From the
In Escherichia coli, inorganic orthophosphate regulates
cAMP levels by acting at two separate loci. First, adenylyl cyclase
activity measured in permeabilized cells of E. coli is
substantially stimulated by physiological concentrations of inorganic
phosphate. This stimulation does not require the presence of cAMP
phosphodiesterase activity. Second, measurements of cAMP
phosphodiesterase activity in permeabilized cells show a dose-dependent
inhibition of that activity by inorganic orthophosphate. A model is
proposed in which inorganic orthophosphate serves as a multifaceted
regulator of cAMP levels by both stimulating synthesis and inhibiting
degradation of the nucleotide.
In Escherichia coli, cAMP complexed to the cAMP
receptor protein serves as an important regulator of the transcription
of a number of inducible genes
(1) . The intracellular level of
the cyclic nucleotide is determined by the relative rates of synthesis,
degradation, and excretion of cAMP. A number of factors have been shown
to influence these three processes.
The enzyme adenylyl cyclase,
responsible for the synthesis of cAMP, has been shown to be regulated
by a variety of agents. Although the mechanism is not completely
understood, the proteins of the sugar transport system known as the
phosphoenolpyruvate:sugar phosphotransferase system play an important
role in regulation of the enzyme activity
(2) . Of relevance to
the current study, it has been shown that inorganic orthophosphate
(P
The studies presented here show that the previously observed
stimulation of adenylyl cyclase by P
While these results indicate that P
The concentration dependence for the inhibition
by P
There is evidence that cAMP
phosphodiesterase plays some role in regulating cellular levels of cAMP
in E. coli. Fraser and Yamazaki
(8) showed that, under
identical growth conditions, two sets of isogenic strains (one
containing and one deficient in the cAMP receptor protein) differing in
the absence or presence of the gene encoding cAMP phosphodiesterase
(PDE) showed cAMP pool size differences. In both sets, the cAMP pool
size was greater in the PDE
Until this point, there has been little information
available concerning the regulation of E. coli cAMP
phosphodiesterase activity. Previous reports have suggested the
presence of two activities responsible for cAMP
degradation
(10, 11, 12) . One is an enzyme that
hydrolyzes both cAMP and cGMP
(12) ; its activity is stimulated
by Ca
The
unexpected finding, documented in this study, is that P
Under conditions of carbon source starvation, E.
coli cells accumulate P
Fig. 3
presents a model in which cellular
levels of P
Permeabilized cells of a
cpd
) stimulates adenylyl cyclase activity
(3) and
that this stimulatory effect is dependent on the presence of proteins
of the phosphoenolpyruvate:sugar phosphotransferase system
(4) .
is not due to an
effect on the degradation process. However, we show here an independent
and opposite (inhibitory) effect of P
on cAMP
phosphodiesterase activity. The net result is that elevations in
P
concentration in E. coli can lead to substantial
increases in cAMP levels by a combination of the stimulation of
synthesis and inhibition of degradation of the cyclic nucleotide.
Materials
Two isogenic strains of E. coli were used in this
study. AB-257 (met) (strain 62) contains the
gene (cpd) for cAMP phosphodiesterase; AB-257
(cpd
, met
,
trp
) (strain 63) is deficient in cAMP
phosphodiesterase. These strains, from the laboratory of E. A. Adelberg
(5), are from the collection of Philip Hartman at Johns Hopkins
University. [
-
P]ATP and
[
H]cAMP were from DuPont NEN. All other materials
were from standard sources.
Methods
Adenylyl Cyclase Activity
E. coli cells
were grown in LB medium to an A of
0.5-0.6. An aliquot (50 ml) of the culture was harvested by
centrifugation at 3000
g for 10 min. The pelleted
cells were washed with 20 mM Bicine
(
)
(sodium) buffer, pH 8.5, and then resuspended in 1 ml of
the same buffer. The washed cells were then permeabilized by treatment
with 1% toluene for 10 min. Assays for adenylyl cyclase were then
performed as described previously
(3, 6) (see
Fig. 1
).
Figure 1:
Effect of inorganic orthophosphate on
adenylyl cyclase activity in cells of strains with and without cAMP
phosphodiesterase (PDE). Permeabilized cells of
cpd (strain 62) and cpd
(strain 63) strains (see ``Experimental Procedures'')
were tested for adenylyl cyclase activity in the absence and presence
of P
(20 mM) as described under
``Methods.'' Incubation mixtures (500 µl) contained
Bicine, pH 8.5 (25 mM), dithiothreitol (1 mM),
MgCl
(10 mM), [
-
P]ATP
(1 mM, 34 cpm/pmol). After addition of permeabilized cells
(strain 62, 0.639 mg of protein; strain 63, 0.553 mg of protein), the
mixtures were incubated at 30 °C. At the indicated times, aliquots
(0.1 ml) were removed and processed for determination of cAMP produced
from [
-
P]ATP as described previously (3).
