ADP-ribosylation of Rhizobium meliloti Glutamine Synthetase III in Vivo

doi:10.1074/jbc.270.4.1624

Volume 270, Number 4, Issue of January 27, 1995 pp. 1624-1628
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

ADP-ribosylation of Rhizobium meliloti Glutamine Synthetase III in Vivo(*)

(Received for publication, July 22, 1994; and in revised form, November 11, 1994)

Yuan Liu (1), (3), (§), Michael L. Kahn (1) (2)(¶)

From the (1)Institute of Biological Chemistry, Departments of (2)Microbiology and (3)Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-6340

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

The control of glutamine synthetase (GS), the first enzyme in the main pathway used by Rhizobium meliloti to assimilate ammonia, is central to cellular nitrogen metabolism. R. meliloti is unusual in having three distinct types of GS, including a unique GS, GSIII, that differs considerably from both GSI, which resembles other bacterial GS proteins and GSII, which resembles the GS found in eukaryotes. We show here that GSIII can be post-translationally modified in vivo by ADP-ribosylation at an arginine residue. PO (4) attached to GSIII during bacterial growth as part of the modifying group could be removed by treatment with snake venom phosphodiesterase or by turkey erythrocyte ADP-ribosylarginine hydrolase. Treatment of modified GSIII with hydroxylamine at neutral pH releases a chromophore that has the retention time of ADP-ribose when analyzed by reversed-phase high performance liquid chromatography. ADP-ribosylation inhibits GSIII activity.

INTRODUCTION

Symbiotic nitrogen fixation takes place in root nodules that form when roots of some legumes are infected by soil bacteria from the genera Rhizobium, Azorhizobium, or Bradyrhizobium. In symbiosis, the bacteria receive carbon compounds from the host plant, and instead of assimilating ammonia that they produce by nitrogen fixation, they release the ammonia to the host plant. This behavior differs from that of free-living, nitrogen-fixing bacteria, which tightly couple nitrogen fixation and ammonia assimilation. Ammonia assimilation in Rhizobium uses the glutamate synthase cycle, in which glutamine synthetase (GS) ()acts in conjunction with glutamate synthase to produce glutamate, at the expense of ATP and reducing power (Miflin and Lea, 1980). Glutamine and glutamate produced by these enzymes can be used in the synthesis of other nitrogen-containing compounds. Regulation of the GS-glutamate synthase pathway occurs primarily by changing the amount or specific activity of GS, and, in enteric bacteria, the control of GS synthesis and specific activity are central to the regulation of cell nitrogen metabolism (Reitzer and Magasanik, 1987).

Many rhizobia differ from enteric bacteria by having more than one GS (Darrow, 1980). Rhizobium meliloti has three distinct GS proteins (DeBruijn et al., 1989; Shatters et al., 1993), and although these GS isozymes are regulated in response to available nitrogen sources, to oxygen concentration, and to the presence of the other enzymes, the contribution of each isozyme to total GS activity is still poorly understood.

GSI resembles the single GS found in enteric bacteria in its structure, sequence, and regulation by post-translational modification (Darrow, 1980; Somerville and Kahn, 1983). Escherichia coli glutamine synthetase is a classic example of an enzyme whose activity is regulated post-translationally (Rhee et al., 1985; Stadtman et al., 1990). Each subunit of the dodecameric GS complex can be adenylylated at a single tyrosine near the carboxyl terminus, and the bound AMP inhibits the activity of that subunit. Adenylylation and deadenylylation of E. coli GS is carried out by the P protein, which in turn is regulated by proteins that ultimately are sensitive to the ratio of glutamine to alpha -ketoglutarate (Reitzer and Magasanik, 1987). This regulatory cascade also controls GS synthesis at the level of transcription by influencing the phosphorylation of NR (I) (NtrC), a transcriptional activator (Stock et al., 1989). There is evidence that Rhizobium GSI is also modified, probably by adenylylation (Darrow, 1980). GSI-type proteins in other bacteria are also modified by adenylylation (Woehle et al., 1990).

