PHYSIOLOGY AND METABOLISM

Purification of PII and PII-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in Rhodospirillum rubrum

Magnus Johansson, Stefan Nordlund
DOI: 10.1128/JB.181.20.6524-6529.1999

ABSTRACT

The PII protein from Rhodospirillum rubrumwas fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni2+-chelating chromatography. The uridylylated form of the PII protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by PII, PII-UMP, glutamine, and α-ketoglutarate were studied in extracts from R. rubrumgrown under different conditions. PII and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by α-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. colisystem. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.

The photosynthetic purple bacteriumRhodospirillum rubrum is a free-living diazotroph, in which nitrogenase activity is regulated at the metabolic level by reversible covalent modification, ADP-ribosylation (17). Under nitrogen-fixing conditions, ammonium is assimilated via the glutamine synthetase-glutamate synthase pathway (2, 25), as in other diazotrophs. The regulation of glutamine synthetase in R. rubrum or other phototrophs has not been as extensively studied as regulation in the enteric bacteria, but interesting differences have been reported, indicating a more complex system.

In enteric bacteria, glutamine synthetase is one of the key enzymes in nitrogen assimilation, catalyzing the ATP-dependent formation of glutamine from NH4+ and glutamate. The enzyme is a dodecamer of the glnA gene product and is subject to tight regulatory control at three different levels (22). In addition to feedback inhibition by several end products of nitrogen metabolism and transcriptional regulation by the Ntr system, enzyme activity is regulated by covalent modification. This process involves two bifunctional enzymes, uridylyltransferase (UTase) and adenylyltransferase (ATase), and the regulatory protein PII, which works as a signal transducer between the two enzymes. UTase, encoded by glnD, senses the nitrogen level and catalyzes uridylylation and deuridylylation of PII(14, 26). When the nitrogen level is high, PII, a trimer of the glnB product (22, 31), is unmodified and stimulates ATase to catalyze the inactivation of glutamine synthetase by adenylylation. PII-UMP is required for the deadenylylation activity of the enzyme, leading to activation of glutamine synthetase (a phosphorylysis reaction which requires Pi and produces ADP) (22). It has recently been demonstrated that ATase in Escherichia coli, encoded byglnE, consists of two homologous domains (8, 30). The N-terminal part is responsible for the deadenylylation activity which is activated by α-ketoglutarate, dependent on PII-UMP, and inhibited by PII. The C-terminal part of ATase catalyzes the adenylylation of glutamine synthetase and is activated by glutamine and PII (8).

Recently the glnK gene in E. coli, encoding a PII homologue, was identified (29). The structure of GlnK is similar to that of PII, and it can also be modified by reversible uridylylation catalyzed by UTase (29). GlnK can complement PII and affects both ATase and NtrB (1). In several organisms, a second gene encoding a protein similar to PII has now been identified (3, 4, 18, 19, 21), but in R. rubrum, it has not yet been found.

Among the anoxygenic photosynthetic bacteria, glutamine synthetase inR. rubrum has been most extensively characterized (6, 20, 25, 32). Studies at the metabolic level have demonstrated that the enzyme is regulated in a way partly similar to that of theE. coli enzyme; however, several interesting differences have also been observed (6, 20, 25, 32). Adenylylated glutamine synthetase from R. rubrum shows lower γ-glutamyltransferase activity in both the presence and absence of 60 mM Mg2+ (20), whereas for the adenylylatedE. coli enzyme, a decrease in this activity is observed only in the presence of 60 mM Mg2+ (27).

In R. rubrum, glutamine synthetase can also be modified by ADP-ribosylation, but the effect of this modification has not yet been clarified and the enzyme(s) responsible for the modification has not yet been identified. Furthermore, each subunit can, at one time, be modified only by AMP or ADP-ribose (32). Glutamine synthetase also shows a switch-off behavior, which seems to be coordinated with nitrogenase activity in response to both ammonium and darkness (5, 20). Several in vivo studies have shown that the glutamine synthetase activity is recovered after ammonium switch-off and that this process is slow compared to inactivation (5, 12).

We have previously shown that PII in R. rubrumis modified by reversible uridylylation in response to nitrogen status (13). PII was demodified by the addition of ammonium, glutamine, or NAD+ and partially demodified by the addition of glutamate. Oxygen and/or darkness had no effect on uridylylated PII. We also showed that the UTase in R. rubrum responds to the glutamine concentration (13).

