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(Received for publication, June 23, 1995; and in revised form, August 30, 1995) From the
The carbon storage regulator gene csrA has been shown
previously to dramatically affect the biosynthesis of intracellular
glycogen in Escherichia coli through its negative control of
the expression of two glycogen biosynthetic operons and the
gluconeogenic gene pckA (Romeo, T., Gong, M., Liu, M. Y., and
Brun-Zinkernagel, A. M.(1993) J. Bacteriol. 175,
4744-4755). Examination of the effects of csrA on
several enzymes, genes, and metabolites of central carbohydrate
metabolism now establishes a more extensive role for csrA in
directing intracellular carbon flux. Phosphoglucomutase and the
gluconeogenic enzymes fructose-1,6-bisphosphatase and
phosphoenolpyruvate synthetase were found to be under the negative
control of csrA, and these enzyme activities were maximal
during the early stationary phase of growth. The enzymes
glucose-6-phosphate isomerase, triose-phosphate isomerase, and enolase
were positively regulated by csrA. Thus, csrA exerts
reciprocal effects on glycolysis versus gluconeogenesis and
glycogen biosynthesis. The glycolytic isozymes pyruvate kinase F and A
(encoded by pykF and pykA, respectively) and
phosphofructokinase I and II (pfkA and pfkB,
respectively) exhibited differential regulation via csrA.
Since the individual members of these isozyme pairs are allosterically
regulated by different cellular metabolites, csrA is also
capable of fine-tuning the allosteric regulation of glycolysis. In
contrast, the expression of genes of the pentose phosphate pathway was
weakly or negligibly affected by csrA.
The central routes of intermediary carbohydrate metabolism in Escherichia coli include the constitutive Embden-Meyerhof and
pentose phosphate pathways along with the inducible Entner-Douderoff
pathway(1) . The Embden-Meyerhof pathway is an amphibolic
system and functions in both glycolysis and gluconeogenesis. Most of
the reactions of the Embden-Meyerhof pathway are reversible in
vivo, with the notable exceptions of the 6-phosphofructokinase (EC
2.7.1.11) (Pfk) ( Although
the allosteric regulation of central carbohydrate metabolism has been
well studied in E. coli and in other bacteria, its genetic
regulation has not. The structural genes of these pathways are
generally regarded to be constitutively expressed(1) . While it
is true that the levels of these enzymes do not change dramatically in
response to various physiological requirements, it is also clear that
the levels of many, if not all, of these enzymes respond to conditions
such as oxygen availability and growth
rate(11, 12, 13, 14, 15, 16) ,
suggesting that the genetic regulation of these pathways is also
physiologically significant. During the transition from exponential
growth into stationary phase the demand for biosynthetic metabolism
decreases and E. coli as well as many other bacteria rapidly
convert available carbohydrate into glycogen, which appears to function
as a source of stored carbon and energy. The regulation of glycogen
synthesis is complex and includes both allosteric and genetic
components (reviewed in (17, 18, 19, 20) ). Our laboratory
recently discovered a regulatory gene, csrA, which
dramatically affects the biosynthesis of glycogen. A csrA::kanR insertion mutation results in the accumulation of approximately
20-fold higher levels of glycogen, which can reach a level of 1.6 mg of
glycogen/mg of protein in the early stationary phase in this
mutant(21) . The csrA gene was found to negatively
control the expression of the two structural genes of the glycogen
biosynthetic pathway, glgC encoding ADP-glucose
pyrophosphorylase (EC 2.7.7.27) and glgB encoding glycogen
branching enzyme (EC 2.4.1.18), as well as the gluconeogenic gene
phosphoenolpyruvate carboxykinase (EC 4.1.1.49) (Pck). The gene csrA was mapped at 58 min on the E. coli genome,
between alaS and serV(22) , and was shown to
encode a 61-amino acid protein, CsrA(21) . Recent studies on
the mechanism of csrA-mediated regulation of glgC have shown that the CsrA protein greatly enhances the decay of glgC mRNA, an effect that involves the region overlapping or
close to the ribosome binding site of glgC(23) . The
deduced amino acid sequence of the CsrA gene product was found to
contain a KH domain, which has been proposed to function as an
RNA-binding region of a diverse subset of RNA-binding proteins (24) . Because the csrA::kanR mutation also affects
cell surface properties, as exhibited by the adherence of mutant cells
to glassware, and because the regulation of glycogen biosynthesis by csrA is mediated independently of the known global regulators
of the glycogen operon glgCAP, cAMP, and ppGpp (25, 26, 39) , it was previously suggested
that csrA may encode a component of a novel global regulatory
system(21) . The present study is consistent with this
possibility and firmly establishes a role for csrA in the
regulation of central carbohydrate metabolism.