A second kind of modification, ADP-ribosylation, has recently been shown for Rhodospirillum rubrum GS, although the physiological importance of the modification is not yet clear (Woehle et al., 1990). ADP-ribosylation of proteins is common (Moss and Vaughn, 1990) and is involved in regulating enzyme activity, protein synthesis, cell proliferation, and both DNA and RNA metabolism. Arginine, cysteine, and glutamate residues can serve as ADP-ribose acceptors. Many ADP-ribosyl transferases are produced by parasites and are involved in disrupting host metabolism. For example, cholera toxin ADP-ribosylates an intestinal guanine nucleotide-binding regulatory protein (Fishman, 1990), activating an adenylate cyclase activity, disrupting ion transport, and ultimately leading to diarrhea. In bacteria, one of the best described modifications of a protein used within the cell is ADP-ribosylation of nitrogenase. Reversible ADP-ribosylation of a specific arginyl residue of the dinitrogenase reductase subunit inhibits nitrogenase activity when ammonia becomes available (Pope et al., 1985). Although E. coli GS is not ADP-ribosylated in vivo, Moss et al. (1990) have shown that the enzyme can be inactivated in vitro by ADP-ribosylation at arginine 172.

GSII, which is similar to eukaryotic GS enzymes in its structure and sequence (Carlson and Chelm, 1986; Shatters and Kahn, 1989), is regulated transcriptionally and possibly post-translationally (Manco et al., 1992). GSII enzymes are uncommon in bacteria and have been reported only in the plant associated bacteria, (Brady)rhizobium and Agrobacterium (Darrow and Knotts, 1977; Fuchs and Keister, 1980), and in actinomycetes, including Frankia, a genus that can carry out symbiotic nitrogen fixation with various plants (Edmands et al., 1987).

GSIII is a newly described type of GS (Espin et al., 1990, Shatters et al., 1993) that is abundant in R. meliloti mutants that lack GSI and GSII. Purified GSIII from R. meliloti has unusual kinetic properties, including a very low affinity for ammonium and glutamate and no transferase activity (Shatters et al., 1993). Analysis of the amino acid sequence of GSIII (Chiurazzi et al., 1992) ()shows that GSIII can be distinguished from both the GSI and GSII families of GS proteins but that amino acids that are highly conserved in comparisons between GSI and GSII proteins are also found in GSIII. Transcription of the glnT gene that encodes GSIII is regulated, and the amount of GSIII activity differs in different growth media. We report here that R. meliloti GSIII can be modified by ADP-ribosylation and that the modification appears to inactivate GSIII biosynthetic activity. Post-translational control could therefore play a role in regulating the contribution of GSIII to cellular glutamine synthesis.

MATERIALS AND METHODS

Strains and Media

E. coli strains MX727 glnA::Tn5 pro ilv thi recA and ET8051 Delta

(glnA-rha) have been described (Shatters et al., 1993). Plasmid pFB6162 is a chloramphenicol-resistant plasmid derived from pACYC184 that carries the glnT region (DeBruijn et al., 1989). WSU650 is a glnA glnII mutant of R. meliloti 104A14 (Somerville et al., 1989). M-9, minimal mannitol (MM), LB, and YMB media were prepared as described by Shatters et al.(1993). For

labeling, M-9 and MM media were modified by replacing their phosphate buffer with 10 mM MOPS (pH 6.8). Approximately 10 µM

was added by inoculating these media (1:5000) with cells that had been grown overnight at 30 °C in conventional medium containing glutamate and ammonium. LB medium was supplemented with chloramphenicol at 100 µg/ml.

Enzymes and Chemicals

Snake venom phosphodiesterase was purchased from Sigma. Alkaline phosphatase and calf spleen phosphodiesterase were purchased from Boehringer Mannheim. H (3)

32PO

was from DuPont-NEN. Other chemicals were from Sigma unless otherwise stated.