In this communication, we report studies of the effects of PII, PII-UMP, glutamine, and α-ketoglutarate on adenylyltransferase in extracts from R. rubrum cells grown under different conditions.

MATERIALS AND METHODS

Purification of PII and PII-UMP.TheglnB gene was PCR amplified with designed primers, including a NdeI site at the 5′ end of the gene. The plasmid pMJET was constructed by using the NdeI and BamHI sites located downstream of glnB and cloning the product into the plasmid pET15b (Novagen). In this construct, the 5′ end ofglnB was fused in frame with a base sequence encoding a polyhistidine sequence at the 5′ end and a thrombin restriction protease site (Leu-Val-Pro-Arg-Gly-Ser) at the 3′ end. The construct was verified by sequencing.

The histidine-tagged PII was overexpressed in E. coli Bl21(DE3)pLysS in a rich medium containing 34 μg of ampicillin and 100 μg of chloramphenicol per ml. The induction of the T7-lac promoter was initiated by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of 0.6, and the culture was incubated at 30°C for 3 h at an OD600 of 1.2. To obtain the uridylylated form of PII, induction was made with 0.5 mM IPTG for 30 min. The culture was then washed three times with M9 minimal medium with NH4Cl omitted and incubated for 3 h at 30°C in minimal medium supplemented with 18 mM α-ketoglutarate, 1 mM methionine sulfoximine, and 1 mM UTP.

Cell extracts were produced and the protein was purified by Ni2+-chelating chromatography according to the manufacturer’s instructions (Novagen). The purified PIIprotein precipitated when the elution buffer, containing 1 M imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9), was removed. To overcome this problem, the histidine-rich sequence was removed by thrombin cleavage in the elution buffer, and the buffer was changed to 0.1 M Tris-HCl (pH 7.8). The purified protein was analyzed by sodium dodecyl sulfate–18% polyacrylamide gel electrophoresis (SDS–18% PAGE) and visualized by staining with Coomassie blue before and after digestion (15).

Preparation of extracts. R. rubrum S1 cultures were grown diazotrophically to an OD600 of 1.5 (13). Extracts were produced from cultures grown and treated in either of the following ways: (i) grown with N2 as N source (N2 grown), (ii) grown with N2, then switched off with 10 mM NH4Cl 30 min prior to harvest (switch off), or (iii) grown with N2, followed by nitrogen starvation under argon for 6 h prior to harvest (N starved). Cultures were harvested by centrifugation, washed once anaerobically in buffer A (1 mM dithiothreitol, 1 mM MnCl2, 100 mM Tris-HCl [pH 7.8]), and then frozen in liquid nitrogen in pellet form. Crude extracts were prepared anaerobically by osmotic shock (16). Unbroken cells were removed by centrifugation at 6,000 × g and the extract containing the soluble fraction and chromatophores was frozen in liquid nitrogen. In some experiments, chromatophores were removed by centrifugation at 100,000 × g for 90 min. Low-molecular-mass molecules were removed from this supernatant by gel filtration with PD10 columns (Pharmacia).

Enzyme assays.ATase activity was determined as the change in glutamine synthetase activity after incubation under different conditions. A 50-μl extract with a protein concentration of 13 mg/ml was mixed with effectors and diluted to a final volume of 100 μl. Ten-microliter samples were removed after 0, 10, 30 and 60 min, and glutamine synthetase activity was determined by the transferase assay described previously (13). In the inactivation (adenylylation) experiments, the mixture contained final concentrations of 2 mM ATP and 6 mM MgCl2. The activation (deadenylylation) mixtures instead contained 10 mM Pi. Final concentrations of effectors like glutamine, PII, PII-UMP, and α-ketoglutarate are given in the figure legends.

Immunoblotting of glutamine synthetase and PII.Extracts were analyzed by SDS–10% PAGE, and glutamine synthetase was detected by immunoblotting (28), with a polyclonal antibody raised against the purified protein. PII was analyzed by SDS–18% PAGE and detected with an antibody against a peptide identical to the N-terminal part of PII, as described previously (13).

RESULTS AND DISCUSSION

Purification of PII and PII-UMP.Extracts from 100 ml of E. coli Bl21(DE3)pLysS cells, harboring the pMJET plasmid, generated 2 mg of purified histidine-tagged PII protein after one Ni2+-chelating affinity column step. The pure protein, before and after digestion with thrombin, was analyzed by SDS–18% PAGE (Fig. 1A). The identity of the protein was also verified with our PII antibody (data not shown).