The data was fit to a linear regression using Origin Version 3.5
(Microcal, Inc.).
cAMP Phosphodiesterase Activity
E. coli cells were washed and prepared as for adenylyl cyclase assays. The
toluene-treated cells were incubated at 30 °C with reaction
mixtures essentially identical to those for adenylyl cyclase assays
(Bicine buffer (25 mM, pH 8.5), dithiothreitol (1
mM), MgCl (10 mM)) except that the
substrate was [
H]cAMP (0.1 mM, 1700
cpm/nmol). At the indicated time intervals, aliquots (0.1 ml) were
withdrawn from master incubation mixtures and processed by the
two-column procedure (7) for purification of cAMP. Radioactivity in the
purified [
H]cAMP samples was determined by
scintillation counting. The data were fit to a linear regression using
Origin, Version 3.5 (Microcal, Inc., Northampton, MA) (see
Fig. 2
and ).
Figure 2:
Effect of inorganic orthophosphate on cAMP
phosphodiesterase activity in cells of strains with and without cAMP
phosphodiesterase (PDE). Permeabilized cells of
cpd (strain 62) and cpd
(strain 63) strains (see ``Experimental Procedures'')
were tested for cAMP phosphodiesterase activity in the absence and
presence of P
(20 mM) as described under
``Methods.'' The data was fit to a linear regression as
described under ``Methods.'' Each 500-µl reaction mixture
contained 0.639 mg of protein of strain 62 cells or 0.553 mg of protein
of strain 63 permeabilized cells.
Inorganic Orthophosphate Stimulates Adenylyl Cyclase
Activity Independent of an Effect on cAMP Phosphodiesterase
Activity
In agreement with the results of previous studies on
adenylyl cyclase
(3) , Fig. 1shows that permeabilized
cells of a wild-type strain of E. coli (strain 62) contain
adenylyl cyclase activity that is stimulated by P. The
possibility that this stimulatory effect, apparently on AC, might
actually be due to an inhibitory effect on cAMP phosphodiesterase was
evaluated. The data in Fig. 1show that permeabilized cells of a
strain (strain 63) deficient in cAMP phosphodiesterase also contains
P
-activated adenylyl cyclase. The observed stimulation by
P
in this experiment is only about half as great in the
cAMP phosphodiesterase-deficient strain as in the wild-type strain. As
pointed out previously
(3) , the extent of activation by P
can vary significantly from one preparation of toluene-treated
cells to another. Therefore, no significance is attributed to the
observed 2-fold difference in P
-activation in this
experiment.
stimulation of adenylyl cyclase does not require the
participation of cAMP phosphodiesterase, the experiments do not address
the question of a possible effect of P
on phosphodiesterase
activity.
Inorganic Orthophosphate Inhibits cAMP Phosphodiesterase
Activity
An independent evaluation of the effect of P on the cAMP phosphodiesterase activity in E. coli strain
62 permeable cells was carried out. The data shown in
Fig. 2
indicate that there is a substantial inhibition of the
degradation of cAMP by 20 mM P
. Control
experiments using a strain (strain 63) deficient in cAMP
phosphodiesterase activity indicate that the assay method used and the
inhibitory effect of P
are specific for cAMP
phosphodiesterase.
of cAMP phosphodiesterase activity was studied
(). Approximately 40% inhibition of cAMP phosphodiesterase
activity was observed at a P
concentration of 1
mM.
DISCUSSION
A major element in the regulation of the activity of E.
coli adenylyl cyclase is P. While it has been shown
that P
inhibits the enzyme activity in either broken cell
extracts
(4) or with the purified enzyme, there is a substantial
stimulation of the activity by P
in permeable cell
preparations
(3) . The interpretation of these findings is that
P
modulates the activity of adenylyl cyclase when the
enzyme is in a physiologically relevant complex with other proteins.
Numerous studies suggest that adenylyl cyclase activity is regulated by
interaction with one or more of the proteins of the
phosphoenolpyruvate:sugar phosphotransferase system. Consistent with
this interaction is the observation that, while permeable cells of a
wild-type strain of E. coli contain adenylyl cyclase that is
stimulated by P
, the adenylyl cyclase in a strain deficient
in phosphoenolpyruvate:sugar phosphotransferase system proteins is not
stimulated by P
(4) .
than in the
PDE
strains. Other evidence that the pool size of cAMP
is greater in cpd
strains comes from the experiments
of Alper and Ames
(9) . They showed that cpd
strains of Salmonella typhimurium are extremely
sensitive to high levels of cAMP and that such strains have a 10-fold
reduced requirement for exogenous cAMP for the expression of catabolic
operons.
