Regulation of Escherichia coli adenylate cyclase activity by the phosphoenolpyruvate:sugar phosphotransferase system

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1 FEMS Microbiology Reviews 63 (1989) Published by Elsevier 103 FER Regulation of Escherichia coli adenylate cyclase activity by the phosphoenolpyruvate:sugar phosphotransferase system Alan Peterkofsky, ngrid Svenson and Niranjana Amin Laboratory ofbiochemical Genetics, National Heart, Lungand Blood nstitute, National nstitutes ofhealth, Bethesda, MD, U.S.A. 1. NTRODUCTON Received 20 October 1988 Accepted 20 October 1988 Key words: PTS; norganicphosphate; Phosphorylation Since the variety of genes that are controlled by camp respond to different concentrations of the regulatory nucleotide, it is of vital importance to the cell to control the cellular concentration of camp. An important aspect of the regulation of camp concentration deals with the factors that control the activity of the enzyme adenylate cyclase [1]. t has been well documented that this enzyme is subject to a complex regulatory mechanism that involves both the sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as well as the cellular pool of inorganic orthophosphate (Pi) [2-4]. The mechanism of the regulation of the activity of adenylate cyclase is the subject of this review. 2. REGULATON OF ADENYLATE CYCLASE ACTVTY 2.1. Effect of glucose on adenylate cyclase activity When glucoseis added to a washed cell suspension of E. coli, there is a transient drop in the Correspondence to: A. Peterkofsky, Laboratory of Biochemical Genetics, National Heart, Lung and Blood nstitute, National nstitutes of Health, Bethesda, MD 20892, U.S.A. cellular concentration of camp; at the time that the glucose is depleted from the medium as a result of uptake of the sugar into the cells, the cellular level of camp returns to the original concentration [5]. To a very great degree, this sugar-dependent variation in cyclic nucleotide concentration reflects an effect of the sugar on the adenylate cyclase-catalyzed synthesis of camp. Using preparations of E. coli that were made permeable to small molecules by treatment with toluene, Harwood and Peterkofsky [2) showed that glucose elicited a rapid and substantial inhibition of adenylate cyclase activity. Since the inhibitory effect could be observed by the addition of glucose, but not glucose-6-phosphate, extensive metabolism of the sugar was clearly not required. The observation that glucose did not inhibit adenylate cyclase activity of either broken cell preparations [2] or purified enzyme [6) suggested that the effect of glucose was not directly on the enzyme itself. Experiments using permeable cell preparations of strains of E. coli carrying mutations in the sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (FTS) provided additional support for that idea. A strain of E. coli that was mutated in the membrane-associated glucose-specific Enzyme components of the PTS was tested for adenylate cyclase activity in the absence or presence of sugar substrates for these Enzyme species. While a /89/$03.50 ~ 1989 Federation of European Microbiological Societies

2 104 Table 1 Adenylate cylase activity in enzyme mutants Strain Genotype Adenylate cyclase activity" 1100 wild-type 2000 SB2260 pts (leaky) pts (leaky) pts (deletion) 86 N2680 ptsl (deletion) 165 Cultures of the indicated strains were grown in salts medium supplemented with nutrient broth (1%). Cell suspensions at midlogarithmic phase were processed for adenylate cyclase activity in toluene-treated cells [2]... pmol of camp formedymg of protein/h. From [7]. wild-type control had adenylate cyclase activity that was markedly inhibited by glucose, a-methylglucoside or 2-deoxyglucose, the mutant deficient in the Enzyme components specific for these sugars (GPT-MPT-) had adenylate cyclase activity that was resistant to inhibition by these sugars [4]. t was concluded that inhibition by glucose of adenylate cyclase activity required a functional PTS Enzyme and that the inhibitory effect of the sugar was either due to an inhibitory complex of the sugar, Enzyme and adenylate cyclase or some other feature of the PTS-mediated transport of the sugar. The data presented in Table 1 suggest that the regulatory effect of glucose on adenylate cyclase activity is not due to a complex solely involving glucose, Enzyme ngl c and adenylate cyclase but must also involve one or more of Table 2 Adenylate cyclase activity in permeable cells and extracts Additions Adenylate cyclase activity" Permeable cells Extracts E. coli strain 433 (wild-type) was grown in salts medium supplemented with nutrient broth (1%) and glucose (0.2%). At midlogarithmic phase, suspensions of cells were either processed for preparation of permeable cells or extracts. Adenylate cyclase activity was measured in these preparations in either the presence or absence of 20 mm K 2HP04... nmol