Figure 1:
Effect of csrA on the specific activities of fructose-1,6-bisphosphatase,
phosphoenolpyruvate synthetase, and phosphoglucomutase. Specific
activities of Fbp (A), Pps (C), and Pgm (D)
were determined in strains BW3414 (csrA
Both
gluconeogenesis and the uptake of glucose via the
phosphoenolpyruvate-dependent phosphotransferase system generate
glucose 6-phosphate in the cell, which must be converted by the enzyme
Pgm into glucose 1-phosphate to be used as a biosynthetic precursor of
polysaccharides such as glycogen. The specific activity of Pgm was up
to 4-fold higher in the csrA::kanR strain relative to the
isogenic csrA
Fig. 2shows that Pgi (A),
Tpi (B), and Eno (C) activities were from 1.5- to
3-fold higher in the csrA
Figure 2:
Effect of csrA on enzymes
catalyzing physiologically reversible reactions of the Embden-Meyerhof
pathway. Specific activities of Pgi (A), Tpi (B), and
Eno (C) were determined throughout the growth curves of BW3414 (csrA
In E. coli,
phosphofructokinase exists as two isozymes, PfkI and PfkII, which
differ in their allosteric properties(1, 2) . The
effect of csrA on each isozyme was examined, and the specific
activity of the major isozyme, PfkI, was found to be significantly
higher (up to 7.4-fold) in the csrA
Figure 3:
Effects of csrA on allosterically
regulated glycolytic enzymes. Specific activities of the two isozymes
of phosphofructokinase, PfkI (A) and PfkII (B), and
of the pyruvate kinase isozymes, PykF (C) and PykA (D), were monitored in BW3414 (csrA
Fig. 3(C and D) also shows the
specific activities of the pyruvate kinase isozymes PykF and PykA in
the csrA In order to test whether the changes observed in
the levels of these enzymes are due to the effect of csrA on
the expression of their structural genes,
Figure 4:
Effects of csrA on the expression
of pykF`-`lacZ and pykA`-`lacZ translational fusions.
The pleiotropic phenotype of a csrA::kanR insertion
mutant originally indicated that the role of csrA in E.
coli is broader than the control of glycogen synthesis (21) . The primary goal of the current study was to explore the
potential involvement of csrA in the regulation of
intracellular carbon flux by observing the effects of disrupting or
overexpressing csrA on the enzymes of central carbohydrate
metabolism. The csrA gene has now been shown to affect several
of these essential enzymes and thereby modulate intracellular carbon
flux on a wide scale in E. coli. As summarized in Fig. 5, csrA exerts reciprocal effects on enzymes of
glycolysis versus those of gluconeogenesis and glycogen
biosynthesis. The studies on adenylate energy charge and metabolite
levels presented here provide additional evidence of the role of csrA in determining central carbohydrate flux.