, Fe
, or Co
.
Its M
was estimated to be 31,000. The second is an
enzyme that hydrolyzes cAMP, but not cGMP
(11) ; its
M
was suggested to be 28,000. In surveying various
buffers for estimating the pH optimum, it was noted that both phosphate
and succinate ions inhibit the activity, but no physiological
significance for this observation was suggested
(11) . Purified
preparations of this enzyme were dependent for activity on the presence
of a proteinaceous activator and a reducing agent
(11) .
regulates not only adenylyl cyclase, but also cAMP
phosphodiesterase. The data presented in Fig. 2and
establish that the same type of permeable cell preparations
that exhibit a P
-dependent stimulation of adenylyl cyclase
activity also show a P
-dependent inhibition of cAMP
phosphodiesterase activity. It is worth noting that, while P
stimulates adenylyl cyclase activity in permeable cells but not
broken cell extracts, the inhibitory effect of P
on cAMP
phosphodiesterase activity is observed both in permeable cells and
broken cell extracts. A concentration of 20 mM P
was shown to cause a 77% inhibition of cAMP phosphodiesterase
activity in an extract of strain 62 prepared by passage of cells
through a French pressure cell (data not shown). While the cAMP
phosphodiesterase activity(ies) in E. coli appear to be
somewhat more sensitive to inhibition by P
in permeable
cells than in broken cells, the data suggest that the inhibitory effect
of P
on cAMP phosphodiesterase activity does not absolutely
require a complex of cAMP phosphodiesterase with other proteins as is
the case with the stimulation of adenylyl cyclase activity by P
(see above). The precise mechanism by which P
regulates cAMP phosphodiesterase activity remains to be
established.
to levels of approximately
20-30 mM(13) . After addition of glucose to a
suspension of carbon-starved E. coli, the cellular P
level decreases substantially
(14) . Under typical growth
conditions, there are numerous controls (the Pho regulon)
(15) that maintain the cytoplasmic P
at a high
concentration (approximately 10 mM). Our approach to studying
the concentration dependence for inhibition of cAMP phosphodiesterase
activity was to eliminate, by toluene treatment, the permeability
barrier in the cells. The studies shown in indicate that
the P
concentrations that are effective in inhibition of
cAMP phosphodiesterase activity range from 0.5 to 5 mM. This
correlation suggests strongly that physiological variations in the
P
level are an important factor in regulating cellular
levels of cAMP.
operate by a dual mechanism to fine tune cAMP
levels. Under conditions of carbon starvation, cellular levels of
P
increase. This is accompanied by an increase in cellular
cAMP levels, due to both a stimulation of adenylyl cyclase activity and
an inhibition of cAMP phosphodiesterase activity. The presence of an
excess of a metabolizable carbon source is associated with a decrease
in cellular P
concentration and a corresponding decrease in
the combined activation of adenylyl cyclase and inhibition of cAMP
phosphodiesterase. Thus, the phenomenon of catabolite repression,
involving the regulation of cAMP levels, appears to be crucially
dependent on variations in cellular P
. The regulation of
the activity of the enzymes involved both in the synthesis and
degradation of cAMP comprises a complex metabolic cascade that confers
on E. coli a highly sensitive response to the availability of
carbon sources.
Figure 3:
A model for the dual regulation of levels
of cAMP in E. coli. Consistent with the data shown in Figs. 1
and 2 and Table I, P stimulates the accumulation of cAMP by
both stimulating the activity of adenylyl cyclase and inhibiting the
activity of cAMP phosphodiesterase
(PDE).
Table:
Effect of inorganic orthophosphate concentration
on cAMP phosphodiesterase activity
strain (strain 62) of E. coli were tested for cAMP phosphodiesterase activity at the indicated
concentrations of added inorganic orthophosphate as described under
``Methods.'' Incubation mixtures were in a total volume of 1
ml and contained 1.164 mg protein of permeabilized cells. Samples were
removed from the master incubation mixtures at 5-min intervals over a
period of 30 min. The data was fit to a linear regression as described
under ``Methods.'' The activity in the absence of added
P
was 4.456 nmol of cAMP degraded in 30 min/0.1 ml of
reaction mixture.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.