(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) ()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) .
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)
(
) 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]
(
).
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 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) .
Fig. 2shows that Pgi (A),
Tpi (B), and Eno (C) activities were from 1.5- to
3-fold higher in the csrA 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.
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) and TR1-5BW3414 (csrA::kanR). Sample identities are as shown for Fig. 1A.
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 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.
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)
and TR1-5BW3414 (csrA::kanR) during the growth curve.
Sample identities are as shown in Fig. 1A.
Fig. 3(C and D) also shows the
specific activities of the pyruvate kinase isozymes PykF and PykA in
the csrA 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.
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, -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 csrA
strain 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.
Figure 4:
Effects of csrA on the expression
of pykF`-`lacZ and pykA`-`lacZ translational fusions.
-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.
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 () by csrA. Metabolic pathways that have been examined to date are
shown in bold lettering.
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 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.
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 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.