Fig. 1.
Fig. 1.

SDS-PAGE analysis of R. rubrumPII purified from E. coli. Lanes: A1, E. coli extract with induced PII; A2, purified histidine-tagged PII; A3, the same as A2, but with PII after thrombin digestion; B1, histidine-tagged PII-UMP; B2, mixture of histidine-tagged PIIand PII-UMP; B3, histidine-tagged PII. PII-UMP was purified from E. coli treated with glutamine, methionine sulfoximine, and UTP, as described in the text. M, molecular weight markers. His-tag, histidine-tagged.

Interestingly, we found that the E. coli UTase could catalyze uridylylation of the R. rubrum protein in vivo. InE. coli cultures incubated in M9 salts with NH4Cl omitted, a partially (approximately 50%) uridylylated PII could be demonstrated. To increase the signalling of nitrogen depletion to UTase (by increasing the α-ketoglutarate/glutamine ratio), α-ketoglutarate and the glutamine synthetase inhibitor methionine sulfoximine were added. However, this did not generate a totally modified PII, possibly due to the limited intracellular UTP levels. Addition of UTP to the cultures (probably taken up as a degradation product) generated a situation where essentially all of the PII was in the modified form, as shown by SDS–18% PAGE (Fig. 1B). The histidine tag could also be removed from PII-UMP by thrombin digestion, without any detectable effect on the modification state.

Effects on glutamine synthetase activity in extracts.Although some studies of the regulation of glutamine synthetase activity by reversible adenylylation in photosynthetic bacteria have been reported (11, 32), the role of PII and the effectors has not been clearly established. The importance of determining the involvement of PII, PII-UMP, and other effectors in anoxygenic photosynthetic bacteria is further emphasized by the situation in Azospirillum brasilense, where neither PII nor PZ is essential for the reversible adenylylation of glutamine synthetase, although the degree of adenylylation responds to the status of nitrogen in that organism (3). In R. rubrum, a glnB mutant would be preferable to fully investigate the in vivo functions of PII. Repeated attempts to generate glnB mutants have been unsuccessful, indicating that PII could be essential for growth. Therefore, we have now used an in vitro system to study the actions of PII, PII-UMP, and other effectors on the adenylylation and deadenylylation of glutamine synthetase. The methods are similar to those used for extracting cells from enteric bacteria (23, 24); however, no glutamine synthetase was added, since the level of enzyme activity was sufficient in the R. rubrum extracts.

The endogenous PII in the extracts used for this investigation was not removed, as we believe its effect can be negligible, compared to the amounts of PII and PII-UMP added. However, the modification status of endogenous PII in the extracts was also examined by SDS-PAGE and Western blotting, followed by evaluation with laser densitometry. In the extracts from N-starved cells, 50% of PII was uridylylated; in the extracts from N2-grown cultures, 10% of PII was uridylylated (data not shown). This was different from the in vivo situation, where we observed that PII was completely modified during these growth conditions (13). This difference can probably be explained by the presence of uridyl-removing activity during the preparation of the extracts. PII in the switch-off extracts was 100% unmodified. Importantly, no change in the state of modification of the endogenous PII in any of the extracts was observed during the incubations in this study.

Adenylylation of glutamine synthetase in extracts.Changes in glutamine synthetase activity were studied in extracts from three different growth conditions (Fig. 2). Adenylylation was dependent on the addition of ATP and MgCl2 and showed essentially the same characteristics in all three extracts used in the results depicted in Fig. 2, although the initial activity of glutamine synthetase was about half of that in the extract from switch-off cells. Adenylylation was stimulated by the addition of glutamine or by addition of (unmodified) PIIbut was totally inhibited by α-ketoglutarate; instead, a small increase in glutamine synthetase activity was observed. On the other hand, the addition of PII counteracted the effect of α-ketoglutarate. The addition of purified uridylylated PII had neither a stimulating nor an inhibiting effect on the adenylylation reaction in any extract examined (data not shown). The observation that no inactivation was obtained when ATP and MgCl2 were excluded from the extract is in agreement with the hypothesis that adenylylation is the cause of the lower activity of glutamine synthetase. Further support was obtained from SDS-PAGE and Western blotting of the incubation mixtures from the experiments shown in Fig. 2. In the extracts from N2-grown and N-starved cells, glutamine synthetase was essentially in the unadenylylated, faster-migrating form (Fig. 3, lanes A1 and B1). After incubation with glutamine and MgATP, the adenylylated, slower-migrating form could be demonstrated (Fig. 3, lanes A3 and B3). In the switch-off extract, both forms were present in about equal amounts; after incubation in the presence of glutamine and MgATP, the adenylylated form increased (Fig. 3, lanes C1 and C3). Previous reports have shown that glutamine synthetase from R. rubrum can be modified by adenylylation and ADP-ribosylation (32), but we found no effect on glutamine synthetase activity when ATP was replaced with NAD+, the donor of the ADP-ribose moiety, with or without the addition of glutamine or PII (data not shown).