Figure 5:
Effects of csrA on carbohydrate
metabolism. Results of the current study and previous studies (22) are summarized to depict the scope of the regulatory
effects which csrA exerts on central carbohydrate metabolism. Circles indicate enzyme specific activities and/or genetic
fusions that have been examined and found to be negatively regulated
(-), positively regulated (+) or unaffected (
Previous
experiments had suggested that csrA negatively regulates
gluconeogenesis(21) . This hypothesis is supported by the
current study, which indicates that Fbp, Pps, and Pgm are negatively
regulated by csrA. This study also shows that these activities
are induced in the stationary phase, as was shown previously to be true
for Pck by Goldie(43) . An earlier study had also indicated
that Fbp activity is higher in stationary phase than in exponential
phase in glucose-grown cells, although the difference was less than
observed here, approximately 2-fold(31) . In the current study,
gluconeogenic enzyme activities were found to increase sharply in early
stationary phase and thereafter decrease to pre-stationary phase
levels. Since this response was not known previously for Fbp, the
greater stationary phase induction of Fbp observed in our studies
(7-10-fold) may be explained by the possibility that in the
earlier experiment (31) the culture was harvested later in the
stationary phase, after Fbp levels had dropped. The experiments of the
current study were conducted under conditions which allow optimum
glycogen synthesis in the early stationary phase. An important
implication of this study is that under these conditions
gluconeogenesis occurs during a fairly restricted period of time,
coincident with glycogen biosynthesis, indicating that the primary role
of gluconeogenesis in glucose-grown cells is to enhance glycogen
synthesis. The growth-phase response documented here for the
gluconeogenic enzymes should help the cell to conserve energy. A futile
cycle of gluconeogenesis and glycolysis would be prevented or at least
minimized in the exponential phase; the conversion of carbohydrate into
endogenous glycogen should be favored as cells enter the stationary
phase; and later in the stationary phase, during glycogenolysis, a
futile cycle of glycolysis and gluconeogenesis would again be avoided. The decrease in the activities of the gluconeogenic enzymes which
occurs later in stationary phase indicates that enzyme inactivation is
also an important determinant of the gluconeogenic capacity of a cell.
This may involve the specific inactivation of these enzymes later in
the stationary phase or these enzymes may be labile throughout the
growth cycle and their patterns of their activity in the growth curve
reflect genetic expression. The induction profile of a pckA`-`lacZ transcriptional fusion (21, 43) favors the latter
alternative. In fact, the cell regulates the levels and activities of
the gluconeogenic enzymes in a variety of ways, including (i)
regulation at the level of transcript initiation via cAMP and the
repressor of the PEP:fructose phosphotransferase system, FruR (43, 44
and references therein), (ii) allosteric control of Fbp by AMP and
potential allosteric control of Pck by calcium ions and by ATP and PEP (10, 45, 46) , and (iii) based upon
extrapolation from studies on the mechanism of glgC regulation
via CsrA(23) , post-transcriptional control of mRNA stability
may also be an important determinant of the gluconeogenic capacity of
the cell. A surprising finding of these studies was that the
isozymes which catalyze the phosphofructokinase and pyruvate kinase
reactions are differentially affected by csrA. In both cases
the individual isozymes have been shown to be
allosteric(2, 3, 4, 5, 6, 7, 8, 9) .
They exhibit distinct and fairly complex regulation via several
intracellular metabolites and are also expressed differently in
response to oxygen levels and carbohydrate
sources(11, 12, 13, 14) . In the
case of Pyk, the cellular levels of the of PykF were somewhat higher
than those of PykA in the csrA In viewing our findings in relation to those of
others(2) , it was noted that in addition to being positively
regulated via csrA, the isozymes PfkI and PykF are related in
other ways. (i) They are allosterically strongly regulated by
glycolytic intermediates, PEP in the former case and FBP in the latter.
