Regulation of Autophosphorylation of Escherichia coli Nitrogen ...

JOURNAL OF BACTERIOLOGY, Mar. 1999, p. 1906–1911 0021-9193/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 6

Regulation of Autophosphorylation of Escherichia coli Nitrogen Regulator II by the PII Signal Transduction Protein PENG JIANG

AND

ALEXANDER J. NINFA*

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan Received 30 October 1998/Accepted 14 January 1999

The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in Escherichia coli. The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated. The phosphatase activity of NRII is activated by the PII signal transduction protein. We showed that PII was also an inhibitor of the kinase activity of NRII. The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated. The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII. We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleavage of ATP by NRII. The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities. Under these conditions, the kinase activity of NRII is dominant. At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity. However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII’s kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).

activators of the autophosphatase activity of the phosphorylated receiver. The phosphorylation of the receiver protein may be used to transmit signals in two different ways. In some cases, the phosphorylated receiver domain serves as a source of phosphoryl groups for a phosphorelay system that phosphorylates another receiver protein at the end of the chain (6). In other cases, the change in conformation associated with phosphorylation of the receiver (21) is used to control the activity of other protein domains, either in the same protein or in a different protein or macromolecular complex (reviewed in references 38 and 39). The phosphorylation of the N-terminal receiver domain of NRI (NRI-N) brings about the oligomerization of NRI dimers, activating an ATPase activity and transcriptional activation activity of the central domain of NRI (18, 40, 41; for reviews, see references 23 and 26). The NRII (NtrB)-NRI (NtrC) two-component system regulates the transcription of nitrogen-regulated (ntr) genes in response to signals of nitrogen and carbon sufficiency (reviewed in references 16, 23, 24, 27, 33, 34). The transmitter protein NRII is both an NRI kinase and a phosphorylated-NRI (NRI;P) phosphatase (30), as depicted in Fig. 1. The phosphatase activity of NRII is activated by the PII signal transduction protein (30), which binds to NRII (17). Previous studies of this system suggested that the kinase activity of NRII was unregulated while the phosphatase activity was regulated, i.e., that these activities were not coordinately regulated (15). For example, in several different assays, the kinase activity of NRII seemed to continue unabated in the presence of PII, even though the rapid dephosphorylation of NRI;P could be readily detected in the similar reaction mixtures. In one set of experiments, NRII and NRI-N were combined with a coupling system, consisting of pyruvate kinase and lactate dehydrogenase, that measured the production of ADP from ATP (15)

Two-component signal transduction systems use the regulated cleavage of ATP to drive conformational changes resulting in signal transduction (shown for the NRI-NRII system in Fig. 1). These regulatory systems contain two conserved protein domains, which may be referred to as the transmitter and receiver domains (32). The transmitter domains transduce signals by controlling the phosphorylation state of the receiver domains (Fig. 1) (for reviews, see references 5, 25, 26, 32, 38, and 39). The transmitter proteins bind ATP and catalyze the phosphorylation of single conserved histidine residues on themselves (7, 29, 41). The receiver proteins bind the phosphorylated histidine and catalyze the transfer of the phosphoryl groups to conserved aspartate residues on themselves (36, 37). The acyl phosphate present on different receiver proteins has been reported to be either stable (half-life, ;5 h) or quite unstable (half-life, ;5 s), depending on the particular receiver (reviewed in reference 25). That is, some receiver proteins catalyze their own dephosphorylation (18). The different receiver proteins may be thought of as phosphatases whose covalent intermediate has a characteristic stability (36). In addition to bringing about the phosphorylation of the receiver protein, some transmitter proteins also bring about the rapid dephosphorylation of the receiver protein in response to signals. For example, the transmitter proteins nitrogen regulator II (NRII or NtrB) (Fig. 1), EnvZ, PhoR, UhpB, FixL, and KdpD, among others, have this property (for reviews, see references 25, 26, 32, and 38). It is not known whether these transmitters are actually phosphatases or act as

* Corresponding author. Mailing address: Department of Biological Chemistry, University of Michigan Medical School, 1301 E. Catherine, Ann Arbor, MI 48109-0606. Phone: (734) 763-8065. Fax: (734) 7634581. E-mail: [email protected]. 1906

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REGULATION OF E. COLI NRII BY PII

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FIG. 1. ATPase activity of the NRI-NRII two-component signal transduction system. The roles of the transmitter protein, NRII, and the receiver protein, NRI, in catalyzing the cleavage of ATP are depicted. The phosphorylated form of NRI, NRI;P, is a transcriptional activator of nitrogen-regulated genes. The dephosphorylation of NRI;P is catalyzed by NRII in a reaction activated by PII. In this study, we showed that PII was also an inhibitor of the autophosphorylation of NRII.