Analytical Methods

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done as described by Laemmli(1970). Protein concentration was determined using a modified Bradford assay (Bio-Rad).

Immunological Methods

Anti-GSIII antiserum was described in Shatters et al.(1993). Western blotting was performed according to Sambrook et al.(1989). The chemiluminescent substrate (ECL detection reagent) was purchased from Amersham Corp. Pre-stained molecular weight standards were from Life Technologies, Inc.

Enzyme Assays

The semi-biosynthetic assay for GSIII was described by Shatters et al.(1993). Units are defined as nanomoles of

-glutamylhydroxamate formed per min and were normalized to total cellular protein. Cultures were started by inoculating the appropriate medium to a final absorbance of 0.05 at 600 nm with a stationary culture grown in the same medium and were grown to mid-log phase. One-ml samples were centrifuged to harvest the cells, washed in 10 mM Tris, pH 7.5, then recentrifuged and resuspended in 50 µl of 10 mM Tris buffer. Cells were broken by freezing at -80 °C and thawing at 44 °C three times. Reactions were run at 42 °C for 30 min. To prepare large amounts of cell extract, cells were centrifuged at 4,000 times

g for 8 min and washed with Tris buffer. Cells were then sonicated for 8 min (four 2-min sonications interrupted by chilling on ice) and centrifuged at 15,000 times

g for 15 min at 4 °C. The supernatant was used for further experiments.

Immunoprecipitation of P-labeled GSIII

WSU650 was grown in MM medium containing glutamate and ammonium and modified by replacing the phosphate salts with 10 mM MOPS. Two mCi of H (3)

32PO

were added to a 250-ml culture, and the cells were harvested at stationary phase (3-4 days). After washing the cells in 10 mM Tris, pH 7.5, they were broken by sonication, and the lysate was centrifuged. Anti-GSIII antiserum was added to the supernatant, and the mixture was incubated overnight at 4 °C. Protein A-Sepharose that had been prewashed with 10 mM Tris buffer, pH 8.0, was added, and the mixture was incubated for 4 h at 4 °C. The complex of protein A-Sepharose, antibody, and GSIII was recovered by centrifugation at 10,000 times

g for 2 min and washed 5 times with 10 mM Tris buffer, pH 8.0. Proteins were released from the protein A-Sepharose by incubating with 100 mM glycine, pH 2.6. After removing the protein A-Sepharose by centrifugation, the pH of the supernatant was adjusted by adding 1 M Tris, pH 8.0, and the samples were lyophilized.

Enzyme Treatment of P-Labeled GSIII

Immunoprecipitated

P-labeled GSIII was treated with snake venom phosphodiesterase, alkaline phosphatase, or spleen phosphodiesterase for 1 h at 37 °C in the presence of 50 mM MOPS (pH 8.6) and 10 mM Mg

. The reaction was terminated by adding 0.33 volume of 100% (w/v) trichloroacetic acid, and the precipitated protein was collected by centrifugation. The precipitates were washed with 300 µl of ether then solubilized in SDS-gel sample buffer (15% glycerol, 5% beta

-mercaptoethanol, 2.3% SDS, 0.001% bromphenol blue, 62.5 mM Tris-HCl, pH 6.8), subjected to 12% SDS-polyacrylamide gel electrophoresis, and analyzed by autoradiography. The trichloroacetic acid-soluble products of snake venom phosphodiesterase treatment were applied to a polyethyleneimine-cellulose F plate (EM Science), and ascending chromatography was conducted using 1 M ammonium formate, pH 3.5 (Bochner et al., 1981). Following chromatography, the plates were soaked in methanol for 5 min to remove residual salts and then dried at room temperature. Phosphate and nucleotides run in adjacent lanes were detected using the spray reagent described by Bochner et al.(1981).