The isozymes which are either under negative control or unregulated by csrA, PfkII and PykA, respectively, are allosterically
regulated by other kinds of metabolites or show very weak effects of
glycolytic intermediates. (ii) The expression of PfkI and PykF was also
regulated according to the carbohydrate source used for growth, glucose
being superior to pyruvate in both cases(2) . The expression of
the other two isozymes shows at best minimal effects of different
carbon sources. Clearly, positive regulation of gene expression via csrA favors that glycolysis is responsive to intermediates of
carbohydrate metabolism. Neither the pentose phosphate genes nor the
glycolytic isozyme PykA, which can be allosterically activated by
ribose phosphate, are substantially regulated via csrA. We
have previously hypothesized that csrA is part of an adaptive
response pathway(21) , which carries the implicit assumption
that the csrA::kanR mutation reflects a physiological
condition under which this pathway is inactive. When csrA is
inactivated glycolysis decreases, but it is still needed to provide ATP
for the cell, while flux through the gluconeogenesis and glycogen
biosynthesis pathways is increased. Thus, it is significant that when csrA is inactivated, PfkII becomes the major Pfk isozyme. PfkI
is activated by ADP or other NDPs and is strongly inhibited by low
concentrations of PEP(2) . It has been shown that PEP levels
can become quite high under gluconeogenic conditions (38) and
in fact PEP was elevated in the csrA::kanR mutant. Thus, PfkI
may not fulfill the demand for glycolysis under this condition. On the
other hand, PfkII shows at best weak allosteric regulation ( (1) and references therein). Perhaps the preferential
expression of PfkB avoids the synthesis of high levels of PfkI which
would be required for glycolysis in the presence of elevated levels of
PEP. Inactivation of csrA caused PykF to decrease while
PykA was not affected, which should also alter the allosteric
regulation of glycolysis. Although both of these Pyk isozymes are
inhibited by ATP and succinyl-CoA, PykF is activated by FBP, while PykA
is activated by nucleotide monophosphates, ribose phosphate, and other
sugar phosphates (3) . Interestingly, the allosteric regulation
of the committed step of glycogen synthesis, the ADP-glucose
pyrophosphorylase reaction, responds to two of these metabolites, FBP
serving as a strong activator and AMP as an inhibitor(20) . It
is significant that PykF decreases when csrA is inactivated,
since this should prevent FBP from activating the pyruvate kinase
reaction of glycolysis. Thus, more FBP should accumulate if glycolytic
flux is limited by the Pyk reaction and should activate ADP-glucose
pyrophosphorylase in a csrA mutant. This prediction appears to
have been borne out, since FBP levels were significantly higher in the csrA::kanR mutant. Therefore, the effect of csrA on
the allosteric regulation of the pyruvate kinase reaction apparently
represents another way in which csrA negatively influences the
regulation of glycogen biosynthesis and the inactivation of csrA favors glycogen synthesis. Clearly, the physiological
parameters to which the csrA-mediated regulatory system
responds still need to be elucidated. However, two potential
physiological conditions can be excluded from being involved.
Regulation via csrA does not involve differences in anaerobic versus aerobic conditions, since glycogen biosynthesis was
shown previously to be strongly regulated via csrA under both
aerobic and anaerobic conditions (21) and since PfkI, which is
positively regulated via csrA, and PykA, which is not
regulated, are the representative isozymes which are expressed better
under anaerobic conditions. It is also clear that csrA does
not regulate gene expression in response to the growth phase, since the
disruption of csrA did not alter the temporal regulation of
the glg genes (21) or the gluconeogenic enzymes. An
additional relevant observation is that csrA regulates some
genes that are induced in the stationary phase and others that are
expressed only in the exponential phase. The product of the csrA gene is capable of exerting either positive or negative effects on
gene expression and is thus able to regulate gluconeogenesis and
glycolysis in an opposite fashion. Studies are in progress to determine
whether csrA regulates additional metabolic pathways. While
the CsrA gene product is directly involved in the negative regulation
of gene expression, a direct role in the positive regulation of genes (e.g. pykF) remains to be established. The complementation
studies described herein present one kind of evidence suggesting that
genetic activation via csrA may be intrinsically different
from inhibition. When the csrA gene was overexpressed from the
plasmid pCSR10, the negatively regulated genes were consistently found
to be expressed at levels lower than in the csrA
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29096-29104
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)and pyruvate kinase (EC 2.7.1.40) (Pyk)
reactions, which are glycolytic and which provide key points of control
for glycolysis. In E. coli both of these reactions are
catalyzed by pairs of isozymes which are encoded by distinct genes and
which respond allosterically to different cellular
metabolites(2, 3, 4, 5, 6, 7, 8, 9) .
During gluconeogenesis, these two steps of the Embden-Meyerhof pathway
are dependent upon the enzymes fructose 1,6-bisphosphatase (EC
3.1.3.11) (Fbp) and phosphoenolpyruvate synthetase (EC 2.7.9.2) (Pps).
Fbp activity is also allosterically regulated(10) .