(Fig. 2). When NRI-N was limiting in this enzyme system, the production of ADP was limited by the slow rate of dephosphorylation of NRI-N;P. Under these conditions, the rate of ADP generation was increased upon addition of PII (15). Since the rate of cleavage of ATP to ADP was increased by the addition of PII, this result suggested that PII did not inhibit the kinase activity of NRII (15). In a second set of experiments, the rates of autophosphorylation of NRII in the presence and absence of PII were directly examined, and no significant difference was observed (15). Since in related experiments the very-rapid dephosphorylation of NRI;P and NRI-N;P by NRII could be observed upon addition of PII, it was concluded that the kinase activity of NRII was unaffected by PII and that the phosphatase activity of NRII was activated by PII (15). However, we have shown in the present study that, as depicted in Fig. 1 and 2, PII regulates both the kinase and phosphatase activities of NRII. The issue of whether the kinase and phosphatase activities of the transmitter proteins are regulated separately or coordinately has been addressed both genetically and biochemically (3, 4, 8, 9, 14, 15, 20, 35). Coordinate regulation of these activities is suggested by several observations. (i) Some of the mutations that reduce the kinase activity of the transmitter proteins also result in an elevation of the basal phosphatase activity (4, 8, 14). Specifically, certain mutations mapping in the C-terminal D, F, and G boxes of the transmitter domain (reviewed in reference 38) have this property as well as some changes at the histidine site of autophosphorylation. In addition, a transmitter domain truncation mutant lacking the D, F, and G boxes lacked kinase activity and had elevated basal phosphatase activity (14). (ii) The kinase and phosphatase activities of purified FixL are reciprocally regulated by oxygen (20). (iii) Mutations in the H box region containing the histidine site of phosphorylation often affect the phosphatase activity, suggesting that the H box plays a critical role in both activities (3, 9, 14). Thus, it has been suggested that the H box may assume a kinase or a phosphatase conformation and that signals control the conformation of the H box region (9, 38). Recently, much has been learned about the interaction of

FIG. 2. Coupled enzyme system for the measurement of ATP cleavage by the NRII-NRI two-component system. On the left, approximate rates for the autophosphorylation of NRII (10/min), transfer of phosphoryl groups from NRII;P to NRI (.100/min), and spontaneous dephosphorylation of NRI-N;P (in the absence of PII, 0.15/min) are shown (reviewed in references 25 and 38, and unpublished data). The steps that are rate limiting under the various reaction conditions are shown on the right. PK, pyruvate kinase; PEP, phosphoenolpyruvate; LDH, lactate dehydrogenase.

NRII and PII and the role of small-molecule effectors in controlling this interaction (11, 12, 17, 19). Specifically, 2-ketoglutarate, an allosteric effector of PII, greatly increases the binding of PII to NRII when present at low concentration (17, 19). At higher concentrations, 2-ketoglutarate inhibits the binding of PII to NRII (17, 19). This behavior is due to the nature of the allosteric regulation of the trimeric PII by 2-ketoglutarate (Fig. 3). The first effector molecule, binding at a very low concentration (kd, ;5 mM), exerts strong negative cooperativity on the binding of additional effector molecules (11). This negative cooperativity is overcome at high concentrations of effector (Kd, ;150 mM [11–13]). PII apparently adopts one of two distinct conformations, depending on whether one or more than one molecule of 2-ketoglutarate is bound to it. In the present study, we reexamined the regulation of the kinase activity of NRII by PII. In retrospect, the earlier experiments (15), performed in the absence of 2-ketoglutarate, used conditions under which only a fraction of the NRII should have been complexed to PII, even though very high PII concentrations were used. Thus, the conclusions drawn from those experiments should be reevaluated. Here, we showed that PII was a potent inhibitor of NRII autophosphorylation and that this activity of PII was allosterically regulated by 2-ketoglutarate.

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FIG. 4. Inhibition of NRII autophosphorylation by PII. Reactions were performed and the results were analyzed as described in Materials and Methods. The data point plotted on the y axis is the rate obtained in the absence of PII. Vo, initial rate of NRII autophosphorylation.