Phosphoamino Acid Analysis

Phosphoamino acid analysis using two-dimensional-TLC was performed according to Farmer et al. (1991). Immunoprecipitated GSIII was hydrolyzed in 200 µl of 6 N HCl at 110 °C for 3 h in a tightly sealed tube. Hydrolysates were dried in a vacuum and then dissolved in 5 µl of acetic acid/formic acid/H (2)

O (78:25:897) containing 1 µg/ml O-phospho-L-serine, O-phospho-L-threonine, and O-phospho-L-tyrosine as nonradioactive standards. Two-dimensional separation of phosphoamino acids by electrophoresis and solvent chromatography was carried out according to Cooper et al.(1983) on 160-µm plastic-backed cellulose sheets (Eastman Kodak Co. 13255). The plates were then air-dried for 3 h, sprayed with ninhydrin (0.2% in acetone) to detect amino acid standards, and subsequently exposed to Kodak XAR film.

Chemical Treatment of P-Labeled GSIII

The sensitivity of the modification to various chemical treatments followed procedures described by Cervantes-Laurean et al.(1993). Immunoprecipitated

P-labeled GSIII was solubilized in 200 µl of 50 mM NaOAc, pH 6.0. 50-µl aliquots of resuspended GSIII protein were treated at 37 °C for 1 h with 50 µl of water, 0.2 M HCl, or 2 mM Hg(OAc) (2)

, or for 3 h with 50 µl of 2 M NH (2)

OH, pH 7.0. The reactions were stopped by adding 25 µl of 100% (w/v) trichloroacetic acid, and the samples were incubated on ice for 15 min. After centrifugation at 10,000 times

g for 10 min at 4 °C, the precipitated proteins were washed with 300 µl of ether, dissolved in 50 µl of 2XTSB (2 M urea, 5% SDS, 20 mM Tris-HCl, pH 8.0, 162 mM dithiothreitol, 0.05% bromphenol blue), boiled for 10 min, and loaded onto a 12% SDS-PAGE gel. After electrophoresis, the gel was dried and analyzed by autoradiography.

To prepare supernatants from the trichloroacetic acid precipitation for HPLC analysis, 250 mM ammonium acetate, 10 mM EDTA, pH 9.0, was added to the supernatant to give a final volume of 5 ml. Each sample was loaded onto a 0.5 ml of dehydroboronyl-Bio-Rex 70 column (obtained from Dr. Michael Jacobson, University of Kentucky) and washed with 10 ml of 250 mM ammonium acetate, 10 mM EDTA, 1 M guanidine, pH 9.0, and 10 ml of 250 mM ammonium acetate, 10 mM EDTA. Samples were eluted with 1 ml of H (2) O, and the volume was reduced to approximately 200 µl in a vacuum centrifuge (Savant). Each sample was then analyzed by reversed-phase HPLC on a 3.9 times 150-mm Nova-Pak C18 column with 100 mM KH (2) PO (4) , pH 6.0, 2% methanol eluent at 0.5 ml/min flow rate. Reaction products were detected by UV A. Standards were run under the same conditions.

Partial Purification of GSIII Protein

In some experiments, an abbreviated version of the GSIII purification protocol of Shatters et al.(1993) was used to enrich the GSIII protein prior to treatment with ADP-ribosylarginine hydrolase. WSU650 was grown in YMB at 30 °C, and ET8051 (pFB6162) and MX727 (pFB6162) were grown in LB at 37 °C. Cells from 250-ml cultures were harvested at stationary phase, washed in IMG buffer (20 mM imidazole, 1 mM MgCl (2)

), pH 7.4, and broken by sonication. The crude extract was treated with DNase (15 µg/ml) and RNase (15 µg/ml) at room temperature for 30 min and then incubated at 50 °C for 5 min. The cell extract was centrifuged at 10,000 rpm in a microcentrifuge for 15 min at 4 °C to remove debris. 29% (NH (4)

)

(w/v) was added to the supernatant, and the (NH (4)