camp ormedy'mg of proteinjh. From [8]. the 'soluble' components of the PTS. Permeabilized cells of strains of E. coli characterized as leaky or deletion mutants in the gene for Enzyme of the PTS were assayed for adenylate cyclase activity. While a typical wild-type strain had a specific activity of 2000, the mutants had adenylate cyclase activities that ranged from 5 to 15% of the wild-type. These studies established that Enzyme ngl c is necessary for the glucose-dependent regulation of adenyl ate cyclase activity and Enzyme of the PTS is necessary for maximal adenylate cyclase activity Effect of inorganic phosphate on adenylate cyclase activity The studies of Harwood and Peterkofsky [2] established an additional requirement, inorganic orthophosphate (Pi), for optimal adenylate cyclase activity. The mechanism by which Pi stimulates adenylate cyclase activity is complex. Purified preparations of the enzyme are not stimulated by Pi [6]. The data in Table 2 expand on that finding. Adenylate cyclase activity was measured in permeable cells and crude extracts of a wild-type strain of E. coli. t was observed that, when assays were performed in the absence of Pi, the adenylate cyclase activity was about 6 times higher in the broken cell extracts than in the permeable cells. However, the addition of Pi to the assays brought the level of activity in the permeable cells up to the level observed in the extracts. n contrast, the addition of Pi inhibited the enzyme activity in the extracts by about 50%. The interpretation of these findings [8] was that the physiologically important form of adenylate cyclase as observed in permeable cells is a complex with some factor or factors that depresses the intrinsic adenylate cyclase activity. t was proposed that this complex must be dissociable since breakage of cells restores adenylate cyclase to a high activity form. The mechanism of regulation of the activity of adenylate cyclase by Pi also involves a return of the adenylate cyclase to a high activity form; therefore, it is possible that Pi can modulate adenylate cyclase activity by either breaking the inhibitory complex of the enzyme with the inhibitory factors or changing the nature of the complex to make it more active.

3 There is reason to believe that the inhibition of adenylate cyclase by glucose is physiologically related to the stimulation of the activity of the enzyme by Pi. Studies in Gram-positive bacteria have indicated that cells starved for sugar nutrients have relatively high cellular concentrations of Pi (up to 40 mm); when such cells are fed sugar, the concentration of Pi drops precipitously and many phosphorylated metabolites accumulate [9,10]. A similar study, using 31p_NMR spectroscopy, was carried out in a strain of E. coli in which it is possible to demonstrate a glucose-dependent inhibition of adenylate cyclase activity. There is a pronounced decrease in the cellular level of Pi associated with the uptake of glucose into the cells. t was estimated that the concentration of Pi changes from mm before the addition of glucose to 5-10 mm after the addition of glucose [11]. A reasonable interpretation of these findings in the context of adenylate cyclase activity regulation is that glucose inhibits adenylate cyclase activity indirectly by causing a decrease in the cellular concentration of the essential activator, Pi. The data in Table 2 established that the stimulation of adenylate cyclase activity by Pi is not a direct effect on the adenyl ate cyclase protein. t is noteworthy that the concentration range in which adenylate cyclase responds to Pi is close to the physiological concentration range for this ion. The pattern of response to Pi is different in a strain of E. coli in which the genes for the PTS proteins Enzyme and Hgl e are deleted. n this case, Pi inhibits the activity of the enzyme as it does in broken cell extracts or with purified enzyme. These data are therefore in keeping with the idea that the physiologically responsive form of adenylate cyclase is a complex of the enzyme with one or more of the PTS proteins; it is that complex that has an activity that is stimulated by Pi. Another property of adenylate cyclase in permeable cells of wild-type E. coli is that, in the absence of Pi, the activity of adenylate cyclase is lower than that in cell extracts (see Table 2). t has also been observed that, when assayed in the absence of Pi, permeable cells of the wild-type strain show a considerably lower activity than permeable cells of a PTS deletion mutant. This 105 constitutes further evidence for the idea that the normal form of adenylate cyclase in cells is a complex with PTS proteins and that this complex has an activity that can be positively modulated by Pi. Some insight into the mechanism by which Pi promotes a stimulation of adenylate cyclase activity in permeable cells came from a comparison of the concentration