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Regulation of Glutamine Synthetase Activity Byadenylylationin the Gram-Positive Bacterium Streptomyces Cattleya

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Regulation of Glutamine Synthetase Activity Byadenylylationin the Gram-Positive Bacterium Streptomyces Cattleya Proc. NatL Acad. Sci. USA Vol. 78, No. 1, pp. 229-233, January 1981 Biochemistry Regulation ofglutamine synthetase activity by adenylylation in the Gram-positive bacterium Streptomyces cattleya (nitrogen metabolism/covalent modification ofproteins) STANLEY L. STREICHER AND BONNIE TYLER Merck Sharp & Dohme Research Laboratories, Merck & Company, P.O. Box 2000, Rahway, New Jersey 07065 Communicated by Boris Magasanik, October 9, 1980 ABSTRACT The enzymatic activity of glutamine synthetase bacterium has not been reported previously. [A preliminary re- [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] port ofthis work has been presented (12).] from the Gram-positive bacterium Streptomyces cattleya is regu- lated by covalent modification. In whole cells containing high lev- els of GS the addition of ammonium chloride leads to a rapid de- MATERIALS AND METHODS cline in GS activity. Crude extracts prepared from such ammonia- shocked cells had very low levels ofGS activity as measured by bio- Bacterial Strain and Culture Conditions. For all experi- synthetic and y-glutamyltransferase assays. Incubation of the ments the original soil isolate ofS. cattleya was used (11). Cells crude extracts with snake venom phosphodiesterase restored GS were grown in a mineral salts medium (D medium) supple- activity. In cell extracts, GS was also inactivated by an ATP- and mented with 1% glucose and 20 mM sodium glutamate. D me- glutamine-dependent reaction. Radioactive labeling studies dem- dium contains, per liter; 0.3 g ofK2HPO4, 0.5 g ofNaCl, 0.5 g of onstrated the incorporation of an AMP moiety into GS protein upon modification. Our results suggest a covalent modification of MgSO4-7H2O, 19.5 g of 2-(N-morpholino)ethansulfonic acid GS in a Gram-positive bacterium. This modification appears to be (Mes) at pH 7, 10 mg of CoCl2, 25 mg of FeSO4-7H2O, and 10 adenylylation of the GS subunit similar to that found in the Gram- mg ofZnSO4-7H20. Spores were inoculated into the medium at negative bacteria. aconcentration of10' per ml and incubated at 370C with shaking for24-36 hr. Cells were harvested by centrifugation orfiltration Glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP- and stored at -800C. forming), EC 6.3.1.2] is responsible for the synthesis of gluta- Preparation of Crude Extracts. Frozen cells were resus- mine from glutamic acid and ammonia: pended in cold buffer A (20 mM imidazole-HCl, pH 7.5/1 mM MnCl2) ataconcentration of0.2-0.5 g ofcells per ml. Cells were glutamate + NH3 + ATP -- glutamine + ADP + Pi. disrupted by intermittent sonic oscillation for a total time of 1 The enzyme occupies a central position in nitrogen metabolism min. Debris was removed by centrifugation for 30 min at because the amide nitrogen ofglutamine is used for the synthe- 20,000 X g. Extracts were stored at either 00C or -80'C. Fro- sis ofmany metabolites (1). In the enteric bacteria such as Esch- zen extracts maintained GS activity for several months. erichia coli, the ability ofthe cell to synthesize glutamine is reg- GS Assays. The y-glutamyltransferase assay was performed ulated at two levels. The amount of GS protein in the cell is essentially as described by Bender et al. (13) at pH 6.9. The "for- regulated at the level oftranscription ofthe glnA gene (2). The ward" biosynthetic assay, which measures the formation of y- rate ofglnA transcription is inversely proportional to the avail- glutamylhydroxamate from glutamate, hydroxylamine, and ATP ability ofnitrogen (3). The ability ofthe GS protein to synthesize was performed at pH 7.3 as described (13). The "radioactive" glutamine is controlled through covalent modification. The ad- biosynthetic assay measuring the formation of [3H]glutamine dition and removal ofan AMP moiety (adenylylation/deadenyl- from [3H]glutamate, NH3, and ATP was performed at pH 7.3 as ylation) alters the biosynthetic activity of the enzyme (4, 5). described (14). One enzyme unit (U) equals 1 ,umol of y-gluta- High levels of adenylylation and low biosynthetic activity are mylhydroxamate or [3H]glutamine formed per min. normally found when nitrogen is in excess and the converse is Adenylylation Reaction. Modification ofGS in crude extracts found when nitrogen is limiting (3-5). In several other Gram- was performed as described by Foor et al. (15). Where indicated negative bacteria GS activity is also regulated through adenylyl- the concentrations of ATP and glutamine were altered. Reac- ation (3, 6, 7). This is in contrast to the situation in the Gram- tions were started by addition ofcrude extract to prewarmed re- positive bacilli. In both Bacillus subtilis (8) and B. stearother- action mixture. At indicated times samples were removed and mophilis (9, 10), from which GS has