)

precipitate was collected at 4 °C by centrifugation at 10,000 rpm. The precipitate was resuspended in IMG buffer, pH 6.8, dialyzed against IMG buffer at pH 6.8, and loaded onto a 10-ml Affi-gel blue (Bio-Rad) column that had been pre-equilibrated with IMG buffer, pH 6.8. GSIII was eluted using a pH step gradient from pH 6.8 to 7.6 (3 ml each). GSIII is released from the column at pH 7.2 and pH 7.3, and its purity was examined on a 12% SDS-PAGE gel. In the preparation from WSU650, only the GSIII band was prominent, accounting for about 75% of the protein. The two E. coli preparations were not as clean, with the GSIII band accounting for 25-50% of the total protein.

ADP-ribosylarginine Hydrolase Treatment

Turkey erythrocyte ADP-ribosylarginine hydrolase (ARH) (Moss et al. 1990) was a gift from Dr. Joel Moss. Assays were conducted according to Moss et al.(1990). 50 µl of 2 times

reaction buffer (100 mM potassium phosphate, pH 7.5, 10 mM dithiothreitol, 20 mM MgCl (2)

, and 0.2 µg of ovalbumin) was added to 49 µl of partially purified protein or crude cell extract. 1 µl of ARH (0.18 µg/µl) or 1 µl of water was added to start the reaction. Following incubation at 37 °C, the biosynthetic activity of GSIII was measured. Data were normalized to total protein concentration, and the ratio of the activities of treated and untreated samples was determined.

RESULTS

GSIII Is Labeled by PO in Vivo

Purified GSIII contains two related proteins that run at 46.5 and 49 kDa (Shatters et al., 1993), and we thought it possible that these represented post-translationally modified forms of the same protein. Since many potential modifications (phosphorylation, nucleotidylylation, ADP-ribosylation) contain phosphorus, R. meliloti WSU650 was grown in the presence of

, and GSIII was precipitated from cell lysates using GSIII-specific antiserum (Shatters et al., 1993) and protein A-Sepharose (Sambrook et al., 1989). GSIII was released from protein A-Sepharose using low pH. SDS-polyacrylamide gel electrophoresis of the released protein revealed one Coomassie Blue-stained band that corresponded to purified GSIII (molecular mass = 46-49 kDa) together with the 50-kDa IgG heavy chain. No other bands were detected. Autoradiography showed that this band was radioactively labeled (Fig. 1, lane A).

Figure 1: PO (4) is associated with GSIII in vivo and can be removed by snake venom phosphodiesterase in vitro. GSIII protein labeled in vivo was immunoprecipitated with GSIII-specific antibody and protein A-Sepharose. Protein released from the protein A at low pH was treated with various enzymes and analyzed by SDS-PAGE and autoradiography. P-Labeled GSIII was treated with water (lane A), alkaline phosphatase (lane B), snake venom phosphodiesterase (lane C), calf spleen phosphodiesterase (lane D), and both alkaline phosphatase and snake venom phosphodiesterase (lane E).

Enzymatic Treatment of PO-Labeled GSIII

To determine the type of linkage between the phosphate and GSIII, immunoprecipitated,

-labeled GSIII was treated with snake venom phosphodiesterase, calf spleen phosphodiesterase, or bacterial alkaline phosphatase. The protein was then analyzed by SDS-PAGE (Fig. 1). All radioactive label was removed from GSIII by snake venom phosphodiesterase but alkaline phosphatase and spleen phosphodiesterase had no effect. The venom phosphodiesterase result indicates that the label is associated with a phosphate diester on the 5` side of a nucleoside. The alkaline phosphatase and spleen phosphodiesterase results suggest that the modification is not a phosphate monoester or a 3` nucleoside diester or that it is not accessible to the hydrolytic enzyme. The acid-soluble fraction produced by treatment with snake venom phosphodiesterase was analyzed by TLC (Fig. 2).

and [

P]AMP were identified as possible products, suggesting that the modification could be either AMP or ADP-ribose. Since the chromatographic behavior on TLC is largely determined by the number of phosphates on the nucleotide, it is also possible that another base might be present.