Bacterial Strains, Bacteriophage, and
Plasmids
The relevant properties of the strains, phage, and
plasmids used in the present study are listed in Table 1.
Media, Growth Conditions, and General
Procedures
Cultures were inoculated for growth curves by adding
1 volume of an overnight culture to 400 volumes of freshly prepared
Kornberg medium (containing 0.5% glucose(26) ) and were grown
at 37 °C with rapid gyratory shaking. For detection of endogenous
glycogen, cultures were streaked onto Kornberg agar medium containing
1% glucose, grown overnight at 37 °C, and stained with iodine
vapor(21) . MacConkey agar (Difco) was used to determine the
Lac phenotype. Ampicillin (100 µg/ml), kanamycin (100 µg/ml),
and diaminopimelic acid (50 µg/ml) were sterilized by filtration
and were included as needed in the media. To study enzyme activities
throughout the growth curve, cells were harvested at the indicated time
points by centrifugation at 4 °C, washed twice in the appropriate
buffer, as described in the corresponding references below, weighed,
and stored at -80 °C until use. The cells (0.5 g) were thawed
and suspended in 10 ml of lysis buffer and disrupted using an ice-cold
French pressure cell at 10,000 p.s.i. The cell lysate was collected on
ice and centrifuged for 10 min at 14,000 
g. The
supernatant solution was used to determine enzyme activity on the day
of preparation and was assayed for total protein. Each of these
experiments was performed in entirety at least twice.Enzyme and Protein Assays
Previously published
methods were used to assay the activities of
fructose-1,6-bisphosphatase(31) , phosphoenolpyruvate
synthetase(32) , phosphoglucomutase (Pgm) (EC
5.4.2.2)(33) , glucose-6-phosphate isomerase (Pgi) (EC
5.3.1.9)(34) , phosphofructokinases I and II(2) ,
triose-phosphate isomerase (Tpi) (EC 5.3.1.1)(35) , enolase
(Eno) (EC 4.2.1.11) (36) and pyruvate kinases F and
A(2) . Each of the respective reactions was started by the
addition of enzyme extract and was coupled to the reduction of
NAD or the oxidation of NADPH, following the change in
absorbance at 340 nm in a Gilford recording spectrophotometer, with the
exception of the phosphoenolpyruvate synthetase assay, which was
measured as the rate of ATP-dependent disappearance of pyruvate. Values
for enzyme activities were determined within the linear range with
respect to the amount of extract added, as determined for each and
every sample. One unit of enzyme activity is defined as 1 µmol of
product generated or substrate utilized per min under the published
reaction conditions. Total protein was measured by the bicinchoninic
acid method using bovine serum albumin as the standard(37) .
-Galactosidase specific activity was assayed and calculated as
described previously(25) .Determination of Intracellular Metabolites
The
metabolites ATP, ADP, AMP, fructose 1,6-bisphosphate (FBP), and
phosphoenolpyruvate (PEP) were extracted and were quantified
fluorimetrically using a Shimadzu RF 5000U fluorometer, according to
previously published methods(38) . The reproducibility of these
measurements was established by conducting at least three
determinations for each sample.Genetic and Molecular Biology
Techniques
Transformation, P1 transduction, and other molecular
biology techniques were conducted as described
previously(26, 21) .