Fig. 2.
Fig. 2.

Adenylylation of glutamine synthetase in extracts. Extracts were supplemented with 2 mM ATP, 6 mM MgCl2, and other effectors (final concentrations as follows). ⊞, no addition; ▵, 5 mM α-ketoglutarate; ■, 5 mM α-ketoglutarate and 0.6 μM PII; ◊, 0.6 μM PII;Embedded Image, 10 mM glutamine and incubated at 30°C. Extracts are from an N-starved culture (A), an N2-grown culture (B), and a switch-off culture (C). The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

Fig. 3.
Fig. 3.

The two forms of glutamine synthetase in extracts, shown by SDS–10% PAGE and Western blotting. (A) N-starved cells; (B) extract from N2-grown cells; (C) extract from switch-off cells. Lanes: 1, no addition; 2, Pi added and extract incubated at 30°C for 1 h; 3, glutamine, MgCl2, and ATP incubated at 30°C for 1 h. GS, unmodified glutamine synthetase; GS-AMP, adenylylated glutamine synthetase.

The extracts in these experiments contained chromatophores, probably having ATPase activity, which could deplete the level of ATP in the assays. This could explain why no further adenylylation was observed after 30 min of incubation. When additional ATP was added after 20 min, a further decrease in glutamine synthetase activity was observed. Only 28% of initial activity remained after 60 min, compared to 42% in the extract without the second addition of ATP. When the chromatophores and low-molecular-mass compounds were removed from the extracts, inactivation of glutamine synthetase was more rapid and the final activity after 1 h was 13% (data not shown). These experiments confirm that the decrease in the rate of inactivation after 30 min is due to depletion of the ATP added. However, as the removal of chromatophores by centrifugation led to a loss of the deadenylylation (activating) reaction (see below), we continued using extracts containing chromatophores in this investigation, although complete inactivation of glutamine synthetase was not obtained.

The effect of α-ketoglutarate on adenylylation was investigated in extracts from N2-grown cultures. As shown in Fig.4, 2.5 mM α-ketoglutarate was sufficient to totally inhibit adenylylation, and even in the presence of 1 mM α-ketoglutarate, the activity was very low. However, when increasing amounts of (unmodified) PII were included, the rate of inactivation increased (Fig. 5A). Furthermore, glutamine was also shown to counteract the effect of α-ketoglutarate (Fig. 5B). These results suggest that the intracellular levels of both α-ketoglutarate, PII, and glutamine play a role in the regulation of the adenylylation reaction in R. rubrum. This is similar to the situation in E. coli, where the unmodified PII and glutamine stimulate adenylylation by ATase (8, 22); recent reports have shown that binding of α-ketoglutarate and ATP to PII is required for interaction with its targets (14). The binding of small molecules to PII might be important as a sensor of the cellular nitrogen level (9, 10).

Fig. 4.
Fig. 4.

The effect of α-ketoglutarate on glutamine synthetase activity. The extract from a N2-grown culture was supplemented with 2 mM ATP, 6 mM MgCl2, and α-ketoglutarate (final concentrations as follows). ⊞, no addition;Embedded Image, 0.2 mM; □, 0.5 mM; ▵, 1 mM;Embedded Image, 2.5 mM; ▾, 5 mM. Extracts were incubated at 30°C. The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

Fig. 5.
Fig. 5.