Figure 2: The modification of GSIII may be AMP or ADP-ribose. P-Labeled GSIII was treated with snake venom phosphodiesterase, and the trichloroacetic acid-soluble products were analyzed by thin layer chromatography as described. The positions of unlabeled ATP, ADP, AMP, and PO (4) standards in adjacent lanes are indicated.

Chemical Nature of P Label on GSIII

If GSIII was phosphorylated at tyrosine, serine, or threonine, treatment of

P-labeled immunoprecipitated GSIII with 6 N HCl would hydrolyze the peptide bonds and release the phosphorylated derivatives of these amino acids (Cooper et al., 1983). Hydrolysis products were resolved by two-dimensional thin layer chromatography. Spots corresponding to phosphate and unknown radiolabeled compounds were detected, but the mobilities of the labeled hydrolysis products were different from those of phosphotyrosine, phosphoserine, and phosphothreonine standards (data not shown). Therefore,

was not originally attached to tyrosine, serine, or threonine as a phosphate monoester. These hydrolysis conditions also convert the tyrosine-AMP in adenylylated E. coli GS to phosphotyrosine (Foster et al., 1989), so the lack of phosphotyrosine also suggests that the label was not present in tyrosine-AMP.

Immunoprecipitated P-labeled GSIII protein was subjected to chemical treatments known to distinguish ADP-ribose linked to various amino acids, and the released products were analyzed by SDS-PAGE and reversed-phase HPLC. HCl treatment can release P-label in ADP-ribose that is linked to glutamate or aspartate, Hg treatment can release P-label in ADP-ribose from cysteine and hydroxylamine (NH (2) OH), pH 7.0, treatment can release P-label in ADP-ribose from arginine (Cervantes-Laurean et al., 1993). NH (2) OH treatment completely removed P-label from immunprecipitated GSIII (data not shown), but the other treatments had no effect. This suggests that the modification was at an arginine residue. HPLC analysis of the trichloroacetic acid-soluble products produced by HCl, Hg, or NH (2) OH treatment of labeled GSIII revealed that only the NH (2) OH treatment produced a new compound (Fig. 3). One product of NH (2) OH treatment had a retention time (5.68) similar to that of an ADP-ribose standard (5.72) suggesting that GSIII was modified by ADP-ribose. The additional peak seen in the HPLC profile of the NH (2) OH treated sample was also observed when an ADP-ribose standard was treated with NH (2) OH. We suggest that this second peak is an ADP-ribose derivative (probably inosine diphosphate ribose) created during the NH (2) OH treatment.

Figure 3: Trichloroacetic acid-soluble products of chemical treatments. Soluble products released from GSIII by treatment with (A) water, (B) 0.1 M HCl, (C) 1 mM Hg(OAc) (2) , or (D) 1 M NH (2) OH (pH 7.0). Each sample was analyzed by reversed-phase HPLC on a 3.9 times 150-mm Nova-Pak C18 column with 100 mM KH (2) PO (4) , pH 6.0, 2% methanol eluent at 0.5 ml/min flow rate. Reaction products were detected by UV A. The mobilities of AMP and ADP-ribose standards are shown on the left.

Inhibition of GSIII by ADP-ribosylation

We have reported that the glutamine auxotrophy of ET8051, a glnA ntrB ntrC deletion mutant of E. coli, could not be complemented on M-9 medium in the presence of plasmid pFB6162, which carries the R. meliloti 1021 glnT region that encodes GSIII (Shatters et al., 1993). But when we used MX727, a glnA::Tn5 mutant, as the host strain for pFB6162, cells could be rescued on the M-9 medium, and a higher GSIII activity was found in the cells grown in LB medium. We investigated the amount of GSIII protein present in these two strains using GSIII-specific antiserum (Fig. 4). Several proteins from E. coli ET8051 react with GSIII antiserum, but E. coli ET8051 (pFB6162) contains much more antibody-reactive material with the molecular weight of GSIII than E. coli MX727 (pFB6162) does. The total GS activity of these extracts was measured and included in Fig. 4. Taken together, the Western blot and activity data show that the specific activity of GSIII in ET8051 (pFB6162) is much lower than in MX727 (pFB6162). The specific activity of GSIII in WSU650, the mutant of R. meliloti from which GSIII was purified, is more similar to that in ET8051 (pFB6162). The two extra bands from WSU650 that cross-react with GSIII antiserum were not labeled.