FIG. 3. Model for the regulation of PII activity by 2-ketoglutarate. The PII trimer is proposed to exist in three distinct conformations, depending on the number of 2-ketoglutarate molecules bound. Under cellular conditions, the synergistic ligand ATP is saturating and one ATP molecule is bound to each PII subunit (not depicted). In the absence of 2-ketoglutarate, PII cannot interact with NRII. The binding of a single molecule of 2-ketoglutarate to a PII trimer (Kd, ;5 mM [11, 17]) results in a conformation that favors the interaction of PII with NRII. The binding of additional molecules of 2-ketoglutarate to PII (Kd, ;150 mM) results in a distinct conformation that does not interact with NRII. The dissociation constants for the binding of the second and third ligand molecules to PII are estimated from kinetic studies (12, 13, 17) and preliminary, unpublished binding studies. The transition between singly bound PII and triply bound PII occurs at physiological 2-ketoglutarate concentrations.

MATERIALS AND METHODS The purified proteins used in our experiments were described previously (1, 10, 15), except that PII was further purified by phenyl Sepharose chromatography, as suggested by Quan Sun, as follows: PII in 50 mM KPi (pH 7.0)–1.2 M (NH4)2SO4 was applied to a phenyl Sepharose CL-4B column (Pharmacia Biotech) equilibrated in the same buffer. Under these conditions, PII is soluble and binds avidly to the column. After extensive washing of the column with the same buffer, PII was eluted with a gradient of 1.2 to 0 M (NH4)2SO4 in 50 mM KPi, pH 7.0. PII eluted from the column at ;0.4 M (NH4)2SO4 in a broad peak. The purest fractions were identified by gel electrophoresis and pooled, and the PII was concentrated by precipitation with (NH4)2SO4 at 60% saturation, resuspended, and dialyzed against storage buffer as described previously (15). The combination of the purification methods described previously (15) and the phenyl Sepharose step described above resulted in purified PII completely lacking contaminating ATPase activity, as determined by thin-layer chromatographic analysis of reaction mixtures in which purified PII was incubated with [a-32P]ATP (data not shown) and by the use of the coupled assay system described below (data not shown). The purified NRI, NRI-N, NRII, PII, and PII-Q39E proteins used here were each at least 90% pure, as judged by gel electrophoresis (data not shown). The PII-Q39E preparation used in our experiments demonstrated no contaminating ATPase activity, as determined with the coupled assay system described below (data not shown). Protein concentrations were determined by the method of Lowry et al. (22). The coupled assay system in which the production of ADP is coupled to the oxidation of NADH by pyruvate kinase and lactate dehydrogenase (Sigma Chemical Co.) was described previously (15, 28, 31) (Fig. 2). The coupling system was in 10-fold or greater excess in all experiments, as indicated by spiking with 0.43 mM ADP at the end of each experiment; this resulted in the immediate and complete oxidation of NADH (data not shown). The oxidation of NADH was measured at 340 nm in a Beckman DU65 spectrophotometer at 30°C. Data were recorded by hand; time was monitored with a stopwatch. Reaction mixtures (0.35-ml volumes) contained 50 mM Tris-Cl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM phosphoenolpyruvate, 0.3 mM NADH, 6.9 U of pyruvate kinase, 23 U of lactate dehydrogenase, 1 mM ATP, and various concentrations of NRI-N, NRII, PII, and 2-ketoglutarate. In this assay system, rates of NADH oxidation, which were stable and linear for several minutes, were determined directly from primary plots of NADH concentration (A340) versus time. 2-Ketoglutarate was found to cause detectable oxidation of the NADH by lactate dehydrogenase when present at a concentration of 1 mM or higher (data not shown). This oxidation of NADH was linear for several minutes, the velocity of

