of gene
expression at the mRNA degradation level. RNase E is believed to
play a central role in the degradation of a variety of mRNAs in
E. coli cells (37, 38). The enzyme exists as a
macromolecular complex called degradosome, a multi-enzyme RNA
degradation complex that also contains polynucleotide phosphorylase, RhlB helicase, enolase, and other minor components (39-42). The presence of enolase in the degradosome complex suggests that this glycolytic enzyme would be involved somehow in the regulation of
degradation of ptsG mRNA in response to the metabolic
blocks in the glycolytic pathway.
Although the present study has clarified an important aspect regarding
the regulation of ptsG mRNA degradation, the molecular mechanism underlying this phenomenon remains largely unknown. There are
several important questions that should be addressed with a priority.
First, how does the accumulation of glucose-6-P or fructose-6-P lead to
the rapid degradation of ptsG mRNA by RNase E? The
increased level of glucose-6-P in mutant strains may stimulate the
metabolic flows from glucose-6-P to glucose 1-phosphate and
6-P-gluconolactone catalyzed by phosphoglucomutase (pgm) and
glucose-6-P dehydrogenase (zwf), respectively (Fig. 1). The
increased flows in these two pathways would alter the levels of downstream metabolites that somehow could affect the stability of ptsG mRNA. If this were the case, mutations
in the pgm and/or zwf genes should affect the
degradation of ptsG mRNA in the pgi strain.
However, the rapid degradation of ptsG mRNA in
the presence of glucose still occurred in the pgm pgi or
zwf pgi double mutant
strains.2 This indicates that
the accumulation of glucose-6-P itself but not the possible metabolic
change in the pentose phosphate pathway and in the glycogen
biosynthetic pathway is responsible for the rapid degradation of
ptsG mRNA. It is highly possible that some molecules
that transduce the signal to RNase E also monitor the accumulation of
glucose-6-P or fructose-6-P. It is certainly important to identify
additional genetic components that might be involved in this process.
One important molecule that could modulate the ptsG mRNA
degradation would be host factor I, an Hfq that mediates RNA-RNA
interaction (43-45). In fact, host factor I is known to modulate
different aspects of RNA metabolism including the stability of several
mRNAs (46-48). Second, because the rapid degradation of mRNA
in response to the metabolic block is observed so far specifically in
ptsG mRNA, there should be some specific features within
ptsG mRNA that are recognized by RNase E and/or some
other factors. Further genetic and biochemical studies are necessary to
answer these questions and to understand fully the molecular mechanism
underlying this novel feedback regulation in the central metabolic pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Red recombinase (14). The FLP
recognition target-flanked resistance gene was eliminated by
using an FLP expression plasmid, pCP20 (14). To transfer the ts8
fda allele to other backgrounds by P1 phage, the cat
gene was inserted 2.7 kb downstream of the chromosomal ts8
fda gene in CAG417. IT1568 carrying a frameshift mutation in the
mlc gene was isolated spontaneously. The zwf gene including the promoter region was amplified by PCR using primers 5'-CCCAAGCTTGTGCCGCACTTTGCGCGCT-3' and
5'-CCCAAGCTTGGCCTGTAACCGGAGCTCA-3'. The amplified DNA fragment was
digested with HindIII and cloned into
HindIII-digested pBR322 to construct plasmid pTM6. Cells were grown at 37 °C in LB medium or TB medium (15) supplemented with
kanamycin (15 µg/ml), tetracycline (15 µg/ml), chloramphenicol (15 µg/ml), and/or ampicillin (50 µg/ml) when needed. Bacterial growth
was monitored by determining the optical density at 600 nm.
Bacterial strains used in this study
70 °C for 30 min and centrifuged at 14,000 × g
for 30 min at 4 °C. The resulting supernatant was dried up,
dissolved in 20 µl of H2O, and used for cAMP assay. The
determination of cAMP by gel mobility shift assay has been described
previously (18). The cellular concentration of cAMP was calculated on
the assumption that an A600 of 1.4 corresponds to 109 cells/ml (15) and that the volume of a cell is
2 × 10
12 ml (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Relevant metabolic pathways
of several sugars in E. coli. The
pathways shown illustrate glycolysis.
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Fig. 2.