Negative Regulation of Gluconeogenesis via
csrA
In the absence of exogenous hexose, and even in the
presence of glucose(31) , the gluconeogenic enzymes are present
and may be used to form glucose 1-phosphate, which in turn may be
converted to a variety of compounds, including the precursor of
bacterial glycogen, ADP-glucose. We previously obtained two kinds of
preliminary evidence suggesting that csrA negatively regulates
gluconeogenesis. First, the expression of a `lacZ transcriptional fusion for the gene encoding phosphoenolpyruvate
carboxykinase, pckA, was elevated approximately 2-fold in a csrA::kanR mutant. Second, a multicopy plasmid which
overexpresses the csrA gene, pCSR10, resulted in the inability
of strains to grow on gluconeogenic compounds as sole sources of
carbon(21) . To further explore the potential role of csrA in regulating gluconeogenesis, the effects of csrA on the
specific activities of Fbp and Pps were determined. Fig. 1(A and B) shows that Fbp specific
activity increased from 7- to 10-fold as cultures progressed from the
exponential phase into early stationary phase of growth and thereafter
declined to mid-log levels. During the early stationary phase, the
levels of Fbp were approximately 1.5-2-fold higher in the csrA::kanR mutant than in the isogenic csrA strain. As shown in Fig. 1B, the effects of the csrA::kanR mutation on Fbp were complemented by pCSR10. In fact,
pCSR10-containing cells possessed significantly lower levels of Fbp
activity than the csrA
strain. The specific
activity of Pps was 2-4-fold higher in the csrA::kanR mutant, and similar to that of Fbp, it was highest in both strains
during the early stationary phase (Fig. 1C).
)
(
) and TR1-5BW3414 (csrA::kanR) (). Growth (A
) is shown for BW3414 (
) and
TR1-5BW3414 (
). B compares Fbp activity in strains
BW3414[pUC19] (), TR1-5BW3414[pUC19]
(), and TR1-5BW3414[pCSR10] (
). Growth is
shown for BW3414 ([circo), TR1-5BW3414 (), and
TR1-5BW3414[pCSR10]
(
).
strain (Fig. 1D). Pgm activity in both strains increased
approximately 2-fold as cultures entered the stationary phase. The
kinetics of expression of Fbp, Pps, and Pgm in the csrA::kanR mutant were approximately parallel to those of the isogenic csrA
strain, as was shown previously for the
expression of the glycogen biosynthetic genes and pckA,
indicating that the regulation of these systems by csrA is
superimposed upon the stationary phase control (21) .
Positive Regulation of Glycolysis by csrA
Carbon
flux through the Embden-Meyerhof pathway must be regulated if a
glycolytic/gluconeogenic futile cycle is to be avoided. In view of the
impact of csrA on glycogen biosynthesis and on the
gluconeogenic enzymes, it was of interest to determine whether csrA affects the activities of the glycolytic enzymes. The specific
activities of three of the bidirectional enzymes of this pathway, Pgi,
Tpi, and Eno, were determined throughout the growth curve, as were the
unidirectional, allosterically regulated glycolytic enzymes, PfkI,
PfkII, PykA, and PykF. strain relative to
the csrA::kanR strain throughout the growth curve, indicating
that these enzymes are under positive control of csrA.
Comparison of Fig. 1and Fig. 2also shows that the
specific activities of the glycolytic enzymes were much higher than the
gluconeogenic enzymes. This is consistent with the idea that levels of
Embden-Meyerhof enzymes generally are present in significantly higher
levels to meet the glycolytic requirements relative to the
gluconeogenic requirements and indicates that csrA is a
positive regulator of glycolysis.
) and TR1-5BW3414 (csrA::kanR). Sample identities are as shown for Fig. 1A.
parent
strain relative to the csrA::kanR mutant (Fig. 3A). In contrast, the activity of PfkII (Fig. 3B) was higher (1.3-4-fold) in the mutant
than in the wild type strain. Therefore, these two glycolytic isozymes
exhibited reciprocal regulation by csrA. In the wild type
strain, PfkI represented approximately 80% of the total Pfk activity
throughout the growth curve. In the csrA::kanR mutant, PfkII
constituted approximately 30% of the total activity in mid-exponential
phase and increased to 80% of the total Pfk activity as the culture
entered stationary phase. Therefore, under conditions that were optimal
for glycogen synthesis, i.e. in the csrA::kanR mutant
during the early stationary phase of growth, PfkII was the predominant
isozyme.
)
and TR1-5BW3414 (csrA::kanR) during the growth curve.
Sample identities are as shown in Fig. 1A.
and the csrA::kanR strains.
PykF activity was 4.3-9.4-fold higher in the wild type strain
during the growth curve. However, PykA levels were similar in the two
strains. Therefore, under the conditions of these studies, csrA appears to positively regulate the levels of PykF without altering
the level of PykA.