this oxidation of NADH was linear with respect to the concentration of 2-ketoglutarate used, and lactate dehydrogenase and NADH were the only nonbuffer components of the coupled assay system necessary for this activity (data not shown). It is unlikely that this activity was due to pyruvate contamination of the commercial 2-ketoglutarate, since spiking of reaction mixtures with low concentrations of pyruvate resulted in a small burst of NADH oxidation lasting only a few seconds (data not shown). It was not determined whether this activity was due to slow reduction of 2-ketoglutarate by lactate dehydrogenase or by an unknown contaminating enzyme activity in the lactate dehydrogenase preparation. This low background rate of NADH oxidation due to 2-ketoglutarate was determined for every concentration of 2-ketoglutarate used in the coupled system and subtracted from the rates observed in the complete system. Genetically expressed and purified NRI-N (15) was used in the coupled assay system in place of NRI, since full-length NRI;P is an ATPase (40). This ATPase activity, which is due to the central domain of NRI, was not observed with NRI-N;P (15, 18, 40). The autophosphorylation of NRII was measured on ice. Reaction mixtures contained 50 mM Tris-Cl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 50 mM 2-ketoglutarate, 0.5 mg of bovine serum albumin/ml, 0.5 mM [g-32P]ATP, 2 mM NRII, and various concentrations of PII. Reactions were initiated by the addition of the [g-32P]ATP. At various time points, aliquots were removed and spotted onto nitrocellulose filters, which were immediately immersed in, and extensively washed in, 0.1 M Na2CO3 (pH, ;11), dried, and counted by liquid scintillation, as described elsewhere (15). Initial rates of autophosphorylation were determined by fitting of primary data to the first-order rate equation, using the Enzfit software. In preliminary experiments, we observed that NRII;P seemed to stick to standard plastic microtubes, since addition of innocuous proteins such as bovine serum albumin (Sigma Chemical Co.) appeared to increase the rate of NRII autophosphorylation. The recovery of NRII;P was optimal in the presence of bovine serum albumin at 0.5 mg/ml (data not shown), which was used for all experiments.

RESULTS AND DISCUSSION In previous experiments, the rate of NRII autophosphorylation was not apparently affected by the presence of PII at high concentrations (15). In retrospect, only a very small fraction of the NRII used in those experiments was complexed to PII, since the experiments were performed in the absence of 2-ketoglutarate (12, 17, 19). We therefore reexamined the effect of PII on the rate of autophosphorylation of NRII in the presence of 2-ketoglutarate at 50 mM. This concentration of 2-ketoglutarate was chosen because previous studies indicated that it was optimal for the activation of the NRII phosphatase activity by PII (12, 17). As shown in Fig. 4, NRII autophosphorylation was strongly inhibited by PII under these conditions (Ki, ;1.0 mM PII). However, since the concentration of NRII was 2 mM in the experiment (to permit accurate measurement of the rate of NRII autophosphorylation), the dissociation constant for the binding of PII to NRII is probably much lower. In a separate experiment, PII;UMP was used in

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place of PII, and no inhibition (or activation) of NRII autophosphorylation was observed even at 5 mM PII;UMP (data not shown). We hypothesized that in previous experiments using a coupling system consisting of pyruvate kinase and lactate dehydrogenase to measure the cleavage of ATP, only a small fraction of the NRII was complexed to PII, since 2-ketoglutarate was absent (15). The small fraction of NRII complexed to PII, by virtue of its high phosphatase activity, could bring about the dephosphorylation of NRI-N, providing a sink for phosphorylgroup transfer from uncomplexed NRII. This would allow the uncomplexed NRII to cleave more ATP. Thus, the addition of PII increased the rate of ADP production (15). This hypothesis supposes that in the presence of PII, the phosphatase activity of the NRII-PII complex is far more robust than the kinase activity of uncomplexed NRII. This is reasonable based on previous results (reviewed in references 16, 27, and 38). The experiments presented earlier (15) did not test whether PII inhibits the NRII kinase activity, although this was not realized at the time. If PII inhibits the kinase activity of NRII, then when all of the NRII is present in complex with PII, the cleavage of ATP in the coupled assay system should cease. The hypothesis presented above makes the following detailed prediction for the coupled assay system in which ADP production from ATP is measured (Fig. 2): for experiments in which NRII is in excess over NRI-N, the rate of ADP production is limited by the slow rate of dephosphorylation of NRIN;P. If 2-ketoglutarate is at a concentration favoring the formation of PII containing a single effector molecule (50 mM), then ADP production will first rise and then fall as the concentration of PII is increased. This is because when PII is at a very low concentration, its effect on the autophosphorylation of NRII is minimal (since few molecules of NRII are complexed), but the robust phosphatase activity of the small number of NRII molecules present in complex with PII will provide a sink for phosphoryl groups from ATP. However, as the concentration of PII is increased, a significant fraction of NRII becomes complexed and the rate of cleavage of ATP will be reduced, even though NRI-N is dephosphorylated. Furthermore, the hypothesis predicts that when NRI-N is in excess and NRII is limiting, the addition of PII will simply reduce the rate of ATP cleavage, because under these conditions the kinase activity of NRII is always limiting (Fig. 2). That is, the rate curve in the first experiment (NRII in excess) should be biphasic while that in the second experiment (NRI-N in excess) should be monophasic. These predictions were tested experimentally and found to hold true (Fig. 5). The results shown in Fig. 5B again indicate that PII inhibits the autophosphorylation of NRII. In this experiment, the concentration of NRII was 0.4 mM and the apparent Ki for PII was also ;0.6 mM, suggesting a dissociation constant for the interaction of PII and NRII of ;0.4 mM under these conditions (assuming a bimolecular reaction in which one PII trimer binds to one NRII dimer). This estimate is an approximation, since variation of the PII concentration in our experiments at the fixed 2-ketoglutarate concentration of 50 mM resulted in a variation in the fractional saturation of PII with 2-ketoglutarate. Previous studies of the regulation of PII activity by the effector 2-ketoglutarate have suggested that the trimeric PII may exist in multiple states, depending on its degree of saturation by 2-ketoglutarate (Fig. 3). When the synergistic PII ligand ATP is present at a saturating concentration (as in all of the experiments discussed here), the first molecule of 2-ketoglutarate binds the PII trimer with a dissociation constant of ;5 mM (11, 17). This form of PII is very active in binding to NRII and