Effects of mutations in glycolytic genes on
the expression of ptsG mRNA. W3110
(lane 1), TM54 (lane 2), TM84 (lane
6), TM61 (lane 7), TM81 (lane 8), and TM154
(lane 9) were grown in LB medium with 1% glucose at
37 °C. Cellular RNAs were prepared at A600 = 0.6. In the case of TM191 (lanes 3 and 4) and
TM234 (lane 5), cells were grown in LB medium with 1%
glucose at 30 °C to A600 = 0.5, and
the temperature was shifted to 42 °C (lanes 4 and
5) or kept at 30 °C (lane 3). Cellular RNAs
were prepared after further incubation for 10 min. The RNA samples (15 µg) were subjected to Northern blot analysis. WT, wild
type.
Intracellular cAMP levels in exponentially growing cells
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Fig. 3.
Effect of ptsI mutation on
the degradation of ptsG mRNA. IT1568
(lanes 1 and 2), TM162 (lanes 3 and
4), and TM145 (lanes 5 and 6) were
grown in LB medium with and without 1% glucose to
A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot
analysis.
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Fig. 4.
Effects of various carbon sources on the
degradation of ptsG mRNA. TM162
(A) and IT1165 (B) were grown in TB medium with
and without 0.1% compounds indicated to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA
samples was subjected to Northern blot analysis. The compounds used are
glucose (Glc), lactose (Lac), mannitol
(Mtl), xylose (Xyl), fructose (Fru),
glycerol (Gly), glucose-6-P (G6P), and
fructose-6-P (F6P).
Intracellular levels of glucose 6-phosphate
Glucose 6-phosphate levels in cells growing in various conditions
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Fig. 5.
Effects of the addition of glucose on the
glucose-6-P level and on the degradation of ptsG
mRNA. IT1568 (mlc) and TM162 (mlc
pgi) were grown in TB medium to A600 = 0.4. Glucose was added at a final concentration of 1%, and the incubation
was continued. Intracellular glucose-6-P levels were determined at the
indicated times as described under "Experimental Procedures"
(A). Each value is the average of three independent assays.
Cellular RNAs also were prepared at the indicated times and analyzed by
Northern blotting using 15 µg of each of the RNA samples
(B).
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Fig. 6.
Effects of glycolytic intermediates on the
degradation of ptsG mRNA in the mlc pgi
pfkA triple mutant. Cells were grown in TB medium with
and without 0.1% compounds indicated to A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA
samples was subjected to Northern blot analysis. G6P,
glucose-6-P; F6P, fructose-6-P.
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Fig. 7.
Effect of overexpression of glucose-6-P
dehydrogenase on the degradation of ptsG
mRNA. TM54/pBR322 (lane 1), TM54/pTM6
(lane 2), TM238/pBR322 (lane 3), and TM238/pTM6
(lane 4) were grown in LB medium with 1% glucose to
A600 = 0.6. Cellular RNAs were prepared, and 15 µg of each of the RNA samples was subjected to Northern blot
analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methyl glycoside, in the
wild-type strain (1). This means that accumulation of phosphorylated
-methyl glycoside also could act as a signal to break down the
ptsG mRNA. On the other hand, the data suggested that
accumulation of fructose-1,6-P2 itself may not be a signal
for the rapid degradation of ptsG mRNA in the ts8
fda strain.
-methyl glucoside. The
metabolic block that leads to the accumulation of glucose-6-P and/or
fructose-6-P also may occur if certain substances inhibit the activity
of phosphoglucose isomerase, phosphofructokinase A, or
fructose-1,6-P2 aldolase. For example, it is known that Zn2+ acts as an inhibitor of the rat liver
phosphofructokinase (30). Furthermore, it is known that the levels of
glycolytic intermediates including glucose-6-P,
fructose-1,6-P2, and phosphoenolpyruvate vary markedly
depending on the nature of carbon and nitrogen sources and also after
the addition of a new nutrient to cultures (31). It is certainly
interesting to examine whether and how these changes in the levels of
metabolites affect the degradation of ptsG mRNA.
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ACKNOWLEDGEMENTS |
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We thank C. A. Gross for providing the ts8 fda strain (CAG417). T. M. is grateful to Masami Oguchi for continuous encouragement during this work.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Ajinomoto Co., Inc.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.: 81-52-789-3653;
Fax: 81-52-789-3001; E-mail: i45346a@nucc.cc.nagoya-u.ac.jp.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300177200
2 T. Morita, W. El-Kazzaz, Y. Tanaka, T. Inada, and H. Aiba, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PTS, phosphoenolpyruvate-dependent sugar phosphotransferase system; EII, enzyme II complex of the PTS; CRP, cAMP receptor protein; Fda, fructose-1,6-diphosphate aldolase; TB, tryptone broth; LB, Luria-Bertani broth.
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