-galactosidase activity
expressed from chromosomally encoded pykF`-`lacZ or pykA`-`lacZ translational fusions was measured in isogenic csrA and csrA::kanR strains (Fig. 4).
-Galactosidase expressed from the pykF`-`lacZ fusion was up to 3.8-fold higher in the csrAstrain versus the csrA::kanR mutant (Fig. 4A). On the other hand, no
appreciable difference was observed in the expression of the pykA`-`lacZ fusion in the relevant strains (Fig. 4B). Therefore, csrA determines PykF
specific activity by enhancing pykF gene expression, rather
than by altering PykF enzymatic activity.
-Galactosidase activities expressed from chromosomally encoded pykF`-`lacZ (A) and pykA`-`lacZ fusions (B) were monitored in the csrA strains SB589 (pykF`-`lacZ) and SB588 (pykA`-`lacZ) (
) and the isogenic csrA::kanR mutants TR1-5SB589 and TR1-5SB588 (). Growth (A
) is shown as
for the parent strains and
for the csrA::kanR mutants.
Complementation of the csrA::kanR Mutation by
pCSR10
It was possible that the effects of the csrA::kanR mutation on the glycolytic and gluconeogenic enzymes could be a
direct consequence of the inactivation of csrA or could result
from polarity effects on genes distal to csrA. Therefore, the
capacity of pCSR10 (a pUC19-derived plasmid that contains a
0.5-kilobase insert encoding only the csrA gene, (21) ) to complement the effects of the csrA::kanR mutation on the levels of several enzymes was
tested in the isogenic strains BW[pUC19] (csrA
), TR1-5BW[pUC19] (csrA::kanR), and TR1-5BW3414[pCSR10] (csrA-overexpressing). Table 2summarizes the results of
these experiments, which showed that the introduction of pCSR10 into
the csrA::kanR strain caused the enzymes that were observed to
be negatively regulated via csrA, i.e. Fbp, Pps, Pgm,
and PfkII, to decrease below their (csrA
)
wild type levels. The plasmid pCSR10 also complemented the effect of
the csrA::kanR mutation on the levels of the enzymes that were
observed to be positively regulated via csrA, Pgi, Tpi, PfkI,
Eno, and PykF. However, overexpression of csrA from pCSR10 did
not result in greater than wild type expression of these enzymes. It
may be concluded from the results of this experiment that the
inactivation of the csrA gene itself was responsible for the
effects of the csrA::kanR mutation on gluconeogenic and
glycolytic enzyme levels.
Effect of csrA on Enzymes Expressed in a
To determine whether the csrA::kanR mutation
alters levels of glycolytic and gluconeogenic enzymes as an indirect
consequence of the dramatic effect that it has on carbon flux into
glycogen(21) , the csrA::kanR mutation was transduced
into G6MD3, a strain in which the glgBX and glgCAP operons have been deleted and which is incapable of synthesizing
either glycogen or ADP-glucose. Levels of glycolytic and gluconeogenic
enzymes were assayed in the resulting isogenic strains. Table 3summarizes these experiments and shows that at 8 h the
specific activities of Fbp, Pps, Pgm, and PfkII were 2.3-, 2.5-, 1.6-,
and 2-fold higher in the csrA::kanR mutant than in the csrA
-glg
Mutant strain. Specific activities of Pgi,
PfkI, Tpi, Eno, PykF, and PykA were 2.1-, 6.0-, 1.4-, 2.0-, 2.8-, and
1.1-fold higher in the wild type strain as compared with the csrA::kanR mutant. These values are reasonably consistent with
values from the strains that synthesize glycogen (Fig. 1Fig. 2Fig. 3) and show that the effects of csrA on glycolytic and gluconeogenic enzyme levels is not a
secondary regulatory response to the enhanced carbon flux toward
glycogen in the csrA::kanR mutant. Of course, these results do
not exclude the possibility that intracellular metabolites other than
those affected by glycogen synthesis may serve to indirectly mediate
the effects of csrA on the levels on one or more of these
enzymes.