dependence for Pi effects on both adenylate cyclase and PTS activities [8]. Permeable cells of a strain of E. coli with a leaky mutation in Enzyme of the PTS were assayed for both adenylate cyclase and PTS activities in reaction mixtures supplemented with phosphoenolpyruvate at a variety of concentrations of Pi. Maximum stimulation of both activities was observed between 5 and 15 mm Pi. This data is consistent with the idea that Pi interacts with some component(s) of the PTS converting it (them) to a form that relieves the inhibition of adenylate cyclase activity imposed on the enzyme by interaction with the PTS component(s) Effect of phosphorylation of PTS proteins on adenylate cyclase activity The PTS proteins can exist in both phosphoand dephospho-forms. The mechanism of the interconversion of these protein forms involves a phosphoenolpyruvate-dependent protein phosphorylation and a sugar-substrate-dependent dephosphorylation of the proteins [12,13]. An experiment was carried out with a strain of E. coli that contained a form of Enzyme that has a poor affinity for PEP. The data in Fig. 1 show that permeable cells of this strain have adenylate cyclase activity that is substantially stimulated by PEP and that the PEP-stimulated activity is blocked by glucose. These opposing effects of glucose and PEP on adenylate cyclase activity are consistent with the idea that stimulation of adenylate cyclase activity requires that PTS proteins be in their phosphorylated forms. These findings lead to the conclusion that inhibition of adenylate cyclase activity by glucose is the result of the compounding of two effects: (1) glucose addition to E. coli results in a decrease in the cellular concentration of Pi, an essential activator of adenylate cyclase, (2) glucose addition also

4 PEP / o ~ / t Glucose Glucose 0+ 0,- /? /J / 20 TME minol t PEP -_0 o --0 Fig. 1. Opposing effects of phosphoenolpyruvate and glucose on adenylate cyclase activity. Strain 1103 (a leaky Enzyme mutant) was assayed for adenylate cyclase activity in permeable cells. At the indicated times, glucose or PEP was added to the incubation mixtures. From (14). promotes the dephosphorylation of the phosphorylated forms of the PTS proteins; Pi-dependent promotion of adenylate cyclase activity requires that the PTS proteins be in their phosphoforms [14] Models for regulation ofadenylate cyclase activity by PTS proteins and Pi A number of possible models by which the PTS might regulate adenylate cyclase activity have been considered. Model suggests that the phosphorylated form of mgl e can interact with and activate adenylate cyclase in the presence of Pi [12]; a ramification of this model is that sugar transport via the PTS results in the inhibition of adenylate cyclase activity by a combination of a decrease in the levels of both Pi and phospho-hlf". An alternative model that has been considered is that the dephosphorylated form of Hg le is an inhibitor of adenylate cyclase activity [15,16]; according to this model, PTS sugars inhibit adenylate cyclase activity by promoting the formation of the inhibitor, glc A third model, suggesting a functional complex of the PTS proteins Enzyme, HPr and glc interacting with adenylate cyclase has also been proposed [17]. n this scheme, PTS sugars inhibit adenylate cyclase activity by decreasing the cellular concentration of Pi as well as that of the functional complex of phospho-pts proteins Pi stimulation of adenylate cyclase activity requires the presence oftu»: A strain of E. coli deficient in nglc was shown to have adenyl ate cyclase activity that is not stimulated by Pi (Fig. 2). When this strain is transformed with a plasmid harboring the gene for lplc, the adenylate cyclase activity measured in permeable cells was stimulated substantially by Pi. This finding eliminates from consideration any regulatory models for adenylate cyclase activation that do not include ngle, but does not specifically eliminate any of the models outlined above Dephosphorylated iuv: promotes inhibition of adenylate cyclase activity A strain of E. coli deficient in the PTS proteins Enzyme, HPr and g ic had adenylate cyclase activity that was inhibited by Pi to the small degree observed with purified enzyme. When the strain was transformed with a plasmid carrying genes for Enzyme and HPr, the pattern of response to Pi was unchanged (Fig. 3). However, when the host strain was transformed with a plasmid harboring the gene for glc, Pi caused a 2000 o Control [J +Phosphate p8r322 pds 45 Fig. 2. Requirement for 1lgl c for Pi-dependent stimulation of adenylate cyclase activity in a strain expressing Enzyme and HPr. A strain of E. coli deficient in the gene for mgl c (HK759) was transformed either with pbr322 or pds45 (which expresses the gene for gle). The two constructs were assayed for adenylate cyclase activity in permeable cells in the absence or presence of Pi (15 mm). From [16].