Figure 4: GSIII protein in various strains. WSU650, ET8051 (pFB6162), and MX727 (pFB6162) were grown in the media indicated. Lysates were prepared and GSIII enzymatic activities were measured. Proteins were separated by SDS-PAGE, and a nitrocellulose blot of the gel was analyzed using anti-GSIII serum. The molecular mass of protein standards are indicated.

Does modification activate or inactivate GSIII? We reasoned that if ADP-ribosylated GSIII was the active form, ET8051 (pFB6162) and MX727 (pFB6162) would have comparable amounts of radioactivity when labeled with PO (4) , but that if ADP-ribosylated GSIII was the inactive form, there should be much more radioactivity in GSIII from ET8051 (pFB6162) since a smaller fraction of this protein appears to be active. When both strains were grown in PO (4) , the immunoprecipitated GSIII from ET8051 (pFB6162) contained much more P-label than MX727 (pFB6162) at a molecular weight that corresponds to GSIII. These results suggest that GSIII in ET8051 (pFB6162) was mostly in modified form and the modified GSIII is less active.

ADP-ribosylarginine Hydrolase Treatment

To test directly the hypothesis that ADP-ribosylation of GSIII was inhibitory, we used turkey erythrocyte ARH (Moss et al., 1990), an enzyme that specifically removes ADP-ribose from ADP-ribosylarginine. ARH was incubated with partially purified GSIII and GSIII activity was measured (Fig. 5). In some early time points, the absolute activity of GSIII increases, but because GSIII was unstable under the conditions used to carry out the ARH reaction, the ratio of GS activities in the presence and absence of ARH was calculated. In addition to controls in which ARH was omitted, control reactions were also carried out in the presence of glycerol, a stabilizing agent added with the ARH, and the results were similar to those shown in Fig. 5. The ratio of GSIII activities increased by approximately 3-fold when partially purified GSIII from WSU650 grown in YMB, or from ET8051 (pFB6162) and MX727 (pFB6162) grown in LB were treated with ARH (Fig. 5). Similar treatment of crude extracts from WSU650 grown in YMB or minimal mannitol medium with glutamate and ammonium increased the relative GSIII activities 1.5-2.0-fold (data not shown). The increase in GS activity when GSIII was treated with ARH strongly supports the conclusions that GSIII was ADP-ribosylated at an arginine residue and that the modification inhibits GSIII activity.

Figure 5: ADP-ribosylarginine hydrolase treatment of GSIII. Partially purified GSIII proteins were treated with ARH as described then analyzed for GS activity as a function of time of incubation with ARH. The ordinate is the ratio of GS activity in samples incubated with ARH divided by the activity in samples treated with ARH buffer.

DISCUSSION

GSIII from R. meliloti is an unusual GS that is found in a glnA glnII double mutant of R. meliloti 104A14. The discovery that GSIII could be labeled in vivo with [P]phosphate led us to investigate the form of this post-translational modification and its role in GSIII regulation. Our results suggest that ADP-ribosylation of GSIII at an arginine inhibits GS activity. Two lines of evidence lead us to conclude that the modifying group is ADP-ribose, the release of a compound with the mobility of ADP-ribose from hydroxylamine-treated protein and the sensitivity of the protein to ARH. The sensitivity of the modification to ARH and to hydroxylamine but not to other chemical treatments suggests that arginine is the modified amino acid. Arginine-specific ADP-ribosylation of proteins is widely distributed in bacterial viruses and prokaryotic and eukaryotic cells (Moss and Vaughan, 1990) and is often involved in post-translational modification of enzyme activity.

