©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Dual Mechanism for Regulating cAMP Levels in Escherichia coli(*)

Niranjana Amin (§) , Alan Peterkofsky (¶)

From the (1) Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) stimulates adenylyl cyclase activity (3) and that this stimulatory effect is dependent on the presence of proteins of the phosphoenolpyruvate:sugar phosphotransferase system (4) .

The studies presented here show that the previously observed stimulation of adenylyl cyclase by P 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.


EXPERIMENTAL PROCEDURES

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.




RESULTS

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.

While these results indicate that P 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.

The concentration dependence for the inhibition by P 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) .

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 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.

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, 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) .

The unexpected finding, documented in this study, is that P 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.

Under conditions of carbon source starvation, E. coli cells accumulate P 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.

Fig. 3 presents a model in which cellular levels of P 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

Permeabilized cells of a cpd 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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 36, Rm. 4C-11, Bethesda, MD 20892. Tel. 301-496-2408; Fax: 301-402-0270; E-mail: alan@codon.nih.gov.

The abbreviation used is: Bicine, N,N-bis(2-hydroxyethyl)glycine.


REFERENCES
  1. Peterkofsky, A., Reizer, A., Reizer, J., Gollop, N., Zhu, P., and Amin, N.(1993) Prog. Nucleic Acids Res. Mol. Biol. 44, 31-65 [Medline] [Order article via Infotrieve]
  2. Peterkofsky, A.(1977) Trends Biochem. Sci. 2, 12-14
  3. Harwood, J. P., and Peterkofsky, A.(1975) J. Biol. Chem. 250, 4656-4662 [Abstract]
  4. Liberman, E., Reddy, P., Gazdar, C., and Peterkofsky, A.(1985) J. Biol. Chem. 260, 4075-4081 [Abstract]
  5. Bachmann, B. J.(1987) in Escherichia coli and Salmonella typhimurium, (Ingraham, J. L., Low, K. B., Magasanik, B., Schaecter, M., and Umbarger, H. E., eds) pp. 1190-1219, American Society for Microbiology, Washington, D.C.
  6. Amin, N., and Peterkofsky, A.(1992) Biochem. Biophys. Res. Commun. 182, 1218-1225 [Medline] [Order article via Infotrieve]
  7. Salomon, Y., Londos, C., and Rodbell, M.(1974) Anal. Biochem. 58, 541-548 [Medline] [Order article via Infotrieve]
  8. Fraser, A. D. E., and Yamazaki, H.(1978) Can. J. Microbiol. 24, 1423-1425 [Medline] [Order article via Infotrieve]
  9. Alper, M. D., and Ames, B. N.(1975) J. Bacteriol. 122, 1081-1090 [Medline] [Order article via Infotrieve]
  10. Nielsen, L. D., Monard, D., and Rickenberg, H. V.(1973) J. Bacteriol. 116, 857-866 [Medline] [Order article via Infotrieve]
  11. Nielsen, L. D., and Rickenberg, H. V.(1974) Methods Enzymol. 38, 249-256 [Medline] [Order article via Infotrieve]
  12. Iwasa, Y., Yonemitsu, K., and Miyamoto, E.(1981) FEBS Lett. 124, 207-209 [CrossRef][Medline] [Order article via Infotrieve]
  13. Rosenberg, H., Russell, L. M., Jacomb, P. A., and Chegwidden, K. (1982) J. Bacteriol. 149, 123-130 [Medline] [Order article via Infotrieve]
  14. Reddy, P., Liberman, E., Gazdar, C., and Peterkofsky, A.(1985) in Gene Manipulation and Expression (Glass, R. E., and Spizek, J., eds) pp. 318-338, Croom Helm, Kent, United Kingdom
  15. Rao, N. N., Kar, A., Roberts, M. F., Yashphe, J., and Torriani-Gorini, A.(1994) in Phosphate in Microorganisms (Torriani-Gorini, A., Yagil, E., and Silver, S., eds) pp. 22-19, ASM Press, Washington, D.C.

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.