The effects of PII and glutamine on glutamine synthetase activity. The extracts were supplemented by 2 mM ATP and 6 mM MgCl2. (A) Extract from N2-grown cells with 1 mM α-ketoglutarate and PII (final concentrations as follows). ⊞, no addition;Embedded Image, 0.09 μM; ■, 0.17 μM; ▴, 0.43 μM;Embedded Image, 0.85 μM; □, 1.31 μM; ▵, 1.97 μM; ◊, 2.56 μM. The boxed insert shows glutamine synthetase activity after 10 min at different PIIconcentrations. (B) Extract from N-starved cells with 2 mM α-ketoglutarate and glutamine (final concentrations as follows). ⊞, no addition; Embedded Image, 1 mM; ■, 5 mM; ▴, 10 mM;Embedded Image, 20 mM. The boxed insert shows glutamine synthetase activity after 10 min at different glutamine concentrations. Experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

Deadenylylation of glutamine synthetase in extracts.To study the deadenylylation (activation) of glutamine synthetase in R. rubrum, extracts from switch-off cultures were prepared and incubated in the presence of 10 mM phosphate. The addition of phosphate to the extracts was required, indicating that deadenylylation occurs as a phosphorylysis reaction, as in E. coli. The effects on deadenylylation by different effectors are shown in Fig.6. Interestingly, the reaction was much slower than the adenylylation, α-ketoglutarate had no stimulatory effect, and glutamine did not inhibit the reaction at the concentrations used. The addition of PII-UMP or PII-UMP together with α-ketoglutarate did not change the rate of activation. It should be emphasized that there is no endogenous PII-UMP in these extracts, as shown by SDS-PAGE and Western blotting.

Fig. 6.
Fig. 6.

Activation of glutamine synthetase in extracts. (A) Extract from switch-off culture supplemented by 10 mM Piand effectors (final concentrations as follows). ⊞, no addition; ▵, 10 mM α-ketoglutarate; ■, 10 mM α-ketoglutarate and 0.85 μM PII-UMP;Embedded Image, 0.85 μM PII-UMP; ◊, 10 mM glutamine. (B) Extract from an N2-grown culture supplemented by 2 mM ATP and 6 mM MgCl2. After 20 min, the extract was divided into tubes containing 10 mM Pi and other effectors as in (A). The experiments were carried out several times. The data are from one representative experiment with duplicate samples. Glutamine synthetase activity is given in micromoles of γ-glutamyl hydroxamate/minute · milligrams of protein.

Extracts from nitrogen-fixing cultures were also examined. After 20 min of adenylylation in the presence of MgATP, the sample was divided into different tubes containing phosphate and different effectors. A similar pattern was observed, i.e., no major difference was observed with any of the effectors added (Fig. 6B). Unmodified PII also did not have any effect (data not shown). In addition, the deadenylylation activity seems to be much more sensitive than the adenylylation activity; it was lost upon centrifugation of the extracts and could not be recovered in the supernatant, the pellet, or a combination of the two, although high adenylylation activity was still present in the supernatant. The native E. coli enzyme has a molecular mass of 115 kDa, but a protease fragment with the molecular mass of 70 kDa, containing only the adenylylation activity, has also been purified (7). We did not, however, observe any difference in the stability of the deadenylylation activity when protease inhibitors were included during extract preparation.

As shown by SDS-PAGE and Western blotting (Fig. 3), the increase in glutamine synthetase activity after incubation with phosphate coincided with a conversion of the slower- to the faster-migrating band, i.e., the adenylylated to the unmodified form.

Our results also show that the regulation of the deadenylylation reaction is very different from that in E. coli, where α-ketoglutarate stimulates activity and PII-UMP is absolutely required. Besides phosphate, which is required for the reaction, we have in fact not been able to find any compound that has an effect on the deadenylylation reaction.

In summary, we have presented results showing that the inactivation of glutamine synthetase by adenylylation is regulated by a process that is stimulated by glutamine and (unmodified) PII. Inactivation is inhibited by α-ketoglutarate. However, the activation of glutamine synthetase (deadenylylation) seems to be different from the situation in enteric bacteria, since it is affected by neither glutamine nor α-ketoglutarate and is independent of PII-UMP. The fact that PII-UMP does not effect deadenylylation is very interesting and might indicate that this reaction is regulated in a way similar to that in A. brasilense.

ACKNOWLEDGMENTS

This work was supported by grants to S.N. from the Swedish Natural Science Research Council.

FOOTNOTES

    • Received 9 March 1999.
    • Accepted 27 July 1999.

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