5 107 severe inhibition of adenylate cyclase activity. This finding provides support for the model that, in the presence of Pi, dephosphorylated Ugl c binds to and inhibits adenylate cyclase activity. t should be not ed, however, that these findings do not eliminate either of the other two models described above. Further studies will be required to fully describe the physiological pattern of regulation of adenylate cyclase activity by the combination of Pi and one or more of the proteins of the PTS A suggested model for regulation of adenylate cyclase activity A working model for the regulation of adenylate cyclase activity by Pi and the PTS that we favor at the moment is summarized in Fig. 4. n this scheme, the PTS proteins Enzyme, HPr and Us lc interact with one another to form a functional complex that may reside at the cytoplasmic membrane in the region of the specific carriers for PTS sugars, for example n g1c n the absence of transportable sugars, the condition of these PTS proteins is supposed to be phosphorylated. Further, under these conditions, cellular Pi levels are high such that the ion interacts with a PTS protein (designated here as US lc ). This constitutes the active form of adenylate cyclase in the cell. When cells are exposed to a transportable PTS sugar, the condition of the adenylate cyclase complex is assumed to change dramatically and quickly. The PTS proteins are shifted to their dephosphorylated forms and the cellular level of Pi decreases to a range so low that little Pi is associated with the 120 &OO(b(ph.l-crr)/pDS20) t:lsd2(a{ptsl-crr)/ pkc7al mp) 40 S "4(A(pl.'''"rr}/pBR322} 581(4(P. crr)/pds484crr).513(6(pu-crr)/pds48) 120 PHOSPHATE CONCENTRATON(mM) Fig. 3. Requirement for ale {or Pi-dependent inhibition of adenylate cyclase activity in a strain not expressing Enzyme 1. A PTS deletion strain was transformed with pds20, which expresses Enzyme [ and HPr, pds48, which expresses n alc or some control plasmids, The constructs were assayed for adenylate cyclase activity in permeable cells at the indicated concentrations of Pi. From [16]. ae 40 ACTVE NACTVE Fig. 4. A model for regulation of adenylate cyclase activity by Pi and the PTS. PTS protein complex. This is the less active physiological form of adenylate cyclase. 3. CONCLUSON The focus of attention in this article has been on the involvement of the PTS and Pi in the activation of adenylate cyclase. t is important to point out that the model described here is probably an oversimplification of the factors regulating adenylate cyclase activity. Evidence has been previously presented that adenylate cyclase activity is regulated by GTP [18], the protein synthesis factor EF-Tu [19] and some aspect of the protonmotive force in E. coli [20]. t is also noteworthy that the effect of Pi in modulating camp levels may be the result of an amplification cycle composed of a Pi-dependent stimulation of adenylate cyclase activity (as outlined here) and a Pi-dependent inhibition of camp phosphodiesterase ([21], and recent studies in this laboratory).