been purified and exam- added to chilled GS assay mixtures. ined, no evidence was obtained to suggest covalent modifica- Snake Venom Phosphodiesterase (SVPDE) Treatment. Ex- tion. tracts were incubated at 37°C with SVPDE (Boehringer Man- We have been investigating nitrogen metabolism in the nheim) at 100 ,ug/ml and samples were removed for analysis as Gram-positive filamentous spore-forming bacterium Strepto- described in the text. myces cattleya (11) and have initially examined the GS in this Radioactive Labeling Studies. Cells were labeled with inor- organism. We describe here results demonstrating that the ac- ganic [32P]phosphate in minimal medium modified to contain 1/ tivity of GS in S. cattleya is regulated through a covalent modi- 10th the usual concentration ofphosphate. When the cell den- fication that appears to be similar to that found in the enteric sity reached 100 Klett units (no. 54 filter), 0.25 mCi (1 Ci = 3.7 bacteria. We believe that adenylylation ofGS in a Gram-positive x 10'0 becquerels) of32pi was added to 100 ml ofculture. After 15 min, 50 ml of the culture was harvested and the remainder The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: GS, glutamine synthetase; SVPDE, snake venom phos- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. phodiesterase; U, enzyme unit. 229 Downloaded by guest on October 1, 2021 230 Biochemistry: Streicher and Tyler Proc. Natl. Acad. Sci. USA 78 (1981) received ammonium chloride (20 mM). After an additional 15 min these cells were harvested. GS present in crude extracts *100 was labeled by performing the standard adenylylation reaction )-. 8 0 in the presence of either [a-32P]ATP or [adenine-3H]ATP (0.25 mCi per 3-ml reaction mixture) for 30 min. Gel Electrophoresis. Two-dimensional gel electrophoresis 80 o-~~0 was carried out by using the procedures of O'Farrell (16). Pro- teins were visualized with either Coomassie blue staining or au- 70 toradiography, using standard procedures. was Fluorography 0 60 done with En3Hance (New England Nuclear). NaDodSO4 gels 6ox were run by using the procedure of Bender and Streicher (17), 0- 50 L1 and nondenaturing gels by the method ofLudwig (18). 0 Protein Determination. Protein concentration was deter- 40 mined by the procedure of Lowry et al. (19) or by the Bio-Rad dye binding method (according to the supplier's instructions). 30 Chemicals. All reagents were ofthe highest quality commer- /00n - - - .S -0 cially available. Radioactive compounds were obtained from 20 0' ~ M New England Nuclear. 100o- ... - _ RESULTS 0o I I Modification of S. cattleya GS in Whole Cells. Crude ex- 0 10 20 30 40 50 60 70 80 90 tracts ofS. cattleya grown in a minimal salts medium containing Incubation Time (min) FIG. 2. Effect of SVPDE on GS transferase activity in crude ex- tractsofammonia-shocked andunshockedcells. Aculture ofS. cattleya cells growing in glucose/glutamate minimal medium was split intotwo parts. One part was harvested while the other received ammonium chloride (to 20 mM). After a 30-min "ammonia shock"periodthese cells were harvested. Crude extracts were prepared and samples were treated with SVPDE. At the indicated time points 5-Al samples were removed and added to cold transferase assay mixture. Samples were assayed at 37"C for 10 min. Ammonia-shocked extract: m, no SVPDE; n, with SVPDE. Unshocked control extract: 0, no SVPDE; o, with SVPDE. glucose and glutamate as the sources of carbon and nitrogen have high levels ofGS as determined by the y-glutamyltransfer- ase assay (about 7-10' U/mg of protein). Upon the addition of 60 ammonium chloride to a mid-logarithmic culture we observed 0-) a rapid decline in whole cell transferase activity (Fig. 1) to a level 600 about 1/7th that of the initial one. Crude extracts of these am- monia-shocked cells also had a lower level oftransferase activity , 50 compared to the control extracts (about 1 U/mg ofprotein) and had a much lower level offorward biosynthetic activity. These results suggested that modification of GS occurred in response 40 to the addition of ammonium chloride. In Gram-negative bac- teria such as E. coli addition of ammonium chloride to a nitro- gen-limited culture results in covalent modification of GS by 30 0- adenylylation (4). Adenylylation decreases the biosynthetic ac- tivity ofE. coli GS and alters the pH profile for transferase activ- ity (4, 5). In the yeast Candida utilis ammonia shock inactivates 20 GS by causing the conversion of native octameric GS into less active tetramers and then into inactive monomers (20). 10-~~~~~~~~ We initially characterized the modification of S. cattleya GS by examining the pH profile and divalent metal requirements of 10 extracts from active and ammonia-shocked cells. Although the amount of transferase activity was low in extracts of ammonia- -5 0 5 10 15 shocked cells, the pH profile was essentially the same as that of the control extract.* Similarly there was no effect of adding Time (min) Mg2e (60 mM) to the transferase reaction with either extract.
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