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FIG. 5. Effect of PII on the rate of ATP cleavage in the coupled assay system. Reactions were performed and the results were analyzed as described in Materials and Methods. For both panels, the data point plotted on the y axis is the rate obtained in the absence of PII. (A) Effect of PII when NRI-N was limiting. Reaction mixtures contained 1 mM NRII, 0.75 mM NRI-N, 50 mM 2-ketoglutarate, the indicated concentrations of PII, and other components as detailed in Materials and Methods. (B) Effect of PII when NRII was limiting. Reaction mixtures contained 0.4 mM NRII, 12 mM NRI-N, 50 mM 2-ketoglutarate, the indicated concentrations of PII, and other components as detailed in Materials and Methods. The apparent Ki for this experiment is ;0.6 mM PII. Vo, rate of ATP cleavage.

in activating the phosphatase activity of NRII (12, 17, 19). Also, as shown already (Fig. 4), this form of PII inhibits the kinase activity of NRII. The remaining two 2-ketoglutarate sites on PII are occupied only at effector concentrations of .100 mM, as deduced from kinetic studies of the interaction of PII with NRII and with the adenylyltransferase that controls the glutamine synthetase adenylylation state (12, 13). This form of PII does not bind well to NRII or adenyltransferase, and its rate of activation of these receptors is greatly reduced (11–13, 17, 19). Thus, at a high concentration, 2-ketoglutarate is an inhibitor of the NRII phosphatase activity, which acts by an antiactivator mechanism. The above-described model predicts that in the coupled assay system, 2-ketoglutarate at certain concentrations should be an activator of the NRII kinase activity, which acts by an anti-inhibitor mechanism. Thus, in a reaction mixture with excess NRII and PII, limiting NRI-N, and no 2-ketoglutarate, a low rate of ATP cleavage should be observed. This is because in the absence of 2-ketoglutarate, PII is known to be a poor

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FIG. 6. Effect of 2-ketoglutarate on the rate of ATP cleavage in coupled assay systems with limiting NRI-N. Reactions were performed and the results were analyzed as described in Materials and Methods. Reaction mixtures contained 1 mM NRII, 0.75 mM NRI-N, 3 mM PII (■) or PII-Q39E (h), the indicated concentrations of 2-ketoglutarate, and other components as detailed in Materials and Methods. The data points plotted on the y axis represent the results obtained in the absence of 2-ketoglutarate. Vo, rate of ATP cleavage.