6 108 REFERENCES [] Peterkofsky, A. (1976) Cyclic Nucleotides in Bacteria, in (Greengard, P. and Robison, G.A., Eds.), Advances in Cyclic Nucleotide Research, Yol. 7, pp. 1-48, Raven Press, New York. [2] Harwood, J.P. and Peterkofsky, A. (1975) Glucose-sensitive AdenyJate Cyclase in Toluene-treated Cells of Escherichia coli B. J. Bio. Chern. 250, [3] Peterkofsky, A. and Gazdar, C. (1915) nteraction of Enzyme of the Phosphoenolpyruvate:sugar Phosphotransferase System with Adenylate Cyclase of Escherichia coli. Proc, Natl. Acad. Sci. U.S.A. 72, [4] Harwood, J.P., Gazdar, C., Prasad, c., Peterkofsky, A., Curtis, S.l and Epstein, W. (1976) nvolvement of the Glucose Enzymes of the Sugar Phospho transferase System in the Regulation of Adenylate Cyclase by Glucose in Escherichia coli. J. Biol, Chern, 251, [5] Peterkofsky, A. (1977) The Mechanism of Regulation of Escherichia coli Adenylate Cyclase, in (Abou-Sabe, M. ed.), Cyclic Nucleotides and the Regulation of Cell Growth. pp , Dowden, Hutchinson and Ross, Stroudsburg, PA. [6J Yang, J.K. and Epstein, W. (1983) Purification and Characterization of Adenylate Cyclase from Escherichia coli K12. J. Bio. Chern. 258, [7J Peterkofsky, A., Gonzalez, J.E. and Gazdar, C. (1978) The Escherichia coli Adenylate Cyclase Complex. Regulation by Enzyme of the Phosphoenolpyruvate.sugar Phosphotransferase System. Arch. Biochern. Biophys. 188, [8J Liberman, E., Reddy, P., Gazdar, C. and Peterkofsky, A. (1985) The Escherichia coli Adenylate Cyclase Complex: Stimulation by Potassium and Phosphate. J. Bio. Chern. 260, [9] Mason, P.W., Carbone, D.P., Cushman, R.A. and Wag goner, A.S. (1981) The mportance of norganic Phosphate in Regulation of Energy Metabolism of Streptococcuslactis. J. Bio. Chem. 256, [10] Thompson, J. (1987) Sugar Transport in the Lactic Acid Bacteria, in (Reizer, J. and Peterkofsky, A., eds.), Sugar Transport and Metabolism in Gram-positive Bacteria, pp , Ellis Horwood, West Sussex, England. [11] Reddy, P., Liberman, E., Gazdar, C. and Peterkofsky, A. (1985) Factors Regulating the Activity of Escherichia coli Adenylate Cyclase, n (Glass, R.E. and Spizek, 1., eds.), Gene Manipulation and Expression. pp , Croom-Helm, Kent, United Kingdom. [12] Postma, P.W. and Lengeler, J.W. (1985) Phosphoenolpyruvate.carbohydrate Phosphotransferase System of Bacteria. Microbiol, Revs. 49, [13] Meadow, N.D., Kukuruzinska, M.A. and Roseman, S. (1984) The Bacterial Phosphoenolpyruvate:sugar Phosphotransferase System, in (Martonosi, A., ed.), The Enzymes of Biological Membranes, Vol. 3, pp , Plenum Press, New York. [14] Peterkofsky, A. and Gazdar, C. (1978) The Escherichia coli Adenylate Cyclase Complex: Activation by Phosphoenolpyruvate. J. Supramolecular Structure 9, [15] Postma, P.W. and Scholle, Rl. (1979) Regulation of Sugar Transport in Salmonella typhimurium, in (Quagliariello, E. et al., eds.), Function and Molecular Aspects of Biomernbrane Transport. pp , Elsevier, New York. [161 Liberman, E., Saffen, D., Roseman, S. and Peterkofsky, A. (1986) nhibition of E. coli Adenylatc Cyclase Activity by norganic Orthophosphate is Dependent on P'c of the Phosphoenolpyruvate:g1ycose Phosphotransferase System. Biochem. Biophys. Res. Commun. 141, [17J Reddy, P., Meadow, N., Roseman, S. and Peterkofsky, A. (1985) Reconstitution of Regulatory Properties of Adenylate 'Cyclase in Escherichia coli Extracts. Proc. Natl. Acad. Sci. U.S.A. 82, [18J Stein, J.M., Kornberg, H.L. and Marlin, B.R. (19'85) Effects of GTP, GDP1:beta S] and Glucose on Adenylate Cyclase Activity of E. coli B. FEBS Lett. 25, [19] Reddy, P., Miller, D. and Peterkofsky, A. (1986) Stimulation of Escherichia coli Adenylate Cyclase Activity by Elongation Factor Tu, a GTP-binding Protein Essential for Protein Synthesis. J. Bio. Chern. 261, [20] Peterkofsky, A. and Gazdar, C. (1979) Escherichia coli Adenylate Cyclase Complex: Regulation by the Proton Electrochemical Gradient. Proc. Natl. Acad. Sci. U.S.A. 76, [21] Nielsen, L.D., Monard, D. and Rickenberg, H.Y. (1973) Cyclic 3 /,5 '-Adenosine Monophosphate Phosphodiesterase of Escherichia coli. J. Bacteriol. 116,