In R. meliloti, we have found GSIII only in a glnA glnII mutant that lacks both GSI and GSII, but large amounts of GSIII protein can be detected in this strain (Shatters et al., 1993). GSIII activity in these cells is different in different growth media, and both transcriptional and post-translational regulation are implicated in controlling GSIII activity. Expression of GSIII protein requires a functional NtrA protein, and transcription of the glnT region of R. meliloti 1021 is strongly dependent on the NtrC protein (DeBruijn et al., 1989), suggesting that glnT is transcriptionally regulated by the nitrogen regulatory system. The results presented here showing that GSIII can be inhibited by ADP-ribosylation suggest that GSIII activity can also be controlled post-translationally.

Woehle et al.(1990) reported that GS in R. rubrum can be adenylylated or ADP-ribosylated in vivo, but the physiological significance of ADP-ribosylation was not established. Although the DNA sequence of R. meliloti GSIII predicts a tyrosine near the site of the tyrosine modified by adenylylation of GSI-type enzymes, we do not detect the phosphotyrosine that would be expected after acid hydrolysis of tyrosine-AMP (Foster et al., 1989). The growth rate of WSU650, the strain from which GSIII was taken for this analysis, is still dependent on both glutamate and ammonium concentrations (Shatters et al., 1993), so the mutant could be nitrogen-limited. Under these circumstances, adenylylation may not be favored, and we cannot eliminate the possibility that GSIII is also adenylylated under some conditions.

Based on a comparison of the specific activities of GSIII in WSU650, ET8051 (pFB6162), and MX727 (pFB6162), it appeared that the GSIII in WSU650 was not as active as it could have been. It may be that ADP-ribosylation of GSIII is not responding directly to a measure of the cell's nitrogen status or that GSIII modification occurs as the cells are being harvested and growth is limited.

Ammonium assimilation and its regulation are very different in R. meliloti and E. coli. The most obvious difference is that there are three glutamine synthetases in R. meliloti while E. coli has only one. The three types of GS in Rhizobium are regulated differently and may serve different purposes in the cell. GSI, the glnA gene product, is post-translationally modified by adenylylation; GSII, the glnII gene product, is transcriptionally regulated by the nitrogen regulatory system and there may be some post-translational regulation of this enzyme (Manco et al., 1992). glnT is apparently also transcriptionally regulated by the nitrogen regulatory system (Shatters et al., 1993) and we have shown here that its product, GSIII, can be inhibited by ADP-ribosylation. The complexity of these regulatory mechanisms may enable the cell to respond to different growth conditions and illustrates the importance of glutamine as a crucial biosynthetic precursor and ammonium donor for cellular nitrogen metabolism.

FOOTNOTES

*: This work was supported by grants from the United States Department of Agriculture-National Research Initiative-Competitive Research Grants Office, a Loyal H. Davis fellowship, and by funds provided by the Agriculture Research Center at Washington State University. 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.
§: Current address: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111.
¶: To whom correspondence should be addressed. Tel.: 509-335-8327; Fax: 509-335-7643.
(): The abbreviations used are: GS, glutamine synthetase; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; ARH, ADP-ribosylarginine hydrolase; HPLC, high performance liquid chromatography.
(): Y. Liu and M. L. Kahn, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Dr. Joel Moss (National Institutes of Health) for providing us with ADP-ribosylarginine hydrolase. We also thank Dr. Michael Jacobson and Donna Coyle (Division of Medicinal Chemistry and Pharmacology, College of Pharmacy, University of Kentucky) for their advice on chemical treatment of ADP-ribosylated proteins and for the dehydroboronyl-Bio-Rex 70 resin, and Dr. Edward Farmer for helpful suggestions.

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