activator of the NRII phosphatase activity and a poor inhibitor of the NRII kinase activity, and since NRI-N is limiting, NRII;P and NRI-N;P accumulate and the rate of ATP cleavage is slow (Fig. 2). As the level of 2-ketoglutarate is increased, at very low concentrations the rate of ATP cleavage should be increased. This is because 2-ketoglutarate will activate the binding of PII to NRII, and in this phase of the reaction only a small fraction of the NRII molecules are complexed with PII. This small fraction of the NRII molecules, by virtue of their robust phosphatase activity, will dephosphorylate the NRIN;P that is present. The rest of the NRII molecules, not complexed with PII, can transfer their phosphoryl groups to the dephosphorylated NRI-N and cleave more ATP. As the 2-ketoglutarate concentration is further increased, however, the binding of PII to NRII will be increased and the rate of ATP cleavage will be greatly reduced since eventually all of the NRII becomes complexed with PII. Further addition of 2-ketoglutarate beyond this concentration, however, should cause an increase in the rate of ATP cleavage. This is because as 2-ketoglutarate binds the already-once-complexed PII, it reduces its ability to bind NRII, permitting a fraction of the NRII molecules to cleave ATP. However, as the level of 2-ketoglutarate is raised further, all PII activity eventually becomes inhibited and the rate will return to that seen in the absence of 2-ketoglutarate. This is because the limiting NRI-N will become phosphorylated and NRII;P will again have no sink for its phosphoryl groups. Thus, a four-phase saturation kinetics curve is predicted by the hypothesis. Since four-phase saturation kinetics curves are rather unusual, this constitutes a strong test of the hypothesis. The experiment was performed, and a four-phase saturation curve was obtained (Fig. 6). We hypothesize that in the third (ascending) phase of the curve, 2-ketoglutarate activates the cleavage of ATP by an anti-inhibitor mechanism. We recently described a mutant form of the PII protein, PII-Q39E, in which the glutamine residue found at position 39 has been altered to glutamate (10). This form of PII protein is partially defective in binding 2-ketoglutarate (10, 13). In experiments in which the NRII phosphatase activity was directly measured in the presence of PII-Q39E, activation by 2-ketoglutarate required very high concentrations of effector (activation constant, 250 mM 2-ketoglutarate) and the rate of the

J. BACTERIOL.

NRII phosphatase activity was not reduced at very high (10 mM) effector concentrations (data not shown). Thus, PIIQ39E displayed properties suggesting that under the conditions used, only a single effector molecule could be bound, and this occurred only at elevated effector concentrations (10) (data not shown). If the effects of 2-ketoglutarate described so far for wild-type PII (Fig. 6) were due to the binding of this effector by PII, then these effects should be altered if the PII-Q39E protein is used in place of PII. The results of this experiment are also shown in Fig. 6. As evidenced, the first and second phases of the saturation curve were shifted to higher 2-ketoglutarate concentrations and the third and fourth phases were not seen. These results are consistent with the hypothesis that PII-Q39E binds the first molecule of 2-ketoglutarate at a higher effector concentration than does PII and is not fully occupied by three effector molecules even at quite high effector concentrations. The experiments reported here indicate that PII is an inhibitor of the autophosphorylation of NRII and that this activity of PII is allosterically regulated by 2-ketoglutarate. Since PII is known to be an activator of the NRII phosphatase activity (30), our experiments are consistent with the hypothesis that the kinase and phosphatase activities of NRII are regulated concertedly. However, our experiments do not prove this hypothesis. These experiments may explain the regulation of glnA expression, which requires NRI;P, in cells lacking the glnDencoded uridylyltransferase/uridylyl-removing enzyme (UTase/ UR), which regulates PII activity by covalent modification (11). In such cells, PII will be completely unmodified, as in our experiments, yet glnA expression is regulated about fivefold by the presence of ammonia (3). (We have recently constructed an utterly gutted glnD allele and confirmed this observation [2].) In intact cells, the concentration of NRI may be limiting, as in our experiments, and the concentration of PII may be in excess, as in the experiments shown in Fig. 6. Changes in the intracellular concentration of 2-ketoglutarate may be responsible for this regulation by controlling the activity of PII, as evidenced in Fig. 6. This hypothesis predicts that in the absence of ammonia the concentration of 2-ketoglutarate must be high enough to inhibit PII activity and that ammonia results in a decrease in 2-ketoglutarate to a level sufficient to activate PII. Also, the regulation of glnA expression in a strain lacking UTase/UR should be altered if a glnB mutant encoding a PII protein that is altered in its ability to bind 2-ketoglutarate is used. If the Q39E mutant form of PII is used, this hypothesis predicts that glnA expression may be lower in the absence of ammonia than in its presence. These predictions are currently being investigated. ACKNOWLEDGMENTS This work was supported by grant MCB9318792 from the National Science Foundation and grant GM47460 from the National Institutes of Health. We thank James A. Peliska for helpful discussions and suggestions during the course of this work. REFERENCES 1. Atkinson, M. R., E. S. Kamberov, R. L. Weiss, and A. J. Ninfa. 1994. Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC). J. Biol. Chem. 269:28288– 28293. 2. Atkinson, M. R., and A. J. Ninfa. Unpublished data. 3. Atkinson, M. R., and A. J. Ninfa. 1992. Characterization of Escherichia coli glnL mutations affecting nitrogen regulation. J. Bacteriol. 174:4538–4548. 4. Atkinson, M. R., and A. J. Ninfa. 1993. Mutational analysis of the bacterial signal-transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB). J. Bacteriol. 175:7016–7023.


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