Effect of csrA on the Expression of Genes of the Pentose
Phosphate Pathway
The pentose phosphate pathway provides NADPH
and biosynthetic intermediates for the cell, and two genes of the
pathway, zwf, encoding glucose-6-phosphate dehydrogenase, and gnd, encoding 6-phosphogluconate dehydrogenase, have been
shown to exhibit growth rate-regulated
expression(28, 40) . The possibility that csrA regulates the expression of these two genes was tested by
determining -galactosidase specific activities expressed from the
chromosomally encoded gnd`-`lacZ transcriptional and
translational fusions of strains HB354 and HB582, respectively, and zwf`-`lacZ transcriptional and translational fusions of
strains HB301[DR52] and HB301[
DR104],
respectively, in isogenic csrA
and csrA::kanR strains throughout their growth curves (data not
shown). The gnd`-`lacZ translational fusion exhibited very
weak but reproducible positive regulation by csrA, i.e. slightly higher expression was observed in the csrA
strain. No appreciable effect was
observed for the zwf`-`lacZ transcriptional or translational
fusions or the gnd`-`lacZ transcriptional fusion, indicating
that at least under our experimental conditions, csrA is
probably not a significant regulator of the pentose phosphate pathway.
Effects of csrA on Metabolite Levels and Energy
Charge
The above observations established that csrA is
an important modulator of intermediary carbohydrate metabolism in E. coli. Further evidence for such a role was obtained by
measuring the levels of five key intracellular metabolites in csrA and csrA::kanR strains. Table 4and Table 5show the effects of the csrA mutation on adenylates and energy charge, FBP and PEP. The
metabolite levels and energy charge in the csrA
strain were similar to those previously determined for E.
coli(38, 42) . On the other hand, the csrA::kanR mutant had lower levels of ATP and higher levels of
AMP and ADP throughout the growth curve, resulting in a lower energy
charge. The intracellular concentrations of FBP and PEP were both
elevated in the csrA::kanR mutant. The decrease in energy
charge in the mutant is consistent with a decrease in glycolysis and an
increase in gluconeogenesis and glycogen synthesis and was predictable
based upon the effect of the mutation on enzyme specific activities.
The elevated level of PEP in the mutant reflects enhanced synthesis of
this metabolite via Pps and Pck, as well as a decrease in its
utilization by Pyk. The elevated FBP levels appear to result from a
decrease in glycolytic flux following the Pfk reaction, and in
particular, the effect of the csrA::kanR mutation on the
expression of pykF is probably important.
) by csrA. Metabolic pathways that have been examined to date are
shown in bold lettering.
strain, and
under our experimental conditions PykF exhibited strong positive
regulation by csrA, while PykA was not regulated. The PfkI
isozyme was the predominant form in the csrA
strain, as has been observed previously(2) . PfkI is an
essential enzyme in the cell, and the inactivation of the structural
gene encoding PfkI (pfkA) alone has been shown to cause a
growth defect on substrates which must be metabolized via this step of
glycolysis. On the other hand, PfkII has generally been considered to
be dispensable to the cell, since a pfkB mutant has no
observable growth defect. However, it should be emphasized that the
PfkII isozyme is capable of performing this step of glycolysis, and
supressors of the pfkA mutation have been isolated and shown
to result from the overexpression of pfkB(47) . The
current studies show that the ``minor'' form of Pfk (PfkII)
predominates when csrA has been disrupted. We may conclude
that a pfkB
cell has the capacity to produce
more PfkII than normally is present and that the elevation of PfkII in
the csrA::kanR strain is not a compensatory response to the
decrease in PfkI.
strain. On the other hand, none of the genes which are apparently
subject to positive regulation by csrA were expressed at
higher levels in the pCSR10-containing strain than in the wild type csrA
strain. These and many other questions
concerning the biological function and mechanism of csrA remain to be answered.
)
We are grateful for the generous gifts of strains by
the individuals listed in Table 1, especially Stefan Bledig (NSC
Technologies), who provided strains prior to publication. We thank Bob
Gracey and Paul Cook and Bill Karsten who provided instrumentation
and/or advice for conducting some of the enzyme and metabolite assays.
We also thank Wayne Nicolson, Umit Yuksel, and Mark Hart for providing
critiques of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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